AFAB-Volume2-Issue3

Volume 2, Issue 32012

ISSN: 2159-8967www.AFABjournal.com

158 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 3 - 2012 159

Sooyoun Ahn University of Florida, USA

Walid Q. AlaliUniversity of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Claudia S. Dunkley University of Georgia, USA

Lawrence GoodridgeColorado State University, USA

Leluo GuanUniversity of Alberta, Canada

Joshua GurtlerERRC, USDA-ARS, USA

Yong D. HangCornell University, USA

Divya JaroniOklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael JohnsonUniversity of Arkansas, USA

Timothy KellyEast Carolina University, USA

William R. KenealyMascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

W.K. KimUniversity of Manitoba, Canada

M.B. KirkhamKansas State University, USA

Todd KostmanUniversity of Wisconsin, Oshkosh, USA

Y.M. Kwon University of Arkansas, USA

Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain

Melanie R. MormileMissouri University of Science and Tech., USA

Rama NannapaneniMississippi State University, USA

Jack A. Neal, Jr.University of Houston, USA

Benedict OkekeAuburn University at Montgomery, USA

John PattersonPurdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos RostagnoLBRU, USDA-ARS, USA

Roni ShapiraHebrew University of Jerusalem, Israel

Kalidas ShettyUniversity of Massachusetts, USA

EDITORIAL BOARD


EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA

EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA

Cesar CompadreUniversity of Arkansas for Medical Sciences, USA

Philip G. CrandallUniversity of Arkansas, USA

MANAGING AND LAYOUT EDITOREllen J. Van LooGhent, Belgium

TECHNICAL EDITORJessica C. ShabaturaFayetteville, Arkansas, USA

ONLINE EDITION EDITORC.S. ShabaturaFayetteville, Arkansas, USA

ABOUT THIS PUBLICATION

Agriculture, Food & Analytical Bacteriology (ISSN

2159-8967) is published quarterly, beginning with

this inaugural issue.

Instructions for Authors may be obtained at the

back of this issue, or online via our website at

www.afabjournal.com

Manuscripts: All correspondence regarding pend-

ing manuscripts should be addressed Ellen Van Loo,

Managing Editor, Agriculture, Food & Analytical

Bacteriology: [email protected]

Information for Potential Editors: If you are interested

in becoming a part of our editorial board, please con-

tact Editor-in-chef, Steven Ricke, Agriculture, Food &

Analytical Bacteriology: [email protected]

Advertising: If you are interested in advertising with

our journal, please contact us at advertising@afab-

journal.com for a media kit and current rates.

Reprint Permission: Correspondence regarding re-

prints should be addressed Ellen Van Loo, Managing

Editor, Agriculture, Food & Analytical Bacteriology

[email protected]

Ordering Print Copies: print editions of this journal

may be purchased and shipped internationally from

our website order form at www.afabjournal.com

Subscription Rates: Subscriptions are not available

at this time. To be advised when subscriptions plans

are made available, please join our newsletter at

www.afabjournal.com

Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701 Website: www.AFABjournal.com

EDITORIAL STAFF


TABLE OF CONTENTS

Age and Diet Effects on Fecal Populations and Antibiotic Resistance of a Multi-drug Resistant Escherichia coli in Dairy Calves T. S. Edrington, R. L. Farrow, B. H. Carter, A. Islas, G. R. Hagevoort, T. R. Callaway, R. C. Anderson, and D. J. Nisbet

162

Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus

S. Datta, I. G. Shah, A. Akter, K. Fatema, T. H. Islam, A. Bandyopadhyay, Z. U.M. Khan, and D. Biswas

187

Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking ProcessJ. R. Pittman, T. B. Schmidt, A. Corzo, T. R. Callaway, J. A. Carroll, and J. R. Donaldson

195

Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of SweetgumA. C. Djioleu, E. M. Martin, M. H. Pelkki, and D. J. Carrier

175

Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens And Food Spoilage BacteriaN. Murali, G. S. Kumar-Phillips, N. C. Rath, J. Marcy, and M. F. Slavik

209

ARTICLES

Instructions for Authors233

Introduction to Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

Prevalence of foodborne pathogens and effectiveness of washing or cooking in reducing microbiological risk of contaminated Red amaranth Md. A. A. Mamun, H. A. Simul, A. Rahman, N. N. Gazi, and Md. L. Bari

222


www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Dairy calves are colonized at a very young age by a multi-drug resistant Escherichia coli (MDR EC) and

research studies indicate that the prevalence is not related to recent use of antimicrobials, but that diet

and other environmental factors are likely involved. To further investigate the occurrence of this bacterium,

we sampled dairy calves on southwestern United States farms at one week of age through 6 months, and

determined not only prevalence, but fecal concentrations of the MDR EC. The influence of feeding pas-

teurized (PWM) versus non-pasteurized (NPWM) waste milk was examined, and the effect of weaning was

investigated. The number of fecal samples positive for MDR EC as well as their populations decreased

(P < 0.01) with increasing calf age. Slight differences were observed when comparing PWM and NPWM

feeding, with MDR EC concentration and prevalence in the latter group generally decreasing at younger

ages. No significant differences were observed in the fecal concentrations of MDR EC due to weaning. No

clear differences were observed in resistance when comparing calves fed PWM or NPWM. Approximately

41% of the MDR EC isolates collected throughout the study were resistant to 10 or more antibiotics, with

two primary phenotypes: ACSSuT and MDR-AmpC. Based on the results herein, it appears that neither

pasteurization of the waste milk or weaning, has a significant effect on the prevalence or concentration of

MDR EC, and based on the age-associated decline in prevalence, they survive in an immature digestive

system with limited bacterial diversity and competition for resources.

Keywords: E. coli, multi-drug resistance, dairy calves, age, weaning

Correspondence: T.S. [email protected]: +1-979-260-3757 Fax: +1-979-260-9332

Age and Diet Effects on Fecal Populations and Antibiotic Resistance of a Multi-drug Resistant Escherichia coli in Dairy Calves†

T. S. Edrington1, R. L. Farrow1, B. H. Carter2, A. Islas2, G. R. Hagevoort3, T. R. Callaway1, R. C. Anderson1, and D. J. Nisbet1

1Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, USDA - ARS, College Station, TX 77845

2Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM 880033Agricultural Experiment Station, New Mexico State University, Clovis, NM 88101

†Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.

Agric. Food Anal. Bacteriol. 2: 162-174, 2012


INTRODUCTION

Antimicrobial resistant bacteria are a growing con-

cern worldwide for both veterinary and human medi-

cine (National Academy of Science, 1999). While

the increased resistance in pathogenic bacteria is

of utmost concern, commensal bacteria can also be

highly resistant to a wide variety of antimicrobials

and are considered by some as a potential reservoir

of resistance elements for the pathogenic strains

(Shoemaker et al., 2001; Summers, 2002). More spe-

cifically, the presence of multi-drug resistant (MDR)

non-pathogenic commensal bacteria such as Esch-

erichia coli on dairy farms could theoretically provide

a pool of transferable resistance genes for important

pathogens such as Salmonella and E. coli O157:H7

(Schmieger and Schicklmaier, 1999; Winokur et al.,

2001; O’Brien 2002; Hoyle et al., 2004).

The general consensus is that antimicrobial-re-

sistant bacteria, to include commensals in humans

and animals, are produced, maintained and dis-

seminated due to the selection pressure induced

by exposure to antimicrobial drugs (van den Bog-

arrd and Stobberingh, 2000). Research examining

E. coli in calves reported that exposure to antibiotic

in the feed resulted in the development of not only

resistance to the fed antibiotic but several other an-

tibiotics as well (Wierup et al., 1975). Others have

reported that the discontinuation of feeding an an-

tibiotic-medicated milk replacer to dairy calves re-

sulted in an increase in tetracycline susceptibility in

E. coli and Salmonella isolates during the first three

months that a non-medicated milk replacer was fed

(Kaneene et al., 2008). While exposure to antibiotics

certainly contributes to resistant bacteria, other non-

antibiotic influences have been reported (Sogaard

1973; Smith 1975; Gellin et al., 1989; Gilliver et al.,

1999).

Younger animals generally harbor more resistant

enteric flora than older animals (Wierup, 1975; Mar-

tel and Coudert, 1993). Pre-weaned calves have

been reported with higher MDR levels in enteric flo-

ra, possibly a result of increased fecal-oral transmis-

sion, higher strain turnover within the gastrointesti-

nal tract, or higher levels of antimicrobial drug use

in younger animals (Howe and Linton, 1976; Hinton

et al., 1985). Mature dairy cows sampled in 21 states

and cultured for E. coli and Salmonella found that the

majority of isolates (greater than 80%) were suscep-

tible to all antibiotics examined (Lundin et al., 2008).

Houser and colleagues (2008) reported that 62% of

the E. coli isolated from healthy lactating dairy cows

were susceptible to all antibiotics examined and 21%

were resistant to only one antibiotic, ampicillin. We

reported similar results when examining dairy cattle

of various ages for MDR Salmonella (Edrington et al.,

2008). In this research we found that young calves,

prior to weaning, were more likely to harbor MDR

Salmonella than all other classes of dairy animals

(heifers, lactating and dry cows) examined. The pri-

mary exception was cows in the hospital pen, as they

also exhibited significant levels of MDR Salmonella.

We hypothesized that the reasons for the high inci-

dence of MDR Salmonella in these two groups was

a result of previous antimicrobial treatment, as these

two groups of cattle are the most likely to receive an-

tibiotic therapy, and/or due to a disturbed or under-

developed gastrointestinal microflora. In the case of

young calves, their intestinal microflora is develop-

ing and changing with the introduction of new feed-

stuffs, weaning, environmental exposure, and other

factors, whereas the cows in the sick pen are gener-

ally off-feed resulting in a disturbed microflora vul-

nerable to competition from new bacterial species.

Several studies have documented the prevalence

of a highly resistant E. coli in dairy calves; however,

the results did not provide for a complete description

of the early temporal shifts or compare calves from

different geographic regions and management sys-

tems (Wierup, 1975; Howe and Linton, 1976; Hinton

et al., 1984; Khachatryan et al., 2004). Interestingly,

these MDR E. coli do not appear to be specific to a

geographic region or management practice, having

been reported in Washington (DeFrancesco et al.,

2004), Pennsylvania (Houser et al., 2008), and the SW

United States (Edrington, unpublished data). Others

have documented that pre-weaned calves had the

greatest prevalence of resistant E. coli, with levels

decreasing with increasing animal age (Khachatryan

et al., 2004). Healthy dairy calves were reported to


be rapidly colonized by antibiotic-resistant strains

of E. coli shortly after birth (Donaldson et al., 2006)

with the highest prevalence observed in 2-week old

calves. Calves were reported to shed MDR bacte-

ria resistant to 9 and 10 antibiotics as early as one

day of age (Donaldson et al., 2006) with similar ob-

servations reported by others (Orden et al., 2000;

Werckenthin et al., 2002). The question then arises:

Are the levels of resistance in these calves a result

of previous/current antibiotic exposure? Berge and

co-workers (2006) reported higher levels of MDR E.

coli in calves fed antimicrobials compared to those

on non-medicated feed. Isolates cultured from

older calves not fed antimicrobials (14 and 28 d old),

had higher levels of resistance compared to day old

animals with 14-day old calves most likely to shed

increasingly resistant bacteria (Berge et al., 2006). In

contrast to this, others have reported that the main-

tenance of the E. coli SSuT resistance phenotype in

dairy calves was due to environmental components

independent of antibiotic selection (Khachatryan

et al., 2006a). Further research by this same group

(Khachatryan et al., 2006b) reported that the antimi-

crobial resistant genes are not responsible for the

greater fitness advantage of antimicrobial-resistant

E. coli in calves, but that the farm environment and

the diet clearly exert critical selective pressures re-

sponsible for the maintenance of antimicrobial resis-

tance genes. Others have also reported that hous-

ing and dietary changes, occurring at weaning, may

affect the prevalence of antibiotic-resistant strains by

altering the calf’s exposure to other animal stock and

bacterial strains that in turn change the E. coli com-

position of their gut microflora (Hoyle et al., 2004).

Therefore, the objectives of the current research

were to evaluate the effect of age, diet (pasteurized

or non-pasteurized waste milk), weaning and farm

origin on fecal populations and prevalence of MDR

E. coli in dairy calves. Antimicrobial susceptibility

patterns were also examined.

MATERIALS AND METHODS

Animals and Sample Collection

This research was conducted on several large

commercial dairies (greater than 3000 head) in the

southwestern United States. Four collections were

made for this research project. The first sampled

calves on two farms representing six age groups (1

week, 2 weeks, 1, 2, 4 and 6 months of age). Fecal

samples (approximately 20 g) were collected from

freshly voided, undisturbed fecal pats from 15 ani-

mals per age group on each farm (n = 90 samples/

farm; 180 total samples). Both farms utilized waste

milk to feed the calves prior to weaning, one farm

pasteurizing the milk prior to feeding, the other us-

ing non-pasteurized waste milk. A second similar

collection was made, the only difference being that

a different farm utilizing pasteurized waste milk was

sampled. A total of 360 samples were collected and

cultured for multi-drug resistant E. coli (MDR EC). A

third collection was made in order to evaluate the in-

fluence of weaning on the prevalence of MDR EC in

dairy calves and was part of a larger study examining

the role of weaning on the prevalence of a number

of important bacteria (Edrington et al., 2011). Two

groups of calves were utilized, the first weaned at

approximately 12 weeks of age (avg. BW = 122 kg)

and the second group at approximately 10 weeks of

age (were not weighed at weaning; estimated BW =

110 kg). Fecal samples were collected from all calves

via rectal palpation on two occasions, two days pre-

and again two days post-weaning for bacterial cul-

ture described below. The fourth collection sampled

newborn calves (1 to 3 days of age) from four dif-

ferent dairies during their first week of arrival at a

central calf rearing facility. Rectal fecal samples were

collected into sterile palpation sleeves from 38, 45,

69 and 40 calves (n = 192 total samples) representing

each of the four farms over a four-week period.

Bacterial Culture and Isolation

All fecal samples were collected into sterile pal-

pation sleeves, placed on ice and shipped to our

laboratory in College Station, Texas for process-

ing the day following collection. For culture and

quantitation of MDR EC populations, 10 g of fecal

material was diluted in 90 mL of tryptic soy broth


and plated on MacConkey’s agar containing 32 µg/

mL chloramphenicol, using a commercially avail-

able spiral plater. Following incubation (24 h, 37º C),

colonies exhibiting typical E. coli morphology were

manually counted to determine colony forming units

(CFU)/g feces. This was converted to CFU (log10)/g

feces for statistical analysis and data presentation

below. A portion of the isolates from each collection

were confirmed as E. coli using the API 20E test kit

(BioMerieux, Durham, NC). Isolates were stored as

glycerol stocks (10% v/v) in TSB at - 80ºC. All media

and agar were from Difco Laboratories (Detroit, MI).

Reagents and antibiotics were obtained from Sigma

Chemical Co. (St. Louis, MO).

Determination of Antimicrobial Suscep-tibility

Antimicrobial susceptibility was determined using

the Sensititre automated antimicrobial susceptibility

system according to the manufacturer’s directions

(Trek Diagnostic Systems, Westlake, OH). Broth

microdilution was used according to methods de-

scribed by the National Committee for Clinical Labo-

ratory Standards (CLSI 2005) using the NARM’s panel

for gram-negative isolates. Resistance breakpoints

were determined using the CLSI (CLSI 2005) inter-

pretive standards unless unavailable, in which case

breakpoints in the NARMS 2000 Annual Report (FDA

2000) or those provided by Trek Diagnostic were

Table 1. Fecal prevalence of MDR EC (number and populations) in dairy calves of multiple ages, housed on two commercial dairy farms and feeding pasteurized (PWM) or non-pasteurized (NPWM) waste milk through weaning

Calf Age

Item 1 wk 2 wks 1 mo 2 mos 4 mos 6 mos

Collection 1

Farm A - PWM

no. positive 15/15 15/15 15/15 15/15 13/15 9/15

CFU(log10)/g feces 2.7bB 5.4A 6A 5.2aA 3.7aB 2.4B

Farm B - NPWM

no. positive 15/15 15/15 15/15 10/15 8/15 4/15

CFU(log10)/g feces 5.1aA 5.2A 5.8A 3.1bB 2.2bBC 1.8C

Collection 2

Farm A - PWM

no. positive 14/15 15/15 15/15 15/15 15/15 14/15

CFU(log10)/g feces 5.3B 6.3aA 5B 5.4aB 5aB 3.8aC

Farm B - NPWM

no. positive 15/15 15/15 15/15 10/15 14/15 9/15

CFU(log10)/g feces 5.4AB 5.9bA 4.9B 2.9bC 2.9bC 2.2bC

abCFU within collection and age column with different superscripts differ (P < 0.05).ABCCFU within collection and farm row with different superscripts differ (P < 0.05).

Culture negative samples assigned value of 1.0.


used. Escherichia coli ATCC 25922, E. coli ATCC

35218, and Enterococcus faecalis ATCC 29212 were

used as quality control organisms.

Statistical Analysis

Data were analyzed using SAS Version 8.02 (SAS

Inst. Inc., Cary, NC, USA). Quantitative data ex-

pressed as CFU (log10)/g feces were subjected to

analysis of variance appropriate for a completely

randomized design. A value of 1.0 was assigned

to all negative samples for statistical analysis. Pen

prevalence was subjected to Chi-square analysis us-

ing the PROC FREQ procedure. Means were consid-

ered different at a 5% level of significance.

RESULTS

Influence of Age on Prevalence and An-timicrobial Susceptibility of MDR EC

The prevalence and concentration of MDR EC

is presented by age and by farm [feeding pasteur-

ized (PWM) or non-pasteurized waste milk (NPWM)]

in Table 1 for the two collections. The number of

fecal samples positive for MDR EC decreased with

increasing calf age during both collections, with the

decrease being more pronounced when comparing

the farm feeding NPWM versus the two farms feed-

ing PWM. Fecal concentration of MDR EC likewise

decreased (P < 0.01) with increasing age on all farms

for both collections (Table 1). When comparing type

of waste-milk fed, MDR EC concentration decreased

more rapidly with increasing age in the farms feed-

ing NPWM (Table 1).

Antimicrobial susceptibility was examined in

MDR EC isolates (six isolates/age group/collection;

n = 72 total MDR EC isolates). In general, during the

first collection, more resistance was observed in the

farm using NPWM compared to collection 2, when

the opposite trend was observed, therefore the data

was pooled across farm and presented by collection

date in Table 2. All isolates were resistant to chlor-

amphenicol and tetracycline and all but one were

resistant to sulfisoxazole, whereas the majority of the

isolates were susceptible to ciprofloxacin and ceftri-

axone. The number of isolates resistant to all other

antibiotics examined decreased with increasing calf

age at each collection time (Table 2).

Multi-drug resistance and resistance phenotypes

are presented in Table 3. One isolate was resistant

to two antibiotics with all other isolates resistant to

four or more antimicrobials. Thirty-eight percent of

the isolates were resistant to 10 or more antibiotics,

the majority of which were cultured in the Novem-

ber collection. Primary resistance patterns observed

were ACSSuT and MDR-AmpC, the first of which

was more prevalent in the second collection and the

frequency of the MDR-AmpC pattern similar among

collections (Table 3).

Influence of Weaning on Prevalence and Antimicrobial Susceptibility of MDR EC

Samples were collected from two groups of calves

immediately prior to and following weaning and cul-

tured for MDR EC (Table 4). No significant differenc-

es were observed in the fecal concentrations or in

the number of MDR EC positive pens in either group

or when data was combined across groups. There

was a tendency (P = 0.06) for fewer MDR EC positive

pens in the second group of calves post-weaning.

Twenty MDR EC isolates were examined for anti-

microbial susceptibility (five per group pre- and post-

weaning). All of the isolates were susceptible to ami-

kacin, ceftriaxone, ciprofloxacin and naladixic acid,

and all but one isolate susceptible to amoxicillin/cla-

vulanic acid, cefoxitin and ceftiofur. All isolates were

resistant to kanamycin, sulfisoxazole and tetracycline

and all but one resistant to chloramphenicol (data

not shown). Half of the isolates were resistant to four

or five antibiotics and most of the remaining half of

the isolates (nine isolates) resistant to six, seven, or

eight antibiotics (Table 5). One isolate was resistant

to 10 antibiotics. Several patterns of resistance were

observed, the most prevalent being ACSSuT. One

isolate demonstrated the MDR-AmpC pattern of re-

sistance (Table 5). Weaning did not appear to have

any influence on antimicrobial resistance in these

isolates.


Table 2. Antimicrobial resistance profiles of MDR EC isolates cultured from fecal samples, by col-lection, from dairy calves of multiple ages on commercial dairy farms. Data represents the num-ber of isolates resistant to the minimum inhibitory concentration (MIC) listed for each antibiotic

Calf Age Combined

Item MIC Collection 1 wk 2 wks 1 mo 2 mos 4 mos 6 mos Ages

No. isolates examined 1 6 6 6 6 6 6 36

2 6 6 6 6 6 6 36

Antibiotic

Amikacin > 64 1 2 3 3 2 0 0 10

2 3 2 1 1 0 0 7

Gentamicin > 16 1 4 5 3 2 1 0 15

2 5 5 5 4 1 2 22

Kanamycin > 64 1 6 6 6 6 2 3 29

2 6 6 6 6 5 4 33

Streptomycin > 64 1 6 6 6 4 5 3 30

2 6 6 6 6 4 5 33

Ceftiofur > 8 1 3 3 3 0 0 0 9

2 4 3 4 4 1 1 17

Ceftriaxone > 64 1 0 0 0 0 0 0 0

2 1 0 0 0 0 0 1

Cefoxitin > 32 1 3 3 4 0 0 0 10

2 4 4 5 4 1 0 18

Ampicillin > 32 1 4 4 4 1 0 1 14

2 6 6 6 5 1 2 26

Chloramphenicol > 32 1 6 6 6 6 6 6 36

2 6 6 6 6 6 6 36

Ciprofloxacin > 4 1 0 0 0 0 0 0 0

2 1 2 0 0 1 2 6

Nalidixic acid > 32 1 0 0 0 0 2 0 2

2 2 2 2 0 1 1 8

Sulfisoxazole > 256 1 6 6 6 6 6 5 35

2 6 6 6 6 6 6 36

Tetracycline > 16 1 6 6 6 6 6 6 36

2 6 6 6 6 6 6 36

Trimethoprim/ 1 4 4 4 3 3 0 18

sulfamethoxazole > 4/76 2 6 5 2 3 2 1 19

Amoxicillin/ 1 3 3 4 0 0 0 10

clavulanic acid > 32/16 2 4 3 5 4 1 0 17


Table 3. Multi-drug resistance and patterns of resistance in MDR EC isolates by collection, cul-tured from fecal samples of dairy calves of multiple ages on commercial dairy farms

Calf Age Combined

Item Collection 1 wk 2 wks 1 mo 2 mos 4 mos 6 mos Ages

No. isolates examined 1 6 6 6 6 6 6 36

in each animal class 2 6 6 6 6 6 6 36

Resistant to:

2 antibiotics 1 0 0 0 0 0 1 1

2 0 0 0 0 0 0 0

4 to 6 antibiotics 1 1 0 2 3 5 5 16

2 0 0 0 1 5 4 10

7 to 9 antibiotics 1 2 4 1 3 1 0 11

2 2 2 1 1 0 1 7

>10 antibiotics 1 3 2 3 0 0 0 8

2 4 4 5 4 1 1 19

At least:

ACSSuTa 1 1 1 0 1 0 0 3

2 2 4 6 3 0 2 17

MDR-AmpCb 1 3 3 4 0 0 0 10

2 4 2 0 2 1 0 9

aACSSuT = resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline.

bMDR-AmpC = resistant to ACSSuT plus amoxicillin/clavulanic acid and ceftiofur, and a decreased susceptibil-ity to ceftriaxone (MIC > 2 µg/ml).

Table 4. MDR EC [fecal concentration = FC; CFU (log10)/g feces] and pen prevalence [% pens with calf culture positive for MDR EC (% Pens)] in two groups of dairy calves on a commercial dairy farm, sampled two days pre- and post-weaning (by group and combined)

Pre-weaning Post-weaning

Group No. samples No. Pens FC % Pens FC % Pens

1 69 18 3.3 83 3.8 89

2 75 19 3.7 89 2.9 63

Combined 144 37 3.5 86 3.4 76


Farm Origin and Influence on Antimi-crobial Susceptibility of MDR EC

In general, farms were similar in regards to sus-

ceptibility/resistance to individual antibiotics. The

majority of all MDR EC isolates (greater than 80%)

were resistant to chloramphenicol, streptomycin, sul-

fisoxasole, and tetracycline, while approximately half

displayed resistance to amoxicillin/clavulanic acid,

cefoxitin, ceftiofur, ceftriaxone, gentamicin, kanamy-

cin, naladixic acid, and trimethoprim/sulfisoxasole

(Table 6). Multi-drug resistance (2 to 14 antibiotics)

was observed in all 192 isolates examined with most

(69%) resistant to 8 or more antibiotics (Table 7). The

most prevalent resistance phenotypes were ACSSuT

and MDR Amp-C, both found in 36% of the isolates.

Multi-drug resistance was similar among farms with

the exception of Farms A and C, in which fewer AC-

SSuT and more MDR AmpC phenotypes were ob-

served on Farm A (Table 7).

DISCUSSION

A few years ago, while investigating a suspected

outbreak of salmonellosis, we cultured MDR EC from

a relatively large number of young dairy calves. Sub-

sequent examination of the literature revealed that

the occurrence of MDR EC had been documented

in young dairy calves in other regions of the United

States (DeFrancesco et al., 2004; Houser et al., 2008)

and that this particular E. coli, or the maintenance

of resistance in this species, was thought to be re-

stricted to very young calves. The prevalence of re-

sistant organisms is typically higher in younger ani-

mals (Brophy et al., 1977; Hinton et al., 1985; Zhang

et al., 1998; Mathew et al., 1999). This at first would

seem counter-intuitive if the development of anti-

microbial resistance is related to previous antibiotic

therapy. However, young animals are typically more

susceptible to disease and receive antibiotics for the

treatment or prevention of such diseases. Even so,

it would stand to reason that as age increases, expo-

sure to antibiotics would also increase, and therefore

the prevalence of resistant isolates would be greater

in older animals. However, as this is not the case

in dairy cattle (Edrington et al., 2008; Houser et al.,

2008; Lundin et al., 2008), researchers have specu-

lated that perhaps this increased resistance in dairy

calves is due to their exposure to more antibiotics

for medication and/or growth promotion compared

to mature cows. Khachatryan and coworkers (2004)

reported just the opposite however, in that the resis-

tant E. coli demonstrated a greater fitness in the calf

intestinal tract environment that was independent of

exposure to antimicrobial drugs and that drug use

was not required to maintain a high prevalence of

this resistant strain of E. coli. Others reported that

the clustering of MDR EC in calves 2 to 4 weeks of

age, on both dairies and calf ranches, suggest there

are host-specific factors influencing the emergence

of resistance that may not be associated with anti-

biotic use (Berge et al., 2005). Taken together, this

suggests that the development or maintenance of

the resistance of E. coli in dairy calves is not depen-

dent on exposure to antibiotics, but was an environ-

mental or diet induced phenomenon.

Table 5. Multi-drug resistance and patterns of resistance in fecal MDR EC isolates cultured from dairy calves. Data combined from two groups of dairy calves on a commercial dairy farm, two days pre- and post-weaning.

Time

Item Pre-wean Post-wean

No. isolates examined 10 10

No. isolates resistant to:

0 - 3 antibiotics 0 0

4 or 5 antibiotics 4 6

6 - 10 antibiotics 6 4

Phenotypes

ACSSuTa 3 2

MDR-AmpCb 0 1

aACSSuT=resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline.

bMDR-AmpC = resistant to ACSSuT plus resistant

to amoxicillin/clavulanic acid and ceftiofur and de-

creased susceptibility to ceftriaxone

(MIC > 2 µg/mL).


Table 6. Antimicrobial resistance profiles of fecal MDR EC isolates from dairy calves originating from multiple dairy farms upon arrival at a central heifer raising facility. Data represents the num-ber of isolates resistant to the minimum inhibitory concentration (MIC) listed for each antibiotic.

