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Classification of enterococci and their roles inspoilage of pork products and as sanitary indicatorsin pork processingLinda Marie KnudtsonIowa State University

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Classification of enterococci and their roles in spoilage of pork products and as sanitary indicators in pork processing

Knudtson, Linda Marie, Ph.D.

Iowa State University, 1992

U M I 300N.ZeebRd. Ann Arbor, MI 48106

Classification of enterococci and their roles in spoilage

of pork products and as sanitary indicators in pork processing

by

Linda Marie Knudtson

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department: Microbiology, Immunology and Preventive Medicine Major: Microbiology

Approved:

In Charge of Major Work

For the Major

Major Department

For the Graduate College

Iowa State University Ames, Iowa

1992

Signature was redacted for privacy.

Signature was redacted for privacy.

Signature was redacted for privacy.

Signature was redacted for privacy.

il

TABLE OF CONTENTS

GENERAL INTRODUCTION 1

Dissertation Format 2

LITERATURE REVIEW 4

History of the Enterococci 4

Origin of the species of the genus Enterococcus 5

Importance of the Enterococci 12 Habitat of the enterococci 12 Enterococci as fecal indicators in food and water 14

Enterococci in Meats 18 Enterococci as a cause of food spoilage 18 Hazard analysis critical control points 19 Meat sampling techniques 21

Enterococci in Food Poisoning 23

Clinical Importance of the Enterococci 24

Isolation and Identification Methods for the Enterococci 30 Selective media 30 Rapid methods for identification 36 Molecular approaches to identification 42

PAPER 1. ROUTINE PROCEDURES FOR ISOLATION AND IDENTIFICATION OF ENTEROCOCCI AND FECAL

STREPTOCOCCI 45

ABSTRACT 47

INTRODUCTION 48

MATERIALS AND METHODS 50

RESULTS AND DISCUSSION 52

ACKNOWLEDGMENTS 57

LITERATURE CITED 58

iii

PAPER 2. ENTEROCOCCI IN PORK PROCESSING 76

ABSTRACT 78

INTRODUCTION 79

MATERIALS AND METHODS 81

RESULTS 84

DISCUSSION 88

ACKNOWLEDGMENTS 91

LITERATURE CITED 92

PAPER 3. COMPARISON OF fGTC AND KF AGARS TO ENUMERATE ENTEROCOCCI AND FECAL STREPTOCOCCI IN MEATS 105

ABSTRACT 107

INTRODUCTION 108

MATERIALS AND METHODS 110

RESULTS AND DISCUSSION 112

ACKNOWLEDGMENT 116

LITERATURE CITED 117

PAPER 4. ANTIBIOTIC RESISTANCE AMONG ENTEROCOCCAL ISOLATES FROM CLINICAL AND ENVIRONMENTAL SOURCES 124

ABSTRACT 126

INTRODUCTION 127

MATERIALS AND METHODS 129

RESULTS AND DISCUSSION 130

ACKNOWLEDGMENT 133

iv

LITERATURE CITED 134

PAPER 5. COMPARISON OF FOUR LATEX AGGLUTINATION KITS TO RAPIDLY IDENTIFY LANCEFIELD GROUP D ENTEROCOCCI AND FECAL STREPTOCOCCI 141

ABSTRACT 142

LITERATURE CITED 148

GENERAL SUMMARY AND DISCUSSION 150

LITERATURE CITED 157

ACKNOWLEDGMENTS 171

APPENDIX A; MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM PORK CARCASSES SAMPLED IN THREE PLANTS 172

APPENDIX B: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM TWO PORK PROCESSING PLANTS 176

APPENDIX C; MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM RETAIL SAMPLES 179

APPENDIX D: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM WATER SAMPLES 181

APPENDIX E; SOURCES AND IDENTIFICATIONS OF CLINICAL ISOLATES FROM MERCY HOSPITAL MEDICAL CENTER, DES MOINES, IOWA 183

1

GENERAL INTRODUCTION

The classification of the enterococci has undergone significant changes

in recent years. In 1984, Schleifer and Kilpper-Balz (132) used DNA-DNA

and DNA-rRNA hybridization studies to show that the enterococcus group of

the streptococci was a separate genus, Enterococcus. Currently, 19 species

belong to this genus; E. avium (26), E. casseliflavus (26), £ cecorum (152), E.

columbae (39), E. dispar (27), E. durans (26), E. faecalis (132), E. faecium

(132), E. flavescens (121), E. gallinarum (26), E. hirae (58), E. malodoratus

(26), E. mundtii (25), E. pseudoavium (24), E. raffinosus (24), E.

saccharolyticus, E. seriolicida (89), E. solitarius, (24) and E. sulfureus (102).

Generally, the habitat of the enterococci is the intestinal tracts of warm

blooded animals (111), including domestic (126) and wild animals (110).

Enterococci also have been isolated from the feces of reptiles (110) and birds

(37) as well as from insects (113). Some enterococci can establish an

epiphytic relationship with plants (113) and they have been isolated from the

soil, where they are considered contaminants (25, 26).

Despite their ubiquity in nature, the natural habitat of the enterococci is

the intestinal tracts of man and animals and their occurrence in food or water

implies either indirect or direct fecal contamination (62). The relative

resistance of these organisms to adverse conditions, such as tolerance to

extremes in temperature, pH, and salinity is advantageous in the examination

of sea water, soft drinks and dried, frozen and processed foods where

conforms might not have survived (108). Although the enterococci are not

entirely host specific, they do show some degree of host specificity (70). The

proportions of enterococcal species differ in various animal and human feces.

2

Therefore, the identification of enterococci rather than their enumeration was

proposed to pinpoint the origin of fecal contamination (124). The enterococci

also cause spoilage of pork and other meat products (64), and they have been

implicated as a cause of diarrhea in suckling rats (48) and possibly humans

(71). Besides possible food poisoning, enterococci cause a number of other

human infections, such as urinary tract infections, bacteremia, and

endocarditis (114).

In this study, a schema for differentiating 13 species of the genus

Enterococcus and S. bovis and S. equinus by using rapid test kits and a

minimum of supplemental tests was proposed. Using this schema,

identifications of isolates from pork slaughtering plants and processed pork

were made; counts also were compared. Two media selective for enterococci,

fGTC and KF agars, were compared for recovery of enterococci from pork

carcasses, and processed pork, beef, and poultry. Identities of isolates on

both media also were compared. Antibiotic resistances of enterococci

isolated from pork, water, and clinical infections were examined. Species

comparisons of antibiotic resistance were made for 13 enterococcal species

and S. bows and S. equinus. Finally, four Lancefield grouping kits were

compared for their ability to accurately group the enterococcal isolates, most

of which are Lancefield group D.

Dissertation Format

This dissertation includes five papers that are manuscripts. Paper 1

appeared in the September 1992 issue (Volume 9) of Applied and

Environmental Microbiology. Paper 2 has been accepted for publication in

the Journal of Food Protection. Paper 3 has been submitted to Applied and

3

Environmental Microbiology as a research note. Paper 4 has been submitted

to the Journal of Food Protection. Paper 5 has been submitted to the Journal

of Rapid Methods and Automation in Microbiology as a research note. All

papers follow the style of the journal to which they were submitted. A general

literature review precedes the first manuscript, and a general summary and

discussion follows the fifth manuscript. There are separate lists of references

for each of the manuscripts as well as a separate list for the Introduction,

Literature Review, and Summary and Discussion.

The doctoral candidate, Linda M. Knudtson, was the principal

investigator in these studies, but worked in conjunction with several other

investigators in the collection of the pork carcass samples.

4

LITERATURE REVIEW

History of the Enterococcl

The term "Enterococcus" was first used by Theircelin and Jouhaud in

1903 as a generic name to describe a Gram-positive diplococcus of intestinal

origin. The type species was called Enterococcus proteiformis. In 1906,

Andrewes and Horder renamed this type species Streptococcus faecalis (3).

They believed that it belonged in the genus Streptococcus because it formed

chains under some cultural conditions. In later classifications, the term

"enterococcus" was used by many to designate Streptococcus species of fecal

origin which had in common some of the outstanding characteristics of

Streptococcus faecalis. In 1937, Sherman (138) proposed a classification

scheme which separated the streptococci into four divisions. One of these

divisions, the enterococcus division, was comprised of organisms that grew at

10 and 45°C, in 6.5% NaCI, and at pH 9.6, and which survived 60°C for 30

min. The ability to hydrolize esculin was also noted. Although the

classification has changed, some of these characteristics are still used today

to help identify enterococcl. In 1970, Kalina (82) recommended that current

nomenclatural and taxonomic decisions should be reconsidered because

chain formation was observed to be the result of unfavorable environmental

conditions and the main type of cell arrangement was the diplococccus form.

The pleomorphic nature of the enterococcl also set them apart from the rest of

the streptococci. In 1984, Schleifer and Kilpper-Balz (132,133) used DNA-

DNA and DNA-rRNA hybridization to show that Streptococcus faecalis and

Streptococcus faecium were so distantly related to other streptococci.

5

including Streptococcus bovis, that they should be transferred to another

genus. They proposed that the genus Streptococcus should be divided into

three genera. The first genus, Streptococcus sensu stricto, comprised the

majority of the known species, particularly the pyogenic and oral streptococci.

The second genus, Lactococcus, encompassed all lactic Lancefield group N

streptococci. Finally, the genus Enterococcus, as previously proposed by

Kalina, contained the enterococcal streptococci. It should be noted that the

fecal species S. bovis and Streptococcus equinus, though they reacted with

group D antisera and shared several characteristics with the enterococci,

were not considered members of the new genus Enterococcus, as they were

more closely related to the streptococci by 16S rRNA studies.