Farm of Origin Combined

Item MIC A B C D Ages (%)

No. isolates examined 38 45 69 40 192

Antibiotic

Amikacin > 64 1 1 1 1 4 (2.1)

Gentamicin > 16 21 30 23 20 94 (49)

Kanamycin > 64 30 32 36 27 125 (65)

Streptomycin > 64 30 34 53 32 149 (78)

Ceftiofur > 8 18 21 22 18 79 (41)

Ceftriaxone > 64 19 23 29 23 94 (49)

Cefoxitin > 32 19 27 22 19 87 (45)

Ampicillin > 32 35 45 61 38 179 (93)

Chloramphenicol > 32 38 45 69 39 191 (99)

Ciprofloxacin > 4 11 17 20 17 65 (34)

Naladixic acid > 32 19 23 32 23 97 (51)

Sulfisoxazole > 256 38 45 68 40 191 (99)

Tetracycline > 16 38 45 68 40 191 (99)

Trimethoprim/sulfamethoxazole > 4/76 18 24 32 23 97 (51)

Amoxicillin/clavulanic acid > 32/16 20 17 25 24 86 (45)

Table 7. Multi-drug resistance and patterns of resistance (number of isolates and percentage in parentheses) in fecal MDR EC isolates cultured from newborn calves, originating from four differ-ent dairies, upon arrival at a central heifer raising facility

Farm of Origin Across

Item A B C D Farms

No. isolates examined 38 45 69 40 192

No. isolates resistant to:

0 - 3 antibiotics 0 0 1 (1.5) 0 1 (0.5)

4 - 7 antibiotics 11 (29) 8 (18) 30 (43) 9 (23) 58 (30)

8 - 14 antibiotics 27 (71) 37 (82) 38 (55) 31 (78) 133 (69)

Phenotypes

ACSSuTa 10 (26) 16 (36) 28 (41) 15 (38) 69 (36)

MDR-AmpCb 17 (45) 19 (42) 18 (26) 15 (38) 69 (36)

a ACSSuT=resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracyclineb MDR-AmpC = resistant to ACSSuT plus resistant to amoxicillin/clavulanic acid and ceftiofur and decreased

susceptibility to ceftriaxone

(MIC > 2 µg/mL)


Dairy calves experience a number of changes dur-

ing a relatively short time frame that may explain the

age related decrease for this bacterium. Adaptation

and eventual weaning from a liquid, milk based diet

to a diet composed of hay and grain, and the associ-

ated changes in gastrointestinal microflora could ex-

plain these age-related changes. Results of the cur-

rent research demonstrated an age-related change in

fecal populations and prevalence of MDR EC in dairy

calves as reported by others and discussed above.

We did however culture MDR EC from a substan-

tial number of calves at 6 months of age, older that

most of the calves examined in previous research.

Examination of calves pre- and post-weaning found

no significant differences in MDR EC prevalence or

populations. Taken together, these results suggest

that the disappearance of MDR EC in dairy calves is

a gradual process that is not strongly influenced by

changing diet or other animal husbandry factors as

we originally hypothesized. If these changes were

in fact a result of changing diet and maturation of

the digestive system, then we would expect to see a

more substantial decline prior to six months of age,

as diet changes significantly early in age but are very

subtle later (4 and 6 months).

Pasteurization of the waste milk used to feed the

calves appeared to have slight influence on MDR EC

populations in these dairy calves. Both the number

of MDR EC positive samples and the concentration

of MDR EC were lower in calves fed the NPWM com-

pared to PWM. Significant reductions (90 to 95%) in

total bacterial counts as well as for specific patho-

gens such as Salmonella have been reported follow-

ing pasteurization of waste milk (Stabel et al., 2004;

Ruzante et al., 2008). However, milk that is not prop-

erly chilled following pasteurization provides a warm

environment for rapid bacterial growth, increasing

the number of cells as much as 8-fold per hour. Over-

all bacterial counts in PWM prior to feeding, were

reported to range from 500,000 to 100 million CFU/

ml, which was not different from 60% of the farms

pasteurizing the milk (Ruzante et al., 2008). Possibly

the differences that were observed in this research

are a result of competitive exclusion as influenced

by the pasteurization process. Pasteurization may

have reduced the bacterial species that are more

able to compete with the MDR EC, thus providing

MDR EC a competitive advantage in the calves fed

PWM. Some researchers have hypothesized that the

presence of MDR EC in calves fed waste milk is due

to a selection pressure maintained through the feed-

ing of low concentrations of antibiotics contained in

the milk (Berge et al., 2005). Subsequent examina-

tion of the waste milk failed to confirm the presence

of antibiotics in the milk and led to the conclusion

that feeding hospital milk had no observable impact

on antibiotic resistance in E. coli. In the current re-

search, if antibiotics in the milk were responsible for

the MDR EC, then we would expect to see higher

levels in calves fed NPWM, assuming the pasteuriza-

tion process affected antibiotic residues in the milk.

On the other hand, if pasteurization had no affect on

the antibiotics in the milk, then we would expect to

see similar levels among the feeding groups, not the

subtle differences we observed.

Possibly the differences we observed were due to

some other farm related factor and not pasteuriza-

tion of the waste milk. This is certainly plausible and

a drawback from the experimental design. Unfor-

tunately, conducting research on commercial dairy

farms, while providing for “real-world” settings,

does have short-comings; in this case the dairy-

man pasteurizing waste milk was not willing to feed

some of the calves on his farm non-pasteurized milk

due to health concerns and labor issues. Therefore

the next best scenario was to sample calves on dif-

ferent farms, similar in most all aspects, except for

pasteurization of the waste milk. While other fac-

tors may have influenced the results, the widespread

dissemination of MDR EC among dairy calves and

similarity of resistance phenotypes, as observed in

the first three collections as well as the fourth col-

lection, comparing calves from four different farms,

suggests this is unlikely and the differences are likely

due to handling of the waste milk.

Contrary to the research of Khachatryan et al.

(2004), who reported a greater prevalence of SSuT

resistance in milk-fed calves, Hinton et al. (1984)

found that fecal E. coli from calves were more likely

to develop MDR resistance during and immediately


after weaning from a medicated milk replacer. In our

research, inclusion of the MDR EC isolates collected

pre- and post-weaning in this discussion confounds

the interpretation. The MDR EC isolates cultured

from the weaning study were resistant to fewer dif-

ferent antibiotics (11) and displayed two patterns of

resistance (ACSSuT and MDR-AmpC) than isolates

from younger calves in the first collection. However,

in comparing these two groups of isolates, it must be

taken into account that they were collected from dif-

ferent farms with different management techniques

and at different times of the year.

Results of this research indicate that the persis-

tence of MDR EC in dairy calves is a function of age.

Furthermore, the decline in populations and preva-

lence does not appear to directly correspond to

changes in diet and may be a more subtle indication

of gastrointestinal maturation or other factors yet to

be determined. While E. coli is present in mature

cows, it is not reported to be MDR, indicating that

maternal transfer is not responsible for its presence

in calves but some other environmental factor(s).

The gradual disappearance with age, suggest diet

may be a limiting factor, although if entirely respon-

sible for the presence and/or disappearance of the

bacteria then we might expect bigger decreases in

its populations when diet is significantly changed,

such as at weaning, and not the steady decline we

observed when diet was not changed. We hypoth-

esize that the survival and disappearance is simply

a matter of the competitive fitness of this species

within the developing gastrointestinal microflora of

the calf. Results of this research and of others sup-

port this conclusion. Berge and colleagues (2005)

suggested that in the young calf-gastrointestinal en-

vironment, E. coli with multiple antibiotic resistance

exhibits a higher fitness compared to susceptible E.

coli. The intestinal microbiota is very different in a

young milk fed calf compared to an adult animal,

which the MDR EC appear to find more suitable for

survival (Khachatryan et al., 2004). This would sug-

gest that the presence of resistance elements may

give the MDR EC a survival advantage over suscep-

tible strains in the developing gastrointestinal tract.

However, as resistance generally comes at a cost to

the bacteria, we suggest that while the gut is un-

developed in terms of bacterial diversity, the MDR

EC is able to successfully compete, however as the

bacterial flora diversifies and increases in numbers,

the MDR EC loses its competitive advantage due,

at least in part, to being MDR and is slowly removed

from the gastrointestinal tract. Khachatryan and col-

leagues (2004) presented a similar explanation. Their

research suggested a direct benefit of the resistance

genes themselves or linkage to other genes that are

adaptive in this environment. However, they went on

to say that relative absence of a diverse bacterial fau-

na, due in part to the milk diet, is indicative that the

MDR EC compete effectively only when significant

competition is lacking and as the animal ages and

the gut matures, the resistance becomes a burden

and the MDR EC is excluded from the system. Pre-

vious research examining MDR Salmonella in dairy

calves supports this idea. Similar to these results, we

found MDR Salmonella only in young calves or sick

cows, suggesting that its ability to compete within

the gastrointestinal tract depends on an immature or

disturbed microflora (Edrington et al., 2008).

The impact of this population of MDR EC on

overall calf health appears to be minimal if any, how-

ever the potential transfer of resistance elements to

pathogenic bacteria such as Salmonella cannot be

ruled out. Research into the origin or transmission

source of this bacteria as well as methods to hasten

the elimination from the gastrointestinal tract of the

calf could theoretically reduce the potential devel-

opment of MDR pathogenic bacteria, leading to im-

proved calf health and in the long term, improved

herd health. Reducing the “load” of pathogenic

bacteria in the production setting has significant

food safety implications.

ACKNOWLEDGEMENTS

Portions of the above research were funded by

the Food Animal Concerns Trust.


REFERENCES

Berge, A.C.B., E.R. Atwill, and W.M. Sischo. 2005.

Animal and farm influences on the dynamics of

antibiotic resistance in faecal Escherichia coli in

young dairy calves. Prev. Vet. Med. 69:25-38.

Berge, A.C.B., D.A. Moore, and W.M. Sischo. 2006.

Field trial evaluating the influence of prophylactic

and therapeutic antimicrobial administration on

antimicrobial resistance of fecal Escherichia coli

in dairy calves. Appl. Environ. Microbiol. 72:3872-

3878.

Brophy, P.O., P.J. Caffrey, and J.D. Collins. 1977.

Sensitivity patterns of Escherichia coli isolated

from calves during and following prophylactic

chlortetracycline therapy. Br. Vet. J. 133:340-345.

Clinical and Laboratory Standards Institute. 2005.

Performance Standards for Antimicrobial Suscepti-

bility Testing: Fifteenth Informational Supplement.

CLSI/NCCLS document M100-S15. Wayne, PA.

DeFrancesco, K.A., R.N. Cobbold, D.H. Rice, T.E.

Besser, and D.D. Hancock. 2004. Antimicrobial re-

sistance of commensal Escherichia coli from dairy

cattle associated with recent multi-resistant salmo-

nellosis outbreaks. Vet. Micro. 98:55-61.

Donaldson, S.C., B.A. Straley, N.V. Hegde, A.A.

Sawant, C. DebRoy, and B.M. Jayarao. 2006. Mo-

lecular epidemiology of ceftiofur-resistant Esche-

richia coli isolates from dairy calves. Appl. Environ.

Microbiol. 72:3940-3948.

Edrington, T.S., T.R. Callaway, R.C. Anderson, and

D.J. Nisbet. 2008. Prevalence of multi-drug resis-

tant Salmonella on commercial dairies utilizing a

single heifer raising facility. J. Food Prot. 71:27-34.

Edrington, T.S., B.H. Carter, R.L. Farrow, A. Islas, G.R.

Hagevoort, T.H. Friend, T.R. Callaway, R.C. Ander-

son, and D.J. Nisbet. 2011. Influence of weaning

on fecal shedding of pathogenic bacteria in dairy

calves. Foodborne Path. Dis. 8:395-401.

Food and Drug Administration. 2000. National An-

timicrobial Resistance Monitoring System Annual

Report. Available: http://www.cdc.gov/narms/an-

nual/2000/tables/table2.htm. Accessed January 5,

2007.

Gellin, G., B.E. Langlois, K.A. Dawson, and D.K. Aar-

on. 1989. Antibiotic resistance of gram-negative

enteric bacteria from pigs in three herds with dif-

ferent histories of antibiotic exposure. Appl. Envi-

ron. Microbiol. 55:2287-2292.

Gilliver, M.A., M. Bennett, M. Begon, S.M. Hazel,

and C.A. Hart. 1999. Antibiotic resistance found

in wild rodents. Nature. 401:233-234.

Hinton, M., A.H. Linton, and A.J. Hedges. 1985. The

ecology of Escherichia coli in calves reared as dairy-

cow replacements. J. Appl. Bacteriol. 58:131-138.

Hinton, M., P.D. Rixson, V. Allen, and A.H. Linton.

1984. The persistence of drug resistant Escherich-

ia coli strains in the majority faecal flora of calves.

J. Appl. Bacteriol. 58:131-138.

Houser, B.A., S.C. Donaldson, R. Padte, A.A. Sawant,

C. DebRoy, and B.M. Jayarao. 2008. Assessment

of phenotypic and genotypic diversity of Esch-

erichia coli shed by healthy lactating dairy cattle.

Foodborne Path. Dis. 5:41-51.

Howe, K., and A.H. Linton. 1976. A longitudinal

study of Escherichia coli in cows and calves with

special reference to the distribution of O-antigen

types and antibiotic resistance. J. Appl. Bacteriol.

40:331-340.

Hoyle, D.V., H.I. Knight, D.J. Shaw, K. Hillman, M.C.

Pearce, J.C. Low, G.J. Gunn, and M.E. Woolhouse.

2004. Acquisition and epidemiology of antimicro-

bial-resistant Escherichia coli in a cohort of new-

born calves. J. Antimicrob. Chemother. 53:867-

871.

Kaneene, J.B., L.D. Warnick, C.A. Bolin, R.J. Erskine,

K. May, and R.A. Miller. 2008. Changes in tetra-

cycline susceptibility of enteric bacteria following

switching to nonmedicated milk replacer for dairy

calves. J. Clin. Microbiol. 46:1968-1977.

Khachatryan, A.R., D.D. Hancock, T.E. Besser, and

D.R. Call. 2004. Role of calf-adapted Escherichia

coli in maintenance of antimicrobial drug resis-

tance in dairy calves. Appl. Environ. Microbiol.

70:752-757.

Khachatryan, A.R., T.E. Besser, D.D. Hancock, and

D.R. Call. 2006a. Use of a nonmedicated dietary

supplement correlates with increased prevalence

of streptomycin-sulfa-tetracycline-resistant Esch-

erichia coli on a dairy farm. Appl. Environ. Micro-


biol. 72:4583-4588.

Khachatryan, A.R., D.D. Hancock, T.E. Besser, and

D.R. Call. 2006b. Antimicrobial drug resistance

genes do not convey a secondary fitness advan-

tage to calf-adapted Escherichia coli. Appl. Envi-


Lundin, J.I., D.A. Dargatz, B.A. Wagner, J.E. Lom-

bard, A.E. Hill, S.R. Ladely, and P.J. Fedorka-Cray.

2008. Antimicrobial drug resistance of fecal Esch-

erichia coli and Salmonella spp. isolates from Unit-

ed States dairy cows. Foodborne Path. Dis. 5:7-19.

Martel, J.L., and M. Coudert. 1993. Bacterial resis-

tance monitoring in animals: the French national

experiences of surveillance schemes. Vet. Micro-

biol. 35:321-338.

Mathew, A.G., A.M. Saxton, W.G. Upchurch, and S.E.

Chattin. 1999. Multiple antibiotic resistance pat-

terns of Escherichia coli isolates from swine farms.

Appl. Environ. Microbiol. 65:2770-2772.

National Academy of Science. 1999. The use of

drugs in food animals: benefits and risks. National

Academy Press, Washington, D.C.

O’Brien, T.F. 2002. Emergence, spread, and envi-

ronmental effect of antimicrobial resistance: how

use of an antimicrobial anywhere can increase re-

sistance to any antimicrobial anywhere else. Clin.

Infect. Dis. 34(Suppl. 3):S78-S84.

Orden, J.A., J.A. Ruiz-Santa-Quiteria, S. Garcia, D.

Cid, and R. de la Fuenta. 2000. In vitro suscepti-

bility of Escherichia coli strains isolated from diar-

rhoeic dairy calves to 15 antimicrobial agents. J.

Vet. Med. B 47:329-335.

Ruzante, J.M., I.A. Gardner, J.S. Cullor, W.L. Smith,

J.H. Kirk, and J.M. Adaska. 2008. Isolation of My-

cobacterium avium subsp. paratuberculosis from

waste milk delivered to California calf ranches.

Foodborne Path. Dis. 5:681-686.

Shoemaker, N.B., H. Vlamakis, K. Hayes, and A.A.

Salyers. 2001. Evidence for extensive resistance

gene transfer among Bacteroides spp. and among

Bacteroides and other genera in the human colon.


Schmieger, H., and P. Schicklmaier. 1999. Transduc-

tion of multiple drug resistance of Salmonella en-

terica serovar Typhimurium DT 104. FEMS Micro-

biol. Lett. 170:251-256.

Smith, H.W. 1975. Persistence of tetracycline resis-

tance in pig E. coli. Nature. 258: 628-630.

Sogaard, H. 1973. Incidence of drug resistance and

transmissible R factors in strains of E. coli isolat-

ed from faeces of healthy pigs. Acta Vet. Scand.

14:381-391.

Stabel, J.R., S. Hurd, L. Calvente, and R.F. Rosen-

busch. 2004. Destruction of Mycobacterium para-

tuberculosis, Salmonella spp., and Mycoplasma

spp. in raw milk by a commercial on-farm high-

temperature, short-time pasteurizer. J. Dairy Sci.

87:2177-2183.

Summers, A.O. 2002. Generally overlooked funda-

mentals of bacterial genetics and ecology. Clin.

Inf. Dis. 34:S85-92.

Van den Bogaard, A.E., and E.E. Stobberingh. 2000.

Epidemiology of resistance to antibiotics. Links

between animals and humans. Int. J. Antimicrob.

Agents. 14:327-335.

Weirup, M., K. Larsson, P. Holtenius, S.O. Jacobsson,

and I. Meansson. 1975. The effect of antibiotic

supplementation on antibiotic resistance, transfer-

able antibiotic resistance, morbidity, and growth in

calves. Nord. Vet. Med. 27:253-265.

Werckenthin, C., S. Seidl, J. Riedl, E. Kiossis, G. Wolf,

R. Stolla, and Q.R. Kaaden. 2002. Escherichia coli

isolates from young calves in Bavaria: in vitro sus-

ceptibilities to 14 anti-microbial agents. J. Vet.

Med. B 49:61-65.

Winokur, P.L., D.L. Vonstein, L.J. Hoffman, E.K.

Uhlenhopp, and G.V. Doern. 2001. Evidence for

transfer of CMY-2 AmpC beta-lactamase plasmids

between Escherichia coli and Salmonella isolates

from food animals and humans. Antimicrob.

Agents Chemother. 45:2716-2722.

Zhang, X.L., F. Wang, D.M. Zhu, S. Wu, P.C. Wu, Y.D.

Chen, Y.Q. Wang, and L Zhou. 1998. The carriage

of Escherichia coli resistant to antibiotics in healthy

populations in Shanghai. Biomed. Environ. Sci.

11:314-320.




ABSTRACT

The possibility of using sweetgum from southern pine dominated forests as a biobased refinery feed-

stock was investigated. Sweetgum wood and bark were pretreated with 0.98% (v/v) sulfuric acid at 140˚C

for 30, 40, 50, 60 or 70 min and at 160˚C for 30, 40, 50 or 60 min. The water insoluble solid (WIS) fraction was

hydrolyzed with a cellulase enzyme cocktail. Maximum xylose and glucose yields from the wood were 82

and 86%, respectively. Similarly, the respective maximum yields of xylose and glucose from the bark were

93 and 24%. Acid based pretreatment also produced fermentation inhibitory compounds such as furfural,

hydroxymethylfurfural (HMF), formic acid and acetic acid in concentrations ranging from 0.1 to 32.3 g/ 100

g of raw dry biomass. Sweetgum bark was more recalcitrant to enzymatic hydrolysis than wood and also

led to higher concentrations of formic acid. Sweetgum wood could be a good source of carbohydrate for

a biobased refinery, but the removal of bark might be necessary to achieve better yields.

Keywords: Dilute acid pretreatment, Enzymatic hydrolysis, Xylose, Glucose, Yields, Inhibitors,

Sweetgum

INTRODUCTION

Southern pine forests produce nearly 60% of the

softwood lumber in the U. S.; in Arkansas, nearly 75%

of all produced timber is from pine-dominated for-

ests (Arkansas Forestry Commission, 2008). However,

hardwood competition in the pine forest understory

is a major impediment to pine forest growth. There-

Correspondence: D. J. Carrier - [email protected]: +1 -479-575-2542; Fax: +1-479-575-2689.

fore, southern pine forests are managed intensively

(Wear and Greis, 2002). Annually, more than $150

million are spent reducing or eliminating competi-

tion in southern pine forests, primarily through the

use of herbicides (Siry, 2002).

The hardwood understory is composed of sweet-

gum (Liquidambar styraciflua L.), among others,

which are competitors with pine for site resources.

In Arkansas, the quantity of logging residue ranges

from 1.71 to 2.03 million dry tons annually, and total

Sugar Yields from Dilute Acid Pretreatment and Enzymatic Hydrolysis of Sweetgum (Liquidambar styraciflua L.)

A. C. Djioleu1, E. M. Martin1, M. Pelkki2, D. J. Carrier1

1 Department of Biological and Agricultural Engineering, University of Arkansas, 203 Engineering Hall, Fayetteville, AR 72701

2 School of Forest Resources, University of Arkansas, Monticello, Monticello, AR 71656



forest based biomass resources are approximately

50 million dry tons annually (Gan and Smith, 2006;

Jackson, 2007). Instead of being a nuisance, this

hardwood understory growth could become an

important source of biomass for biobased refiner-

ies, especially because sweetgum is a fast-growing

hardwood. Capturing biomass from fuel-reduction

thinning and understory harvests could raise forest

based biomass production from 2.3 million to 5 mil-

lion dry tons annually in the state of Arkansas alone

(Pelkki, 2007).

In a standard biorefinery, lignocellulosic biomass

is deconstructed into simple sugars that can be used

to produce either biofuels or other biochemical

products (Wyman, 1994). The deconstruction pro-

cess consists of a pretreatment step, which is impor-

tant in altering biomass structure and facilitates sub-

sequent enzymatic hydrolysis of pretreated biomass.

There are several methods for pretreatment, but di-

lute acid pretreatment has gained considerable im-

portance over the years (Sannigrahi et al., 2011). In

most studies involving dilute acid pretreatment, the

efficiency of pretreatment is measured by the digest-

ibility of the pretreated biomass, and less attention

is paid to the other attributes of a good pretreat-

ment process such as limiting the formation of com-

pounds, which could inhibit subsequent enzymatic

hydrolysis, or fermentation steps. In addition, for a

long time, glucose was the primary sugar of interest.

However, to improve the overall biorefinery opera-

tion costs, it is necessary to consider hemicellulose

sugars, especially xylose, which can also be ferment-

ed into ethanol by the appropriate microorganism

(Chung et al., 2005; Saha et al., 2005).

Investigations focusing on the digestibility of

sweetgum biomass after dilute acid pretreatment

have been reported by Torget et al. (1990; 1991). An

80% digestibility of cellulose was obtained with a 60

min 140˚C dilute acid (pH 1.35 to 1.45) pretreatment

of debarked sweetgum wood. Sweetgum bark also

was investigated as a feedstock; dilute acid pretreat-

ment did not improve enzymatic digestibility (Torget

et al., 1991). At 140˚C, dilute acid (pH 1.35 to 1.45)

pretreatments released 40% of the sweetgum bark

carbohydrates, but the remaining biomass was recal-

citrant to subsequent enzymatic attack; the percent

digestibility remained at 25% throughout the dura-

tion of enzymatic treatment (Torget et al., 1991).

Although sweetgum has been investigated as a

biorefinery feedstock, explicit data on sugar yields

obtained with cutting edge saccharification enzyme

cocktails and the formation of inhibitory compounds

have not been reported. The purpose of this effort

was to study the deconstruction of sweetgum wood

and bark using dilute acid pretreatment and enzy-

matic hydrolysis. Xylose and glucose yields, as per-

centages of the theoretical amount in non-pretreat-

ed (raw) dry biomass, were investigated, as well as

the formation of inhibitory products such as furfural,

hydroxymethylfurfural (HMF), formic acid and acetic

acid.


Raw biomass

Chipped wood and bark from mature sweetgum

trees were obtained from Matthew Pelkki and Philip

Tappe, School of Forest Resources, University of Ar-

kansas, Monticello, AR. The biomass was milled to

pass through a 20 mesh (0.84 mm) screen using a

Wiley Mini Mill (Thomas Scientific, Swedesboro, NJ)

and samples were dried in a 105˚C oven until sample

moisture was less than 5% as determined using an

MB45 Moisture Analyzer (Ohaus Corporation, Pine

Brook, NJ). Analyses for structural carbohydrate and

acid insoluble lignin (AIL) content of the raw biomass

were conducted as described in NREL LAP/TP-510-

42618 (Sluiter et al., 2008), except the biomass was

subjected to a 24 h ethanol extraction rather than a

water extraction followed by an ethanol extraction

(Table 1).

Pretreatment

One g of raw biomass (wood or bark) was soaked

in 10 mL of 0.98% (v/v) sulfuric acid (H2SO4) in a 50

mL centrifuge tube for 12 h. The mixture was placed

in a 32 mL stainless steel pretreatment tube (14.22


mm inner diameter, 5.59 mm wall thickness, 200 mm

length) with an additional 10 mL of H2SO4. Pretreat-

ment tubes containing raw biomass and acid were

heated in a fluidized sand bath (Techne Incorpo-

rated, Burlington, NJ) at 140˚C for 30, 40, 50, 60 or

70 min and at 160˚C for 30, 40, 50 or 60 min. After

pretreatment, tubes were immediately submerged

into cold tap water for 1 min; slurry contents were

poured into 15 mL centrifuge tubes for separation

into liquid fraction (prehydrolysate) and solid frac-

tions (pretreated biomass). The pretreated biomass

was washed by stirring in 30 mL of Millipore filtered

water on a stir plate, set at 300 rpm for 30 min. The

water-insoluble-solid (WIS) fraction was separated

from the wash water by vacuum filtration through a

Büchner funnel containing Whatman No. 1 filter pa-

per. The WIS fraction was stored at 4˚C for a maxi-

mum of 3 days until used for enzymatic hydrolysis.

Prehydrolysate and wash water were recovered and

stored for a maximum of 3 days at 4˚C before xylose,

glucose, and degradation compounds determina-

tion. Each pretreatment experiment was performed

in triplicate.

Enzymatic hydrolysis

An industrialized enzyme cocktail, Accelle-

rase®1500, provided by Genencor (Danisco US Inc.,

Rochester, NY) was used to hydrolyze the WIS frac-

tion. The enzyme cocktail had an endoglucanase ac-

tivity of 2200 to 2800 CMC U/g and a ß-glucosidase

activity of 525 to 775 pNPG U/g (provided by the

manufacturer). The WIS fraction was mixed in a 50 mL

amber bottle with 5 mL of citrate buffer (pH = 4.8),

0.5 mL of enzyme and 4.5 mL of Millipore filtered wa-

ter. The amber bottle was placed in a shaking water

bath (Thermo Electron Corporation, Winchester, VA)

at 55˚C and 100 rpm for 24 h. The resulting slurry was

poured into a 15 mL centrifuge tube, submerged in

boiling water to stop the reaction, and centrifuged

at 3000 g for 2 min. The volume of the supernatant

(enzymatic hydrolysate) was measured and the liq-

uid was stored at 4˚C for a maximum of 3 days until

analyzed for sugar content; the pellet was discard-

ed. Each enzymatic hydrolysis experiment was per-

formed in triplicate.

Analysis

Five mL aliquots of prehydrolysate, wash water

and the enzymatic hydrolysate were neutralized with

calcium carbonate and filtered through a 0.2 µm

filter for xylose and glucose analyses with a Waters

2695 Separations module (Milford, MA) equipped

with Shodex precolumn (SP-G, 8 µm, 6 x 50 mm) and

Shodex column (SP0810, 8 µm x 300 mm). Millipore

filtered water (0.2 mL/min) was the mobile phase

and the column was heated to 85˚C with an external

heater. Sugars were detected with a Waters 2414 Re-

fractive Index Detector (Milford, MA).

Aliquots of the prehydrolysate and wash water

were analyzed for degradation compounds with a

Waters 2695 Separations module equipped with a

Bio-Rad Aminex HPX-87H Ion Exclusion 7.8 mm X 30

mm column, heated to 55˚C. The mobile phase was

0.005 M H2SO4 flowing at 0.6 mL/min. Compounds

were detected with a UV index using the Waters

2996 Photodiode Array detector. Furfural and HMF

were detected at 280 nm; whereas, formic acid and

acetic acid were detected at 210 nm.

RESULTS AND DISCUSSION

Sweetgum wood

Dilute acid pretreatment

Figure 1 represents the yields of xylose and glu-

cose recovered in the prehydrolysate and wash water

using various pretreatment times at 140˚C and 160˚C.

Table 1. Composition of raw sweetgum bio-mass in % dry basis

Biomass Glucan Xylan AILa

Wood 42.2 19.9 22.5

Bark 18.2 6.0 31.9

a Acid insoluble lignin


Although the two liquid streams were analyzed sep-

arately, their carbohydrate contents were combined

to calculate xylose and glucose yields as percentages

of the theoretical amount in the dried raw biomass.

Xylose was the primary sugar recovered in the pre-

hydrolysate and wash water, indicating hydrolysis of

the hemicellulosic fraction of wood during pretreat-

ment. At 140˚C (Figure 1A), xylose yield increased

with pretreatment time up to a maximum value of

79% after 60 min. Conversely, at 160˚C (Figure 1B)

hemicellulose hydrolysis released its maximum (71%)

within 40 min of pretreatment, at which time xylose

yield decreased. Glucose also was detected in the

prehydrolysate and wash water. Pretreatment time

did not affect glucose recovery at 140˚C, with less

than 5% of the glucose recovered. However, at 160˚C

glucose yield increased with pretreatment time.

Pretreatment at lower temperatures is ideal in

achieving a high xylose recovery. More elevated

temperatures, especially for prolonged periods of

time, will result in considerable loss of xylose and

premature hydrolysis of the cellulosic fraction, which

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70

Yie

lds

(%)

Pretreatment time (min)

Xylose

Glucose

(A)

0

10

20

30

40

50

60

70

80

90

30 40 50 60

Yie

lds

(%)


Xylose

Glucose

(B)

Figure 1. Prehydrolysates of sweetgum wood: xylose and glucose yields. Pretreatment occurru-reed at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval.


can result in glucose degradation. These findings are

in agreement with studies performed on other feed-

stock with dilute acid pretreatment (Cara et al., 2008;

Lloyd and Wyman, 2005; Torget et al., 1990).

An inherent and undesirable property of dilute

acid pretreatment is the production of sugar and

lignin degradation compounds which are inhibitory

to enzymatic hydrolysis and detrimental to micro-

organisms used in sugar fermentation (Palmqvist

and Hahn-Hagerdal, 2000). Furfural, HMF, formic

acid and acetic acid were detected in prehydroly-

sate and wash water from the wood pretreatment

(Table 2). Furfural and HMF result from xylose and

glucose degradation, respectively, and both can fur-

ther degrade into formic acid; acetic acid is released

from the acetyl group of the hemicellulose polymer

(Palmqvist and Hahn-Hagerdal, 2000). Concentra-

tions (g/100 g of dried raw biomass) of degradation

products increased with time and severity of pre-

treatment (Table 2). Sugar degradation was less se-

vere at 140˚C than at 160˚C; the increase in degrada-

tion compounds, especially furfural and formic acid,

at 160˚C coincided with a decrease in xylose recov-

ery. Even though there was a slight degradation of

xylose at 140˚C, xylose recovery did not decline with

pretreatment time because, at lower temperature,

the rate of xylan hydrolysis is higher than its degra-

dation rate (Lloyd and Wyman, 2005).


The effects of pretreatment time on xylose and

glucose yields from the enzymatic hydrolysis of

sweetgum wood pretreated at 140˚C and 160˚C are

depicted in Figure 2. Xylose and glucose yields were

calculated as percentages of the theoretical amount

in the dried raw biomass and should be differentiated

from the cellulose digestibility reported in the study

done by Torget et al. (1990). As expected, most of

the glucose was solubilized during enzymatic hydro-

lysis for both pretreatment temperatures; however,

biomass pretreated at 140˚C (Figure 2A) was less

responsive to enzymatic attack than the one pre-

treated at 160˚C (Figure 2B), shown here by a higher

glucose recovery at 160˚C than at 140˚C. Although

most of the xylose was solubilized during pretreat-

Table 2. Degradation compounds (g/100g of raw biomass dry basis) produced from 0.98% (v/v) sulfuric acid pretreatment of sweetgum wood

Pretreatment conditions

Prehydrolysate Wash water a

Te m p . (˚C)

Time (min)

Acetic Acid

FurfuralFormic Acid

HMFAcetic Acid

FurfuralFormic Acid

HMF

140 30 4.6 ± 1.6 0.1 ± 0.1 1.8 ± 0.6 0.0 ± 0.0 2.5 ± 0.4 0.1 ± 0.1 0.8 ± 0.4 0.0 ± 0.0

140 40 2.9 ± 0.8 0.2 ± 0.2 1.8 ± 1.0 0.0 ± 0.0 2.7 ± 0.5 0.1 ± 0.1 0.9 ± 0.5 0.0 ± 0.0

140 50 2.9 ± 0.4 0.2 ± 0.0 1.8 ± 0.3 0.0 ± 0.0 3.1 ± 0.5 0.1 ± 0.0 0.9 ± 0.1 0.0 ± 0.0

140 60 3.6 ± 0.4 0.3 ± 0.2 3.4 ± 2.1 0.0 ± 0.0 2.5 ± 0.9 0.2 ± 0.2 1.5 ± 1.4 0.0 ± 0.0

140 70 3.0 ± 1.2 0.3 ± 0.1 3.1 ± 0.7 0.0 ± 0.0 3.3 ± 1.1 0.4 ± 0.3 3.0 ± 2.0 0.0 ± 0.0

160 30 4.8 ± 3.5 0.7 ± 0.3 8.1 ± 4.4 0.1 ± 0.0 3.2 ± 0.7 0.6 ± 0.2 5.4 ± 2.8 0.0 ± 0.0

160 40 4.3 ± 0.1 1.2 ± 0.2 10.4 ± 1.4 0.1 ± 0.0 2.7 ± 0.1 0.7 ± 0.1 4.8 ± 0.3 0.0 ± 0.0

160 50 5.0 ± 0.7 1.3 ± 0.2 10.6 ± 1.4 0.2 ± 0.1 3.2 ± 0.3 1.0 ± 0.1 5.6 ± 0.4 0.1 ± 0.0

160 60 3.7 ± 1.3 1.6 ± 0.7 6.6 ± 2.2 0.2 ± 0.1 3.7 ± 1.1 1.8 ± 0.6 5.5 ± 2.5 0.2 ± 0.1

Means ± standard deviation of three replications a Water used for washing biomass after pretreatment


ment at 140˚C, when it comes to sweetgum wood,

nearly complete removal of the hemicellulose during

pretreatment does not translate to a highly digest-

ible biomass. It is possible that performing the en-

zymatic hydrolysis for more than 24 h could improve

the glucose yield; however, we showed that glucose

yield increased only 10% after 48 h of enzymatic hy-

drolysis. Moreover, 24 h was the time recommended

by the enzyme manufacturer for maximum activity of

the enzyme.

Seventy four percent of glucose was recovered in

the enzymatic hydrolysate of the biomass pretreated

at 160˚C (Figure 2B) and better digestibility of the

pretreated wood was observed with an increasing

Figure 2. Enzymatic hydrolysates of pretreated sweetgum wood: xylose and glucose yields. Pre-treatment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval

0

5

10

15

20

25

30 40 50 60 70

Yie

lds

(%)


Xylose

Glucose

(A)

0

10

20

30

40

50

60

70

80

90

30 40 50 60

Yie

lds

(%)


Xylose

Glucose

(B)


pretreatment time. Obtaining more digestible ma-

terial from pretreatment conducted at harsher con-

ditions has been observed and explained in the lit-

erature (Foston and Ragauskas, 2010); hydrolysis of

the amorphous section of the cellulose, observed

in this work, resulted in higher glucose concentra-

tions during prolonged pretreatment at 160˚C. Kabel

et al. (2007) attribute the relationship between high

temperature and cellulose degradability to the dis-

ruption of the lignin structure during pretreatment;

however, the lignin structure in raw and pretreated

sweetgum wood was not analyzed in our work.

Overall yields

The dilemma between maximizing xylose recov-

ery during pretreatment and producing a highly

digestible cellulosic material occurred because the

conditions for maximum xylose recovery do not cor-

respond to the condition for maximum glucose re-

covery. Similar results had been observed (Lloyd and

Wyman, 2005). One solution to this issue could be to

maximize the yields of total fermentable sugars (TFS

= xylose + glucose) from pretreatment and enzy-

matic hydrolysis of the biomass as reported by Lloyd

and Wyman (2005). Yields of xylose, glucose and TFS

expressed as percentages of theoretical amounts in

the dried raw wood (sugar yields) or as the amount

of sugars (g) produced from 100 g of dried raw bio-

mass (raw biomass yields) are depicted in Table 3.

In general, at 140˚C xylose, glucose, and TFS yields

increased with pretreatment time. Up to 47% of TFS

was recovered after 70 min of pretreatment; these

pretreatment conditions yielded maximum xylose re-

covery of 82%. Any sugar cocktail (xylose + glucose)

obtained at 140˚C contained mainly xylose and, for

a fermentation process, this is not the ideal sugar

stream. Pretreatment at 160˚C yielded a maximum

TFS of 72% after 60 min of pretreatment; these pre-

treatment conditions also gave maximum glucose

recovery of 86%. At 160˚C, an increase in pretreat-

ment time did not have an effect on TFS yields; how-

Table 3. Sugars produced from 0.98% (v/v) sulfuric acid pretreatment and enzymatic hy-drolysis of sweetgum wood


Sugar yieldsa Raw biomass yieldsb

Temp. (˚C)

Time (min)

Xylose Glucose TFSc Xylose Glucose TFSc

140 30 68.9 ± 8.8 13.4 ± 7.8 31.5 ± 5.2 15.6 ± 2.0 6.3 ± 3.7 21.9 ± 3.6

140 40 74.1 ± 7.6 20.8 ± 3.6 38.2 ± 4.7 16.8 ± 1.7 9.8 ± 1.7 26.5 ± 3.3

140 50 71.3 ± 9.4 23.1 ± 1.2 38.8 ± 3.7 16.1 ± 2.1 10.8 ± 0.6 27.0 ± 2.6

140 60 82.1 ± 6.8 27.4 ± 1.7 45.2 ± 3.2 19.9 ± 2.4 15.8 ± 5.5 35.7 ± 7.2

140 70 82.0 ± 3.6 30.4 ± 0.7 47.2 ± 1.6 18.6 ± 0.8 14.3 ± 0.3 32.8 ± 1.1

160 30 71.4 ± 5.6 55.0 ± 5.5 64.6 ± 8.7 16.2 ± 1.3 28.7 ± 6.6 44.9 ± 6.1

160 40 72.1 ± 12.1 66.8 ± 3.9 68.5 ± 1.4 16.3 ± 2.7 31.3 ± 1.8 47.7 ± 1.0

160 50 54.0 ± 14.1 74.8 ± 3.9 68.1 ± 1.9 13.1 ± 2.1 35.1 ± 1.8 47.3 ± 1.4

160 60 41.9 ± 9.8 86.2 ± 1.0 71.8 ± 3.8 9.5 ± 2.2 40.4 ± 0.5 49.9 ± 2.6

Mean ± standard deviation of three replicationsa Percentage of the amount of individual sugar present in one g of the raw biomass. Xylose: 0.23 g; glucose: 0. 47 g; TFS: 0.7 g.b Yields in g/100 g of raw materialc Total fermentable sugars


ever, the sugar stream obtained at times before 40

min had a higher percentage of xylose than streams

obtained after 40 min, which had a higher percent-

age of glucose. This occurred because the xylose

concentration in the sugar stream decreased while

the glucose concentration increased with pretreat-

ment time.

Sweetgum Bark

Dilute acid pretreatment

In assessing biomass as feedstock for a biorefin-

ery, tree bark usually is not considered an ideal can-

didate, mainly because it is not a substantial source

of carbohydrate when compared to tree wood.

Figure 3. Prehydrolysates of sweetgum bark: xylose and glucose yields. Pretreatment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glu-cose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval.

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60 70

Yie

lds

(%)


Xylose

Glucose

(A)

0

10

20

30

40

50

60

70

80

90

100

30 40 50 60

Yie

lds

(%)


Xylose

Glucose

(B)


However, using the whole tree would simplify sup-

ply chain processing. The sweetgum bark used for

this study contained on a dry basis 18.2% glucan and

6.0% xylan (Table 1). Even though bark should be in-

tegrated in the biomass conversion process, wood

will dictate process parameters; therefore, sweet-

gum bark in this study was submitted to the same

pretreatment and enzymatic hydrolysis conditions as

sweetgum wood.

The effects of pretreatment time on xylose and

glucose yields from the prehydrolysate and wash

water of the bark pretreated at 140˚C and 160˚C are

shown in Figure 3. Sugar recovery from sweetgum

bark pretreatment did not follow the same trend as

for sweetgum wood pretreatment. Xylose loss oc-

curred faster at 140˚C (Figure 3A) than at 160˚C (Fig-

ure 3B). More xylose was recovered at 160˚C than

at 140˚C; these results were in contrast to results

obtained for sweetgum wood because harsher pre-

treatment conditions of the wood yielded lower xy-

lose recovery. Table 5 includes the sugar recoveries

from the pretreatment and the enzymatic hydrolysis

of sweetgum bark. The issue between maximizing

xylose or glucose yield was nonexistent with the bark

because the maximum recovery for both sugars oc-

curred at 160˚C. Moreover, at 160˚C the pretreatment

time did not affect TFS or glucose yields; therefore,

maximizing xylose recovery could be the only factor

dictating the pretreatment conditions for sweetgum

bark.

Furfural, HMF, formic acid and acetic acid were

present in the prehydrolysate and wash water from

the bark pretreatment (Table 4). Concentrations of

these by-products in pretreatment liquid streams

were lower at 140˚C than at 160˚C. It was expected

concentration of furfural and formic acid would be

higher at 140˚C than at 160˚C given that a higher loss

of xylose occurred at 140˚C. Concentrations of for-

mic acid in the bark prehydrolysate and wash water,

especially at 160˚C, were over 11 g per 100 g of raw

biomass. When combining formic acid recovery in

the prehydrolysate and wash water obtained from

pretreatment at 160˚C for 40 min, formic acid yield

was 43% of the raw biomass. Thus, for sweetgum

bark, reactions other than sugar degradation are

responsible for xylose loss and formation of formic

Table 4. Degradation compounds (g/100g of raw biomass) produced from 0.98% (v/v) sulfuric acid pretreatment of sweetgum bark


Prehydrolysate Wash watera

Temp (˚C)

Time (min)

Acetic Acid

FurfuralFormic Acid

HMFAcetic Acid

FurfuralFormic Acid

HMF

140 30 1.4 ± 0.3 0.0 ± 0.0 16.1 ± 0.7 0.0 ± 0.0 1.6 ± 0.2 0.0 ± 0.0 4.8 ± 1.1 0.0 ± 0.0

140 40 3.0 ± 2.1 0.0 ± 0.0 19.7 ± 3.8 0.0 ± 0.0 1.5 ± 0.2 0.0 ± 0.0 3.9 ± 1.5 0.0 ± 0.0

140 50 1.6 ± 0.8 0.1 ± 0.0 14.5 ± 6.8 0.0 ± 0.0 2.0 ± 0.6 0.1 ± 0.0 7.6 ± 3.4 0.0 ± 0.0

140 60 1.5 ± 0.4 0.1 ± 0.0 12.5 ± 3.7 0.0 ± 0.0 2.0 ± 0.7 0.1 ± 0.1 7.7 ± 3.0 0.0 ± 0.0

140 70 1.1 ± 0.1 0.1 ± 0.0 9.0 ± 0.4 0.0 ± 0.0 2.6 ± 0.3 0.2 ± 0.0 11.9 ± 1.1 0.0 ± 0.0

160 30 3.9 ± 2.1 0.1 ± 0.1 21.6 ± 5.1 0.1 ± 0.0 2.6 ± 0.5 0.2 ± 0.1 14.7 ± 3.3 0.0 ± 0.0

160 40 7.9 ± 4.9 0.4 ± 0.1 32.3 ± 11.1 0.1 ± 0.0 2.0 ± 0.8 0.2 ± 0.1 11.2 ± 4.2 0.0 ± 0.0

160 50 6.3 ± 2.9 0.5 ± 0.1 26.9 ± 2.2 0.1 ± 0.0 2.2 ± 0.5 0.3 ± 0.0 10.8 ± 1.7 0.0 ± 0.0

160 60 2.6 ± 0.9 0.6 ± 0.3 16.7 ± 8.2 0.1 ± 0.0 2.5 ± 0.9 0.5 ± 0.1 10.8 ± 5.3 0.1 ± 0.0

Mean ± standard deviation of three replications

a Water used for washing biomass after pretreatment


acid during pretreatment. The presence of those in-

hibitory compounds at such elevated concentrations

in the pretreatment liquid streams could be another

reason why bark is not an ideal candidate as a feed-

stock for a biorefinery.


The enzymatic hydrolysis of the sweetgum bark

(Figure 4) was not as successful as the hydrolysis of

the sweetgum wood. A maximum of 11% of glucose

was recovered in bark enzymatic hydrolysate com-

pared to 74% for wood. An increase in pretreatment

time or temperature did not improve glucose yields.

This resistance to enzymatic attack after pretreat-

ment has been reported to be inherent to sweetgum

bark (Torget et al., 1991). Specifically, hot dilute acid

pretreatment promotes lignin condensation, caus-

ing the formation of a lignin barrier that can possibly

impede the access of enzyme binding sites to cellu-

Table 5. Sugars produced from 0.98% (v/v) sulfuric acid pretreatment and enzymatic hydrolysis of sweetgum bark


Sugar yields a Raw biomass yields b

Temp (˚C)

Time (min)

Xylose Glucose TFS c Xylose Glucose TFS c

140 30 60.0 ± 7.5 15.9 ± 2.1 29.5 ± 3.7 5.3 ± 0.7 3.2 ± 0.4 8.5 ± 1.1

140 40 81.4 ± 11.1 16.9 ± 2.4 36.7 ± 5.0 7.2 ± 1.0 3.4 ± 0.5 10.6 ± 1.5

140 50 73.6 ± 5.7 16.9 ± 0.8 34.3 ± 2.2 6.5 ± 0.5 3.4 ± 0.2 9.9 ± 0.6

140 60 65.3 ± 12.5 16.9 ± 2.2 31.8 ± 3.7 5.8 ± 1.1 3.4 ± 0.4 9.2 ± 1.1

140 70 60.2 ± 3.5 13.9 ± 0.6 28.1 ± 0.8 5.3 ± 0.3 2.8 ± 0.1 8.1 ± 0.2

160 30 88.2 ± 5.8 17.8 ± 1.7 40.3 ± 2.1 7.8 ± 0.5 3.6 ± 0.3 11.6 ± 0.6

160 40 93.5 ± 11.3 21.4 ± 1.7 44.5 ± 3.3 8.3 ± 1.0 4.3 ± 0.3 12.9 ± 1.0

160 50 91.8 ± 12.7 22.4 ± 2.4 44.9 ± 4.4 8.1 ± 1.1 4.4 ± 0.5 13.0 ± 1.3

160 60 72.7 ± 1.8 24.5 ± 1.2 39.3 ± 1.3 6.4 ± 0.2 4.9 ± 0.2 11.3 ± 0.4

Mean ± standard deviation of three replicationsa Percentage of the amount of individual sugar present in one g of the raw biomass. Xylose: 0.09 g; glucose: 0. 2 g; TFS: 0.29 g. b Yields in g/100 g of raw material00c Total fermentable sugars

lose bonds (Torget et al., 1991). Moreover, Cantarella

et al. (2004) showed that formic acid concentrations

of 11 mg/mL inhibited the cellulose enzymatic cock-

tail; therefore, the formic acid detected in the bark

prehydrolysates of our study could contribute to the

recalcitrance observed in the bark. Insufficient wash-

ing of the pretreated pellet could exacerbate this

recalcitrance. A better understanding of sweetgum

bark structure and composition needs to be estab-

lished to design optimum processing conditions to

maximize saccharification of this feedstock system.

CONCLUSIONS

Dilute acid pretreatment at 160˚C for 60 min cou-

pled with enzymatic hydrolysis of sweetgum wood

yielded a maximum of 72% total fermentable sugars.

Sweetgum wood could be a potential feedstock for

a biochemical based refinery, especially because it is


a fast growing, voluntary tree. However, the addition

of sweetgum bark with the wood could render the

use of the whole tree problematic because it pro-

duces high concentrations of inhibitory compounds

causing resistance to enzymatic hydrolysis. Bark is re-

moved in pulp and paper operations; the removal of

this component also may be necessary in biorefinery

operations.

ACKNOWLEDGEMENTS

The authors thank the University of Arkansas, Divi-

sion of Agriculture, and the Department of Biologi-

cal and Agricultural Engineering for financial assis-

tance. The authors also acknowledge South Central

Sun Grant award # DTOS59-07-G-00053 for finan-

cial support to A. C. Djioleu, Department of Energy

Figure 4. Enzymatic hydrolysates of pretreated sweetgum bark: xylose and glucose yields. Pretreat-ment occurred at (A): 140˚C and (B): 160˚C with 0.98% (v/v) H2SO4. Yields represent the amount of xylose and glucose recovered as a percentage of the theoretical amount in the raw biomass. Error bars are 95% confidence interval.

0

1

2

3

4

5

6

7

8

9

30 40 50 60 70

Yie

lds

(%)


Xylose

Glucose

(A)

0

2

4

6

8

10

12

14

30 40 50 60

Yie

lds

(%)


Xylose

Glucose

(B)


award #08GO88035 for pretreatment equipment

and CSREES National Research Initiative award #

2008-01499 for the HPLC instrument.

REFERENCES

Arkansas Forestry Commission. 2008. Timber sever-

ance tax report for 2007. Arkansas Forestry Com-

mission, Little Rock, AR.

Cantarella, M., L. Cantarella, A. Gallifuoco, A. Spera,

and F. Alfani. 2004. Effect of inhibitors released

during steam-explosion treatment of poplar wood

on subsequent enzymatic hydrolysis and SSF. Bio-

technol. Prog. 20:200-206.

Cara, C., E. Ruiz, J. M. Oliva, F. Saez, and E. Castro.

2008. Conversion of olive tree biomass into fer-

mentable sugars by dilute acid pretreatment and

enzymatic saccharification. Bioresour. Technol.

99:1869-1876.

Chung, Y., A. Bakalinsky, and M. H. Penner. 2005.

Enzymatic saccharification and fermentation of xy-

lose-optimized dilute acid-treated lignocellulosics.

Appl. Biochem. Biotechnol. 121-124:947-961.

Foston, M. and A. J. Ragauskas. 2010. Changes in

lignocellulosic supramolecular and ultrastructure

during dilute acid pretreatment of Populus and

switchgrass. Biomass Bioenergy 34:1885-1895.

Gan J. and C. Smith. 2006. Availability of logging res-

idues and potential for electricity production and

carbon displacement in the USA. Biomass Bioen-

ergy 30:1011-1020.

Jackson, S. 2007. Southeastern Biomass/Bioenergy

Overview. In: W. Hubbard, L. Biles, C. Mayfield,

and S. Ashton. Eds. Sustainable Forestry for Bioen-

ergy and Bio-based Products: Trainers Curriculum

Notebook. Southern Forest Research Partnership

Inc, Athens, GA. p 45-475.

Kabel, M. A., G. Bos, J. Zeevalking, A. G. J. Voragen,

and H. A. Schols. 2007. Effect of pretreatment se-

verity on xylan solubility and enzymatic breakdown

on the remaining cellulose from wheat straw. Bio-

resour. Technol. 98:2034-2042.

Lloyd, T. A., and C. E. Wyman. 2005. Combined sugar

yields for dilute sulfuric acid pretreatment of corn

stover followed by enzymatic hydrolysis of the re-

maining solids. Bioresour. Technol. 96:1967-1977.

Palmqvist, E. and B. Hahn-Hagerdal. 2000. Fermen-

tation of lignocellulosic hydrolysates. II: inhibitors

and mechanisms of inhibition. Bioresour. Tech-

nol. 74:25-33.

Pelkki, M. 2007. Statewide In-Forest Residue Supply

for Arkansas. Presentation for the Arkansas De-

partment of Economic Development August 29,

2007, Little Rock, AR.

Saha, B. C., L. B. Iten, M. A. Cotta, and Y. V. Wu. 2005.

Dilute acid pretreatment, enzymatic saccharifica-

tion and fermentation of wheat straw to ethanol.

Proc. Biochem. 40:3693-3700.

Sannigrahi, P., D. Kim, S. Jung, and A. Ragauskas.

2011. Pseudo-lignin and pretreatment chemistry.

Energy Environ. Sci. 4:1306-1310.

Siry, J. 2002. Intensive Timber Management Practic-

es. In: D. N Wear and J. G. Greis. Eds. Southern

forest resource assessment. Gen. Tech. Rep. SRS-

53, USDA For. Serv. So. Res. Sta., Asheville, NC. p

327-340.

Sluiter, A., B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D.

Templeton, and D. Crocker. 2008. Determination

of Structural Carbohydrates and Lignin in Biomass.

Laboratory Analytical Procedure (LAP). National

Renewable Energy Laboratory, Golden, CO.

Torget, R., P. Werdene, M. Himmel, and K. Grohm-

ann. 1990. Dilute acid pretreatment of short rota-

tion woody and herbaceous crops. Appl. Biochem.

Biotechnol. 24/25:115-126.

Torget, R., P. Werdene, M. Himmel, and K. Grohm-

ann. 1991. Dilute acid pretreatment of hardwood

bark. Bioresour. Technol. 35:239-246.

Wear, D. and J. Greis. 2002. Southern forest resource

assessment: summary report. Gen.Tech. Rep. SRS-

54, USDA For.Serv.So.Res.Sta., Asheville, NC. p

103.

Wyman, C.E. 1994. Ethanol from lignocellulosic bio-

mass: technology, economics, and opportunities.

Bioresour. Technol. 50:3-16.




ABSTRACT

The objective of this study was to assess the microbiological quality of raw meat and processed meat

products in Dhaka city and test the antibiotic susceptibility of the Staphylococcus isolates. A total number

of 79 meat samples were categorized into two groups, viz., Group-1 meat (raw meat), collected from dif-

ferent slaughter yards and meat stalls located in the commercial areas of Dhaka city and Group-2 meat

(processed meat products), collected from ready-to-eat foods. Microbiological quality of the samples

was determined by Total Viable Bacterial Count (TVBC), Total Coliform Count (TCC), Total Salmonella and

Shigella Count (TSSC), Total Staphylococcus aureus Count (TSAC) and Total Fungal Count (TFC). Hetero-

trophic bacteria were recovered from all the meat samples but no Salmonella, Shigella were detected. As

expected, some of the samples were found positive with Staphylococcus spp. and coliform. The statisti-

cal analysis showed that the mean TVBC (log value/g) was significantly greater (P<0.05) in raw meat from

Kawranbazar than all other meat samples studied. TVBC and TSAC exhibited regional significant variation

(P<0.05), whereas TCC did not show any remarkable regional variation. Our present study reveals that the

TVBC, TCC and TSAC of the meat samples were high in those commercial areas and pose potential risk

for public health. From seventy nine samples, 35 isolates of S. aureus were obtained and identified by

standard biochemical tests. All these isolates were tested for their sensitivity against common antibiotics

used in Bangladesh. Percentage resistance of the S. aureus samples to penicillin, ampicillin, streptomycin,

tetracycline, amoxicillin and neomycin were found to be 85.71%, 71.42%, 100%, 71.42%, 100% and 85.71%,

respectively. But no resistance to vancomycin, bacitracin, cefaclor and ciprofloxacin was found in these

isolates. The percentage of multidrug resistant (MDR, resistant against more than three antibiotics) staphy-

lococci was 20%.