The genus Enterococcus consists of gram-positive, facultatively

anaerobic organisms that are ovoid in shape and may appear in a gram stain

in short chains, in pairs, or as single cells. Like streptococci, they are catalase

negative, although some strains do produce pseudocatalase (52,132). Most

(but not all) react with group D antisera and some react with group Q.

Hydrolysis of L-pyrrolidonyl-p-naphthylamide (PYR) is a characteristic feature,

although some species are PYR-negative (39,128,152). Most strains of the

newly defined genus Enterococcus possess the characteristics summarized

by Sherman (138), although there are species of enterococci that grow poorly

at 10 or 45°C. Another key test is the ability to hydrolyze esculin in the

presence of bile (49). However, some enterococci require up to 48 hours for

the correct reaction to occur.

6

Origin of tlie species of the genus Enterococcus

Enterococcus faecalis was among the first Enterococcus species

recognized. Previously Streptococcus faecalis, this group of organisms was

transferred to the genus Enterococcus in 1984 by Schliefer and Kilpper-Balz

(132) as proposed by Kaiina (82). This species was first described by

Thiercelin and Jouhaud in 1903, and named Enterococcus proteiformis. In

1906, Andrewes and Horder (3) grouped this species with the streptococci

and renamed it Streptococcus faecalis. In 1937, Sherman (138) recognized

that several species of streptococci showed only slight variations to S. faecalis

isolates and suggested that these species be considered varieties of 5.

faecalis becoming S. faecalis var. faecalis, S. faecalis var. liquifaciens, and S.

faecalis var. zymogenes. These varieties were differentiated by their ability to

be proteolytic and hemolytic (35). Since that time several investigators have

questioned these varieties (16, 34, 59, 80,111) because hemolysis and

proteolysis were inconsistent within these subspecies. The abilities to liquify

gelatin and hemolyze red cells, upon which these varieties were based, were

later found to be controlled by plasmids (78, 119) which explains the earlier

inconsistencies. Therefore, these subspecies are no longer recognized.

The change from S. faecium to Enterococcus faecium was also

proposed by Schleifer and Kilpper-Balz (132). Streptococcus faecium

isolates were initially considered to be the same species as S.faecalis. In

1919, Orla-Jensen (cited in (34)) proposed dividing the two species on the

basis of fermentation characteristics, tolerance to heat and sodium chloride,

and temperature limits of growth. Many researchers supported this division on

the basis of many different criteria, and eventually it was generally accepted.

7

Shattock (137) supported the distinction between S. faecalis and S. faecium

by reexamining the physiological characteristics of 350 fecal streptococci.

She claimed that S. faecium actually did comprise a legitimate species.

Barnes (5) added another important criterion for differentiation by showing that

S. faecium does not have the ability to reduce tetrazolium to formazan in a

glucose medium at pH 6, whereas S. faecalis does reduce tetrazolium.

Whittenbury (151) stated that the two species could be separated on the basis

of tellurite tolerance and reducing ability. Many subspecies of S. faecium

also existed, but these have all been transferred to other species.

Enterococcus durans is an example of a species that was initially

considered a subspecies of S. faecium. It was isolated from dairy products

and named Streptococcus durans by Sherman and Wing (139). Later, Deibel,

Lake and Niven (35) noted its similarity to S. faecium and suggested a varietal

status for the organism, as did Barnes (6). In 1984, Collins et al. (26)

proposed that S. durans (or S. faecium subsp. durans) be named

Enterococcus durans.

Isolates of another subspecies of S. faecium, S. faecium subsp. mobilis,

were shown to be members of the species Streptococcus casseiifiavus. This

subspecies was first described as the motile strains of S. faecium isolated

from plant material (93). S. casseiifiavus also began as a subspecies of S.

faecium, S. faecium subsp. casseiifiavus. This subspecies was proposed by

Mundt and Graham (112) to describe a group of motile, yellow pigmented

streptococci isolated from vegetation. These strains were elevated to species

level by Vaughan et al. in 1979 (148). It was proposed in 1984 that all strains

of S. casseiifiavus, on the basis of biochemical, chemical and genetic data

8

(59, 80,148), should be transferred to the genus Enterococcus as

Enterococcus casseliflavus (26).

Pette (59) isolated some streptococcal strains from Gouda cheese in

1955 which he named S. faecalis var. malodoratus. A numerical phenetic

study by Jones et al. (81) indicated that these strains were more closely

related to S. faecium than S. faecalis. In 1984, Collins et al. and others (26,

59) determined that S. faecalis var. malodoratus was sufficiently distinct to be

considered a separate species. It was renamed Enterococcus malodoratus.

The species Streptococcus avium was proposed by Nowlan and Deibel

(118) to accommodate streptococci resembling S. faeca/Zs and S. faeciumoi

serological group Q. The numerical phenetic study of Jones et al. (81) in 1972

indicated that some similarity existed between S. avium and S. faecium but

the similarities were not sufficient for S. avium strains to be included in the

species S. faecium. In 1984, Collins et al. (26) also proposed transferring S.

avium to the new genus as Enterococcus avium.

Barnes et al. (8) isolated from the intestines of young chickens a

number of streptococci of serological group D which were apparently distinct

from S. faecalis, S. faecium and S. durans. The DNA homology data

performed by Farrow et al. (59) indicated that the streptococci isolated by

Barnes represented a distinct species. This finding was supported by Bridge

and Sneath (15), who named this group of isolates Streptococcus gallinarum.

This species was transferred by Collins et al. (26) in 1984 to the genus

Enterococcus and renamed Enterococcus gallinarum. This species also

included a variety of motile strains isolated from clinical infections which had

9

been called E. faecalis or £ faecium (although neither is considered motile)

(25).

Schleifer and Kilpper-Balz (132) reported that 3 strains designated E.

faecium showed very little DNA sequence homology (<40%) with the type

strain of E. faecium. These three strains were ancestrally related to a common

strain (NCDO 1258) which also was found to be distinct from E. faecium and

E. durans. They suggested that these strains may represent a new species

(132). Other strains, intermediate between E. durans and E. faecium, which

caused growth depression in young chickens (63) were believed to belong to

a new species. Farrow and Collins (58) studied these "atypical" E. faecium

strains, as well as some unclassified group D streptococci from Sharpe and

Fewins (136). Their results (58) indicated that these strains represented a

new species of enterococcus, for which the name Enterococcus hirae was

proposed.

Until recently a number of "enterococcus-like" strains remained

unclassified. Some of these were yellow-pigmented isolates from plants (148)

and soil (144). Primary studies indicated that they were not related to the

yellow-pigmented E. casselifiavus (148). Other isolates from cows (69) had

previously been shown by Farrow et al. (59) to be distinct from recognized

Enterococcus species. Studies on these isolates by Collins et al. (25) found

them to belong to a new species and the name Enterococcus mundtii was

proposed. E. mundtii has also been isolated from normally sterile body sites

in two septic patients (84); this was the first detailed description of E. mundtii

as a cause of human infection.

10

Unclassified enterococci from clinical sources were studied by Collins

et al. (24). Nucleic acid studies and penicillin-binding protein patterns

demonstrated that 6 human strains, a single clinical isolate and a strain from

bovine mastitis were all genetically distinct from each other and from all

previously described Enterococcus species. For the 6 human strains, the

name Enterococcus raffinosus was proposed due to their ability to metabolize

raffinose. For the strain from bovine mastitis, the name Enterococcus

pseudoavium was proposed. For the single clinical isolate (from ear exudate),

the name Enterococcus solitarius was proposed.

De Vriese et al. (40) described a species. Streptococcus cecorum,

as carboxyphilic strains isolated from the caeca of chickens. It resembles the

enterococci in many respects, but it fails to grow at 10°C and in 6.5 % NaCI,

characteristics generally attributed to enterococci. It also fails to react with

group D antiserum, unlike all other enterococcal species to date. Sequencing

of 16S rRNA by reverse transcriptase was performed by Williams et al. (152)

to clarify its taxonomic position. Streptococcus cecorum was clustered with

the enterococcal species and was only distantly related to the other

streptococci, suggesting that S. cecorum is phylogenetically a member of the

genus Enterococcus. Thus, the name Enterococcus cecorum was proposed.

Further studies on £ cecorum carried out by De Vriese et al. (38) showed that

this species was not limited to growth in chickens. It was isolated from the

intestines of pigs, cattle, horses, a mallard duck, and canaries (where it

appears to predominate).

Reverse transcriptase sequencing of 16S rRNA was performed on

another Streptococcus species to clarify its phylogenetic position.

11

Streptococcus saccharolyticus, first described by Farrow et al. (1984),

included atypical S, bows-like strains isolated from cows and straw bedding.

Although distinct from S. bovis, S. saccharolyticus failed to react with group D

antisera and was placed in the group Streptococcus sensu stricto. It did,

however, more closely resemble members of the genus Enterococcus.

Research by Rodrigues and Collins (128), using reverse transcriptase

sequencing, revealed that S. saccharolyticus was only distantly related to

members of the genus Streptococcus sensu stricto and formed a distinct

group with E. faecalis. Therefore, it was proposed that S. saccharolyticus

should be reclassified as Enterococcus saccharolyticus comb. nov.

A new species of enterococcus, closely related to E. cecorum and E.

avium, was proposed by De Vriese et al. (39), This species, Enterococcus

columbae, dominates in the intestinal flora of domestic pigeons. Another new

species of Enterococcus, Enterococcus dispar, was proposed by Collins et al.

(27). This species was discovered during a survey of atypical enterococci

from human sources. They were isolated from synovial fluid and stool. It is

most closely related to E. hirae in biochemical characteristics.