Keywords: Raw meat, meat products, contamination, microbiological quality, antibiotic, susceptibility, resistance, Staphylococcus, Bangladesh

Correspondence: Suvamoy Datta, [email protected]: +880-17-5554-8324

Microbiological Quality Assessment of Raw Meat and Meat Products, and Antibiotic Susceptibility of Isolated Staphylococcus aureus

S. Datta1, A. Akter1, I. G. Shah1, K. Fatema1, T. H. Islam1 , A. Bandyopadhyay2, Z. U.M. Khan1, D. Biswas3

1Department of Microbiology, Primeasia University, 9 Banani, Dhaka-1213, Bangladesh2 DOEACC Centre, Jadavpur University Campus, Kolkata-700032, India

3Department of Animal and Avian Sciences, University of Maryland College Park, MD, USA



INTRODUCTION

Radical dietary shifts in many developed and

developing nations are supplanting traditional pat-

terns of eating with a western diet high in animal

meat products and refined carbohydrates and low

in whole grains, fruits, and vegetables (Frank et al.,

2008). There are considerable human health con-

sequences with foodborne infections ranging from

protracted illness to death and patients with im-

paired immunity are at greater risk. Microbiological

food borne diseases are typically caused by bacteria

or their metabolites, parasites, viruses or toxins. The

importance of different food borne diseases varies

between countries depending on foods consumed,

food processing, preparation, handling, storage

techniques employed, and sensitivity of the popula-

tion (ICMSF, 2002).

Meat is not only highly susceptible to spoilage,

but also frequently implicated in the spread of food

borne illness. Contaminated raw meat is one of the

main sources of foodborne illness (Bhandare et al.,

2007; Podpecan et al., 2007). During slaughter and

processing, all potentially edible tissues are subject-

ed to contamination from a variety of sources within

and outside animal. In living animals, those surfaces

in contact with the environment, harbor a variety of

microorganisms. The contaminating organisms are

derived mainly from the hide of the animal and also

comprise organisms that originate from both feces.

In addition, processed meat foods are more prone

to contamination with pathogenic microorganisms

during the various stages of processing. Meat and

meat products are important sources of human in-

fections with a variety of foodborne pathogens, i.e.

Salmonella spp., Campylobacter jejuni/coli, Yersinia

enterocolitica, verotoxigenic Escherichia coli and, to

some extent, Listeria monocytogenes. Some patho-

gens in meats (eg. Salmonella spp., Campylobacter

spp.) are most efficiently controlled by the main in-

terventions applied in the primary production com-

bined with the optimization of the slaughter hygiene.

For organisms like, L. monocytogenes, Staphylococ-

cus aureus and Clostridium spp., the main control

measures are focused on later stages of the meat

chain (Norrung et al., 2009). The high prevalence

of diarrheal diseases in many developing countries

suggests major underlying food safety problems

(Food safety and food borne illness, 2009). These

food items can cause serious problems when they

are contaminated with harmful microorganisms due

to lack of proper sanitary condition, hygiene prac-

tices, and proper storage and mishandling (WHO,

2009). Due to unawareness and non-enforcement of

laws often consumers buy meat and meat product

that failed to protect consumers’ right and possess

a potential risk.

In Bangladesh, beef rolls, chicken fries, sandwich-

es are gradually becoming the popular ready-to-eat

foods and there is also a rapid growth in local pro-

duction of chicken fries in recent years. After 1996,

the large foreign franchises were launched especially

Pizza Hut, KFC (Kentucky Fried Chicken). This trend

was followed by local producers and many franchise

companies were formed. However, there are major

differences between local chicken fries and those

franchised. The quality of locally produced and fran-

chise chicken fries should be monitored from time to

time to ensure that the products meet the minimum

requirements of standards and specifications, and

are of acceptable quality to the consumers. Con-

siderable studies have been carried out in different

countries of the world on fast foods and fast food

restaurants with respect to the outbreak of many

gastrointestinal and other diseases (Easa et al., 2010).

The indiscriminate use of antimicrobial drugs in

food animals may result in transfer of resistance to

human, it is unlikely that the so called reverse-an-

timicrobial drug will be restricted to use in human

medicine (Schwartz and Chaslus Dancla, 2001). The

problem may be due to the natural resistance of spe-

cies to certain antibiotics, possible transfer of antibi-

otics resistance among species and the use of sub-

therapeutic doses of antibiotics in animal feeds to

improve animal productivity could also select for re-

sistance strains. Recently, a dramatic increase in the

resistance against antibiotics routinely used in human

as well as in veterinary medicine has been recorded

in the members of the genus Staphylococcus. Devel-

opment of resistant or multi resistant Staphylococcus


strains causes considerable therapeutic problems.

Although, it is not inevitable to prove a direct role

of drug resistance in bacteria contaminating food

items with increased clinical cases of resistant infec-

tions, the presence of such bacteria in food items

and their related environment could play a role on

the spread of antimicrobial resistance amongst food

borne pathogens (Farzana et al., 2009). Therefore,

this study was conducted to investigate the micro-

bial quality of raw meat and meat products available

in most commercial areas in Dhaka City and to deter-

mine the antibiotic resistant pattern of the isolated

S. aureus.


Meat Sample Collections

Samples of raw meat were collected from differ-

ent butcher open shops. The samples collected from

Mohakhali bazar, Kawran bazaar, Badda Rampura ba-

zaar and Khilkhet bazaar were marked as ‘1’, ’2’, ’3’

and’4’, respectively. Fast food samples named ready

packet Bangla meat products, chicken sandwiches,

beef rolls, chicken cutlets, chicken shawarmas and

chicken fries, were collected from six different lo-

cations including Farmgate, Motijheel, Malibagh,

Mouchak, Banani and Uttara, and samples collected

from Farmgate, Motijheel, Malibagh, Mouchak, Ba-

nani and Uttara were marked as ‘5’, ‘6’, ’7’, ’8’, ’9’

and ‘10’, respectively. The collected samples were

immediately transported in insulated ice containers

to the laboratory for microbial analysis.

Meat sample preparation

Ten gram of the solid sample was weighed and

aseptically taken into a sterile jar containing 90 ml

sterile normal saline. It was homogenized with sterile

blender (Retsch, GM 200, Australia) at 3000 rpm for

5-10 min. A 1mL aliquot of homogenate was trans-

ferred to a test tube containing 9 mL sterile distilled

water to make 10-2 dilution and shaken well with vor-

tex mixer (Digosystem, VM-1000, Taiwan). Serial dilu-

tions up to 10-5 were prepared for the microbiologi-

cal analysis.

Microbiological analysis

The microbiological quality and safety of meat

and meat products were assessed on the basis of

Total Viable Bacterial Count (TVBC), Total Coliform

Count (TCC), Total Staphylococcus aureus Count

(TSAC) and Total Salmonella and Shigella Count

(TSSC), and Total Fungal Count (TFC) using Plate

count agar (PCA, Himedia, India), MacConkey agar

(MCA, HiMedia, India), Mannitol Salt agar (MSA, Hi-

Media, India), Salmonella-Shigella agar (SSA, HiMe-

dia, India) and Potato Dextrose agar (PDA, HiMedia,

India), respectively. Diluted meat samples in normal

saline were spread onto these plates and incubated

at 37°C for 24 hr except detection of fungi, which

were incubated at 25°C for 5 days. Staphylococcus

isolates were confirmed by microscopic, cultural and

standard biochemical tests (motility, catalase, coagu-

lase, oxidase, urease, citrate utilization, indole, gela-

tin hydrolysis, MR-VP, TSI test) according to Bergey’s

Manual of Determinative Bacteriology, (9th Edition,

1994) for further analysis.

Antibiotic Susceptibility Testing

The antibiotic susceptibility of the Staphylococcus

isolates was determined using the standard disc-dif-

fusion (Kirby–Bauer, 1997) method. Overnight grown

cultures were used for the test. The antibiotic discs

(Oxoid®, UK) used in this study were: amoxicillin (10

µg), ampicillin (25 µg), bacitracin (10 µg), cefaclor (30

µg), ciprofloxacin (5 µg), neomycin (30 µg), penicillin-

G, (10 units), streptomycin (10 µg), tetracycline (30

µg) and vancomycin (30 µg).


Microbiological analysis

The present study evaluated the microbiological

quality of raw meat and meat products in Dhaka,


Bangladesh. The microbiological condition of safety

and hygiene were then assayed using the methods

recommended by ICMSF (International Commission

on Microbiological Specifications for Foods). The

total viable counts of raw meat and meat products

were determined by standard method. We found

that all samples were contaminated with microor-

ganisms (Table 1).

Total Viable Bacterial Count (TVBC) (log mean value/g)

The TVBC mean log value in sample 1 to sample

10 were 6.47, 8.65, 7.75, 7.63, 7.54, 6.57, 6.29, 5.42,

6.74 and 5.59, respectively. Viable counts of raw meat

were significantly higher (P<0.05) in raw meat sam-

ple ‘2’ followed by sample ‘3’, ‘4’, ‘5’ and significant-

ly lower mean value was observed in chicken cutlets

and chicken fries. However, TVBC of sample 3, 4 and

5 did not differ significantly among them. Similarly,

sample 1, 6, 7 and 9; and sample 8 and 10, did not

differ among them. TVBC found in meat samples of

the present study indicated a remarkable increase.

Hoque et al. (2008) studied raw meat samples and

found 6.03 from slaughter yards and 6.53 from meat

stalls. Another study was carried out by Waliullah et

al. (2011), on the meat based fast foods from Dhaka

University campus areas. The standard plate count

revealed that in that meat based fast food chicken

sandwiches were 5.12, chicken burgers 6.23 and hot

dogs 6.42. Since chicken fries and chicken shawrmas

contain fried meat, low TVBC was observed and our

results agreed with their results.

Total Coliform Count (TCC) (log mean value/g)

The total coliform count (TCC) in meat samples

1, 3, 4, 6 and 8 were 4.31, 4.42, 5.5, 5.58, and 4.8,

respectively (Table 1). None of TCC pathogens were

detected in samples 2, 5, 7, 9 and 10. It was found

that chicken sandwiches contained significantly

(P<0.05) higher numbers of coliform compared to

other open meat samples and meat products. Anal-

ysis of variance revealed significantly lower (P<0.05)

coliform counts from samples 1, 3, and 8. However,

no significant difference was found between the

mean TCC of sample 4 and 6. These counts indicate

the aseptic techniques of food processing.

Total Staphylococcus aureus Count (TSAC) (log mean value/g)

A total of 35 out of 79 meat samples were found

positive in total S. aureus counts (TSAC). TSAC of

samples 1, 2, 4, 5, 6, and 7, were 5.48, 5.75, 4.13, 5.95,

4.34, and 5.97, respectively but no S. aureus was

found in the samples 3, 9 and 10 (Table 1). The mean

staphylococcal counts of raw meat sample 2, sample

5 and beef roll were significantly high (P<0.05) than

other samples. However, there was no significant dif-

ference among sample 4 and 6. Anawar et al. (2004)

isolated 90.63% Staphylococcus spp. from dressed

broilers. Our results showed that 73.68% S. aureus

were isolated from raw meat samples.

Total Salmonella-Shigella Count (TSSC) (log mean value/g)

None of the samples contained Salmonella and

Shigella (Table 1); this is in accordance with the re-

sults of Selvan et al. (2007) who did not recover Sal-

monella from samples of retail meat products. The

absence of Salmonella in the meat product samples

indicate the quality of raw meat and other hygienic

processing including the quality of the water used in

processing.

Total Fungal Count (TFC) (log mean value/g)

No fungus was found in the sample of meat and

meat products except sample ‘2’ and sample ‘4’.

However, total fungus counts of Kawran Bazar and

Khilkhet were 4.65 and 3.65, respectively (Table 1).

It is evident from Table 1, sample 2, i.e. the raw

meat sample collected from Kawran Bazar was not

safe as compared to the other samples with high

TVBC, TSAC, and TCC. Among meat products,

Bangla packet products, followed by beef rolls and


chicken cutlets were significantly (P< 0.05) contami-

nated as compared to the other meat products. Ac-

cording to statistical analysis, it was noted that the

microbiological quality of most of the raw meat and

meat products of Dhaka city was significantly poor.

Our finding showed that raw meat and meat prod-

ucts, which were collected from different location,

were contaminated with pathogenic microorgan-

isms. As observed in the course of the study, the

method of slaughtering of animals is responsible for

the microbial contamination .The study indicated

that the count of enumerated bacteria in raw meat

was higher than acceptable values, making the prod-

uct a potential public health hazard.

Antibiotic sensitivity of the Staphylo-coccus isolates

Multidrug resistant strains of S. aureus will be a

risk factor for the public health of a developing

country such as Bangladesh. To find the prevalence

of drug resistant Staphylococci, assays for suscepti-

bility profiles were performed. High level resistance

of staphylococci isolates to various classes of antibi-

otics was observed. As expected, the staphylococ-

cal isolates from the meat samples were found to be

resistant to many of the antibiotics tested, particu-

larly the ones that are generally used as initial line of

treatment. Our susceptibility results of the isolated

staphylococci from seven different locations showed

that they are highly resistant to penicillin, ampicillin,

amoxicillin, tetracycline, streptomycin, and neomy-

cin. Percentage resistance of the S. aureus samples

to penicillin, ampicillin, amoxicillin, streptomycin

and neomycin were 85.71, 71.42, 100, 100, and 85.71,

respectively. As evident from Table 2, all S. aureus

Table 1. Mean (±SE) and analysis of bacterial counts (log value/g) of different samplesA

Total Count

Meat and Meat Products

Raw meat1

(n=5)

Raw meat2

(n=7)

Raw meat3

(n=3)

Raw meat4

(n=4)

Bangla meat5

(n=10)

TVBC 6.47±0.2101c 8.65±0.065a 7.45±0.093b 7.63±0.088b 7.54±0.101b

TCC 4.31±0.1002b 0 4.42±0.05b 5.5±0.047a 0

TSAC 5.48±0.0675b 5.75±0.0607a 0 4.13±0.0132c 5.95±0.0408a

TSSC 0 0 0 0 0

TFC 0 4.65±0.0612a 0 3.65±0.09b 0

Chicken

Sandwich6

(n=10)

Beef

Roll7

(n=10)

Chicken cutlet8

(n=10)

Chicken

Shawarma9

(n=10)

Chicken fry10

(n=10)

TVBC 6.57±0.097c 6.29±0.08c 5.42±0.07d 6.74±0.07c 5.59±0.09d

TCC 5.58±0.024a 0 4.8±0.3007b 0 0

TSAC 4.34±0.0655c 5.97±0.0599a 0 0 0

TSSC 0 0 0 0 0

TFC 0 0 0 0 0

1-10these number indicates the sample numbers

AData represent the mean values obtained from ten samples, and are expressed in logarithmic colony forming unit per gram (CFU/g). Significant differences in plate count data were established by the least-significant dif-ference at the 5% level of significance. Mean values with the same letter in the same row are not significantly different (P<0.05).


isolates showed no resistance to vancomycin, baci-

tracin, cefaclor and ciprofloxacin, i.e., they are sus-

ceptible, thus giving us some way of treating any

infection caused by the same strains of the S.aureus

isolates. Thirty five of the tested isolates of staphylo-

cocci (47 %) were resistant to only two antibiotics, 25

(33 %) to three antibiotics, and multidrug resistance

(MDR) was confirmed in 15 isolates (20 %) (Figure 1).

Regecová et al. (2009) found 47 % to two antibiotics,

and multidrug resistance was 25 % from fish meat

Staphylococci isolates. In Korea, Heo et al. (2008)

found 7.8% MDR S. aureus isolates from domestic

and imported meats. Waters et al. (2011) found 52%

MDR (resistant against 3 or more drug) S. aureus

from poultry and meats.

CONCLUSIONS

The presence of bacteria in meat has been widely

reported from different parts of the world (Holds

et al., 2007; Kinsella et al., 2008). Some groups rec-

ognized the presence of bacteria especially gram-

negative organisms as an indicator of open air meat

spoilage, while others argued this assertion and

considered the presence of a high number of back-

ground organisms as pathogen-reduction strategy

due to the organisms antagonistic effect against

pathogenic bacteria and thus safe for meat quality.

Our result indicated that the gram negative coli-

forms and gram-positive bacteria were present pre-

dominantly, and the fungus was the least frequent in

the meat and meat samples. In view of the microbial

implication in handling, slaughtering, dressing, pro-

cessing and distribution of meat and meat products

which may endanger human health, the study was

undertaken to determine the extent of microbial

contamination of meat in the commercial areas of

Table 2. Antibiotic Resistance patterns of isolated S. aureusB

SampleNumber of Antibiotic resistant isolates from each sample

Pen Amp Amo Tet Strp Neo Cef Cipro Bac Van

1(n=5) 4 3 5 3 5 5 0 0 0 0

2(n=5) 5 4 5 5 5 5 0 0 0 0

4(n=4) 3 2 4 2 4 3 0 0 0 0

5(n=5) 4 4 5 3 5 4 0 0 0 0

6(n=6) 5 5 6 5 6 6 0 0 0 0

7(n=4) 3 2 4 2 4 3 0 0 0 0

8(n=6) 6 5 6 5 6 4 0 0 0 0

Total 30 25 35 25 35 30 0 0 0 0

Percentage (%)

85.71 71.42 100 71.42 100 85.71 0 0 0 0

BSample ‘3’,’9’ and ‘10’ were omitted since S. auerus was absent in these samples

Figure 1. Percentage of Multidrug Resistance in S. aureus isolates (against two or more than two drugs)


Dhaka city in Bangladesh. Contamination prevention

rather than end-product testing to ensure the safety

of meat is needed. As raw meats were heavily con-

taminated with microorganisms and are potential

sources of food borne infections, therefore raw meat

handlers should receive education in food hygiene.

Meat and poultry processors and regulators should

use process control techniques to ensure that per-

formance standards for meat and poultry are met.

Vaccination, immuno-modulation and pre and probi-

otics need to be considered as alternatives for com-

bating bacterial infection as far as possible, although

they are unlikely to completely replace antimicrobial

drugs (Schwarzt et al., 2001). We therefore, suggest

the application of stringent hygiene practices along

the food chain and prudent use of antibiotics in ani-

mal husbandry which are essential for the control of

further emergence of multidrug resistance.

ACKNOWLEDGEMENTS

A study on the microbiological quality of raw meat

and processed meat products was carried out in the

Department of Microbiology, Primeasia University,

Banani, Dhaka, Bangladesh.

REFERENCES

Anower, A. K. M. M., M. M. Rahman, M. A. Ehsan,

M. A. Islam, M. R. Islam, G. C. Shil, and M. S. Rah-

man. 2004. Bacteriological Profile of Dressed Broil-

ers and Its Public Health Implications. Bangl. J. Vet.

Med. 2:69-73.

Bergey, D. H., J. G Holt, and N. R Krieg. 1994. Ber-

gey’s Manual of Determinative Bacteriology, 9th

Edition. Lippincott Williams & Wilkins. MI, USA.

Bhandare, S. G., A. T. Sherikarv, A. M. Paturkar, V. S.

Waskar, and R. J. Zende. 2007. A comparison of

microbial contamination of sheep/goat carcasses

in a modern Indian abattoir and traditional meat

shops. Food. Contr. 18:854-868.

Easa, S. M. H. 2010. The Microbial Quality of Fast

Food and Traditional Fast Food. Nature and Sci-

ence. 8(10).

Farzana, K., S. Akhter, and F. Jabeen. 2009. Preva-

lence and antibiotic resistance of bacteria in two

ethnic milk based products. Pak. J. Blot. 41:935-

943.

Frank, B. H. 2008. Globalization of Food Patterns and

Cardiovascular Disease Risk. Circulation. 118:1913-

1914.

Heo, H. J., B. K. Kyung, D. H. Bae, C. K. Park, and Y.

Ju Lee. 2008. Antimicrobial resistance of Staphylo-

coccus aureus isolated from domestic and import-

ed raw meat in Korea. Korean J Vet Res. 48:75-81.

Holds, G., A. Pointon, M. Lorimer, A. Kiermeier, G.

Raven, and J. Sumner. 2007. Microbial profiles of

carcasses and minced meat from Kangaroos pro-

cessed in South Australia. Int. J. Food. Microbiol.

123:88-92.

ICMSF (International Commission on Microbiologi-

cal Specifications for Foods). 2002. Microorgan-

isms in Foods 7. Microbiological Testing in Food

Safety Management. New York: Kluwer Academic/

Plenum Publishers.

Kinsella, K. J., D. M. Prendergast, M. S. McCann, I. S.

Blair, D. A. McDowell, and J. Sheridan. 2008. The

survival of Salmonallea enteric serovar Typhimuri-

um DT 104 and total viable counts on beef surfaces

at different relative humidities and temperatures J.

App. Microbiol. 106:171-180.

Kirby-Bauer Method. 1997. Disk Diffusion Suscep-

tibility Testing. Newsletter of Animal Disease Di-

agnostic Laboratory. http://www.addl.purdue.edu/

newsletters/1997/spring/dds.shtml.

Hoque, M. A., M. P. Siddique, M. A. Habib, V. Sarker,

and K.A. Choudhury. 2008. Evaluation of sanitary

quality of goat meat obtained from slaughter yards

and meat stalls at late market hours. Bangl. J. Vet.

Med. 6:87-92.

Norrung, B., J. K. Anderson, and S. Buncic. 2009.

Main Concerns of Pathogenic Microorganisms

in Meat. In Safety of Meat and Processed Meat

(Toldra F.), Part I, Springer New York, pp 3-29.

Selvan, P., R. Narendra Babu, S. Sureshkumar, and V.

Venkataramanujam. 2007. Microbial Quality of Re-

tail Meat Products Available in Chennai City. Am. J.

Food Technol. 2:55-59.


Podpecan, B., A. Pengov, and S. Vadnjal. 2007. The

source of contamination of ground meat for pro-

duction of meat products with bacteria Staphylo-

cocccus aureus. Slov. Vet. Res. 44:24-30.

Regecová, I., M. Pipová, P. Jevinová, P. Popelka, and

I. Kožárová. 2009. Determination of sensitivity of

Staphylococcal isolates from fish meat against se-

lected antibiotics. Folia Veterinaria 53:37-39.

Schwarz, S., and D. E. Chaslus. Use of antimicrobi-

als in veterinary medicine and mechanism if resis-

tance. Vet Res 2001 32:201-225.

Schwarzt, S., C. Kehrenberg, and T. R. Walsh. 2001.

Use of antimicrobials in veterinary medicine and

food animal production. Intern J. Antimicrob

Agen. 17:431-437.

Waliullah, S., and C. R. Ahsan. 2011. Assessment

of microbiological quality of some meat-based

fast foods collected from street vendors. J. Innov.

Strategy 5:44-46.

Waters, A. E., T. Contente-Cuomo, J. Buchhagen, C.

M. Liu, L. Watson, K. Pearce, J. T. Foster, J. Bowers,

E. M. Driebe, D. M. Engelthaler, P. S. Keim, and L.

B. Price. 2011. Multidrug-Resistant Staphylococ-

cus aureus in US Meat and Poultry. Clin Infect Dis.

52:1227–1230.

WHO. 2009. Food safety and food borne illness.

Factsheets of the Programmes and Projects of

WHO. http://www.who.int/mediacentre/fact-

sheets/fs237/en/.




ABSTRACT

Listeria monocytogenes is a dangerous food-borne pathogen and is a frequent contaminant found in

the cold-smoked fish industry. To identify strategies to eliminate this bacterium from the cold-smoking

processing environment, it is imperative to understand how this microorganism tolerates the conditions en-

countered. The aim of this study was to determine whether exposure to conditions likely to be encountered

during the cold-smoking process differentially impacts various strains of Listeria monocytogenes and Liste-

ria innocua. Viability of L. monocytogenes (EGDe, F2365, HCC7, ATCC 15313, and HCC23) and L. innocua

in exponential or stationary growth phase were analyzed following a sequential exposure to conditions that

mimic those utilized in the cold-smoking process: freeze (-20°C)-thaw (25°C), elevated salt, liquid smoke,

and anaerobic storage (2°C). Viability for stationary phase cells exposed to the mock process decreased

(P<0.05) for all strains except EGDe. Viability for exponential phase cells also decreased (P<0.05) for all

strains except for EGDe and HCC7 treated cells. The cell envelope of the avirulent strain HCC23 was al-

tered by all treatments examined, while the cell envelope of the virulent strain HCC7 was altered only after

exposure to liquid smoke and anaerobic storage. Results indicate that both virulent and avirulent strains in

this study, whether in exponential or stationary phase, can tolerate the conditions encountered during the

cold-smoking process, and that virulent strains are more resistant than avirulent strains. Collectively, these

data strongly suggest that differences exist in the mechanisms utilized by virulent and avirulent strains to

adapt to conditions encountered in the cold-smoking process.

Keywords: Listeria monocytogenes, Listeria innocua, cold-smoking, salmon, stress-response, salt, liquid smoke, cold, transmission electron microscopy, L-forms, protoplasts

Correspondence: Janet R. Donaldson, [email protected] Tel: +1 -662-325-9547 Fax: +1-662-325-7582

Effect of Stressors on the Viability of Listeria During an in vitro Cold-Smoking Process†

J. R. Pittman1, T. B. Schmidt2, A. Corzo3, T. R. Callaway4, J. A. Carroll5, and J. R. Donaldson1

1Department of Biological Sciences, Mississippi State University, Mississippi State, MS, 2Animal Science Department, University of Nebraska, Lincoln, NE

3Department of Poultry Science, Mississippi State University, Mississippi State, MS4Food and Feed Safety Research Unit, U. S. Department of Agriculture, Agricultural Research Service, College Station,

TX5Livestock Issues Research Unit, U. S. Department of Agriculture, Agriculture Research Service, Lubbock, TX

† Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The U.S. Department of Agriculture (USDA) prohibits

discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all

or part of an individual’s income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Per-sons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or

(202) 720-6382 (TDD). USDA is an equal opportunity provider and employer.



INTRODUCTION

Listeria monocytogenes is a gram-positive, food-

borne bacterium that causes 2,500 cases of listeriosis

and nearly 500 deaths annually in the United States,

making it one of the most deadly food-borne patho-

gens (Ben Embarek, 1994; Mead et al., 1999). The

costs associated with the annual cases of listeriosis

in the United States is estimated at $8.8 billion USD

for health care alone (Scharff, 2010). Consumption of

food contaminated with L. monocytogenes poses

a serious health risk to the elderly, pregnant wom-

en, immunocompromised individuals, and human

neonates, and can result in life-threatening medical

conditions such as meningitis, meningoencephalitis,

spontaneous abortive pregnancies, as well as febrile

gastroenteritis (Cossart and Toledo-Arana, 2008;

Posfay-Barbe and Wald, 2009; Sleator et al., 2009).

Ready-to-eat foods, such as smoked finfish, are

the most common sources of listeriosis (Gilbreth et

al., 2005; Gombas et al., 2003). The prevalence of L.

monocytogenes in cold-smoked fish products is typi-

cally between 15-20% (Uyttendaele et al., 2009). Vari-

ous aspects within finfish smoking facilities can serve

as sources of contamination, such as contaminated

raw materials entering the plant and contaminated

food-processing equipment (Fonnesbech Vogel et

al., 2001; Rorvik, 2000; Vaz-Velho et al., 2001). Persis-

tent L. monocytogenes strains present in biofilms or

L-forms can also lead to contamination of the pro-

cessed food products (Gandhi and Chikindas, 2007;

Moretro and Langsrud, 2004). The ubiquitous and

recalcitrant nature of L. monocytogenes makes re-

moval or exclusion of this microorganism from the

processing environment difficult (Wulff et al., 2006).

For instance, L. monocytogenes can survive condi-

tions encountered during the cold-smoking process,

such as decreased temperatures (2°C), elevated con-

centrations of salt (3.5 to 6%, w/v), and phenols en-

countered in cold smoke or through the addition of

liquid smoke (Gandhi and Chikindas, 2007; Hwang,

2007; Porsby et al., 2008). Additionally, Listeria is ca-

pable of continued growth even under anaerobic

storage conditions (Cortesi et al., 1997; Guyer and

Jemmi, 1991; Rorvik et al., 1991).