Streptococcicosis, a disease of fish, is the most important disease of

yellowtail fish in Japan. The disease agent, previously identified as a

streptococcus, did not fit into any of the species yet described. A study by

Kusada et al. (89) determined that the causative agent of the disease is a new

species of Enterococcus, Enterococcus seriolicida.

In 1991, Martinez-Murcia and Collins proposed yet another new

species of enterococcus (102). Enterococcus sulfureus was proposed as the

name for 3 unknown non-motile, yellow-pigmented enterococci originating

12

from plants. The isolates were closely related to E. casseliflavus and E.

mundtii, both yellow-pigmented enterococcal species.

The newest members of the genus Enterococcus, Enterococcus

flavescens, are yellow pigment-producing strains of clinical origin. Three

yellow-pigmented strains of enterococci (£ casseliflavus, E. mundtii, and E.

sulfureus) had already been described (25, 26,102). Almost all of these had

been isolated mainly from environmental materials (plants, soil etc.) and rarely

from clinical material (130). Pompei et al. (121) studied four atypical yellow-

pigmented group D isolates from severe human infections (122). By using

DNA-DNA hybridization tests and fatty acid content determinations, they found

these 4 isolates comprised a new species and proposed the name

Enterococcus flavescens.

Importance of the Enterococci

Habitat of the enterococci

Enterococci are found in the feces of most healthy adults. When

enterococci from feces have been identified to species level, many studies

report that E. faecalis is most common and is found in higher numbers than E.

faecium (114). Besides the alimentary tract of humans , enterococci also

reside in many other animals. Enterococcal species have been isolated from

buffalo, cattle, sheep, camels, pigs, horses, mules, donkey, rabbits, chickens

and geese (126). Mundt (109,110) surveyed the incidence of enterococci in

wild animals, reptiles and birds. He noted that many wild mammals and

reptiles excreted enterococci (34). Although Mundt did not associate

enterococci with birds, new species of enterococci have been defined. E.

13

cecorum was isolated from canaries and a mallard duck (37), E. hirae and E.

avium from chickens (26, 58), and E. columbae from domestic pigeons (39).

These species had not been defined at the time of Mundt's survey. Although

enterococci are natural residents of the intestinal tracts of man and other

warm- and cold-blooded animals, their resistance and tolerance, together with

their low minimum and high maximum temperature limits of growth, fit them for

survival and even growth under diverse conditions in nature (138). In

humans, they are infrequently found in vaginal and oral specimens (such as

dental plaques) (114). Mundt (110) studied the distribution of enterococci in

insects and concluded that in the insect digestive tract, enterococci are

transient, and their occurrence on the insect exterior was due most probably to

mechanical transfer. Extensive studies verified the common occurrence of

enterococci on plants, particularly on domestic plants. It was believed that an

epiphytic relationship occurred since the bacteria could establish a cycle in

plants with transmission in the plant seed (113). Contamination of plants

probably occurred through insects and wind. Several species of enterococci

have been isolated from plants and soil. E. casseliflavus, E. mundtii, and E.

sulfureus are all yellow-pigmented enterococci and all have been isolated

from plants and soil (25, 26,102). There is general agreement that

enterococci are not native to soil, and their presence in soil samples

represents contamination from either plant or animal sources. In this

environment, the enterococci are disseminated most probably by wind, rain,

and insects.

14

Enterococci as fecal indicators in water and food

An ideal indicator organism should 1) be associated in high numbers

with feces and/or intestinal pathogens, 2) not multiply in the environment, 3)

die off less rapidly than potential pathogens, and 4) withstand the processing

conditions undergone by the substrate (62). Questions had arisen regarding

the complete reliance on the use of Escherichia coli as the sole indicator

organism. Its relatively rapid death in water, in comparison with pathogenic

organisms (including enteric viruses), had led some public health officials to

question its utility as the sole index microorganism for fecal contamination

(34). In the routine procedures employed , E. coli may be confused with

physiologically similar bacteria. Many workers have emphasized the

importance of enterococci as indicators of fecal pollution (7,108,126,151). In

the past, the acceptance of enterococci as pollution indicators also had been

questioned. E. coli was easier to detect in water, whereas there was a lack of

quantitative recovery methods for enterococci. The absence of information

regarding the sources of enterococci, questions concerning their

classification, and their significance in the water supply also were concerns

(34). Questions regarding their classification were answered with a new

classification system (132). Many studies have been conducted on

enumeration of enterococci and many selective and differential media are

available (72). Although enterococci are widely distributed in nature, the

major natural habitat of these organisms is the intestinal tract of man and

animals (62). Thus, the occurrence of these bacteria in water implies either

direct or indirect fecal contamination. Characteristically, enterococci are not

15

detected in waters free from fecal contamination even though they are found

on plants and insects that could contaminate water.

The ratio fecal coliforms/fecal streptococci was proposed by Geldreich

and Kenner in 1969 (124). It was used to identify the origin of pollution of

surface waters. A ratio above 4 suggests human contamination and a ratio

below 0.7 contamination by animals. The persistence of both indicators after

discharge of feces in water, however, is unclear. According to Bartley and

Slanetz (9), after initial contamination both groups may exhibit a slight

increase in numbers (presumably due to the organic matter in the water and

the temperature) followed by a pronounced decrease. An early study of the

coWiorm-Streptococcus ratio in various water sources indicated a more rapid

decrease in conforms. Recent studies also have shown that E. coli is more

sensitive than enterococci to natural water (124). It is important to note that S.

bovis also dies off rapidly in water. As £ coli numbers decrease the ratio

approaches unity and the ratio is no longer accurate for identification of fecal

origin. It is estimated that the ratio would only be valid for the first 24 hours

(124). The choice of selective media adds variability to the ratio approach.

Many investigators have reported a lack of correlation between

Enterococcus and E. coli counts, and the unreliability of enterococcal counts

as a reflection of fecal contamination is established rather well. Because the

proportions of the various enterococcal species are not the same in different

animal and human feces, the identification of enterococcal species rather than

their enumeration was proposed as a possible solution (124). None of the

enterococci can be considered absolutely host specific, but some species

evidence a degree of host specificity (70). There are reports indicating that

16

while S. faecalis (£ faecalis) predominates in the human intestines, S. bovis

and S. faecium (E. faecium) are the predominant species of animals, including

swine (126). E. faecalis dominated the enterococcal flora of human origin and

birds feces, whereas it was absent or in low numbers in cow, pig, sheep, or

rabbit feces. E. faecium, on the other hand, showed a wider range (124),

Studies on the flora of British pigs revealed that S. faecium (E. faecium) is

commonly present in the pig intestine, while £ faecalis is rare (7). Kjellander

(87) in 1960, also observed that S. faecalis (E. faecalis) was more numerous

in humans, whereas S. faecium (E. faecium) was of animal origin. The

presence of S. bovis in humans would be associated with disease (12, 97).

According to De Vriese et al. (41), the enterococcal flora of poultry comprises

a relatively small number of frequently occurring species. E. faecium, E.

cecorum, E. faecalis, E. hirae and E. durans were regularly isolated, while E.

mundtii, E. casseliflavus, E. gallinarum and E. avium were only rarely isolated.

Another study by De Vriese et al. (43) revealed that no E. avium were isolated

from chickens. This finding is odd since the original strains of E. avium were

isolated from the feces of chickens (26). A study of enterococci isolated from

calves, young cattle, and dairy cows (42) revealed that enterococcal species

are rare in ruminating cattle, but E. faecalis was isolated from nearly half of the

pre-ruminating calves. Variation in the enterococcal flora of animals has been

associated with many factors. Geographical location, diet, age, species of the

animal, and even seasonal effects have been observed to contribute to this

variation (34). Despite this potential for variation, significant differences in the

distribution of the enterococci and fecal streptococci have been observed in

17

host animals. It is believed that identification of enterococci and fecal

streptococci provides valuable information on the origin of pollution (124).

Enterococci are important as fecal indicator organisms in foods

because they are readily isolated in large numbers from human and animal

feces. The relative resistance shown by their cells against adverse conditions,

such as tolerance to extremes in temperature (64), pH, and salinity, proves

advantageous in the bacteriological examination of sea water, soft drinks and

dried, frozen and processed foods (108) where coliforms might not have

survived. The use of enterococci as a fecal indicator in frozen foods was

discussed by Deibel (34). Most, but not all, of these foods undergo some

thermal processing or precooking prior to freezing, so bacteria found in these

products represents recontamination after thermal processing prior to

freezing. Since E. coli is particularly susceptible to freezing and enterococci

are quite resistant, enterococci provide a more reliable index to the sanitary

history of frozen food. Enterococci may occur in comminuted, cured meat

products, either as the result of survival of thermal processing or from

postprocessing contamination. These products are heated to kill most

vegetative bacteria; however, processors assume that raw sausage mix does

not contain excessive bacterial numbers. The high heat and salt resistance of

the enterococci as well as high initial population in the sausage mix are

factors contributing to their survival in marginally processed products. Even

under ideal conditions the products could be recontaminated during

subsequent slicing and prepackaging. In these instances, the occurrence of

enterococci does not necessarily suggest direct fecal contamination (34).

Quite often enterococci become established in food plants and grow in areas

18

far removed from the original source of fecal contamination. Therefore,

caution and discretion must be exercised in attributing significance to the

numbers and species of enterococci and fecal streptococci present in foods

(70).