While previous studies have provided vital infor-

mation on the response of L. monocytogenes to con-

ditions encountered during the cold-smoking pro-

cess, most of these studies have only included the

analysis of one or two strains. Additionally, none of

these prior studies examined the effect of sequential

exposure to each of the steps in the cold-smoking

process on the viability of L. monocytogenes. There-

fore, the goal of this study was to determine whether

exposure to conditions typically encountered during

the cold-smoking process differentially impacts vari-

ous serovars of Listeria of different pathogenic po-

tential.


Bacterial strains and growth conditions

Virulent and avirulent strains of Listeria analyzed

in this study are listed in Table 1. All strains were

routinely cultivated under aerobic conditions in

brain-heart infusion (BHI) medium at 37°C in an or-

bital shaking incubator. The viability of each strain

was monitored throughout the study by viable plate

counts on BHI agar; plates were incubated for 24 h

at 37°C prior to enumeration. All assays were per-

formed with bacteria in either stationary phase or

exponential (mid-logarithmic) phase. To obtain cells

in stationary phase, colonies from each strain were

grown in BHI broth for 8 h, diluted 1:100 in 5 mL BHI,

then allowed to grow at 37°C for 16 h to an OD600

1-1.5. To obtain cells in exponential phase, colonies

from each strain were grown in BHI broth for 16 h,

diluted 1:100 in 5 mL BHI, then allowed to grow at

37°C to an OD600 0.3-0.4.

Mock cold-smoking procedure

Stationary and exponential phase cells were ex-

posed to a mock cold-smoking procedure to de-

termine the effect of the sequential process on cell

viability; this procedure was developed based upon

information obtained through consultations with in-

dividuals from two cold-smoking facilities located in


Alaska, United States. Specifically, cells (5 mL) were

frozen at -20°C for 2 h and then thawed at room

temperature (RT) for 1 h. Following the freeze-thaw

treatment, 1 mL of cells was pelleted by centrifuga-

tion for 2 min at 10,000 x g (TOMY MX 301, TOMY

TECH USA, Freemont, CA) and then re-suspended

in 1 mL BHI supplemented with 6% NaCl (w/v) to

mimic the brining process. Cells were treated with

NaCl for 1 h at 30°C, pelleted by centrifugation and

re-suspended in BHI supplemented with 0.6% liquid

smoke to mimic exposure to phenolic compounds

encountered during the smoking step as previously

described (Faith et al., 2007). Cells were treated with

liquid smoke for 1 h at 30°C, after which cells were

pelleted by centrifugation at 10,000 x g and resus-

pended in 1 mL of BHI broth. Cells were then vacu-

Table 1. Strains utilized

Strain/ Source Serovar Isolation Information ReferencesVirulent

EGDe/ ATCC BAA-679 1/2a Guinea Pig Murray et al., 1926

F2365/ CDC 4bMexican-style soft cheese

Linnan et al., 1988

HCC7/ MSU CVM1 1 Catfish Brain Wang et al., 1998Avirulent

HCC23/ MSU CVM1 4a Catfish Brain Wang et al., 1998ATCC 15313 1 Guinea Pig Barber, 1939; Murray et al., 1926

Listeria innocua ATCC BAA-680 6a Dairy Product Danielsson-Tham et al., 1993

1 HCC7 and HCC23 were isolated from catfish specimens at Mississippi State University’s College of Veterinary Medicine ; isolates were serotyped and characterized (Wang et al., 1998)

Figure 1. Experimental design of mock cold-smoking process. Cells in either mid-log or sta-tionary phase were initially frozen for 2 h, then thawed for 1 h. Cells were then exposed to 6% NaCl for 1 h, 0.6% liquid smoke for 1 h, and then finally to 2°C anaerobic conditions for 16 h. To control for affects within each condition tested, aliquots of cells were exposed to 2°C anaerobic conditions following the initial freeze/thaw or the salt treatment.


um-sealed and stored anaerobically at 2°C for 16 h;

anaerobic conditions were verified by a Mitsubishi

anaerobic indicator. Briefly, a single anaerobic indi-

cator packet was placed in each container immedi-

ately before being sealed and the absence of a color

change indicated that the condition was anaerobic.

Growth was monitored by viable plate counts follow-

ing exposure to each of the conditions examined:

prior to freezing and after thawing, salt treatment,

liquid smoke treatment, and refrigerated storage

(Figure 1). Three independent replicates were per-

formed for each strain. The viable plate counts were

used to calculate the mean log10 proportion reduc-

tion values [log10(Ntreated CFU/mL)/(Noriginal CFU/mL)].

Statistical analysis

Viability data were analyzed as a completely ran-

dom design with repeated measures using the mixed

procedures of SAS to test the effects of the cold-

smoking process on the resistance of six strains of

Listeria to storage conditions (SAS version 9.2, 2008,

SAS Institute Inc., Cary, NC). When F-tests were sig-

nificant (P< 0.05), treatment means were separated

using the method of least significant difference.

Transmission electron microscopy

Exponential phase cultures of the L. monocyto-

genes strains HCC7 and HCC23 were exposed to

the mock cold-smoking process as described above.

For each culture 2 mL of cells were collected and

processed for analysis by transmission electron mi-

croscopy (TEM) prior to the freezing condition and

after thawing, salt treatment, liquid smoke treat-

ment, and storage as previously described (Merritt

et al., 2010). Briefly, cells were pelleted by centrifuga-

tion at 10,000 x g for 2 min, washed with 1X PBS, and

fixed in 2.5% (v/v) glutaraldehyde in 0.1M cacodylate

buffer overnight at 2°C. Samples then were washed

with 0.1M cacodylate buffer, post-fixed in 1% (v/v) os-

mium tetraoxide in 0.1M cacodylate buffer, washed

with distilled water, dehydrated in an ethanol series,

treated in a stepwise resin/acetone series, and em-

bedded overnight in resin at 68-70°C. Samples were

sectioned to 60-80nm in thickness using an ultrami-

crotome (Reichert-Jung Ultracut E); sections were

double stained with uranyl acetate and lead citrate

and viewed under a transmission electron micro-

scope (JEOL JEM-100CXII, JEOL Ltd., Tokyo, Japan).

The widths of the cell membrane, cell wall, and the

entire cell envelope were measured for 20 individual

cells from two independent experiments. The mean

average of the cells at mid-log phase (control) was

compared to the mean average of cells exposed to

each condition using a student-paired t-test. A P-val-

ue < 0.05 indicated that changes in the thickness of

the structures examined were statistically significant.


Previous studies examining the effects of the cold-

smoking process on L. monocytogenes are conflict-

ing, as some studies suggest the process leads to

a reduction in cell viability while others report that

cellular concentrations increase following storage

conditions (Guyer and Jemmi, 1991; Porsby et al.,

2008). To determine whether these differences could

be attributed to strain-to-strain variation, six differ-

ent strains were exposed to a sequential series of

conditions that mimic the cold-smoking fish process

to determine if cell viability was affected differently.

An in vitro model (Figure 1) was used in the present

study in order to determine the direct effect that ex-

posure to these conditions has on the viability of L.

monocytogenes, as opposed to inoculating a single

strain or microbial consortium onto raw salmon fil-

lets as has been performed by others (Hwang, 2007;

Neunlist et al., 2005; Porsby et al., 2008). The in vitro

model was tested on cells in both stationary and ex-

ponential growth phases to determine whether dif-

ferences exist in the resistance capability when cells

are in the more sensitive state of exponential growth

as opposed to the more resilient stationary growth

phase.

Effects of the cold-smoking process on the viability of Listeria in stationary phase


The process of cold-smoking finfish requires an

initial freezing of the fish in order to remove para-

sitic pathogens. The fish are then subjected to a salt

brining process, utilizing 4-6% NaCl and exposure

to smoking conditions. Since the products are not

fully preserved, it is critical that storage temperature

conditions are 4°C or less. To determine whether the

sequential exposure to these conditions affects the

viability of cells when in stationary phase, the mean

log10 proportion reduction values of the virulent

strains EGDe, F2365, and HCC7 and the avirulent

strains HCC23, ATCC 15313, and L. innocua were

analyzed following the cold-smoking procedure pre-

sented in Figure 1.

The first step of the challenge involved exposing

cells in stationary phase to a freeze-thaw condition.

Consistent with previous studies, the psychrotoler-

ant nature of Listeria allowed for viability to remain

relatively unchanged following freeze-thaw (Azizo-

glu et al., 2009; Wemekamp-Kamphuis et al., 2002a)

(Table 2). None of the changes exhibited following

freeze-thaw were significant between the strains

tested in this study.

Following the freeze-thaw condition, cells were

exposed to 6% NaCl at 30°C for 1 h. Exposure to

salt as a means to simulate brining conditions en-

countered during the process has been shown to

result in a significant decrease in cell survival of L.

monocytogenes (Neunlist et al., 2005). However, in

the present study, exposure of stationary-phase cells

to high osmolarity following a previous freeze-thaw

did not significantly alter the viability of any of the

strains tested (P> 0.21).

Following the sequential exposure of stationary

phase cells to a freeze-thaw then salt condition, cells

were then treated with liquid smoke for 1 h at 30°C.

The application of liquid smoke as an alternative

method of smoking fish products has been frequent-

ly used to mimic exposure to the phenols, carbon-

yls, and organic acids present during the smoking

process; it has also been reported to have a detri-

mental effect on the viability of L. monocytogenes

(Faith et al., 2007; Gedela et al., 2007; Guilbaud et

al., 2008; Hwang, 2007; Sunen, 1998; Thurette et al.,

1998; Vitt et al., 2001). In general, exposure to liq-

uid smoke led to an increase in viability (as indicated

by an increase in log proportion reduction values) in

virulent strains and a decrease in viability in avirulent

strains in the present study. Within the treatment,

strain F2365 had the highest viability in comparison

to strains HCC23 and L. innocua (Table 2). In com-

parison to non-treated controls, virulent strain F2365

(P= 0.0253) increased in viability and the avirulent

strains L. innocua (P= 0.0052) and HCC23 (P= 0.0085)

decreased. However, the change in viability exhib-

ited by F2365, L. innocua, and HCC23 was not signifi-

cant in regards to the affect already induced by the

salt treatment. This indicates that viability remained

stable in these strains following the salt treatment.

The final condition in the sequential exposure to

stressors involved in the smoking process was expo-

sure to an anaerobic cold storage (2°C) for 16 h. The

viability of EGDe increased (P= 0.04) in comparison

to non-treated cells, while the viability of both F2365

Table 2. Log10 proportion reduction values for stationary phase cells exposed to a mock cold-smoking procedure

StrainStationary Cells

Freeze-Thaw

Salt Liquid Smoke Storage

EGDe 0.000 a 0.026 a 0.196 b, c 0.185 a 0.271 a

F2365 0.000 a 0.252 a, b 0.220 b, c 0.298 a, c -0.209 b

HCC7 0.000 a 0.109 a 0.132 a, b, c 0.170 a -0.261 b

HCC23 0.000 a -0.046 a -0.185 a, b, c -0.353 b -0.776 c

ATCC 15313 0.000 a 0.127 a 0.018 a -0.118 a, b -0.733 c

L. innocua 0.000 a -0.104 a, c -0.149 a, b -0.376 b -0.701 c

a, b, c Least significant means within column lacking common subscripts differ (P <0.05)


and HCC7 decreased (P= 0.11 and 0.05). Surprising-

ly, the viability of all three avirulent strains decreased

(P< 0.0001; Table 2). Following anaerobic storage

conditions, EGDe had the least log10 CFU/ml pro-

portion reduction (0.271) and was greater (P< 0.05)

than HCC23, which had the greatest log10 proportion

reduction (-0.776; Table 2). Since viability was not al-

tered following the cold exposure portion (first step)

of the cold-smoking procedure, these data suggest

that one of the stressors encountered in this chal-

lenge may actually precondition Listeria to be more

resistant to the cold storage conditions encountered

at the conclusion of the process.

To determine whether exposure to the stressors

individually (as opposed to sequentially) were re-

sponsible for the significant decrease in viability ob-

served among the avirulent strains, the effect that a

pre-treatment with either freeze-thaw or salt had on

viability following the anaerobic 2°C storage was ex-

amined (Figure 2). With the pretreatment of freeze-

thaw conditions, F2365 had a greater (P= 0.04) in-

crease in population growth than all other strains

tested (Figure 2). However, the exposure to salt pri-

or to storage conditions resulted in higher (P< 0.05)

viability of EGDe and F2365 in comparison to the

avirulent strains HCC23, ATCC 15313, and L. innocua

(Figure 2). The decreased viability exhibited by the

avirulent strains in response to osmotic stress could

be due to a defective capability to uptake compat-

ible osmolytes (e.g., peptides glycine betaine) from

the BHI medium (Amezaga et al., 1995). These data

suggest that overall the virulent strains examined

were more resistant to the cold-smoking process

than the avirulent strains when in stationary phase.

Effects of the cold-smoking process on the viability of Listeria in mid-log phase

Variations were evident in the viability of virulent

and avirulent strains following exposure to the mock

cold-smoking process when cells were in stationary

phase. Therefore, the next objective of this study was

to determine whether variations in viability also exist-

ed between the strains when in the more susceptible

state of exponential growth (mid-log growth phase).

The sequential series of conditions tested were the

-1.2

-0.8

-0.4

0

0.4

Log 1

0(N

t/N 0

)

EGDe F2365 HCC7 HCC23 ATCC 15313 L. innocua

a

a b

aac b c bc c d c d

Figure 2. Proportion reduction values (log10 CFU/mL) of stationary phase cells following treat-ment with either freeze-thaw or salt, followed by anaerobic 2°C conditions for 16 h. Cells in stationary phase were pre-treated with either freeze-thaw (green filled) or salt (orange fill) prior to exposure to 2°C anaerobic conditions. Viability was expressed as the mean log10 proportion reduction values [log10 (Ntreated CFU/mL)/(Noriginal CFU/mL)]. Values represent the average log10 CFU/mL proportion reduction values ± standard error from (n = 3) independent replicates. Within each pretreatment condition, values with different superscripts are significantly different (P < 0.05).


same as the conditions presented in Figure 1. The

first step of the cold-smoking process entailed expo-

sure to the freeze-thaw condition. The least signifi-

cant mean log10 proportion reduction values (CFU/

mL) of virulent strains EGDe, F2365, and HCC7 and

the avirulent strains HCC23, ATCC 15313, and L. in-

nocua in mid-log phase following the cold-smoking

process are presented in Table 3. Viability remained

relatively unchanged after exposure to the freeze-

thaw stress and no differences (P > 0.05) were found

to exist between the strains (Table 3), which is consis-

tent with previous studies (Wemekamp-Kamphuis et

al., 2002a) and also the present data obtained when

cells were in stationary phase (Table 2).

Subsequent exposure to high salt conditions re-

sulted in a decrease in viability in HCC23 (P= 0.0002)

and F2365 (P= 0.0103) in comparison to control cells

(Table 3). These data are in contrast to results ob-

tained from stationary phase cells exposed to these

conditions, which indicated that none of the strains

tested exhibited a significant change in viability fol-

lowing exposure to salt. This suggests that these

strains are actually more susceptible to a salt treat-

ment when in exponential growth as compared to

when in stationary phase.

When stationary phase cells were sequentially ex-

posed to liquid smoke, viability remained unaltered

in the virulent strains (Table 2). However, much varia-

tion was observed in the viability of mid-log cells

following the subsequent exposure to liquid smoke

(Table 3). L. innocua had a decrease (P= 0.0012) in

viability in comparison to non-treated cells, suggest-

ing that this strain is more susceptible to these con-

ditions encountered in the cold-smoking process.

HCC23 significantly increased in viability in compari-

son to cells following the salt exposure (P= 0.0115).

The viability of F2365 remained stable following

exposure to liquid smoke, yet decreased (P= 0.01)

in regards to mid-log phase control cells (Table 3).

These data indicate that exposure to liquid smoke

did not affect this strain any more than the exposure

to salt.

Viability of F2365, HCC23, ATCC 15313, and L. in-

nocua significantly decreased after the sequential

exposure to the anaerobic storage in comparison

to non-treated cells. Of all strains tested, only the

viability of ATCC 15313 decreased (P< 0.0001) fol-

lowing storage in comparison to viability prior to

this treatment (log10 proportion reduction values of

0.157 after liquid smoke decreased to -0.567 follow-

ing storage).

To determine which individual condition of the

cold-smoking process impacted Listeria the most

when in mid-log phase, viability following a pre-

treatment with either freeze-thaw or salt followed by

anaerobic storage was analyzed. The log10 propor-

tion reduction values (CFU/mL) for each strain with

statistical analysis specific within each pretreatment

category were analyzed (Figure 3). The effect of the

freeze-thaw pre-treatment on viability following stor-

age conditions resulted in a significant decrease in

viability of L. innocua in comparison to only HCC7

and HCC23. A pretreatment with salt prior to anaer-

obic storage did not significantly alter the viability

Table 3. Log10 proportion reduction values for mid-log phase cells exposed to a mock cold-smoking procedure

StrainMid-log Cells

Freeze-Thaw

Salt Liquid Smoke Storage

EGDe 0.000 a 0.085 a -0.043 a -0.241 a -0.261 a

F2365 0.000 a -0.049 a -0.462 b -0.464 a, c -0.703 b

HCC7 0.000 a 0.018 a 0.111 a 0.178 b -0.037 a, c

HCC23 0.000 a 0.122 a -0.693 b, c -0.238 a -0.354 a, b, c

ATCC 15313 0.000 a 0.068 a 0.004 a 0.157 a, b -0.567 a, b

L. innocua 0.000 a 0.155 a -0.180 a, b -0.594 a, c -0.682 b

a, b, c Least significant means within column lacking common subscripts differ significantly (P <0.05)


-0.8

-0.6

-0.4

-0.2

0

0.2

Log

10(N

t/N

0)

ab a abc abab a ac

ac

abca a b

EGDe F2365 HCC7 HCC23 ATCC 15313 L. innocua

Figure 3. Proportion reduction values (log10 CFU/mL) of mid-log phase cells following exposure to freeze-thaw or salt, followed by anaerobic 2°C conditions. Cells in mid-log phase were pre-treated with either freeze-thaw (green filled) or salt (orange fill) prior to exposure to 2°C anaerobic conditions. Viability was expressed as the mean log10 proportion reduction values [log10 (Ntreated CFU/mL)/(Noriginal CFU/mL)]. Values represent the average log10 CFU/m proportion reduction val-ues ± standard error from (n = 3) independent replicates. Within each pretreatment condition, values with different superscripts are significantly different (P < 0.05).

Table 4. Average thickness (nm) of the cell membrane, cell wall, and cell envelope of HCC7 and HCC23 cells following exposure to the mock cold-smoking process

Mid-log Phase Freeze-Thaw 6% NaCl0.6% Liquid

SmokeStorage

HCC7 HCC23 HCC7 HCC23 HCC7 HCC23 HCC7 HCC23 HCC7 HCC23

Cell membrane 7.22 8.70 7.96* 6.26* 7.24 5.74* 6.60 4.43* 7.17 5.93*

Cell wall 17.84 17.78 17.40 18.20 17.60 14.69* 17.10 19.80* 22.86* 18.04

Cell envelope 24.92 26.48 25.36 24.46* 24.84 20.43* 23.7* 24.23* 30.14* 23.98*

* Indicates significant changes (P < 0.05) in cells exposed to freeze-thaw, salt, liquid smoke, or storage treatments compared to mid-log phase cells


0.5

µm

0.6%

Liq

uid

Smok

e A

naer

obic

Sto

rage

HCC7 HCC23

Mid

-log

Phas

e Fr

eeze

-Tha

w

6% N

aCl

A B

C D

E F

G H

I J

Figure 4. Transmission electron micrographs of HCC7 and HCC23 following exposure to stress-ors encountered during the cold-smoking process. Micrographs of HCC7 (A) and HCC23 (B) in mid-log phase; HCC7 (C) and HCC23 (D) following freeze-thaw. Retraction of the cell membrane from the cell wall of HCC7 following exposure to 6% NaCl is depicted in (E). Abnormal swollen morphology of HCC23 after exposure to 6% NaCl is depicted in (F). Normal appearing cells of HCC7 following exposure to 0.6% liquid smoke is depicted in (G). (H) is representative of proto-plasts observed in HCC23 following exposure to liquid smoke. The presence of filamentous cells of HCC7 following 2°C anaerobic conditions is indicated by (I). Damage to the cell wall of HCC23 following anaerobic storage is depicted in (J). Scale bars represent 0.5um.


between any strains examined (Figure 3; P> 0.19).

This suggests that exposure to either of these con-

ditions prior to anaerobic storage may lead to the

decrease in viability among populations of Listeria

strains.

Effects of the cold-smoking process on cell wall integrity

Changes in the width of the cell membrane, cell

wall, and the entire cell envelope in response to the

stresses of the cold-smoking process were examined

by TEM. Only mid-log phase cells were analyzed, as

the affect of the process on log proportion reduc-

tion was generally higher when cells were in mid-log

phase as opposed to stationary phase. The TEM

analysis was limited to HCC7 and HCC23 because

HCC23 exhibited a significant decrease in viability

following the exposure to the process, while HCC7

remained unaltered, suggesting that the two strains

possess different adaptive mechanisms. Addition-

ally, these two strains were isolated from fish (Wang

et al., 1998), making the strains appropriate model

microorganisms for this analysis. Representative mi-

crographs from each treatment for each strain are

presented in Figure 4.

Following the initial freezing conditions, a major-

ity of HCC7 and HCC23 cells remained undamaged

(65%); the remainder of these cells was elongated

(Figure 4C and D). Freeze-thaw conditions resulted

in a thickening of the cell membrane of HCC7 (P=

0.0033), while the cell membrane and cell envelope

(P< 0.01) of HCC23 decreased in thickness (Table 4).

The increase in the thickness of the cell membrane

of HCC7 could potentially be due to changes in the

lipid membrane composition in order to maintain

membrane fluidity (Annous et al., 1997).

Following exposure to 6% NaCl, the majority of

HCC7 and HCC23 cells remained unchanged (63%

and 60%, respectively). However, detachment of the

cell membrane from the cell wall was observed in

14% of the HCC7 cells and 7% of the HCC23 cells

(Figure 4E, F). Interestingly, 23% of the HCC7 cells

and 25% of the HCC23 cells became elongated fol-

lowing exposure to salt (Figure 4F). It was recently

discovered that YneA is involved in cell elongation

and inhibition of cell division following induction of

the SOS response (van der Veen et al., 2010). This

could potentially lead to the formation of filamen-

tous cells following exposure to 6% NaCl as ob-

served in this study and others (Hazeleger et al.,

2006). Exposure to elevated salt concentrations

caused almost no change in the thickness of the cell

envelope of HCC7, but led to a significant decrease

in the thickness of HCC23’s cell membrane and cell

wall (P<0.01) in comparison to non-treated cells (Ta-

ble 4). Decreased expression or activity of penicillin-

binding proteins (PBP) following exposure to high

salt concentrations could result in reduced pepti-

doglycan cross-links and an abnormal morphology

similar to what was observed in this study (Guinane

et al., 2006), potentially altering the susceptibility of

HCC23 to osmotic stress (Piuri et al., 2005; Popham

and Young, 2003). It has been postulated that one

reason for the increased incidence of listeriosis in hu-

mans is the reduced salt content of food products,

allowing for survival and growth of the organism and

potential likelihood of infection following consump-

tion by susceptible individuals (Goulet et al., 2008).

It will be critical to further examine the expression of

peptidoglycan-associated proteins following expo-

sure to a salt stress.

Following the sequential exposure of mid-log

HCC7 and HCC23 cells to freeze-thaw and salt, cells

were treated with 0.6% liquid smoke. A major pro-

portion (84%) of HCC7 cells appeared normal (Fig-

ure 4G), but the remainder of the cells (16%) exhib-

ited detachment of the cell membrane from the cell

wall. In HCC23 about 39% of the cells were elongat-

ed, 8% exhibited loss of cytoplasmic material, and

11% had a deformed morphology. Liquid smoke ex-

posure caused a decrease in the thickness of the cell

envelope due to a decrease in the cell membrane of

both HCC7 (P= 0.0179) and HCC23 (P< 0.01) (Table

4). Cell wall thickness of HCC23 increased following

liquid smoke treatment (P< 0.01). Damage to the cell

envelope of HCC23 observed in this study is consis-

tent with the ability of liquid smoke to damage the

cell membrane (Guilbaud et al., 2008), causing the

cell membrane to become thinner and the cell wall


to become thicker in comparison to untreated cells.

The increased thickness of the cell wall could be ac-

counted for by changes in the composition of the

cell wall to increase chemical resistance as observed

by others (Santos et al., 2004).

Exposure to liquid smoke also resulted in the po-

tential production of protoplasts (4.5%) (Figure 4H),

indicating either serious damage to the cell wall oc-

curred or the cells were transitioning into L-forms. L.

monocytogenes L-forms have been shown to have

an increased expression of stress-response genes

and are capable of growth and cell division (Dell’Era

et al., 2009), thus providing an adaptive mechanism

to survive environmental stressors (Markova et al.,

2010). It is therefore possible that L-forms of L. mono-

cytogenes are generated during the cold-smoking

process. Because cultivation of L-forms of L. mono-

cytogenes requires the use of specialized media and

a prolonged incubation time, they may go undetect-

ed in the food-processing environment. Therefore,

it is necessary to further determine whether this is

the point in the processing procedure where Listeria

transition into an L-form state.

A previous study reported that exposure of L.

monocytogenes to 7% NaCl for 1 h could provide

cross-protection against 0.1% H2O2 (Lou and Yousef,

1997). Thus the salt treatment in the present study

could have protected the bacterial cells from liquid

smoke damage via a similar mechanism. Although

exposure of Shewanella putrefaciens to increasing

concentrations of NaCl increased sensitivity to liq-

uid smoke (Leblanc et al., 2000), a longer or altered

treatment with smoking conditions might produce

altered results.

The final step of the sequential exposure was an

anaerobic incubation at 2°C for 16 h. HCC7 had an

increased thickness of the cell envelope as indicated

by a thickening of the cell wall (P< 0.01), and 33% of

the cells formed chains of cells with a visible septum

(Figure 4I); the remainder of the cells appeared to be

normal. Following anaerobic storage, 46% of HCC23

cells showed signs of damage to the cell wall, 48%

were elongated or filamentous, and ~16% formed a

division septum towards the pole of the cell. Follow-

ing anaerobic storage conditions, the cell envelope

of HCC7 increased in thickness due to a thicker cell

wall (P< 0.01), while the thickness of the cell mem-

brane and cell envelope of HCC23 decreased (P<

0.01; Table 4). One potential explanation for a de-

crease in the cell membrane thickness of HCC23 is

that under anaerobiosis and low temperature con-

ditions, synthesis of unsaturated fatty acids and

branched-chain fatty acids could be inhibited due

to reduced levels of NADH oxidation (de Sarrau et

al., 2012; Van Der Voort and Abee, 2009). This would

lead to reduced membrane fluidity and an inability

to adapt to cold temperatures.

CONCLUSIONS

In conclusion, the virulent L. monocytogenes

strains examined in this study were more resistant

to storage conditions following salt than the aviru-

lent strains regardless of the growth phase. This re-

sistance could be explained by expression of similar

mechanisms required for survival during exposure

to salt and low temperatures. Previous studies have

reported an overlap in the expression of genes re-

quired for the transport of osmolytes following cold

or osmotic stress in L. monocytogenes (Wemekamp-

Kamphuis et al., 2004; Wemekamp-Kamphuis et al.,

2002b), suggesting freeze-thaw conditions are re-

quired to increase resistance to salt. The ability of

the sub-lethally injured cells to survive these condi-

tions suggests that these strains may be undetected

during sampling in the food-processing environ-

ment and could continue to grow following storage

conditions. Further testing is needed to determine

whether this affect is primarily governed by the ex-

posure to salt.

ACKNOWLEDGEMENTS

We would like to thank Kendrick Currie at Missis-

sippi State University for his assistance with this proj-

ect. This project was supported through the Office

of Research, the Department of Biological Sciences,

and the Research Initiation Program at Mississippi

State University.


REFERENCES

Amezaga, M. R., I. Davidson, D. McLaggan, A. Ver-

heul, T. Abee, and I. R. Booth. 1995. The role of

peptide metabolism in the growth of Listeria

monocytogenes ATCC 23074 at high osmolarity.

Microbiol. 141 ( Pt 1):41-9.

Annous, B. A., L. A. Becker, D. O. Bayles, D. P.

Labeda, and B. J. Wilkinson. 1997. Critical role of

anteiso-C15:0 fatty acid in the growth of Listeria

monocytogenes at low temperatures. Appl. Envi-


Azizoglu, R. O., J. Osborne, S. Wilson, and S. Kath-

ariou. 2009. Role of growth temperature in freeze-

thaw tolerance of Listeria spp. Appl. Environ. Mi-

crobiol. 75:5315-20.

Barber, M. 1939. A comparative study of Listerella

and Erysipelothrix. J. Pathol. Bacteriol. 48:11-23.

Ben Embarek, P. K. 1994. Presence, detection and

growth of Listeria monocytogenes in seafoods: a

review. Int. J. Food Microbiol. 23:17-34.

Cortesi, M. L., T. Sarli, A. Santoro, N. Murru, and T.

Pepe. 1997. Distribution and behavior of Listeria

monocytogenes in three lots of naturally-contami-

nated vacuum-packed smoked salmon stored at 2

and 10 degrees C. Int. J. Food Microbiol. 37:209-

14.

Cossart, P., and A. Toledo-Arana. 2008. Listeria

monocytogenes, a unique model in infection biol-

ogy: an overview. Microbes Infect. 10:1041-50.

Danielsson-Tham, M. L., J. Bille, R. Brosch, C. Bu-

chriesser, K. Persson, A. Schwarzkopf, W. Tham,

and J. Ursing. 1993. Characterization of Listeria

strains isolated from soft cheese. Int. J. Food Mi-

crobiol. 18:161-6.

de Sarrau, B., T. Clavel, C. Clerte, F. Carlin, C. Ginies,

and C. Nguyen-The. 2012. Influence of Anaero-

biosis and Low Temperature on Bacillus cereus

Growth, Metabolism, and Membrane Properties.


Dell’Era, S., C. Buchrieser, E. Couve, B. Schnell, Y.

Briers, M. Schuppler, and M. J. Loessner. 2009. Lis-

teria monocytogenes L-forms respond to cell wall

deficiency by modifying gene expression and the

mode of division. Mol. Microbiol. 73:306-22.

Faith, N. G., A. E. Yousef, and J. B. Luchansky. 2007.

Inhibition of Listeria monocytogenes by liquid

smoke and isoeugenol, a phenolic component

found in smoke. J. Food Safety 12:303-314.

Fonnesbech Vogel, B., H. H. Huss , B. Ojeniyi, P.

Ahrens, and L. Gram. 2001. Elucidation of Liste-

ria monocytogenes contamination routes in cold-

smoked salmon processing plants detected by

DNA-based typing methods. Appl. Environ. Mi-

crobiol. 67:2586-95.

Gandhi, M., and M. L. Chikindas. 2007. Listeria: A

foodborne pathogen that knows how to survive.

Int. J. Food Microbiol. 113:1-15.

Gedela, S., R. K. Gamble, S. Macwana, J. R. Escou-

bas, and P. M. Muriana. 2007. Effect of inhibitory

extracts derived from liquid smoke combined with

postprocess pasteurization for control of Listeria

monocytogenes on ready-to-eat meats. J. Food

Prot. 70:2749-56.

Gilbreth, S. E., J. E. Call, F. M. Wallace, V. N. Scott,

Y. Chen, and J. B. Luchansky. 2005. Relatedness of

Listeria monocytogenes Isolates recovered from

selected ready-to-eat foods and listeriosis patients

in the United States. Appl. Environ. Microbiol.

71:8115-22.

Gombas, D. E., Y. Chen, R. S. Clavero, and V. N.

Scott. 2003. Survey of Listeria monocytogenes in

ready-to-eat foods. J. Food. Prot. 66:559-69.

Goulet, V., C. Hedberg, A. Le Monnier, and H. de

Valk. 2008. Increasing incidence of listeriosis in

France and other European countries. Emerg. In-

fect. Dis. 14:734-40.

Guilbaud, M., I. Chafsey, M. F. Pilet, F. Leroi, H. Pre-

vost, M. Hebraud, and X. Doussett. 2008. Response

of Listeria monocytogenes to liquid smoke. J.

Appl. Microbiol. 104:1744-53.

Guinane, C. M., P. D. Cotter, R. P. Ross, and C. Hill.

2006. Contribution of penicillin-binding protein

homologs to antibiotic resistance, cell morphol-

ogy, and virulence of Listeria monocytogenes

EGDe. Antimicrob. Agents Chemother. 50:2824-8.

Guyer, S., and T. Jemmi. 1991. Behavior of Listeria

monocytogenes during fabrication and storage

of experimentally contaminated smoked salmon.



Hazeleger, W. C., M. Dalvoorde, and R. R. Beumer.

2006. Fluorescence microscopy of NaCl-stressed,

elongated Salmonella and Listeria cells reveals the

presence of septa in filaments. Int. J. Food Micro-

biol. 112:288-90.

Hwang, C.A. 2007. Effect of salt, smoke compound,

and storage temperature on the growth of Listeria

monocytogenes in simulated smoked salmon. J.

Food Prot. 70:2321-8.

Leblanc, L., F. Leroi, A. Hartke, and Y. Auffray. 2000.

Do stresses encountered during the smoked salm-

on process influence the survival of the spoiling

bacterium Shewanella putrefaciens? Lett. Appl.

Microbiol. 30:437-42.

Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May,

C. Salminen, D. W. Hird, M. L. Yonekura., P. Hayes,

and R. Weaver. 1988. Epidemic listeriosis associ-

ated with Mexican-style cheese. N. Engl. J. Med.

319:823-8.

Lou, Y., and A. E. Yousef. 1997. Adaptation to sub-

lethal environmental stresses protects Listeria

monocytogenes against lethal preservation fac-

tors. Appl. Environ. Microbiol. 63:1252-5.

Markova, N., G. Slavchev, L. Michailova, and M. Jour-

danova. 2010. Survival of Escherichia coli under le-

thal heat stress by L-form conversion. Int. J. Biol.

Sci. 6:303-15.

Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S.

Bresse, C. Shapiro, P. M. Griffin, and R. V. Tauxe.

1999. Food-Related Illness and Death in the Unit-

ed States. Emerg. Infect. Dis. 5:607-625.

Merritt, M. E., A. M. Lawrence, and J. R. Donaldson.

2010. Comparative Study of the Effect of Bile on

the Listeria monocytogenes Virulent Strain EGD-

e and Avirulent Strain HCC23 Arch. Clinical Micro.

1:4-9.

Moretro, T., and S. Langsrud. 2004. Listeria mono-

cytogenes: biofilm formation and persistence in

food-processing environments. Biofilms 1:107-121.

Murray, E. G. D., R. A. Webb, and M. B. R. Swann.

1926. A disease of rabbit characterised by a large

mononuclear leucocytosis, caused by a hitherto

undescribed bacillus: Bacterium monocytogenes

(n. sp.). J. Pathol. Bacteriol. 29:407-439.

Neunlist, M. R., M. Ralazamahaleo, J. M. Cappelier,

V. Besnard, M. Federighi, and F. Leroi. 2005. Effect

of salting and cold-smoking process on the cultur-

ability, viability, and virulence of Listeria monocyto-

genes strain Scott A. J. Food Prot. 68:85-91.

Piuri, M., C. Sanchez-Rivas, andS. M. Ruzal. 2005.

Cell wall modifications during osmotic stress in

Lactobacillus casei. J. Appl. Microbiol. 98:84-95.

Popham, D. L. and K. D. Young. 2003. Role of peni-

cillin-binding proteins in bacterial cell morphogen-

esis. Curr. Opin. Microbiol. 6:594-9.

Porsby, C. H., B. F. Vogel, M. Mohr, and L. Gram. 2008.

Influence of processing steps in cold-smoked salm-

on production on survival and growth of persistent

and presumed non-persistent Listeria monocyto-

genes. Int. J. Food Microbiol. 122:287-95.

Posfay-Barbe, K. M., and E. R. Wald. 2009. Listeriosis.

Semin. Fetal Neonatal. Med. 14:228-33.

Rorvik, L. M. 2000. Listeria monocytogenes in the

smoked salmon industry. Int. J. Food Microbiol.

62:183-90.

Rorvik, L. M., M. Yndestad, and E. Skjerve. 1991.

Growth of Listeria monocytogenes in vacuum-

packed, smoked salmon, during storage at 4 de-

grees C. Int. J. Food Microbiol. 14:111-7.

Santos, P. M., D. Benndorf, and I. Sa-Correia. 2004.

Insights into Pseudomonas putida KT2440 re-

sponse to phenol-induced stress by quantitative

proteomics. Proteomics 4:2640-52.

Scharff, R. L. 2010. Health-related costs from

foodborne illness in the United States. George-

town University, Washington, DC.

Sleator, R. D., D. Watson, C. Hill, C. G. and Gahan.

2009. The interaction between Listeria monocyto-

genes and the host gastrointestinal tract. Micro-

biol. 155:2463-75.

Sunen E. 1998. Minimum inhibitory concentration of

smoke wood extracts against spoilage and patho-

genic micro-organisms associated with foods. Lett.


Thurette, J., J. M. Membre, L. H. Ching, R. Tailliez,

and M. Catteau. 1998. Behavior of Listeria spp. in

smoked fish products affected by liquid smoke,

NaCl concentration, and temperature. J. Food

Prot. 61:1475-9.

Uyttendaele, M., P. Busschaert, A. Valero, A. H.


Geeraerd, A. Vermeulen, L. Jacxsens, K. K. Goh, A.

De Loy, J. F. Van Impe, and F. Devlieghere. 2009.

Prevalence and challenge tests of Listeria mono-

cytogenes in Belgian produced and retailed may-

onnaise-based deli-salads, cooked meat products

and smoked fish between 2005 and 2007. Int. J.

Food Microbiol. 133:94-104.

van der Veen, S., S. van Schalkwijk, D. Molenaar, W.

M. de Vos, T. Abee, and M. H. Wells-Bennik. 2010.

The SOS response of Listeria monocytogenes is

involved in stress resistance and mutagenesis. Mi-

crobiol. 156:374-84.

Van Der Voort, M. and T. Abee. 2009. Transcription-

al regulation of metabolic pathways, alternative

respiration and enterotoxin genes in anaerobic

growth of Bacillus cereus ATCC 14579. J. Appl. Mi-

crobiol. 107:795-804.

Vaz-Velho, M., G. Duarte, J. McLauchlin, and P. Gibbs.

2001. Characterization of Listeria monocytogenes

isolated from production lines of fresh and cold-

smoked fish. J. Appl. Microbiol. 91:556-62.

Vitt, S. M., B. A. Himelbloom, and C. A. Crapo. 2001.

Inhibition of Listeria innocua and L. monocyto-

genes in a laboratory medium and cold-smoked

salmon containing liquid smoke. J. Food Safety

21:111-125.

Wang, C., L. F. Zhang, F. W. Austin, and C. R. Boyle.

1998. Characterization of Listeria monocytogenes

isolated from channel catfish (Ictalurus punctatus).

Am. J. Vet. Res. 59:1125-8.

Wemekamp-Kamphuis, H. H., A. K. Karatzas, J. A.

Wouters, and T. Abee. 2002a. Enhanced levels of

cold shock proteins in Listeria monocytogenes

LO28 upon exposure to low temperature and high

hydrostatic pressure. Appl. Environ. Microbiol.

68:456-63.

Wemekamp-Kamphuis, H. H., R. D. Sleator, J. A>

Wouters, C. Hill, and T. Abee. 2004. Molecular and

physiological analysis of the role of osmolyte trans-

porters BetL, Gbu, and OpuC in growth of Listeria

monocytogenes at low temperatures. Appl. Envi-


Wemekamp-Kamphuis, H. H., J. A. Wouters, R. D.

Sleator, C. G. Gahan, C. Hill, and T. Abee. 2002b.

Multiple deletions of the osmolyte transporters

BetL, Gbu, and OpuC of Listeria monocytogenes

affect virulence and growth at high osmolarity.


Wulff, G., L. Gram, P. Ahrens, and B. F. Vogel. 2006.

One group of genetically similar Listeria monocy-

togenes strains frequently dominates and persists

in several fish slaughter- and smokehouses. Appl.

Environ. Microbiol. 72:4313-22.




ABSTRACT

Bacterial foodborne diseases are caused by consumption of foods contaminated with bacteria and/

or their toxins. In this study, we evaluated antibacterial properties of twelve different extracts including

turmeric, lemon and different kinds of teas against four major pathogenic foodborne bacteria including

Campylobacter jejuni, Escherichia coli O157:H7, Salmonella Enteritidis and Staphylococcus aureus and

food spoilage bacteria Pseudomonas aeruginosa and Pseudomonas putida. Of the twelve extracts, lemon

extract was found to be most antibacterial and killed all the bacteria within 24 h of incubation. Among the

bacterial pathogens, E. coli O157:H7 was most susceptible to lemon extract and C. jejuni was the least sus-

ceptible. Turmeric was found to kill all the C. jejuni isolates and MRSA within 36 h but killed E. coli and S. En-

teritidis only after 48 h of incubation. However, turmeric showed maximum activity against P. putida which

was killed within 24 h of incubation, but failed to kill P. aeruginosa even after 48 h of incubation. Among the

different teas tested, green and white tea extracts were found to be the most antibacterial and white tea

killed all the bacteria except C. jejuni 81176 within 48 h of incubation. Other tea varieties including Rose

of Suzhou, Sweet Fruit Garden and Silver Needle had various degrees of bactericidal effects. These results

demonstrate the potential for using plant extracts, especially lemon extracts, as successful antibacterial

agents. These extracts could be used as food additives to certain foods to reduce or eliminate foodborne

bacterial pathogens and food spoilage bacteria.

Keywords: Foodborne bacteria, Tea, Turmeric, Lemon, Plant Extract, Flavonoids, Campylobacter, E. coli, Salmonella, MRSA, Pseudomonas

Correspondence: Geetha S. Kumar-Phillips, [email protected]: +1-479-856-5079

Antibacterial Activity of Plant Extracts on Foodborne Bacterial Pathogens and Food Spoilage Bacteria

N. Murali1, G. S. Kumar-Phillips1, N.C. Rath1,2, J. Marcy1 and M. F. Slavik1

1Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas 727012USDA/ARS

Agric. Food Anal. Bacteriol. 2:209-221, 2012


INTRODUCTION

Some foodborne bacteria responsible for disease

and spoilage are resistant to existing treatments,

generating a need for different methods to elimi-

nate or reduce these bacteria (Sirsat et al., 2009).

Recently, medicinal plants and their extracts have

gained importance as potential antibacterial agents.

Secondary metabolites of plants including tannins,

flavonoids and alkaloids have been found to pos-

sess antimicrobial properties in vitro (Dahanukar et

al., 2000). Many medicinal plants including Clausena

anisata (Rutaceae), Ocimum tenuiflorum (tulsi), Cur-

cuma longa (turmeric), Azardirachta indica (neem),

Eugenia caryophyllata (clove) have been shown to

possess antibacterial effects in vitro (Sher, 2009). In

a recent study, it was shown that flavonoids (or phy-

tochemicals), if used in combination with antibiotics,

were not only effective against Pseudomonas infec-

tion, but also decreased the toxicity of the antibiot-

ics (Jayaraman et al., 2010). Flavonoids are second-

ary plant metabolites used in nutraceutical industries

(Srinivas et al., 2009) as antioxidants and antimicro-

bial agents and are gaining importance in the food

industry (Srinivas et al., 2010).

Of the flavonoids, tea flavonoids have been ex-

tensively studied for almost a decade. The most

common tea flavonoids are catechins and epicat-

echins. Different types of teas (Camellia sinensis)

contain varying amounts of flavonoids. Increased

enzymatic oxidation of tea results in a decrease in

catechin concentration and formation of complexes

like theaflavins. Black tea undergoes complete fer-

mentation, oolong tea undergoes partial fermenta-

tion, while green and white teas are unfermented

(Chou et al., 1999). Black tea has approximately 9%

catechin and 4% theaflavin, while green teas contain

as high as 30% catechins w/v (Wiseman et al., 1997).

Researchers have shown that Asian black teas and

green teas (we have specified Asian teas since black

and green teas are mostly cultivated in asia) exhibit

antibacterial activity in vitro against Staphylococ-

cus aureus, Escherichia coli, Pseudomonas aerugi-

nosa and Enterococcus fecalis (Bancirova, 2010) and

spoilage bacteria in fish and meat (Wenjiao et al.,

2008). A recent study reported that tea polyphenols

altered the integrity of the outer and inner bacterial

membranes as evidenced by transmission electron

microscopy (TEM) and disrupted the cell walls (Yi et

al., 2010). Green, black and white teas were used in

the study since they are the most common variet-

ies of teas consumed by people around the world.

Other teas like Silver Needle, Rose of Suzhou, and

oolong were used since they are specialty teas and

we wanted to compare their antibacterial properties

with the common teas consumed

Most plants contain flavonoids in varying amounts.

Some plants like lemon (Citrus limon) and turmeric

(Curcuma longa) contain high amount of polyphe-

nols. While fruits like lemon contain essential oils and

eriocitrin (a flavonoid) which is bactericidal, turmeric

contains a flavonoid curcumin, known for its antioxi-

dant, anti-inflammatory and antibacterial properties.

Lemon juice contains 5 to 6% citric acid, has a sour

taste and a pH from 2 to 3. This property of lemon

juice can be exploited to make lemon a good an-

tibacterial agent. In one study conducted by Conte

and coworkers (2007) lemon extracts were found to

successfully inhibit vegetative cells and spores of

some food spoilage microorganisms including yeast,

Bacillus species and lactic acid bacteria. Naz and co-

workers (2010) have shown that curcumin and essen-

tial oils of turmeric varieties were effective against

Bacillus and Azotobacter species. Other researchers

have shown that inhibition of the FtsZ assembly dy-

namics (FtsZ is a protein shown to play a critical role

in bacterial cytokinesis) in the Z–ring is a possible

antibacterial mechanism of action of curcumin (Rai

et al., 2008). These in vitro studies show that lemon

extracts and curcumin have potential antibacte-

rial properties against both Gram positive as well as

Gram negative bacteria.

In the present study, we evaluated the antibac-

terial activity of plant extracts including lemon, tur-

meric and different varieties of teas including black,

green, white, oolong, mint, Rose of Suzhou, Sweet

Fruit Garden, Silver Needle, Emerald Princess and

rooibos against foodborne bacteria including C.

jejuni, E. coli O157:H7, S. Enteritidis, S. aureus and

food spoilage bacteria including P. aeruginosa and


P. putida. Although there have been various stud-

ies reporting the effects of tea flavonoids, this study

tests the activities of different kinds of teas including

specialty teas like Rose of Suzhou, Sweet Fruit Gar-

den, Silver Needle, Rooibos and Emerald Princess.

Viable cell counts were performed and viability per-

centages were calculated for each extract to assess

the efficacy of the extracts. Due to the importance of

foodborne bacterial pathogens and food spoilage

bacteria, plant extracts could prove to be efficient

and practical antibacterial treatments.


Bacterial strains used

Pathogenic bacteria included four different iso-

lates of Campylobacter jejuni including human

isolate 81176, chicken isolates from a pre–chilled

chicken carcass (PRCC), from a post–chilled chicken

carcass (POCC), and from a retail chicken carcass

(RECC); Escherichia coli O157:H7 ATCC 43888; Sal-

monella Enteritidis (T1B4E); methycillin resistant

Staphylococcus aureus ATCC 43300. Pseudomonas

aeruginosa ATCC 17485 and Pseudomonas putida

ATCC 10145 were used as the food spoilage bacte-

ria.

Media used

Campylobacter Enrichment Broth (CEB) (Acume-

dia®, Lansing, MI) was used for initial culturing of

C. jejuni strains and Nutrient Broth (NB) (DIFCO®,

Franklin Lakes, NJ) was used for culturing E. coli, S.

Enteritidis, MRSA, P. aeruginosa and P. putida. Muel-

ler Hinton Agar (MHA) (DIFCO®, Franklin Lakes, NJ)

was used for plating for all the bacteria after serial

dilution. For enumeration of C. jejuni, in addition to

MHA, Campylobacter Enrichment Agar (CEA) sup-

plemented with 5% horse blood was used.

Turmeric Extraction

Dried and powdered turmeric was purchased from

a local supermarket (Fayetteville, AR). Ten grams

were boiled in 100 mL sterile water for 20 minutes

and filtered through sterile gauze. After neutralizing

the pH to 7.0±0.2 with 10N NaOH, the filtrate was

autoclaved at 121°C for 15 minutes. The autoclaved

turmeric extract was stored in a dark bottle at -20°C

for further use (Weerasekera et al., 2008).

Lemon Extraction

Fresh lemon fruits were purchased from a local su-

permarket (Fayetteville, Arkansas). The lemons first

were washed with tap water and then with distilled

water. Twenty grams of cut fruits including the rind

and flesh were immersed in 100 ml of 96% ethanol

for 30 mins and ground with a mortar and pestle to

extract soluble material. The ground extracts were

held at room temperature for 48 h, filtered through

Whatman No. 1 paper and placed in sterile petri

dishes for 48h at room temperature to evaporate the

ethanol. Dried extracts were re-suspended in 15 ml

of phosphate buffered saline (PBS) and maintained

at 4°C until used (Valtierra- Rodríguez et al., 2010).

Tea Extraction

Ten different varieties of teas (Camellia sinen-

sis) were included in the study including, black tea,

green tea, white tea, oolong tea, mint tea, Rose of

Suzhou tea, Sweet Fruit Garden tea, Silver Needle

tea, Emerald Princess tea and Rooibos tea (Aspala-

thus linearis). Dried and powdered tea leaves were

purchased from various sources. Ten grams of tea

leaves were suspended in sterile phosphate buff-

ered saline (PBS) 20% concentrations (w/v), held at

room temperature for 3h and centrifuged at 15,000

rpm for 10 minutes. After transferring the superna-

tants to another sterile tube, the pH was adjusted

to 7.0 ± 0.2 and the extracts were stored in a dark

bottle at 4°C (Diker et al., 1991). In order to evaluate

boiling as an extraction method, green tea, oolong

tea, Silver Needle tea and Rose of Suzhou tea leaves

were boiled for 5 minutes each, cooled and centri-

fuged at 15,000 rpm for 10 minutes. The supernatant

was transferred to a sterile tube and pH neutralized.


Measurement of bactericidal activity

Bacterial strains of C. jejuni were grown in Campy-

lobacter Enrichment Broth (CEB) for 18 h at 42°C. E.

coli, S. Enteritidis, MRSA, P. putida and P. aeruginosa

strains were grown in Nutrient Broth (NB) for 18 h at

37°C. The cultures were centrifuged at 8000 x g for

2 minutes at 25°C. The supernatants were discarded

and the pellets were reconstituted in fresh enrich-

ment broth media. Five ml of each bacterial suspen-

sion were mixed with 5 mL of the above extracts and

incubated as follows: 1. 42°C microaerobic condi-

tions for C. jejuni strains and; 2. 37°C for E. coli, S.

Enteritidis, MRSA, P. putida and P. aeruginosa. Five

mL of the bacterial suspensions were mixed with 5

mL of the growth media and were used as controls.

Viable cell counts of the above incubated test sam-

ples and controls were determined by serial dilution

in PBS and plated onto MHA at regular intervals. The

results were expressed in terms of log CFU vs. time

for each isolate and each extract.

Statistical Analysis

All tests were done three times to establish statis-

tical significance. Statistical analysis was performed

using JMP 8.0 provided by University of Arkansas,

Fayetteville. The results were considered statistically

significant with p<0.001.


Antibacterial activity of lemon

Citrus limon contains approximately 5 to 6% citric

acid and the pH of the juice is 2 to 3. The lemon

peel is rich in essential oils. The lemon extract used

for this study contained both the juice and the es-

sential oil. The extract killed all the bacteria within 24

Figure 1. Mean Log CFU vs. time of five different extracts against E. coli O157:H7. Lemon extract was the most effective of the extracts killing the bacteria within 24 h of incubation (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

00h 01h 06h 12h 24h 36h 48h

Mea

n Lo

g CF

U

Lemon extract

Turmeric

Green Tea

White Tea

Silver Needle tea

Time


Table 1. Antibacterial activity of twelve different plant extracts against four major foodborne bacte-

rial pathogens and two spoilage bacteria.

PLANT

EXTRACTS

C. ejuni 81176

C. jejuni PRCC

C. jejuni POCC

C. jejuni RECC

E.coli S.Enteritidis MRSA P. aeruginosa P. putida

Lemon +++ +++ +++ +++ +++ +++ +++ +++ +++

Turmeric ++ ++ ++ ++ + + ++ - +++

Black tea + + + + + + + - -

Emerald Princess tea

+ + + + + + + + -

Sweet Fruit Garden tea

- - - - - - - - -

Rooibos tea ++ ++ ++ ++ ++ ++ ++ + +

Mint tea ++ ++ ++ ++ ++ ++ ++ + +

White tea - + + + + + + + +

Green tea ++ ++ ++ ++ ++ + ++ - +

Silver Needle tea

- - - - - - - - -

Oolong tea + + + + + + + + -

Rose of Su-zhou tea

- - - - - - - - -

Legend: +++ indicates that the extract killed the bacteria within 24 h of incubation; ++ indicates death within 36 h of incubation; + indicates bacteria were killed only after 48 h of incubation while – indicates that the bacteria were not killed even after 48 h of incubation. PRCC-pre chilled chicken carcass; POCC-post chilled chicken carcass; RECC-retail chicken carcass; MRSA-methycillin resistant S. aureus.


h of incubation (Table 1). E. coli O157:H7 (Figure 1)

was the most susceptible to the extract followed by

MRSA, Campylobacter jejuni, Salmonella Enteritidis,

P. aeruginosa and P. putida. Previous experiments

by Fischer and Phillips (2006) have shown that the

zone of inhibition of commercially available lemon

essential oils is greater against Staphylococcus au-

reus than E. coli O157:H7 and Campylobacter jejuni.

The difference in this study is probably either due to

a difference in the strains used or because the cur-

rent study uses lemon juice, rich in citric acid, in ad-

dition to lemon essential oils. In our study, C. jejuni

and MRSA showed approximately a 2-log reduction

after 1h of incubation, while E. coli O157:H7 showed

a 3–log reduction. Among C. jejuni strains, the RECC

isolate showed a greater susceptibility than POCC

strain, with a 5-log reduction after 6 hours as com-

pared to 3-log reduction after 6 h of incubation, re-

spectively. Salmonella Enteritidis also showed a 3-log

reduction after 1 h of incubation which was similar to

the results reported by Xiong and coworkers (1999).

Furthermore, lemon extract killed both spoilage

bacteria P. aeruginosa and P. putida within 24 h on

incubation. P. aeruginosa and P. putida also showed

a 6–log decrease in growth within 1 h of incubation

with lemon extract. These results were similar to

those by Adedeji and coworkers (2007) who tested

the effect of lemon and lime juice on clinical isolates

of P. aeruginosa. Another study also reported that

lemon essential oils showed significant antibacterial

activity against Pseudomonas (Prabuseenivasan et

al., 2006).

Figure 2. Mean Log CFU vs. time of plant extracts against poultry strain of C. jejuni RECC (retail chicken carcass). Green tea was more effective than white tea killing the strain within 36 h of incu-bation. White tea killed the bacteria only after 48 h of incubation (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

11

00h 01h 06h 12h 24h 36h 48h

Lemon extract

Turmeric

Green Tea

White Tea

Silver Needle tea

Mea

n Lo

g CF

U

Time


Antibacterial activity of different types of teas

Teas are aromatic beverages containing large

amounts of flavonoids, which are known antioxidants

and antibacterial agents. Green, white, oolong,

black and herbal teas were tested for their anti-

bacterial activity against different bacteria. Green

and white teas showed highest antibacterial effects

probably because green and white teas contain the

largest amount of flavonoids among the varieties of

teas (Table 1). White tea killed all the C. jejuni iso-

lates except C. jejuni 81176 in 36 h, while black tea

killed all the bacteria only after 48 h of incubation. C.

jejuni POCC and RECC isolates showed a 4-log de-

crease with green and white teas (Figure 2) after 12 h

incubation. Lee and coworkers (2009) reported that

green teas have potential anti-adhesive properties.