Enterococci in meats

Enterococci as a cause of food spoilage

Enterococci present in foods are not only important as indicators of

fecal pollution, they are also a serious cause of food spoilage, especially in

meats. There are many reasons for spoilage in meat and meat products. The

following factors can be important: meat from sick or stressed animals; poor

slaughtering hygiene; inadequate chilling of meat during transport, cutting or

storage; poor hygiene amongst personnel; insufficient cleaning and

disinfection of equipment and food-handling surfaces; processing of heavily

contaminated meat and other ingredients; inadequate heat treatment for

refrigerated products or for canned products that can be stored unrefrigerated;

and inadequate refrigeration of products after purchase (73). Whether the

origin of the enterococci lies with fecal pollution or contamination of the food

plant, the presence of food spoilage organisms greatly decreases the shelf-life

of meat products and other foods. A number of interrelated factors influence

the shelf-life of meat, specifically holding temperature, atmospheric oxygen,

indigenous enzymes, light, and microorganisms. Although some deterioration

of meat will occur in the absence of microorganisms, microbial growth is by far

the most important factor in relation to keeping the fresh quality of meat (91 ).

The initial bacterial load of meat has a major influence on its shelf-life. At

lower cooking temperatures, the shelf life and microbiological safety of the

19

product are reduced because a greater number of organisms survive the

process. The enterococci are the major group of heat-resistant organisms that

survive the processing of cured meat products (64). However, excessively

high numbers of thermoduric enterococci in cured meats indicate inadequate

processing. Both E. faecalis and £ faecium have been implicated in the

spoilage of these products. Enterococci have also been shown to cause food

spoilage in canned hams (116,136). The shelf life of sliced, prepackaged

ham (and sometimes other similarly prepared cured meats) also may be

dictated by the initial numbers of contaminating enterococci (70). These

bacteria produce sour flavors, discoloration, gas, slime, and milky exudates.

Dykes et al. (46) performed quantitation of microbial populations associated

with the manufacture of vacuum-packaged, smoked Vienna sausages. They

concluded that the lactic acid bacteria contaminated sausage surfaces as a

result of manufacturing and handling processes. Complete avoidance of

enterococci in these products is difficult, and control of numbers rather than

avoidance of occurrence must be practiced.

Hazard Analvsis Critical Control Points

Good manufacturing practice (GMP) during slaughter, as described in

the Recommended International Code of Hygienic Practice for Fresh Meat,

includes all necessary measures to produce meat with the lowest possible

microbial contamination. This is attainable only if the whole process is strictly

controlled. The hazard analysis critical control point (HACCP) concept, as

reviewed by Pierson and Corlett, Jr. (120) and Tompkin (146), provides a

systematic approach to achieve this end. If the goal of reduced foodborne

illness is to be achieved, it is necessary to identify the errors which are

20

involved in food preparation. HACCP plans must be customized to the unique

conditions existing within each establishment, but it is possible to generalize

on similar procedures in the production of meat and poultry products.

The first step toward establishing a HACCP plan in a meat or poultry

plant is to conduct a hazard analysis. Hazard has been defined as the

unacceptable contamination, growth or survival by microorganisms of concern

to safety or spoilage: and/or the unacceptable production or persistence in

foods of microbial metabolic products such as toxins, enzymes or amines.

The purpose of hazard analysis is to identify potential problems which could

occur in an operation. Examples of potential hazards in a meat or poultry

plant include; raw materials with a history of causing microbiological

problems; sites of contamination in the process; and the potential for

microorganisms to survive or multiply during production, storage, distribution,

or use.

After hazards have been identified, procedures must be established for

their control. The definition for critical control point (CCP) is important to the

meat and poultry processor and regulator because it defines the limits of what

should be achieved when a HACCP program is established. The current

ICMSF definition for CCP is "a location, practice, procedure or process at

which control can be exercised over one or more factors which, if controlled,

could minimize or prevent a hazard" (146). This definition states the value of a

CCP, while limiting it to steps in a process where some degree of control is

possible. The definition allows for and encourages the adoption of CCPs to

minimize contamination with enteropathogens during the slaughtering

processes and subsequent handling of raw meat and poultry. There are two

21

levels of CCP, depending on the confidence that the hazard can be controlled.

A CCP1 will assure control (ie. cooking to 63°C assures destruction of

salmonellae in raw meat), while a CCP2 can only minimize the hazard (proper

care in eviscerating reduces incidence of salmonellae on fresh meat). Control

means managing the conditions of an operation to maintain compliance with

established criteria. If the criteria for each CCP are met, the hazards will be

reduced or eliminated. A key element of the HACCP system is the use of

measurements to monitor and verify whether established criteria are being

met. All of the monitoring and verification measurements that are taken must

be recorded. Included must be action taken when the established criteria

have been exceeded.

The concept of HACCP is applicable to a wide variety of problems. It is

a common sense approach to preventing problems. It is the best system

currently available for improving the microbiological safety of food. In the

Federal Republic of Germany a new slaughter hygiene monitoring system (CR

monitoring) was tested. It is the German equivalent to HACCP. According to

Schutz et al. (135), during a 7-month experimental period using CR

monitoring, it was found to be an effective means of control and an efficient

monitoring system. If carried through consistently, it causes a noticeable

reduction in visible carcass dirt.

Meat samplino techniques

Sampling of carcass surfaces for microbiological examination should

be used to identify critical control points in slaughterlines. The data should

then be used to monitor the process to attain a good end product. Sampling

techniques have to be accurate and precise to make valid microbiological

22

comparisons of different steps in slaughtering plants. Various techniques

have been suggested for determining bacterial colony counts on carcasses.

Those commonly used are the agar contact technique, the swab technique,

and the excision technique. Snijers et al. (143) compared four sampling

techniques: the excision, the double swab, agar contact and modified agar

contact. In the agar contact technique (called agar sausage technique by

other authors) an agar surface is pressed onto the test area and incubated.

The modified technique involves homogenization of impressed agar slices in

peptone saline and pour plating of the samples. The swab technique relies

on rubbing one swab (or two swabs) on a test surface, transferring it (them) to

a dilution bottle, mixing to release the bacteria from the swab, diluting, and

plating on appropriate media. In the excision technique, pieces of tissue are

removed and homogenized in a solution and plated. Snijers et al. (143)

showed that the agar contact technique cannot be used for determining

contamination of carcasses because plates were overcrowded. The double

swab and modified agar contact techniques yielded significantly higher

standard deviations than did the excision technique. The excision technique

was the most suitable method in view of its high accuracy and precision.

Several other researchers obtained similar results (2, 60). Nortje et al. (117)

also reported that the excision technique was the most reliable. Morgan et al.

(107) stated that the method used to collect microbial samples from carcasses

should be simple, non-destructive, reproducible and economical. When pork

carcasses were sampled, the excision technique was admittedly the most

reliable. It is used as the standard against which all other methods were

evaluated. This technique, however, is not practical in a commercial

23

environment due to carcass mutilation; swabbing, despite its drawbacks, is

still the most universally used method. Lasta and Fonrouge (94) used the

swab technique to ascertain whether small sampling areas (10 to 100 cm^)

from bovine carcasses were characteristic of the hygiene level in the

slaughtering plant. They concluded that the most heavily contaminated areas

of the carcass are generally small; the microorganisms were not evenly

distributed. Contamination is random and comes from touching or rubbing the

carcass with hands, clothes, tools, equipment, hides, and spattering with

different materials (feces, water, pus, etc.). Based on their results, they

concluded that the bacterial count from relatively small areas of the carcass

(less than 100cm2) was not an adequate indicator of hygiene.

Homogenization by blending and stomaching was compared by Dickson for

the recovery of Listeria monocytogenes from inoculated beef tissue (44). No

difference between these two methods was detected. He did find, however,

that phosphate buffer was slightly inferior to buffered peptone as a diluent.

Sampling methods for poultry carcasses are somewhat different.

Lillard (96) compared the whole carcass rinse with the stomaching or

blending of excised skin for sampling broilers. She demonstrated that these

three sampling methods most commonly used resulted in the isolation of

equivalent levels of aerobic bacteria and Enterobacteriaceae, but only a small

portion of the total bacterial load was recovered by any sampling method.

Enterococci in Food Poisoning

Enterococci also have been implicated as a cause of food poisoning.

Murray (114) stated that this was a misconception which probably arose

because of the occurrence of enterococci in the intestinal tract and, therefore.

24

on fecally contaminated food. In a recent study, a strain of Enterococcus hirae

from a stool of a human with diarrhea was implicated as the cause of diarrhea

in suckling rats (48). In 1924, enterococci were suggested as the cause of

food borne illness in an outbreak ascribed to milk. The most convincing

evidence that enterococci cause food poisoning was obtained in feeding tests

on human volunteers by Carey et al. In 1931 (19). These volunteers became

ill when they ingested whole-cell enterococcal cultures. But these results

could not be consistently repeated by other investigators. A collection of

strains implicated in various food-poisoning outbreaks indicated an

approximate equal distribution of the S. faecalis and S. faecium species (34).

Hartman et al. in 1965 (71) suggested that for enterococcal food poisoning to

occur, the conditions must be "right". The question then, is what are these

conditions and how do we avoid them when processing foods implicated in

enterococcal food poisoning? Variations in temperature, pH, freshness of

cultures, and synergism all have been suggested as possible contributing

factors in enterococcal food poisoning (71).

Clinical Importance of the Enterococci

Urinary tract infections (UTI) are commonly caused by enterococci. The

rate of urinary colonization by enterococci rises in patients who have been

instrumented, received antibiotics (especially cephalosporins), have structural

abnormalities, and/or recurrent UTIs (95, 114). In healthy patients enterococci

cause less than 5% of UTIs.

Enterococci also cause an estimated 5-15% of the cases of bacterial

endocarditis (20). As with other human enterococcal infections, most isolates

25

are identified as E. faecalis. However, otfier species also cause this disease.

E. avium, E. casseliflavus, E. durans, E. gallinarum, and E. raffinosus as well

as E. faecium also have been isolated (53). Although enterococcal

endocarditis usually affects older adults (56-59 years of age), it occasionally

occurs in children (20,114). Fifty percent of men with enterococcal

endocarditis have a history of previous enterococcal urinary tract infection

(UTI) or genitourinary tract instrumentation. Forty-three percent of women

have a history of childbirth or abortion in the preceding three months.

Patients with underlying valvular heart disease and IV drug users also are at

risk,

Enterococcal bacteremia is much more common than enterococcal

endocarditis. The incidence of enterococcal bacteremia appears to be

increasing (20, 95,114). There has been a steady increase in patients with

enterococcal bacteremia since 1975, even though there was no increase in

admissions. This increase was entirely due to an increase in nosocomial

bacteremias (100). Possible sources of enteroccoccal bacteremia include

infection or colonization of the genitourinary, gastrointestinal, and

hepatobiliary tracts. The most common source in several studies had been

the urinary tract. Enterococci also have been reported in secondary

bacteremia in patients with postoperative wound infections, pyelonephritis,

and many gynecological infections. Intravascular catheters are also a major

source of enterococcal bacteremia (100). Mortality of enterococcal

bacteremia has generally been high because of underlying complicating

factors. These factors include association with burns, hospital-acquired

infections, and serious underlying illness.

26

Enterococci have been implicated in several other human infections. In

intra-abdominal infections, the role of enterococci is controversial. It has been

suggested that enterococci are pathogenic in these infections only

synergistically with anaerobes (20). Although group B streptococci are the

most common cause of neonatal infections, it has been well documented that

enterococci also can cause infections in neonates. An outbreak of E. faecium

occurred in premature infants with severe underlying disease, nasogastric

tubes, and multiple intravascular devices. Positive blood cultures and CSF

samples were recovered. It was concluded that all the E. faecium isolates

were the same strain, and the outbreak was spread by hospital personnel. In

addition to neonatal meningitis, enterococci can cause central nervous system

(CNS) infections in older children and adults. Most cases are related to an

underlying disorder such as a long-term primary illness, invasive CNS

procedures, prior antibiotic therapy or all three (114).

Enterococcal infections are most often nosocomial. These nosocomial

infections account for as much as 10% of all hospital infections (95). The

numbers are increasing. In 1984, they were the third leading cause of urinary

tract infections and the sixth leading cause of bacteremia (77). To account for

the increased infection rate, attention has been drawn to the growing use of

broad-spectrum antibiotics, cephalosporins in particular. Treatment with these

agents, which lack appreciable activity against the enterococci, would be

expected to provide this organism with a selective growth advantage, leaving

patients vulnerable to superinfection (77). It is also possible that the increase

may be caused in part by factors other than exposure to antibiotic therapy.

Greater reliance on indwelling urinary catheters and intravascular devices in

27

the care of the hospitalized patient may be another possible cause. Patient-

to-patient transmission, and even interhospital spread of the organism can

occur. The epidemiology of nosocomial infection caused by enterococci is

similar to that caused by methicillin-resistant staphylococci. Therefore, control

measures such as those taken for methicillin-resistant staphylococci should

be taken to prevent nosocomial spread of enterococci. The most likely way

these resistant bacteria are spread is from an infected patient by transient

carriage on the hands of personnel. Nosocomial enterococci often show high-

level resistance to aminoglycosides (95).

Enterococci do not possess the potent virulence factors associated with

certain other bacterial species. However, enterococci possess a number of

characteristics which make them particularly capable of surviving and causing

disease in this antibiotic era. They are intrinsically resistant to a number of

antimicrobial agents, including p-lactam antibiotics and other agents that

inhibit cell wall synthesis, the polymixins, and lincosamides (clindamycin and

lincomycin) (47). Two mechanisms are responsible for resistance to p-lactam

antibiotics, low affinity of the penicillin-binding-proteins and production of p-

lactamase (61). They also are intrinsically resistant to clinically low levels of

aminoglycosides. However, cell wall active agents plus aminoglycosides

have been effective in the therapy of serious infections caused by enterococci.

To make matters worse, enterococci have recently acquired resistance to a

number of clinically important antimicrobial agents, including high-levels of

aminoglycosides. In one animoglycoside, gentamicin, high level resistance is

due to the presence of a 2'-phosphorylating enzyme which inactivates the

drug. This enzyme also has 6'-acetylating activity and is mediated by a

28

transmissible genetic determinant comprised of two fused genes. IVIost

isolates studied also have streptomycin adenylating activity and are resistant

to streptomycin (47). One of the major reasons for the rapid dissemination of

antibiotic resistance determinants in enterococci is the ability of these

organisms to exchange genetic elements among one another and with other

bacteria, particularly other gram-positive cocci including staphylococci (22,

104). Aminoglycoside-modifying enzymes responsible for high-level

aminoglycoside resistance to gentamicin in E. faecalis infections have been

shown to be very similar to those isolated from cultures of aminoglycoside

resistant Staphylococcus aureus. Schaberg and Zervos (131) were able to

identify gentamicin resistance genes in strains of gentamicin resistant E.

faecalis when a staphylococcal plasmid encoding a similar resistance

determinant was used as a probe, but not when a deletion mutant of the

identical plasmid was used. Many of the important resistance determinants

have been shown to reside on transmissible plasmids. Resistance to

chloramphenicol, for example, is mediated by a plasmid carrying

chloramphenicol acetyltransferase (114). In addition, enterococci are able to

transfer resistance on transposons, without transmission of plasmids. The

intestinal location of these organisms and their abundance of plasmids and

transposons suggests that they may serve as a significant reservoir of genetic

information available to other gram-positive bacteria residing in the gut (22).

The recent acquisition of plasmid-borne resistance to gentamicin and p-

lactamase-associated penicillin resistance, has left vancomycin as one of the

few remaining drugs to treat serious enterococcal infections. Very recently,

however, plasmid-borne vancomycin resistance was detected in a few clinical

29

isolates (140). Vancomycin resistance was inducible and transferable by

conjugation or mobilization between species of enterococci. A membrane

protein which may block the access of the antibiotic to its peptidoglycan target

is responsible for the resistance (140).

With the increased emergence of antibiotic resistance, rapid and

reliable methods of detecting both low-level resistance and high-level

resistance to a number of antibiotics has become increasingly important.

Routine susceptibility testing (especially for aminoglycosides and |3-lactam

antibiotics) of clinical enterococcal isolates is of primary importance in the

care of patients suffering from enterococcal infections (66). Knowledge of the

prevalence of these resistant strains cultured from a hospital's patient

population can be used as a guide for appropriate antimicrobial therapy (75).

The association of antibiotic resistance and species identification of isolates

has also received considerable attention. With the recent changes in

classification, and the knowledge that the rapid methods used in hospitals are

not adequate to accurately identify the enterococci, many researchers are

reclassifying previously isolated enterococci and performing antibiotic

susceptibilities. They are finding differences in patterns of resistance between

different species of enterococci. Moellering et al. (105) stated that

Enterococcus faecium displays greater resistance than E. faecalis to penicillin

and certain synergistic drugs. Louie et al. (98) reported that E. faecalis and £

faecium are the most predominant species of enterococci encountered in

human infections. In general, E. faecium strains also are less susceptible to p-

lactams and aminoglycosides than are E. faecalis strains. E. faecium strains

also are often more refractory to the synergistic effect of the antibiotic

30

combination. The possible existence of similar susceptibility differences

among the more recently described species of Enterococcus suggests an

asssessment of possible correlations between a certain species and a given

susceptibility pattern could provide valuable information (129). Gray et al. (65)

found several differences in antibiotic resistance patterns between different

species. More than half of E. faecium isolates were resistant to ampicillin, but

all E faecalis strains were sensitive to ampicillin. High-level gentamicin

resistance was seen in E faecalis, but no other enterococcal species. Two

studies on E raffinosus from clinical infections were performed. In one study,

E. raffinosus isolates showed higher levels of resistance to penicillin and

kanamycin than E avium isolates (66). The second study revealed that high-

level ampicillin resistance existed among isolates of E raffinosus, but there

were no significant differences between patients with ampicillin-resistant E

raffinosus and those with ampicillin-sensitive E. raffinosus (21).

Isolation and Identification Methods for the Enterococci

Selective media

As the importance of the enterococci as fecal indicators, in food

spoilage, in clinical infections, and antibiotic resistance emerged, the desire to

isolate and grow these organisms increased. Many media have been

developed to select for enterococcal species and differentiate them from other

types of bacteria. Applications of media parallel to a great extent the

development of taxonomy of the streptococci (72). Initially, most media were

designed to isolate streptococci associated with human and animal infections.

Many of these early media may have been selective for most species of

enterococci, but the nomenclature of the group was not defined well enough

31

to recognize the importance of the discovery. Improvements on these media

were made to adapt them to the isolation of the enterococci.

Krumwiede and Pratt (88), in 1914, found that some streptococci were

more resistant than were other bacteria to gentian violet. This led to the

addition of small amounts of crystal violet to glucose broth to produce a

selective medium. Edwards devised an agar medium containing crystal violet

and esculin. This was to become the predecessor of other esculin-containing

media. It is important to note that the concentrations of these inhibitors must

be carefully chosen. The concentration must be high enough to inhibit other

bacteria, but not so high as to inhibit the enterococci.

Media containing dyes alone were not selective enough, so other

inhibitors were studied, Hartmann (cited in (72)) examined the relative

inhibition of E. coli and streptococci by a variety of compounds, and found that

azide was the only compound that inhibited £ coli more than the streptococci

at a relatively wide range of concentration. Azide exerts its primary function by

inhibiting metalloporphyrin enzyme systems, such as catalases and

cytochrome c oxidases. Electron transport is interrupted. Azide penetrates

some cells only as the disassociated acid, so the pH of the medium can have

a great effect on the selective properties of the medium. The addition of

0.02% sodium azide allowed Mallmann (101) to estimate selectively the

numbers of streptococci in samples of sewage. McKenzie (103) preferred

thallous-crystal violet medium. The use of a single selective ingredient, azide,

leaves much to be desired when grossly contaminated samples are tested.