In an experiment conducted in-vitro with human and

mouse epithelial cell lines, it was shown that green

tea extracts inhibited adhesion of S. aureus and He-

licobacter pylori but not E. coli. In another study by

Cho and coworkers (2008), there was a synergistic ef-

fect between green tea and antibiotics. This combi-

nation was highly effective against various strains of

MRSA. As shown in Figure 3, specialty teas including

rooibos and mint killed S. Enteritidis and C. jejuni,

E. coli, and MRSA isolates (data not shown) within

36 hours of incubation. Rooibos tea showed a 5-log

decrease within 12 h of incubation, while mint tea

showed 4-log decrease within 12 h of incubation with

S. Enteritidis. In addition, with rooibos and oolong

teas all bacteria showed an initial decrease of ap-

proximately 2-log CFU after 1 h of incubation and

Figure 3. Mean Log CFU vs. time S. Enteritidis with various tea extracts. Rooibos and mint tea extracts killed the isolate within 36 hours of incubation while oolong and green tea extracts killed the isolates after 48 hours of incubation. (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

11

00h 01h 06h 12h 24h 36h 48h

Rooibos Tea

Mint Tea

Green Tea

Oolong Tea

Silver Needle tea

Time

Mea

n Lo

g CF

U


rooibos tea killed all the bacteria within 36 h, while

Emerald Princess showed a 1-log decrease within 1

h of incubation and killed all the bacteria within 48 h.

However, with Rose of Suzhou, Sweet Fruit Garden

and Silver Needle after an initial decrease in growth,

a 1-log increase in growth of S. Enteritidis was seen

between 24 h and 36 h of incubation.

With spoilage bacteria, green tea did not kill P.

aeruginosa and P. putida strains even after 48 h of

incubation, but showed a 9–log reduction in count

after 48 h. These findings were consistent with those

of Vandeputte and coworkers (2010). Although many

studies have reported that green tea and green

tea catechins possess bacteriostatic effects against

Pseudomonas species, there have been other re-

ports which indicated little or no effect (Vandeputte

et al., 2010; Yi et al., 2010).

To evaluate boiling as a method of extraction,

green, oolong, Silver Needle and Rose of Suzhou

teas were boiled and their antibacterial activities

were compared (Table 2). Silver Needle tea, a type

of white tea, showed little antibacterial activity in the

current study, until the boiling extraction method was

used. It was found that boiling significantly increased

the antibacterial efficacy of Silver Needle tea and re-

sulted in a 4-log reduction in growth of P. putida af-

ter 6 h of incubation and killed all the bacteria within

48 h of incubation. It was also found that boiling in-

creases the efficacy of antibacterial effect of green

tea which killed all the isolates within 24 h of incuba-

tion, while green tea extracted without boiling killed

the isolates only after 36 h. While boiled green tea

extract showed a 6-log reduction in growth within 12

h of incubation, green tea extracted without boiling

showed only a 4-log reduction with E. coli O157:H7.

Figure 4. Mean Log CFU vs. time with C. jejuni human strain 81176. Turmeric was effective against 81176 and killed C. jejuni within 36 h of incubation. (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

11

00h 01h 06h 12h 24h 36h 48h

Lemon extract

Turmeric

Green Tea

White Tea

Silver Needle tea

Mea

n Lo

g CF

U

Time


Table 2. Comparison of antibacterial activity of different tea extracts with and without boiling against four major foodborne bacterial pathogens and two spoilage bacteria.

Plant ExtractC. jejuni 81176

C. jejuni PRCC

C. jejuni POCC

C. jejuni RECC

E. coli O157:H7

S. Enteritidis MRSAP.

aeruginosaP. putida

Green tea ++ ++ ++ ++ ++ + ++ - +

Boiled Green tea

+++ +++ +++ +++ +++ +++ +++ +++ +++

Silver Needle - - - - - - - - -

Boiled Silver Needle

++ ++ ++ ++ ++ ++ ++ - +

Sweet Fruit Garden

- - - - - - - - -

Boiled Sweet Fruit Garden

- - - - - - - - -

Rose of Su-zhou

- - - - - - - - -

Boiled Rose of Suzhou

- - - - - - - - -

+++ indicates that the extract killed the bacteria within 24 h of incubation; ++ indicates death within 36 h of incubation; + indicates bacteria were killed only after 48 h of incubation while – indicates that the bacteria were not killed even after 48 h of incubation.

PRCC-pre chilled chicken carcass; POCC-post chilled chicken carcass; RECC-retail chicken carcass; MRSA-methycillin resistant S. aureus.

Efficacy of Turmeric

Turmeric was found to be the most bactericidal

against MRSA followed by S. Enteritidis, C. jejuni

and, finally, E. coli O157:H7 (Table 1). Turmeric com-

pletely killed all the bacteria within 36 h of incuba-

tion with a 2–log decrease within 6 h after incuba-

tion with C. jejuni 81176. C. jejuni 81176 (Figure 4)

showed maximum sensitivity, while C. jejuni PRCC

showed least sensitivity to turmeric. Tajbhaksh and

coworkers (2008) also reported similar results using

curcumin, indium curcumin, indium diacetylcurcum-

in, and diacetyl curcumin against S. aureus and E.

coli. Another recent study by Moghaddam and co-

workers (2009) showed that curcumin when coupled

with antibiotics was more effective against S. aureus

than curcumin alone.

With respect to P. aeruginosa (Figure 5) and P.

putida (Figure 6), turmeric was able to produce ap-

proximately a 5–log decrease after 24 h of incuba-

tion with P. aeruginosa, and killed P. putida within 24

h. These results were also similar to Tajbaksh and co-

workers (2008), who reported that indium curcumin

and indium diacetylcurcumin was effective against P.

aeruginosa with a minimum inhibitory concentration

of 23.4 mg/mL. Another recent study also reported

similar results, where curcumin nanoparticles were

tested for their antibacterial efficacy against Pseudo-

monas. The same study also showed that curcumin

nanoparticles inhibited 80% of P. aeruginosa while

curcumin alone inhibited only 60%. They went on

to prove that curcumin nanoparticles were more ef-

fective than curcumin itself since they are more sol-

uble in water (Bhawana et al., 2011). These studies


showed turmeric could be used as an antibacterial

agent worldwide. Curcumin the active ingredient

in turmeric is a polyphenolic compound with many

therapeutic uses including, but not limited to, po-

tential anti-HIV, anti-tumor, anti-amyloid and anti-

inflammatory properties, in addition to being anti-

microbial.

CONCLUSIONS

A total of 12 extracts were tested against four dif-

ferent bacterial foodborne pathogens and two food

spoilage bacteria. Of these plant extracts, lemon ex-

tract was found to be the most effective and killed all

the bacteria within 24 h of incubation, with a 5-log

decrease in count of C. jejuni strains after 6 h of in-

cubation and a 6-log reduction of P. putida, followed

by green tea extract, white tea extract, turmeric ex-

tract and black tea extract. However, three specialty

teas including Rose of Suzhou, Sweet Fruit Garden

and Silver Needle teas were ineffective against all

the organisms and failed to kill the bacteria even af-

ter 48h of incubation. When the teas were boiled to

extract flavonoids and the extraction methods were

compared, it was found that Silver Needle tea killed

all the isolates except P. aeruginosa within 48 h of

incubation with a 3-log reduction in growth of C. je-

juni, E. coli and S. Enteritidis and a 4-log reduction of

P. putida isolates within 6 h of incubation. It was also

Figure 5. Mean Log CFU vs. time with P. aeruginosa. Lemon extract was found to be effective against P. aeruginosa killing the isolate within 24 h of incubation. White tea killed the organism within 48 h of incubation, while turmeric and boiled Silver Needle tea failed to kill even after 48 h. (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

11

12

00h 01h 06h 24h 48h

Lemon extract

Turmeric

White tea

Silver Needle tea

Boiled Silver Needle tea

Mea

n Lo

g CF

U

Time


found that boiling increased the antibacterial effi-

ciency of green tea extract which killed all the bacte-

rial isolates within 24 h of incubation as compared to

36 h with green tea extracts without boiling. Based

on this research, it can be concluded that plant ex-

tracts have great potential to be used as effective

antibacterial agents against foodborne pathogens

and spoilage bacteria.

REFERENCES

Adedeji, G. B., O. E. Fagade, and A. A. Oyelade.

2007. Prevalence of Pseudomonas aeruginosa in

clinical samples and its sensitivity to citrus extract.

Afr. J. Biomed. Res. 10:183–187.

Bancirova, M. 2010. Comparison of the antioxidant

capacity and the antimicrobial activity of black and

green tea. Food Res. Int. 43:1379-1382.

Bhawana, R., K. Basniwal, H. S. Buttar, V. K. Jain, and

N. Jain. 2011. Curcumin nanoparticles: Prepara-

tion, characterization, and antimicrobial study. J.

Agric. Food Chem. 59:2056-2061.

Cho, Y. S., N. L. Schiller, and K. H. Oh. 2008. Antibac-

terial effects of green tea polyphenols on clinical

isolates of methicillin-resistant Staphylococcus au-

reus. Curr. Microbiol. 57:542-546.

Chou, C. C., L. L. Lin, and K. T. Chung. 1999. Anti-

microbial activity of tea as affected by the degree

of fermentation and manufacturing season. Int. J.

Figure 6. Mean Log CFU vs. time with P. putida. Lemon proved to be the most effective against P. putida. Although Silver Needle tea proved to be the least effective and failed to kill the bacteria even after 48 h of incubation; turmeric and white tea were found to be quite effective, killing the isolate within 24 h and 48 h respectively. (p<0.001)

0

1

2

3

4

5

6

7

8

9

10

11

00h 01h 06h 24h 48h

Lemon extract

Turmeric

Green tea

White tea

Silver Needle tea

Boiled Silver Needle tea

Mea

n Lo

g CF

U

Time


Food Microbiol. 48:125-130.

Conte, A., B. Speranza, M. Sinigaglia, and M. A. Del

Nobile. 2007. Effect of lemon extract on foodborne

microorganisms. J. Food Prot. 70:1896-1900.

Dahanukar, S.A., R. A. Kulkarni, and N. N. Rege.

2000. Pharmacology of medicinal plants and natu-

ral products. Indian J. Pharmacol. 32:S8-S118.

Diker, K. S., M. Akan, G. Hascelik, and M. Yurdakok.

1991. The bactericidal activity of tea against Cam-

pylobacter jejuni and Campylobacter coli. Lett.


Fisher, K., and C. A. Phillips. 2006. The effect of lem-

on, orange and bergamot essential oils and their

components on the survival of Campylobacter

jejuni, Escherichia coli O157, Listeria monocyto-

genes, Bacillus cereus and Staphylococcus aureus

in vitro and in food systems. J. Appl. Microbiol.

101:1232-1240.

Jayaraman, P., M. K. Sakharkar, C. S. Lim, T. H. Tang,

and K. R. Sakharkar. 2010. Activity and interactions

of antibiotic and phytochemical combinations

against Pseudomonas aeruginosa in vitro. Int. J.

Biol. Sci. 6:556-568.

Lee, J. H., J. S. Shim, M. S. Chung, S. T. Lim, and K.

H. Kim. 2009. In vitro anti-adhesive activity of green

tea extract against pathogen adhesion. Phytother.

Res. 23:460-466.

Moghaddam, K.M., M. Iranshahi, M. C. Yazdi, and

A. R. Shahverdi. 2009. The combination effect

of curcumin with different antibiotics against

Staphylococcus aureus. Int. J. Green Pharm. Apr-

Jun:141-143.

Naz, S., S. Jabeen, S. Iliyas, F. Manzoor, F. Aslam,

and A. Ali. 2010. Antibacterial activity of Curcuma

longa varieties against different strains of bacteria.

Pak. J. Bot. 42:455-462.

Prabuseenivasan, S., M. Jayakumar, and S. Ignaci-

muthu. 2006. In vitro antibacterial activity of some

plant essential oils. BMC Complement. Altern.

Med. 6:39.

Rai, D., J. K. Singh, N. Roy, and D. Panda. 2008.

Curcumin inhibits FtsZ assembly: An attractive

mechanism for its antibacterial activity. Biochem.

J. 410:147-155.

Sher, A. 2009. Antimicrobial activity of natural prod-

ucts from medicinal plants. Gomal J. Med. Sci.

7:72-78.

Sirsat, S. A., A. Muthaiyan, and S. C. Ricke. 2009. An-

timicrobials for foodborne pathogen reduction in

organic and natural poultry production. J. Appl.

Poult. Res. 18:379-338.

Srinivas, K., J. W. King, J. K. Monrad, L. R. Howard,

and C. M. Hansen. 2009. Optimization of subcriti-

cal fluid extraction of bioactive compounds us-

ing Hansen solubility parameters. J. Food Sci. 74:

E342-54.

Srinivas, K., J. W. King, L. R. Howard, and J. K. Mon-

rad. 2010. Solubility and solution thermodynamic

properties of quercetin and quercetin dihydrate in

subcritical water. J. Food Eng. 100:208-218.

Tajbakhsh, S., K. Mohammadi, I. Deilami, K. Zandi,

M. Fouladvand, E. Ramedani, and G. Asayesh.

2008. Antibacterial activity of Indium curcumin

and Indium diacetylcurcumin. Afr. J. Biotechnol.

7:3832-3835.

Valtierra- Rodríguez, D., N. L. Heredia, S. Garcia, and

E. Sanchez. 2010. Reduction of Campylobacter je-

juni and Campylobacter coli in poultry skin by fruit

extracts. J.Food Prot. 73:477-482.

Vandeputte, O. M., M. Kiendrebeogo, S. Rajaonson,

B. Diallo, A. Mol, M. El Jaziri, and M. Baucher. 2010.

Identification of catechin as one of the flavonoids

from Combretum albiflorum bark extract that re-

duces the production of quorum-sensing-con-

trolled virulence factors in Pseudomonas aerugi-

nosa PAO1. Appl. Environ. Microbiol. 76:243-253.

Weerasekera, D., N. Fernando, L. B. A. E. Bogaha-

watta, R. Rajapakse-Mallikahewa, and D. J. Naulla.

2008. Bactericidal effect of selected spices, medic-

inal plants and tea on Helicobacter pylori strains

from Sri Lanka. J. Natn. Sci. Foundation Sri Lanka.

36:91-94.

Wenjiao, F., C. Yuanlong, and Z. Shuo. 2008. The use

of a tea polyphenol dip to extend the shelf life of

silver carp (Hypophthalmicthys molitrix) during

storage in ice. Food Chem. 108:148–153.

Wiseman, S. A., D. A. Balentine, and B. Frei. 1997.

Antioxidants in tea. Crit. Rev. Food Sci. Nutr.

37:705-718.

Xiong, R., G. Xie, and A. S. Edmondson. 1999. The


APPENDIX - DIFFERENT VARIETIES OF TEAS USED IN THE STUDY AND THEIR SCIEN-TIFIC NAMES

S.No Tea Varieties Scientific Name Composition

1. Green Tea Camellia sinensisFresh tea leaf → Withering → Curling → minimal Oxidation → Drying → Green tea

2. Black Tea Camellia sinensisFresh tea leaf→ Withering → Curling → complete Oxidation → Drying → Black tea

3. White Tea Camellia sinensisFresh tea leaf → Withering → Drying (air drying, solar drying or mechanical drying) → White tea

4. Silver Needle Tea Camellia sinensis

It is a type of white tea. For the production of Silver Needle, only the leaf shoots, i.e. the leaf buds before opening, are plucked, and the buds undergo light oxidation – hence classified as white tea.

5. Rooibos Tea Aspalathus linearis

This tea is usually classified as ‘black’ tea since it undergoes complete oxidation. Recently ‘green’ va-rieties of Rooibos are produced with partial oxidative processing.

6. Mint Tea Camellia sinensis Herbal tea infused with peppermint, Mentha piperita.

7. Oolong Tea Camellia sinensisTraditional Chinese tea produced through a unique process including withering under the strong sun and oxidation before curling and twisting.

8. Enerald Princess Tea Camellia sinensisA blend of green tea with pineapple, citrus pieces, coconut, cornflowers and rose petals.

9. Rose of Suzhou Tea Camellia sinensisThis is a Suzhou (the Venice of China) white tea vari-ety comprised of jasmine flowers, marigold blossoms and a globe amaranth flower.

10. Sweet Fruit Garden Tea Herbal teaHerbal tea with sweet and sour Morello cherries & tart raspberries and a hint of hibiscus, apple, pine-apple and papaya.

fate of Salmonella enteritidis PT4 in home-made

mayonnaise prepared with citric acid. Lett. Appl.

Microbiol. 28:36-40.

Yi, S. M., J. L. Zhu, L. L. Fu, and J. R. Li. 2010. Tea poly-

phenols inhibit Pseudomonas aeruginosa through

damage to the cell membrane. Int. J. Food Micro-

biol. 144:111-117.




ABSTRACT

Red amaranth (lal shak) is one of the main vegetables consumed extensively in Bangladesh. This study

was conducted to monitor the prevalence of microorganisms, heavy metal contents and vitamins in raw

red-amaranth and the impact of cooking on microorganisms, heavy metal contents and vitamins of red

amaranth. The averaged viable bacterial load and coliform count in raw amaranth samples was recorded as

> log 8.0 CFU/g, and approximately 7.0 log CFU/g, respectively throughout this study. Higher prevalence

of pathogenic microorganisms such as Salmonella spp, Escherichia coli, Listeria spp. and Yersinia spp. were

recorded in raw red amaranth samples.

Washing raw amaranth samples with tap water removed some soil and other debris, but did not reduce

the bacterial load. However, washing these vegetables with scallop powder followed by a distilled water

wash could reduce 1.0-1.5 log CFU/g of viable bacterial load and coliform bacterial load. Washing these

vegetables with 200 ppm chlorine water was able to reduce additional 2.0 log CFU/g.

However, cooking these vegetables at a boiling temperature (90°C, for 15 min) did reduce by more than

5.0 log CFU/g the viable bacterial load but was unable to inactivate them. In contrast, cooking successfully

eliminated coliform and all the pathogenic microorganisms including Salmonella spp., E. coli, Listeria spp.

and Yersinia spp. from all the samples tested. In addition, cooking reduced almost 90% vitamin-C content

from 14.2 mg/100 g to 1.5 mg/100 g. In contrast, some metal (Ca, Fe and Zn) contents were increased

significantly after cooking. Therefore, these study results demonstrated that cooking could reduce the

microbiological risk of red amaranth and improve food safety for human consumption. However, cooking

reduced the vitamin-C content and increased some of the metal contents, coming from the supply water.

Keywords: Prevalence pathogens, Red amaranth, microbial risk, scallop powder, washing and cooking

Correspondence: Md. Latiful Bari, [email protected] Tel: 8802-9661920-59 Ext 4721 Fax: 8802-8615583

Prevalence of Foodborne Pathogens and Effectiveness of Washing or Cooking in Reducing Microbiological Risk

of Contaminated Red Amaranth

Md. Arafat Al Mamun1, Hasina Akther Simul2, Asma Rahman3, N. N. Gazi2, and Md. Latiful Bari1

1Food Analysis and Research Laboratory2Environmental Chemistry laboratory,

3Drug Laboratory, Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh



INTRODUCTION

Amaranth (Amaranthus tricolor L.) plays an im-

portant role in nutrition among the leafy vegetables

grown in Bangladesh. Among the leafy types, Ama-

ranthus tricolor L. is the most commonly cultivated

species in Bangladesh. It is cultivated all over the

country in any season due to its adaptability to a

wide range of soil and climate (Alam et al., 2007). Red

amaranth (lal shak) is grown in homestead gardens

using indigenous technologies and ground water

is often used for irrigation and is subsequently har-

vested and sold in local markets without any process-

ing steps between harvest and market. Consumers

buy these vegetables from local markets, bring them

home along with other vegetables in a basket, wash

with tap water, cut into pieces and cooked or stir fry

with spices and consume.

During the past decade, increased frequency of

fresh produce associated outbreaks was reported.

E. coli O157:H7, Salmonella spp., Listeria monocy-

togenes, and Yersinia enterocolitica is of particular

food safety concern, because they are widespread in

the environment (IFT, 2004), grow under refrigeration

conditions (ILSI, 2005), and are frequent residents

in certain food processing establishments (Brandl,

2006). These microorganisms have been isolated

from soil, sewage sludge, vegetation, and water

(NACMCF, 1999) and, therefore, have the potential to

contaminate produce surfaces. Many vegetables, in-

cluding bean sprouts, cabbage, cucumber, potatoes,

and radishes, have been reported to be contaminat-

ed with pathogenic microorganisms (Beuchat, 1996).

The pathogen has been reported to survive long-

term storage on leafy vegetables, has been respon-

sible for numerous product recalls of salads (Wong et

al., 2000), and was identified as being responsible for

an outbreak of foodborne disease due to coleslaw

prepared from contaminated raw cabbage.

Many sanitizers have been evaluated for their ef-

fectiveness in killing pathogenic microorganisms on

different produce (Beuchat et al., 2001). Chlorine as

sodium or potassium salt and calcium hypochlorite

commonly has been used to eliminate microorgan-

isms from food surfaces. Treatment of raw produce

with other disinfectants is partially effective in remov-

ing disease-causing microorganisms from the surface

of raw fruits and vegetables (Beuchat et al., 2001).

Washing produce with sodium-chlorinated water (Na-

ClO) is the most commonly used method to remove

pathogens from fruit and vegetable surfaces. The ac-

tive hypochlorite is believed to lose its effectiveness

by reacting with nitrogen-containing compounds in

foods, resulting in halogenated organic compounds

(Wei et al., 1985). Concern over the carcinogenicity

and toxicity of these compounds, particularly trihalo-

methanes, has prompted consideration of alternative

disinfectants. Inatsu et al. (2005) reported that acidi-

fied sodium chloride solution could be useful as a

sanitizer for surface washing of fresh produce. In this

study scallop powder-a new biodegradable and nat-

ural alkaline sanitizer was used to see its effectiveness

in reducing microorganisms on the produce surface.

The level of sanitation and the populations of

microorganisms are of primary importance to the

quality, shelf stability, and safety of fresh produce

(NACMCF, 1999). Based on a comprehensive survey

produce outbreaks accounted for 13% (713/5,416) of

total outbreaks and 21% (34,049/161,089) of associ-

ated illnesses from 1990 through 2005, according to

data from the Center for Science in the Public Interest

(CSPI) (Smith DeWaal and Bhuiya, 2009). However, in

Bangladesh, unlike other developing countries, such

databases for fresh produce do not exist. Outbreak

data is necessary to improve the hazard analysis for

various food commodities, and to ensure produce

safety.

Nevertheless arsenic contamination of groundwa-

ter for irrigation in the homestead gardens is a widely

based concern (Rahman and Hasan, 2007). Therefore,

use of this contaminated water during vegetable pro-

duction, washing and cooking may introduce arsenic

in the final product. In addition, as there is a lack of

hygiene education in the rural population, therefore,

there are frequent opportunities for microbial con-

tamination of pond and well water. Use of this water

in any stage of vegetables chain may contribute to

contamination of the final products.

Based on the above mentioned facts this study

was designed: 1) to determine the prevalence of mi-


croorganisms and pathogens in raw red amaranth 2)

to determine the effectiveness of scallop powder and

widely used chlorinated water for controlling the nat-

ural microflora and environmental foodborne patho-

gens including Listeria monocytogenes, Salmonella

Enteritidis, Escherichia coli O157:H7, Yersinia entero-

colitica in raw red amaranth; 3) to determine the ef-

fectiveness of cooking in reducing microbiological

risk of heavily contaminated market Red amaranth;

and 4) to determine the heavy metal content and

vitamin-C in fresh and cooked red amaranths.


Samples collection

Commercial red amaranth samples were pur-

chased from the Kawranbazar market of Dhaka city,

Bangladesh and were used within 24 h of collection.

Raw vegetable samples were collected aseptically

in sterile polyethylene bags and transported to the

laboratory. Cracked or dirty red amaranth samples

were discarded.

Washing solution and sample washing

Scallop powder solutions and chlorine water were

used as washing solutions and were prepared imme-

diately prior to application. The final concentration

of scallop powder solutions was adjusted to 0.01%

(w/v) in deionized water. The chlorine solution was

prepared using sodium hypochlorite (Wako Chemi-

cal Co. Ltd, Osaka, Japan) solution to distilled wa-

ter (v/v), and concentration was adjusted to 200

ppm. Distilled water (DW) was used for control ex-

periments. Tap water/supply water was also used to

wash raw red amaranths. For each experimental con-

dition, 50 g of samples was washed with 250 mL of

the washing solution in a sterile beaker (1 L). Wash-

ing was carried out for 5 min at room temperature

with gentle agitation using a glass rod. After wash-

ing, the solutions were decanted, and the samples

were rinsed with 250 mL of distilled water. After that

the red amaranth samples were placed in a sterile

perforated tray to drain off the excessive water and

placed in laminar flow bio-safety cabinet to facilitate

drying for 2 hours.

Cooking of samples and cooling down to room temperature

As the consumer will usually cook or stir fry the

red amaranth samples with spices and subsequently

consumed. Therefore, an experiment was designed

to see the effectiveness of cooking on microorgan-

isms. Washed or non-washed commercial red ama-

ranths samples were boiled at approximately 90°C

for 15 minutes in a bowl and after boiling, the red

amaranth samples were placed on a sterile perforat-

ed tray to drain off the excessive water and placed in

laminar flow bio-safety cabinet to facilitate cooling

down to room temperature.

Sample processing and microbiological analysis

After washing and/or cooking, the 25 g red ama-

ranth samples were placed in a stomacher bag with

225 mL of sterile saline water. The mixture was pum-

meled for 60 s and serial decimal dilutions were

prepared with sterile saline water. The diluted and

undiluted samples (0.1 mL) were then surface plated

on both selective and nonselective agar media. Tryp-

tic soy agar (TSA; Oxoid) was used as a nonselective

media for determination of viable cells number. Coli-

form agar; Sorbitol MacConkey agar (SMAC) supple-

mented with cefixime (0.05 mg/liter) and potassium

tellurite (2.5 mg/liter) (CT23 selective supplement,

Oxoid); Bismuth sulfite agar (BSA; Oxoid); Listeria

selective agar supplemented with SR0227E and Yer-

sinia selective agar supplemented with Yersinia Se-

lective Supplement were used as the selective me-

dia for the determination of coliform bacteria, E. coli

O157:H7, Salmonella spp., L. monocytogenes and

Y. enterocolitica respectively, in the red amaranths

samples.

All the plates were then incubated at 37°C for 24

to 48 h and counted. After incubation from the re-

spective selective media at least five presumptive


colonies of Salmonella, E. coli O157:H7, L. monocy-

togenes and Y. enterocolitica were subjected to con-

firmation tests using a direct immunoassay test kit

(Universal Health Watch, Columbia, MD, USA and/

or API diagnostic Kits, Oxoid, UK). All experiments

were repeated five times to confirm the reproduc-

ibility.

Sample preparation, Digestion and Metal Analysis

Red amaranth samples were brought from a local

market. The samples were washed with water and

half of the samples were subsequently cooked and

the excess water was drained off. The cooked and

uncooked samples were then freeze dried and kept

at room temperature until use. Freeze Dryer (Ilshin

Lab Co, Ltd. Seoul, Korea), Microwave oven (CEM

Corporation, NC, USA, Model Marss Express), and

microwave Teflon sealed vessels were used. A Perkin

Elmer Atomic Absorption Spectrophotometer (Mod-

el A Analyst 800; Illinois, USA) was used for metal

analysis in red amaranth vegetables.

Digested samples were mechanically ground and

weighted approximately 0.125 g into a Teflon vessel;

10 mL of concentrated nitric acid (Sigma pure chemi-

cal Industries, ltd, USA) was added and digested in a

microwave oven. The condition of microwave diges-

tion was: powder 800W; rate 10 min; temp 100°C;

time 10 min and 800W; 10 min; 200°C for 10 min.

Treatment/conditionsPopulation (log CFU/g) a

Total viable bacteria

Total coliform bacteria

E. coli Salmonella spp.

Listeria spp.

Yersinia spp.

1. No treatment 8.7 ± 0.5C 6.9 ± 0.1A 4.6 ± 0.2A 5.8 ± 0.2A 5.8 ± 0.2A 5.7 ± 0.1A

2. Washing with tap water follow 8.5 ± 0.1A 6.1 ± 0.4B 4.0 ± 0.1A 5.7 ± 0.3B 5.9 ± 0.3B 5.7 ± 0.1A

3. Dipped in scallop powder wa-ter for 5 min with periodic stirrer

7.6 ± 0.2A 5.5 ± 0.2A 3.1 ± 0.1A 4.9 ± 0.1A 4.3 ± 0. 2A 4.9 ± 0.1A

4. Dipped in Scallop Powder Water followed by a 2nd wash with DW

7.4 ± 0.3B 4.9 ± 0.1A 3.1 ± 0.1A 4.8 ± 0.1A 4.4 ± 0.1A 4.9 ± 0.2B

5. Washing with 200 ppm chlorine water for 5 min

6.4 ± 0.2B 5.1 ± 0.1A 3.3 ± 0.2A 4.8 ± 0.2A 3.27 ± 0.1A 4.9 ± 0.1A

6. Washing with 200 ppm chlorine water followed by 2nd wash with DW

6.2 ± 0.3C 5.1 ± 0.4B 3.2 ± 0.3B 4.7 ± 0.3B BDL 4.5 ± 0.1A

7. Boiling and Cooling 3.2 ± 0.5C - - - - -

a n=10 for each data, count= Average of ‘∑n’ ± SD; BDL= Below Detection limit. The detection limit was < 1.0 log CFU/g. The mean values in columns with different letters are significantly (P < 0.05) different, while mean values with the same letter are not significantly different

Table 1. Prevalence of viable bacteria, coliform bacteria, and other foodborne pathogens in pre- and post-washing / cooking Red amaranths.


After digestion the content of Teflon vessel was dis-

solved in de-ionized water and filtered into 25 mL

volumetric flask quantitatively and brought up to the

mark with de-ionized water. The digested sample

solutions were subsequently analyzed for the metals

Ca, Cd, Cu, Fe, Mn, Na, Pb and Zn by an automatic

sampler and analyzed by using an air acetylene flame

in combination with single element hollow cathode

lamps into an atomic absorption spectrophotome-

ter, having a detection limit as described in Table 2,

respectively. However, the arsenic (As) was analyzed

by GF-AAS (Graphite Furnace Atomic Absorption

Spectrophotometer; Perkin Elmer, Illinois, USA) with

electrode less discharge lamp of having a detection

limit 0.5 µg/L of arsenic. The wavelength, correlation

coefficient and detection limit of each metal is listed

in Table 1.

Determination of Vitamin C by HPLC

The cooked and fresh Red Amaranth samples

were analyzed for vitamin-C (ascorbic acid) content

using High Performance Liquid Chromatography

(HPLC) according to the method as described in the

ASEAN Manual of Nutrient Analysis, 2011 and Lak-

shanasomya, 1998 as described below:

Instrumentation and Chromatographic condi-

tions

A Prominence HPLC system (Shimadzu Scientific

Instrument, Tokyo, Japan) equipped with two pumps

(LC-20AD), an auto sampler (SIL 20AC HT), UV-Vis-

ible detector (SPD 20A), column oven (CTO-20AC)

and communication bus module (CBM 20A) was

used for this analysis. The data was processed using

LC- solutions software.

Stationary phase: Analytical reversed phase C-18,

Luna 5µ, 250 x 4.6 mm, Phenomenex, Inc., Mobile

phase: 0.3 mM potassium dihydrogen phosphate

in 0.35% (v/v) ortho- phosphoric acid, Diluent: 3%

metaphosphoric acid, Flow rate: 0.5 mL/min., Detec-

tion: 248 nm., Injector: 20 µL.

Preparation of standard solutions and method

calibration

The stock solution of L-ascorbic acid was pre-

pared by diluents having a concentration of 1.0 mg/

mL. The appropriate volume from this stock solution

MetalsWavelength

( hollow Cathode Lamp)

Linear Range

(mg/ L)

Correlation Coefficient

Detection Limit

(mg/ L)

Detection Limit

(mg/ L)

Ca 422.7 0-5.0 0.997050 0.10 0.10

Cd 228.8 0-2.0 0.999616 0.03 0.03

Cu 324.8 0-5.0 0.998286 0.02 0.02

Fe 248.3 0-6.0 0.999012 0.02 0.02

Mn 279.5 0-2.0 0.999877 0.05 0.05

Na 589.0 0-1.0 0.997632 0.01 0.01

Pb 283.3 0-20.0 0.999520 0.45 0.45

Zn 213.9 0-1.0 0.999461 0.02 0.02

Table 2. The wavelength, correlation coefficient and detection limit of each metal


was further diluted with the same diluent to prepare

standards 60, 70, 80, 90 and 100 µg/mL of ascorbic

acid. Each of the five different concentrations men-

tioned previously were injected for three times to

obtain average peak area. The average peak areas

were plotted against concentrations to construct a

linear curve from which the correlation coefficients,

slopes and interception values were calculated (Fig-

ure 1A).

Preparation of sample solution

Fresh or cooked samples (2.5 g) were pulverized

with 3% meta phosphoric acid in a 200 mL volumet-

ric flask and vortexed for 5 minutes to uniformly mix

and the volume was adjusted to 100 mL with same

diluent. The contents were subsequently filtered

through a 0.45 µm membrane and injected into the

system. The chromatogram of standard, fresh and

boiled Red amaranth is shown in Figure 1 (B, C, D),

respectively.

Statistical analysis

All trials were replicated five times. Reported plate

count data represented the mean values obtained

from five individual trials, with each of these values

being obtained from duplicated samples. Data were

subjected to analysis of variance using the Microsoft

Excel program (Redmond, Washington DC, USA.).

Significant differences in plate count data were es-

tablished by the least-significant difference at the

5% level of significance.


The averaged viable bacterial loads and coliform

counts in the raw amaranth samples were recovered

at greater than log 8.0 CFU/g, and approximately

7.0 log CFU/g, respectively throughout this study.

Higher prevalence of pathogens such as Salmonella

spp, E. coli, Listeria spp and Yersinia spp were ob-

served in the raw red amaranth samples. This finding

demonstrated that the red amaranth sold in the lo-

cal market of Dhaka city is heavily contaminated with

pathogenic bacteria.

Washing raw amaranth samples with tap water

removed some soil and other debris, but did not

reduce the bacterial load. However, washing these

vegetables with scallop powder followed by a dis-

tilled water wash did reduce by 1.0 to 1.5 log CFU/g

of the viable bacterial load and coliform bacterial

load. However, washing these vegetables with 200

ppm chlorine water reduced by an additional 2.0 log

CFU/g the viable bacterial load and coliform count.

However, scallop powder and 200 ppm chlorine wa-

ter was unable to inactivate pathogenic bacteria.

Vegetables can act as a vector for transporting

pathogenic bacteria from the farm (Beuchat, 2001).

Although washing produce with tap water may re-

move some soil and other debris, it cannot be re-

lied upon to remove microorganisms and may re-

sult in cross-contamination of food preparation

surfaces, utensils, and other food items (Bari et al.,

2005). To reduce the risk of food poisoning caused

by contaminated vegetables, effective sanitation of

the raw produce is required. Washing raw produce

with water containing sodium hypochlorite (NaClO)

is the most commonly used method for removing

pathogens from the surfaces of vegetables (Wei et

al., 1985). In this study, 200 ppm of chlorinated wa-

ter was used, which is the maximum limit permitted

for washing of raw vegetables by the United States

Food and Drug Administration (FDA). The active hy-

pochlorite is believed to lose its effectiveness after

reacting with nitrogen compounds in foods, result-

ing in halogenated organic compounds (Odabasi,

2008). Concern over the carcinogenicity and toxic-

ity of these compounds, particularly of trihalometh-

anes, has prompted consideration of alternative dis-

infectants (Wei et al., 1985).

In this study, scallop powder-a new biodegrad-

able alkaline sanitizer is used. The inner portion

of the scallop (Patinopecten yessoensis) shell was

baked at 200˚C and then exposed to a heat treat-

ment of 1000°C, and then passed through a micro

sieve to obtain 5 to 15 um particle size of powder.

This powder said to have bactericidal action against

Escherichia coli, Salmonella Typhimurium, Staphylo-

coccus aureus and Bacillus subtilis (vegetative cells).


Figure 1. A) Linearity of curve for standard ascorbic acid and HPLC chromatogram of B) standard ascorbic acid C) fresh red amaranth and D) cooked red amaranth.

y = 107449x + 460448R² = 0.9907

0

2000000

4000000

6000000

8000000

10000000

12000000

0 20 40 60 80 100 120

Are

a

Concentration

A

B

C


The bactericidal action is due to calcium oxide that

is converted by heat treatment from calcium carbon-

ate, which is the main component of the shell pow-

der. As this powder is produced from natural sources

these do not pose any hazard to the environment,

and biodegradable (Sawai et al., 2001).

However, cooking these vegetable at a boiling

temperature (90˚C, for 15 min) was able to reduce by

more than 5.0 log CFU/g of the viable bacterial load

and coliform bacterial load but was unable to inac-

tivate them. In contrast, cooking successfully elimi-

nated all pathogens including Salmonella spp, E.

coli, Listeria spp. and Yersinia spp. from all samples

tested.

The average vitamin C content in raw red ama-

ranths was recorded as 14.2 mg/100g, and after

cooking, the vitamin-C content was reduced signifi-

cantly and recorded as 1.5 mg/100g, which is approx-

imately 90% lower than from fresh samples (Table 3

and Figure 1). This finding suggested that the eating

habit could lead to a lower intake of micronutrients

even though microbiologically safe.

In addition, as there is a lack of hygiene education

in rural population, therefore, there is every chance

of microbial contamination in any stage of food

chain. In addition, lack of processing facilities after

the harvest may also contribute to cross contamina-

tion of transport utensils, storages boxes, and finally

display utensils. Furthermore, consumer purchase

these vegetables from local market, bring them

home with other vegetables in a basket, and store

them in the refrigerator (vegetable boxes) with other

perishable vegetables, consequently cross-contami-

nating the entire process from farm to freeze. These

production and storage practices may contribute to

contaminating other foods in the refrigerator.

To ensure the microbiological quality and safety

of fresh vegetables, processing steps must be intro-

duced to reduce the cross contamination during the

entire process. In addition, good agricultural practic-

es (GAP) must be introduced in the field/agricultural

land when producing fresh vegetable intended for

human consumption.

ACKNOWLEDGEMENTS

This research work is a coordinated research of

different laboratories of the Center for Advanced

Research in Sciences (CARS), University of Dhaka.

The authors express their sincere gratitude to Dr.

Zakir Sultan for his advice and technical support on

HPLC. The authors also express their sincere grati-

tude to Md. Ashraful Islam, Kanik Kumar Sharker,

Md. Harun-or-rashid and Md. Abul-Kalam Azad for

their technical and all-out support and cooperation

D


during this work.

REFERENCES

Alam, M.N., M. S. Jahan, M. K. Ali, M. S. Islam, and S.

M. A. T. Khandaker. 2007. Effect of Vermicompost

and NPKS Fertilizers on Growth, Yield and Yield

Components of Red Amaranth. Austr. J. Basic

Appl. Sci. 1:706-716.

ASEAN Manual of Nutrient Analysis 2011. Editors, P.

Puwastien, T. E. Siong, J. Kantasubrata, G. Craven,

R. R. Feliciano, K. Judprasong. ASEANFOODS

published by Institute of Nutrition, Mahidol Uni-

versity, Thailand, 141-144.

Bari, M. L., D. O. Ukuku, T. Kawasaki, Y. Inatsu, K. Is-

shiki and S. Kawamoto. 2005. Combined effcacy

of nisin and pediocin with Sodium Lactate, Cit-

ric Acid, Phytic Acid and Potassium Sorbate and

EDTA in Reducing Listeria monocytogenes Popu-

lation of Inoculated Fresh-cut Produce. J. Food

Prot. 68:1381-1387.

Beuchat, L. R. 1996. Pathogenic microorganisms as-

sociated with fresh produce. J. Food Prot. 59:204–

216.

Beuchat, L. R., L. J. Harris, T. E. Ward, and T. M.

Kajs. 2001. Development of a proposed standard

method for assessing the efficacy of fresh produce

sanitizers. J. Food Prot. 64:1103–9.

Brandl, M. T. 2006. Fitness of Human Enteric Patho-

gens on Plants and Implications for Food Safety.

Annual Review of Phytopathology 44: 367-392.

IFT. 2004. Institute of Food Technologists: Scientific

Status Summary. August 2004. Bacteria Associated

with foodborne diseases, p. 1-25.

Inatsu, Y., M. L. Bari, S. Kawasaki, K. Isshiki, and S.

Kawamoto. 2005. Efficacy of acidified sodium

chlorite treatments in reducing Escherichia coli

O157:H7 on Chinese cabbage. J. Food Prot.

68:251-255.

Metals Cooked sample (mg/100g)Uncooked sample

(mg/100g)

Ca 127.20 88.10

Cd 0.15 0.18

Cu 0.46 0.46

Fe 15.22 5.12

Mn 0.78 0.42

Na 34.00 37.60

Pb BDL* BDL*

Zn 2.26 1.60

AS 0.002 0.001

Vitamin C

Vit-C 1.5 14.2

Table 3. The metal and vitamin-C content of Red amaranth samples before and after cooking.


ILSI. 2005. ILSI Research Foundation/Risk Science In-

stitute. 2005. Achieving continuous improvement

in reductions in foodborne listeriosis - A risk based

approach. J. Food Prot. 68:1932-1994.

Lakshanasomya, N. 1998. Determination on Vitamin

C in Some Kinds of Food by HPLC. Bull. Dept.

Med. Sci. 40:347-357.

NACMCF (National Advisory Committee on Micro-

biological Criteria for Foods). 1999. Microbiologi-

cal safety evaluation and recommendations on

fresh produce. Food Control 10:117–143.

Odabasi, M. 2008. Halogenated volatile organic

compounds from the use of chlorine-bleach-con-

taining household products. Environ. Sci. Technol.

42:1445-51.

Rahman, I.M.M., and Hasan M.T. 2007. Arsenic In-

corporation into Garden Vegetables Irrigated with

Contaminated Water. J. Appl. Sci. Environ. Man-

age. 11:105–112.

Sawai, J., M. Satoh, M. Horikawa, H. Shiga, and H.

Kojima. 2001. Heated scallop-shell powder slurry

treatment of shredded cabbage. J. Food Prot.

64:1579–1583.

Smith DeWaal, C., and F. Bhuiya. 2009. Center for

Science in the Public Interest (CSPI), Washington,

DC, Available at http://www.cspinet.org/foodsafe-

ty/IAFPPoster.pdf accessed on 4th March 2012.

Wei, C.-I., D. L. Cook, and J. R. Kirk. 1985. Use of

chlorine compounds in the food industry. Food

Technol. 39:107–115.

Wong, S., D. Street, S. I. Delgado, and K. C. Klontz.

2000. Recalls of foods and cosmetics due to micro-

bial contamination reported to the U.S. Food and

Drug Administration. J. Food Prot. 63:1113–1116.



MANUSCRIPT SUBMISSION

Authors must submit their papers electronically

([email protected]). According to instruc-

tions provided online at our site: www.afabjournal.

com. Authors who are unable to submit electroni-

cally should contact the editorial office for assistance

by email at [email protected].

INSTRUCTIONS TO AUTHORS

• Aerobic microbiology

• Aerobiology

• Anaerobic microbiology

• Analytical microbiology

• Animal microbiology

• Antibiotics

• Antimicrobials

• Bacteriophage

• Bioremediation

• Biotechnology

• Detection

• Environmental microbiology

• Feed microbiology

• Fermentation

• Food bacteriology

• Food control

• Food microbiology

• Food quality

• Food Safety

• Foodborne pathogens

• Gastrointestinal microbiology

• Microbial education

• Microbial genetics

• Microbial physiology

• Modeling and microbial kinetics

• Natural products

• Phytoceuticals

• Quantitative microbiology

• Plant microbiology

• Plant pathogens

• Prebiotics

• Probiotics

• Rumen microbiology

• Rapid methods

• Toxins

• Veterinary microbiology

• Waste microbiology

• Water microbiology

CONTENT OF MANUSCRIPT

We invite you to consider submitting your re-

search and review manuscripts to AFAB. The jour-

nal serves as a peer reviewed scientific forum for to

the latest advancements in bacteriology research

on Agricultural and Food Systems which includes

the following fields:


With an open access publication model of this

journal, all interested readers around the world can

freely access articles online. AFAB publishes origi-

nal papers including, but not limited to the types

of manuscripts described in the following sections.

Papers that have been, or are scheduled to be, pub-

lished elsewhere should not be submitted and will

not be reviewed. Opinions or views expressed in pa-

pers published by AFAB are those of the author(s)

and do not necessarily represent the opinion of the

AFAB or the editorial board.

MANUSCRIPT TYPES

Full-Length Research Manuscripts

AFAB accepts full-length research articles con-

taining four (4) figures and/or tables or more. AFAB

emphasizes the importance of sound scientific ex-

perimentation on any of the topics listed in the focus

areas followed by clear concise writing that describes

the research in its entirety. The results of experi-

ments published in AFAB must be replicated, with

appropriate statistical assessment of experimental

variation and assignment of significant difference.

Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.

Manuscripts clearly lacking in language will be re-

turned to author without review, with a suggestion

that English editing be sought before the paper is

reconsidered. AFAB offers a fee based language

service upon request. Please contact [email protected] for more information about our fees

and services.

Rapid Communications

Under normal circumstances, AFAB aims for re-

ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-

cation in the next available issue of AFAB. However,

if an author chooses or requires a much more rapid

peer review, the journal editorial office has the capa-

bility to shorten the review timing to one week or less.

Any type of manuscript whether it be a full length

manuscript, brief communication or review paper can

be submitted as a rapid communication. There will be

additional costs for processing and page charges will

be double the normal rate. Authors who choose this

option must select Rapid Communications as the pa-

per type when submitting the paper and the editors

will judge whether a rapid review is possible and let

the author know immediately.

Brief Communications

Brief communications are short research notes giv-

ing the results of complete experiments but are con-

sidered less comprehensive than full-length articles

with three (3) figures and/or tables or less. Manuscripts

should be prepared with the same subheadings as full

length research papers. The running head above the

title of the paper is “Brief Communications.”

Unsolicited Review Papers

Review papers are welcome on any topic listed in

the focus section and have no page limits. Reviews

are assessed the same pages charges as all other

manuscripts. All AFAB guidelines for style and form

apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter

than 20 pages of double spaced text and references

will be considered mini-reviews with the subhead-

ing above the title on the first page. The running

head above the title of the paper is either “Review”

or “Mini-review”.

Solicited Review Papers

Solicited reviews will have no page limits. The

editor-in-chief will send invitations to the authors

and then review these contributions when they are

submitted. Nominations or suggestions for potential

timely reviews are welcomed by the editors or edito-


rial board members and should be sent to submit@

afabjournal.com. There will be no page charges for

solicited review papers but the solicitation must origi-

nate from the editor-in-chief or one of the editors. Re-

quests from authors will automatically be classified as

unsolicited review papers. The running head above

the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews

AFAB welcomes opportunities to publish papers

from symposia, scientific conference, and/or meet-

ings in their entirety. Conference organizers need

simply to contact AFAB at [email protected]

and a rapid decision is guaranteed. If in agreement,

the conference organizers must guarantee delivery

of a set number of peer reviewed manuscripts within

a specified time and submitted in the same format

as that described for unsolicited review papers. Con-

ference papers must be prepared in accordance with

the guidelines for review articles and are subject to

peer review. The conference chair must decide

whether or not they wish to serve as Special Issue

Editor and conduct the editorial review process. If

the conference chair/organizer chooses to serve as

special issue editor, this will involve review of the pa-

pers and, if necessary, returning them to the authors

for revision. The conference organizer then submits

the revised manuscripts to the journal editorial of-

fice for further editorial examination. Final revisions

by the author and recommendations for acceptance

or rejection by the chair must be completed by a

mutually agreed upon date between the editor and

the conference organizer. Manuscripts not meeting

this deadline will not be included in the published

symposium proceedings but if submitted later can

still be considered as unsolicited review papers. Al-

though offprints and costs of pages are the same

as for all other papers, the symposium chair may be

asked to guarantee an agreed upon number of hard

copies to be purchased by conference attendees. If

the decision is not to publish the symposium as a

special issue, the individual authors retain the right

to submit their papers for consideration for the jour-

nal as ordinary unsolicited review manuscripts.

Book Reviews

AFAB publishes reviews of books considered to

be of interest to the readers. The editor-in-chief ordi-

narily solicits reviews. Book reviews shall be prepared

in accordance to the style and form requirements of

the journal, and they are subject to editorial revision.

No page charges will be assessed solicited reviews

while unsolicited book reviews will be assigned the

regular page charge rate.

Opinions and Current Viewpoints

The purpose of this section will be to discuss, cri-

tique, or expand on scientific points made in articles

recently published in AFAB. Drafts must be received

within 6 months of an article’s publication. Opinions

and current perspectives do not have page limits.

They shall have a title followed by the body of the

text and references. Author name(s) and affiliation(s)

shall be placed between the end of the text and list

of references. If this document pertains to a par-

ticular manuscript then the author(s) of the original

paper(s) will be provided a copy of the letter and of-

fered the opportunity to submit for consideration a

reply within 30 days. Responses will have the same

page restrictions and format as the original opinion

and current viewpoint, and the titles shall end with

“Opinions.” They will be published together. Letters

and replies shall follow appropriate AFAB format

and may be edited by the editor-in-chief and a tech-

nical editor. If multiple letters on the same topic are

received, a representative set of opinions concern-

ing a specific article will be published. A disclaimer

will be added by the editorial staff that the opinion

expressed in this viewpoint is the authors alone and

does not necessarily represent the opinion of AFAB

or the editorial board.

COPYRIGHT AGREEMENT

The copyright form is published in AFAB as space

permits and is available online (www.afabjournal.com).

AFAB grants to the author the right of re-publication

in any book of which he or she is the author or edi-


tor, subject only to giving proper credit to the original

journal publication of the article by AFAB. AFAB re-

tains the copyright to all materials accepted for pub-

lication in the journal. If an author desires to reprint

a table or figure published from a non-AFAB source,

written evidence of copyright permission from an au-

thority representing that source must be obtained by

the author and forwarded to the AFAB editorial office.

PEER REVIEW PROCESS

Authors will be requested to provide the names

and complete addresses including emails of five (5) potential reviewers who have expertise in the research

area and no conflict of interest with any of the authors.

Except for manuscripts designated as Rapid Commu-

nication each reviewer has two (2) weeks to review

the manuscript, and submit comments electronically

to the editorial office. Authors have three (3) weeks

to complete the revision, which shall be returned to

the editorial office within six (6) weeks after which the

authors risk having their manuscript removed from

AFAB files if they fail to ask the editorial office for

an extension by email. Deleted manuscripts will be

reconsidered, but they will have to be processed as

new manuscripts with an additional processing fee as-

sessed upon submission. Once reviewed, the author

will be notified of the outcome and advised accord-

ingly. Editors handle all initial correspondence with

authors during the review process. The editor-in chief

will notify the author of the final decision to accept or

reject. Rejected manuscripts can be resubmitted only

with an invitation from the editor or editor-in chief. Re-

vised versions of previously rejected manuscripts are

treated as new submissions.

PRODUCTION OF PROOFS

Accepted manuscripts are forwarded to the edito-

rial office for technical editing and layout. The manu-

script is then formatted, figures are reproduced, and

author proofs are prepared as PDFs. Author proofs

of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and

checked against the typed manuscript, because the

responsibility for proofreading is with the author(s).

Corrections must be returned by e-mail. Changes

sent by e-mail to the technical editor must indicate

page, column, and line numbers for each correction

to be made on the proof. Corrections can also be

marked using “track changes” in Microsoft Word or

using e-annotation tools for electronic proof correc-

tion in Adobe Acrobat to indicate necessary chang-

es. Author alterations to proofs exceeding 5% of the

original proof content will be charged to the author. All

correspondence of proofs must be agreed to by the

editorial office and the author within 48 hours or proof

will be published as is and AFAB will assume no re-

sponsibility for errors that result in the final publication.

PUBLICATION CHARGES

AFAB has two publication charge options: conven-

tional page charges and rapid communication. The

current charge for conventional publication is $25 per printed page in the journal. There is no additional

charge for the publication of pages containing color

images, micrographs or pictures. For authors who

wish to have their papers processed as a rapid com-

munication, authors will pay the rapid communication

fee when proofs are returned to the editorial office

in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one

week and $100 per journal page.

HARD COPY OFFPRINTS

If you are wishing to obtain a physical hard copy of

the AFAB journal, offprints are available in any quan-

tity at an additional charge: $100/page for black-white

and $150/page for color prints. You may order your

offprints at any time after publication on our website.

Scientific conference organizers may be expected to

agree to a set number of offprints as a part of their

agreement with AFAB.


MANUSCRIPT CONTENT REQUIREMENTS

Preparing the Manuscript File

Manuscripts must be written in grammatically

correct English. AFAB offers a fee based language

service upon request ([email protected]).

Manuscripts should be typed double-spaced, with

lines and pages numbered consecutively. All docu-

ments must be submitted in Microsoft Word (.doc or

.docx, PC or Mac). All special characters (e.g., Greek,

math, symbols) should be inserted using the sym-

bols palette available in this font. Tables and figures

should be placed in separate sections at the end of

the manuscript (not placed in the text). Failure to fol-

low these instructions will cause delays of the pro-

cessing and review of the manuscript.

Title Page

At the very top of the title page, include a title of

not more than 100 characters. Format the title with

the first letter of each word capitalized. No abbre-

viations should be used. Under the title, the authors

names are listed. Use the author’s initials for both first

and middle names with a period (full-stop) between

initials (e.g., W. A. Afab). Underneath the authors, a

list affiliations must be listed. Please use numerical

superscripts after the author’s names to designate

affiliation. If an authors address has changed since

the research was completed, this new information

must be designated as “Current address:”. The cor-

responding author should be indicated with an aster-

isk e.g., * Corresponding author. The title page shall

include the name and full address of the correspond-

ing author. Telephone and e-mail address must also

be provided for the corresponding author, and email-addresses must be provided for all authors.

Editing

Author-derived abbreviations should be defined

at first use in the abstract and again in the body of

the manuscript. If abbreviations are extensive au-

thors may need to provide a list of abbreviations

at the beginning of the manuscript. In vivo, in vitro

and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and

merge such paragraphs appropriately. Authors must

not begin sentences with “Figure or Table shows…”

as these are inanimate objects and cannot “show”

anything. When number are reported in text or in ta-

bles, always put a zero in front of decimal numbers:

“0.10” instead of “.10”.

MANUSCRIPT SECTIONS

Abstract

The abstract provides an abridged version of the

manuscript. Please submit your abstract on a sepa-

rate page after the title page. The abstract should

provide a justification of your work, objectives, meth-

ods, results, discussion and implications of study or

review findings . Your abstract must consist of com-

plete sentences without references to other work or

footnotes and must not exceed 250 words. On the

same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-

ing. Ideally, these keywords should include signifi-

cant words from the title.

Introduction

The introduction should clearly present the foun-

dation of the manuscript topic and what makes the

research or the review unique. The introduction

should validate why this topic is important based on

previously published literature, and the relevance of

the current research. Overall goals and project ob-

jectives must be clearly stated in the final sentence

of the last paragraphs of the introduction.

Materials and Methods

Information on equipment and chemicals used

must include the full company name, city, and state

(country if outside the United States or Province if

in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].


Variability, Replication, and Statistical Analysis

To properly assess biological systems indepen-

dent replication of experiments and quantification

of variation among replicates is required by AFAB.

Reviewers and/or editors may request additional

statistical analysis depending on the nature of the

data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

Discussion

The discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example


“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes.

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

Dissertation and thesis:

Examples:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.


Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft Excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

www.AFABjournal.com

• Free Access

• Print PDFs

• Flip Through Issues

• Search Article Archives

• Order Reprints

• Submit a Paper


Online Publication: www.AFABjournal.com

Documents

AFAB-Volume2-Issue3