Thus, the use of azide alone in media is restricted to preenrichment prior to

confirmation in a more selective medium. One exception to this is M-

32

enterococcus agar devised by Slanetz and Bartley (141). This medium is

used for recovery of enterococci from water samples. Lachica and Hartman

(90) modified this medium by adding Tween 80, KH2PO4 and NaHCOa. They

called this modified medium Tween-carbonate medium and used it to recover

enterococci from frozen foods. Higher counts were obtained by using the new

Tween-carbonate medium than the original M-enterococcus medium of

Slanetz and Bartley, According to Hartman et al. (72) the use of Tween and

carbonate may reverse in part the inhibitory power of the azide, and the

carbonate itself may be contributing to the selectivity of this medium.

In 1961, Kenner et al. (86) described new solid and liquid media for

enumerating enterococci. These media contained, among other ingredients,

sodium azide, bromcresol purple, and 2,3,5-triphenyltetrazolium chloride

(TTC). These media were called KF streptococcal broth and agar. Kenner's

data (86) indicate that typical strains of S. bovis, S. equinus, S. mitis, S.

salivarius, and the enterococcal group and its biotypes produce growth in or

on KF media. Growth on KF agar was counted as all colonies on the plate

with a red or pink color visible with 15X magnification after 48 hours of

incubation. Negative growth was observed with S. cremoris, S. lactis, S.

pyogenes, S. thermophilus, and S. uteris as well as some non-streptococcal

species.

Raibaud et al. (125), also in 1961, devised yet another medium to

enumerate and identify dominant streptococci, this time in pigs. This medium

(AGAT) contained sodium azide and a mixture of sodium glutamate and

acridine orange as inhibitors, plus triphenyl tetrazolium chloride as a redox

indicator. This medium was supposed to allow selective enumeration of

33

Streptococci in the presence of large numbers of lactobacilli. Azide-containing

media possess disadvantages for certain applications because of the

instability of azide (72) and and the failure of some streptococci, such as S.

bovis to initiate growth on azide-containing media (45).

Besides azide, other inhibitory substances have been studied. One of

these was thallium salts. After reports that certain gram-positive cocci were

more resistant than other bacteria to thallium salts, McKenzie examined the

selectivity of thallium salts in detail (103). He developed a thallium acetate

(TA)-crystal violet broth. The selectivity of TA is not affected by minor changes

in pH, although the selectivity of sodium azide is. Barnes (5) incorporated

thallous acetate into a tetrazolium agar medium. The medium was used to

determine numbers and types of fecal streptococci in bacon factories (4). The

thallous acetate suppressed most other organisms and the tetrazolium salt

differentiated Streptococcus faecalis and its variants from other Lancefield

group 0 organisms. S. faecalis reduced the tetrazolium to the insoluble red

formazan so colonies had dark red centers; S. faecium, S. durans, and S.

bovis did not reduce it and formed white to very pale pink colonies.

Thallium salts also were used in conjunction with other ingredients to

increase the selectivity for certain organisms. Thallium acetate was

incorporated into media with 0.5% NaCl, in spite of the fact that McKenzie

(103) mentioned that TA, in concentrations of greater than 0.1%, reacted with

NaCl to yield insoluble and nonselective thallium chloride. Barnes (4),

however, could not find evidence that concentrations of NaCl up to 0.5%

reduced the inhibitory properties of TA. The use of esculin in media

containing thallium salts was also investigated. However, the variability in the

34

ability of an organism to ferment esculin was noted years ago (72); some

organisms were weakened and give a negative result, yet their activity could

be regained on subculture. Lachica and Hartman (90) described the use of

citrate in combination with thallous acetate. Citrate functioned as the primary

energy source, as well as a selective agent. It was observed that citrate

(1.0%) was inhibitory to some lactobacilli but stimulatory to enterococci (18).

Gentamicin had been used by Black and Van Buskirk (14) for isolating

P-hemolytic streptococci; growth of staphylococci and nearly all gram-negative

bacilli was inhibited. Based on these favorable results, Donnelly and Hartman

(45) developed a gentamicin-based medium containing thallous acetate for

the selective isolation of all group D streptococci. Gentamicin and TA were

the major selective agents. NaHCOa, Tween 80, and KH2PO4 were added

as specified by Lachica and Hartman (90) to stimulate the growth of group D

streptococci. Esculin was included because group D streptococci hydrolyzed

it (50), in the presence of ferric citrate, forming dark halos of ferric salts. This

medium was superior to TA, KF, and PSE agars for the enumeration of fecal

streptococci in fecal and surface-water samples (45). This medium also was

tested for its ability to enumerate fecal streptococci in frozen foods (145). In

overall efficiency, the GTC agar recovered significantly greater numbers of

presumptive fecal streptococci. Both Donnelly and Hartman (45) and Thian

and Hartman (145) believed that it was important to incorporate more

differential abilities to this medium, to eliminate false positives and distinguish

between species. This was accomplished by Littel and Hartman (97) in 1983.

GTC medium was modified by incorporating a colorimetric starch substrate

and a fluorogenic substrate, allowing differentiation of colonies on the agar

35

surface. The incorporation of amylose azure and 4-methylumbelliferyl-a-D-

gaiactoside (a-D-MUGAL) allowed differentiation of the fecal streptococci into

three phenotypic groups: starch hydrolysis and fluorescence; no starch

hydrolysis but fluorescence; and no starch hydrolysis or fluorescence (97).

Cooper and Ramadan (30) discovered that a broth containing 0.2%

potassium tellurite was suitable for isolating streptococci from the feces of

various animals. The use of tellurite agar also was proposed. In 1940, the

complimentary action of penicillin and tellurite was devised. In the same year,

it was shown that enterococci were resistant to penicillin and tellurite. All

enterococci tested were resistant to 0.1% tellurite, while resistance of other

streptococci varied (72). The mechanism of growth inhibition by tellurite is not

fully understood. Of the enterococci, E. faecalis is resistant to 0.5% tellurite,

which it reduces to pure tellurium metal (72). This resistance to higher

concentrations of tellurite was used to differentiate the species S. faecalis from

S. faeciunr, S. faecalis could grow in the presence of 0.4% tellurite while S,

faecium could not (35). This information was critical in supporting the belief

that these two were distinct species.

Other inhibiting agents have been incorporated into media to select for

enterococci. Sodium taurocholate, bile salts, phenylethyl alcohol, selenite,

and tetrathionate have all been studied for their usefulness as inhibiting

agents. More information on these agents is available in a review by Hartman

et al. (72).

Rapid methods for identification

In 1933, Rebecca Lancefield detected proteins on the cell walls of

streptococci that were group specific (92). The soluble substances upon

36

which these groups were based were first noted by Hitchcock in 1924 (76).

He called them 'residue antigens' and believed that they were common to

almost all hemolytic streptococci. This view held until the specific nature of

these antigens was discovered by Lancefield. Based on the presence of

these antigens, the streptococci were divided into groups A through V. The

group D antigen, produced by most enterococci, contains glycerol teichoic

acids. Most of these teichoic acids of group D antigen are buried in the cell

wall. The intracellular location of these teichoic acids have made serologic

identification of organisms bearing this type of group-specific antigen difficult.

Nevertheless, the Lancefield grouping scheme became the primary method of

grouping streptococcal isolates, including enterococci. Today, the Lancefield

groups are still widely used to identify groups of streptococci. However, the

original Lancefield grouping procedures have been replaced by more rapid

methods, in which latex beads coated with antibodies to each specific group

antigen are used. Numerous commercial kits are available. Their

applications have been reviewed (127, 149).

Despite the heavy reliance placed by some investigators on serological

grouping, streptococci had been investigated by other techniques, and much

information has been accumulated from a variety of different kinds of study.

Once it was discovered that organisms could be grouped by using simple

biochemical tests, many workers devised classification schemes using

physiological tests. These include the use of improved or additional

biochemical tests, including detection of enzymes (111), resistance to

chemical compounds and antibiotics (36), studies of the peptidoglycan cell

37

wall (133), and studies of biochemical pathways and electron transport

systems (80).

Numerical taxonomy, performed by Bridge and Sneath (16), involved

the use of many biochemical tests, colony morphology, tolerance to

temperature and inhibitory compounds, and antibiotic resistance. Data were

collected on different streptococcal strains and a complicated mathematical

approach was used to determine the degree of relatedness among strains of

streptococci.

Facklam and co-workers (49, 50) presented the results of extensive

studies on identifying the enterococci by using biochemical tests. They used

the bile-esculin test, growth at 45°C and at pH 9.6, reduction of tetrazolium

and resistance to tellurite as well as acid production from a number of

carbohydrates to separate the enterococci and fecal streptococci into 3

divisions. An abbreviated battery of five tests was recommended to

presumptively identify pathogenic streptococci on a routine basis (54). The

bile-esculin reaction and salt tolerance were major determinants to

differentiate enterococci from other pathogenic streptococci. Using these

criteria, 97% of the enterococci were correctly identified. Gross et al. (67)

used Facklam's battery of tests, plus pyruvate and arginine tests, for

presumptive speciation of the group D streptococci. Confirmatory

carbohydrate tests were also recommended. Gross et al. (67) found that

certain differences in fermentation abilities within the species may be

dependent on the source of isolation. In 1985, Facklam et al. (56) described a

test based on the hydrolysis of pyrrolidonly-b-naphthylamide (the PYR test)

that could replace the 6.5% NaCI tolerance test to presumptively identify

38

group D enterococci. They found that the failure of some enterococci to grow

on salt agar led to misidentifications. The PYR test was rapid, convenient and

specific for the group D streptococci. The PYR test will be described in more

detail later.

As the classification of the enterococci changed, schemes had to be

updated to reflect these changes. In 1989, Facklam et al. (53, 57) published a

scheme to identify all of the accepted new species of the genus Enterococcus.

A battery of conventional tube tests was used to distinguish between species.

The type strain of each species was tested and then unknown strains of

previously isolated enterococci were identified by comparing them to the

expected results obtained by using the type strain. Conventional tests and

commercially available systems were used by Ruoff et al. (130) to determine

the species identities of clinical isolates of enterococci. Strict adherence to

the scheme developed by Facklam et al. (53, 57) resulted in misidentification

of lactose-negative E. faecalis as E. solitarius. This problem was overcome by

performing additional tests.

As the number of tests increased, the difficulty of handling conventional

tube tests became obvious. New commercial rapid test systems, utilizing

panels of specific tests, emerged. The commercial systems used to identify

enterococci have been reviewed by several researchers. Colman and Ball

(29) reviewed the APl-20 Strep system (Analytab Products, Plainview, New

York). In this system, dehydrated substrates are incorporated into

microcupules attached to stiff cardboard strips. The inoculum rehydrates the

substrates in the cupules. Colman and Ball (29) examined 965 streptococci

by using the APl-20 Strep (API-20S) and established methods, and found that

39

with supplemental tests the API-20 Strep could identify the streptococci,

Facklam et al. (51) compared the API-20S system with the Automicrobic

Gram-Positive Identification system (GPI; Vitek Systems, Hazelwood, Miss.).

Very few differences in identification were detected between the two systems.

The 20S system was more convenient, but it required more supplemental

testing. The Vitek system required less supplemental testing, but it required

the use of expensive equipment. Another evaluation of the GPI system (17)

revealed two minor difficulties when compared with the conventional scheme

of Facklam and Collins (53). First, three Enterococcus casseliflavus isolates

that were arginine negative, sorbitol negative, sorbose negative and mannitol

positive could not be identified using Facklam's scheme. Secondly, some

strains of E. faecalis were misidentified as E. solitarius. Bryce et al. (17)

recommended that tests for tellurite reduction and ribose fermentation should

be added to Facklam's scheme to avoid these misidentifications. The GPI was

also compared with the API Rapid Strep system (79). The API Rapid Strep

system is very similar to the API-20 Strep system, except that a few tests differ,

and the Rapid Strep system has a 4 hour (like the 20S) or 24 hour incubation.

A comparison of identification of isolates from bovine mammary glands

showed that both systems were accurate in the identification of Streptococcus

species of bovine origin. Facklam et al. (55) also evaluated the API Rapid

Strep System to identify the streptococci. They concluded that the Rapid

Strep system was more rapid and efficient than conventional identification

systems, but improvement in the data base was needed (55). Tritz et al. (147)

evaluated a different panel, the MicroScan panel (Baxter Healthcare, West

Sacramento, Calif.), to identify Enterococcus species. They compared results

40

obtained by using MicroScan panel with those obtained by using

conventional media. Incubation times for conventional media were as long as

96 hours, whereas the MicroScan panel yielded results within 18-24 hours.

The results of tests recommended by Facklam et al. (53) agreed with the

MicroScan results, except for six isolates. Tritz et al. (147) concluded that

MicroScan panels were reliable for the identification of E. faecalis and E.

faeciunr, however, modification of the data base would be necessary to

identify other Enterococcus species.

The traditional technique of identifying an isolate as an enterococcus

was by using bile-esculin and 6.5 % NaCI tolerance tests. This combination of

tests was accurate, but required an incubation period of up to 48 hours (49).

Most enterococci also reduce litmus milk with a species-specific enzyme, but

litmus milk is costly and difficult to prepare. New rapid techniques to identify

group D streptococci have emerged. One such method utilizes the hydrolysis

of L-pyrolidonyl-p-naphthylamide (PYR) by a specific aminopeptidase that is

produced by these organisms when isolated on selective media (32).

Facklam et al. (56) incorporated the substrate into agar. Daly et al. (32)

evaluated a technique in which a synthetic substrate is incorporated onto a

filter paper strip. Hydrolysis of the substrate can usually be detected visually

within 1 minute by adding a coupling dye. A major drawback of this test is that

Streptococcus pyogenes is also PYR-positive. To differentiate them from the

enterococci, the PYR test was combined with a rapid chromogenic test for

beta-glucosidase. A test strip was devised in which a novel beta-glucosidase

test using indoxyl glucoside as one substrate was combined with the PYR

substrate. Only enterococci were positive for both enzymes (33)(85).

41

Study of the murein (peptidoglycan) type was also used to differentiate

enterococcal species. The amino acid sequence of the murein, in particular

the nature of the diamine acid and the sequence of the interpeptide bridge

connecting the stem peptides, define the murein type (133). Most enterococci

possess the murein type Lys-D-Asp, containing D-isoasparagine as a cross-

bridge. The presence of the murein type Lys-Alag-s in E faecalis facilitates a

separation of this species from all other enterococci (83). This test is not

generally used to identify enterococci on a routine basis.

A novel approach to enterococcal speciation is analysis of bacteriolytic

activity patterns (123). By varying media, substrate, pH, and additives, seven

major groups (lyogroups) of bacteriolytic activity against Micrococcus

lysodeikticus and Micrococcus luteus were defined. One to four species fit

into each lyogroup. This method is new and has not yet become readily

available. Cellular fatty-acid analysis has also been used as an aid in the

classification of streptococci and enterococci (1,150). This method requires

cumbersome technology such as gas chromatography and possibly mass

spectrometry. However, it does have potential in laboratories that process

large numbers of samples because it yields results within hours once pure

cultures are available for testing An immunological method was proposed

(68) in which a monoclonal antibody against E. faecalis allowed differentiation

within minutes of £ faecalis from the non-enterococcal fecal streptococci, S.

bovis and S. equinus. This method has not yet been commercialized.

Tests employing fluorogenic substrates are being introduced for

identification of the streptococci. Beighton et al. (10) utilized 4-

methylumbelliferyl-linked fluorogenic substrates to test for the production of a

42

range of glycosidase activities. A combination of conventional fermentation

and liydrolytic tests was used to devise a sclieme for differentiation of oral and

vividans streptococci. The fluorescent tests required only three hours. Tests

employing three fluorogenic 4-methylumbelliferyl-conjugated substrates and

the lectin of Dolichos biflorus were developed for the identification of p-

hemolytic streptococcal colonies from throat cultures (142). It is based of the

generation of fluorescence when free 4-methylumbelliferone is released by

enzyme hydrolysis of the substrate. This identification system has not been

widely accepted. This non-serological method was unique in that it permitted

identification of groups C, F, and G as well as A and B. It is rapid, simple and

specific.

Molecular approaches to identification

Several new methods have been employed within the last decade to

study the relationships between the streptococci and the enterococci. The

application of nucleic acid hybridization and sequencing techniques provided

significant insights into the natural relationships among the streptococci.

Along with DNA-DNA hybridization studies, oligonucleotide cataloguing of

16S rRNA of several species of streptococci showed, as stated previously, that

the streptococci should be divided into three genera (11, 99,133). The first

group, those bacteria which conformed to the enterococcus division of

Sherman (138), was renamed Enterococcus (132). DNA-DNA and DNA-RNA

hybridization studies were used to determine into which species new strains

fell. Collins et al. (28) used reverse transcriptase sequencing of 16S rRNA to

determine the interrelationships of Pediococcus and Aerococcus species

which resemble the enterococci physiologically. The small-subunit rRNA is

43

highly conserved and is recognized as a powerful molecular chronometer

(154). Different degrees of sequence conservation allows comparison of

closely related species as well as those of great geneological distances.

Later the same techniques were used (153) to clarify the intragenic

relationships between species of enterococcl. Several species groups were

revealed. Enterococcus avium, E. malodoratus, E. pseudoavium, and E.

raffinosus formed a distinct group as did E. durans, E. faecium, E. hirae, E.

mundtii, E. casseliflavus, and E. gallinarum. E. cecorum, E. columbae, E.

faecalis, and E. saccharolyticus formed another group. Enterococcus

solitarius displayed a closer affinity with Tetragenococcus fialophiius than with

other enterococci. Therefore, the precise phylogenetic placement of E.

solitarius remains unclear, although its affinity with Tetragenococcus

halophilus merits further investigation.

In 1990, Murray (115) compared genomic DNAfrom different

enterococcal isolates by using restriction endonucleases with infrequent

recognition sites in attempts to aid in species identification and to possibly

identify strains within a species. This "genomic fingerprinting" was also used

on strains of Streptococcus suis (106). The method provides a means of

studying the epidemiology of both swine and human infections.

DNA probes are receiving much attention for their use in identifying

bacteria. Construction of DNA probes for the specific identification of species

of streptococci allowed rapid and confident identification of S. oralis (134),

Acridium ester-labeled, chemiluminescent DNA probe tests also have been

developed for Enterococcus species (31). The probe is a DNA oligomer

complimentary to a rRNA target, and the DNA-RNA hybrids are measured by

44

using a luminometer. Lactococci and enterococci were also identified by

colony hybridization with 23S rRNA-targeted oligonucleotide probes (13).

Specific sequences of the 23S rRNA of one species of Lactococcus and three

species of Enterococcus were identified, and complimentary oligonucleotide

probes were synthesized. The probes were specific when used in a dot blot

assay. The Lactococcus probe was also successfully used for the specific

enumeration of lactococci as CFU in a mixed population in fermented milk.

However, only one strain of each species was used.

45

PAPER 1 ; ROUTINE PROCEDURES FOR ISOLATION AND IDENTIFICATION OF ENTEROCOCCI AND FECAL STREPTOCOCCI

46

Routine Procedures for the Isolation and Identification of

Enterococci and Fecal Streptococci

LINDA M. KNUDTSON AND PAUL A. HARTMAN*

Department of Microbiology, Immunology and Preventive Medicine, Iowa

State University, Ames, Iowa 50011

Telephone: (515)294-8824

FAX: (515)294-6019

^Corresponding author

47

ABSTRACT

Over the past six years, a revised classification of the streptococci and

enterococci, based primarily on molecular techniques such as 16S rRNA

sequencing and DNA-DNA hybridization, emerged. However, little attention

was placed on routine physiological tests that could be used in food and

clinical laboratories to differentiate between species of a new genus

Enterococcus, and fecal Streptococcus spp. The purpose of this study was to

devise a convenient and reliable system to identify the enterococci and fecal

streptococci by using conventional procedures. Fifty-nine strains of 13

Enterococcus spp., including the type strains and many strains used by

previous investigators, were characterized by using conventional tube tests,

the API Rapid Strep system, and MicroScan Pos ID panels. Results were

compared with each other and with previously published results. A

comparison of conventional tube tests versus published tube test results

yielded 17 discrepancies. Although not all tests were done with each of the

three systems, 28 discrepancies between results obtained with the API system

and those obtained with conventional tube tests were found. There were 24

discrepancies between results obtained with the MicroScan Pos ID panel and

those obtained with conventional tube tests. There were 12 discrepancies

between the results with the API Rapid Strep system and those with the

MicroScan Pos ID panels. We devised flow charts of key tests that might be

used to identify cultures without resorting to nucleic acid analysis and other

labor- and equipment-intensive analyses.

48

INTRODUCTION

Enterococcus spp. inhabit the intestinal tracts of warm-blooded animals

and of Insects (16). Therefore, these bacteria have been useful as indicators

of fecal contamination in water and in foods (5). Because of the prevalence of

certain species of enterococci in the intestinal tracts of swine, enterococci are

implicated in the spoilage of pork products (20). Enterococci and fecal

streptococci are also receiving increased attention because of their role in

serious human infections, such as endocarditis and bacteremia (17) and

diarrheal diseases in neonates (7).

In 1984, the genus Streptococcus was divided into three genera:

Enterococcus, Lactococcus, and Streptococcus. (19). The genus

Enterococcus now contains 18 species, differentiated primarily by the results

of 16S rRNA and DNA-DNA hybridization studies. In addition to the 13

species described in Table 1, five new species have been proposed: E.

sulfureus (15), which includes yellow-pigmented strains isolated from plants;

£ columbae (6), a species that dominates the intestinal flora of domestic

pigeons: E. dispar{A), composed of two strains of human origin; E. seriolicida

(14), a fish pathogen; and E. saccharolyticus (18), previously named

Streptor vous saccharolyticus.

Initially, these species were phenotypically characterized by using API

50CH and 20S systems in conjunction with conventional tube tests (1-4, 6,12,

14,15,18). However, the API data base includes only 6 of the 13 tested

enterococcal species, and the data base for another system, the MicroScan

system, contains only 4 of the species. An identification system with

conventional tube tests exclusively was introduced in 1989 by Facklam and

49

Collins (9). However, conventional tube tests are cumbersome, costly, and

labor-intensive, and the results are often difficult to accurately reproduce in

different laboratories.

In this study, we tested 59 strains of 13 species of enterococci and fecal

streptococci. These included selected strains of each enterococcal species

used in the original 16S rRNA studies as well as each type strain. Two

different rapid systems, as well as conventional tube tests were compared. A

scheme was developed to identify 13 species of enterococci, and

Streptococcus bovis and S, equinus. The test scheme uses either the API

Rapid Strep or MicroScan system, plus a minimum of supplemental tests.

50

MATERIALS AND METHODS

Strains. A total of 59 strains of 13 enterococci and S. bovis and S.

equinus were collected (Table 1). Type strains used were obtained from the

American Type Culture Collection (Rockville, Md.) or Dr. Richard Facklam

(Centers for Disease Control, Atlanta, Ga.). Cultures were also obtained from

the National Collection of Food Bacteria (Shinfield, Reading, England),

MicroScan (Baxter Diagnostics, Deerfield, III.), Marcia Etheridge (Baltimore,

Md.) (7) and the culture collection of Paul A. Hartman (Iowa State University,

Ames).

Stock cultures were maintained on Brain Heart Infusion (BHI) slants at

5 to 10°C and transferred monthly. Stock cultures were also frozen in 10%

glycerol and stored at -70 to -lOO'C.

Tests, All cultures were gram stained as they were obtained to verify

that they were gram-positive cocci. Each culture was also tested for the

presence of catalase before being inoculated to the test panels.

The orf/70-nitrophenyl-p-D-galactopyranoside (ONPG) test (21) was

used to determine the presence of p-galactosidase. All other conventional

tube tests were performed as described by Facklam and coworkers (8, 9,10)

and Gross et al (13). Motility was determined by using modified Difco motility

medium (Difco Laboratories, Detroit, Mich.) or wet mounts of cultures grown in

BHI broth at SO'C and 37°C (21). Cultures were monitored for yellow

pigmentation on a cotton swab used to pick up growth from BHI agar plates

incubated overnight.

51

Serogrouping for groups A, B, C, D, F, and G was accomplished by

using the Streptex grouping kit (Wellcome Diagnostics, Research Triangle

Park, N.C.). ^-Hemolysis was determined by observation of 24-h growth on a

plate of tryptone soy agar-5% sheep blood (BBL Microbiology Systems,

Cockeyville, Md.).

Inoculum preparation for the API Rapid Strep system (Analytab

Products, Inc., Plainview, N.Y.) was carried out on blood agar plates as

indicated in the manufacturer's directions but without anaerobic incubation.

The test strips were inoculated, overlayed with mineral oil where specified,

incubated and read as indicated in the manufacturer's directions. After 4 h

incubation, Zyme A and B reagents (Analytab) were added to the enzyme

tests, ninhydrin solution was added to the hippurate test, and reagents A and

B were added to the Vogues-Proskauer (VP) test; the results of these tests

were recorded. The results of the remaining tests were determined after

incubation of the panel for 18-24 h.

Inoculum preparation for the MicroScan Pos ID panels (Baxter

Diagnostics, Deerfield, III.) was carried out according to the log phase

technique specified by the manufacturer. The panels were inoculated,

covered, and incubated at 37°C. After 18-24 h of incubation, appropriate

reagents were added and the tests were interpreted as indicated in the

manufacturer's instructions.

52

RESULTS AND DISCUSSION

Table 2 shows the results of conventional tube tests. A comparison of

our results with published results (9) revealed 17 discrepancies among 87

instances in which comparisons could be made. Every attempt was made to

perform the tests as specified in the literature, but the discrepancies appeared

repeatedly. These discrepancies are probably due to the difficulty in

accurately reproducing tube test results in different laboratories.

There was also a poor correlation between results obtained with the

API and MicroScan systems and conventional tube tests (Table 2). There

were 28 discrepancies between API (see Table 4) and conventional tube test

results (Table 2). There were 24 discrepancies between MicroScan results

(Table 3) and conventional tube test results (Table 2).

Twelve discrepancies between the API Rapid Strep results and

MicroScan Pos ID system results were observed (Tables 2 and 3). VP test

results were more variable when the API Rapid Strep strips were used than

when MicroScan panels were used. Enterococcus cecorum and S. bovis test

results varied unpredictably on the API panels, but they were always positive

on the MicroScan Pos ID panels. On the other hand, S. equinusvias VP

positive when tested with API strips, but VP negative on MicroScan panels.

Because the same a-naphthol and KOH solutions were used for the VP tests

on both panels, these discrepancies are caused by something other than the

detection reagents.

p-Galactosidase results for Enterococcus faecalis and E. pseudoavium

were negative on API Rapid Strep panels, whereas positive results were

53

obtained for botti species with MicroScan Pos ID panels. Alkaline

phosphatase test results also differed. E. faecalis was alkaline phosphatase

negative when run on the API panel but positive when tested with the

MicroScan Pos ID panel. These discrepancies probably are the results of

differences between the methodologies of the API Rapid Strep and MicroScan

system p-galactosidase and alkaline phosphatase tests. The API Rapid Strep

panel utilizes a naphthol-linked substrate, and color development is detected

after the addition of Zyme A and B reagents furnished by API. The MicroScan

Pos ID p-galactosidase and alkaline phosphatase tests use para-nitrophenyl-

p-D-galactopyranoside (PNPG) and para-nitrophenyl phosphate, respectively,

as substrates. Both enzyme reactions release para-nitrophenol which

generates a yellow color. p-Galactosidase tests were also conducted in test

tubes (21) with ONPG. ONPG is very susceptible to cleavage by p-

galactosidase and, as in the MicroScan test, releases a yellow product. The

product in this case, however, is ort/70-nitrophenol. The only difference

between the two tests is the orientation of the nitrogen on the phenyl group,

para or ortho. The ONPG tube test results, except for those with S. bovis and

S. equinus, correlated well with the MicroScan results(Table 2). It seems that

the API Rapid Strep methodology, at least for the p-galactosidase test, is not

as sensitive as either the PNPG methodology of the MicroScan system or the

ONPG tube methodology.

Discrepancies in carbohydrate fermentation tests of the API and

MicroScan systems were also observed. Three discrepancies in the sorbitol

test were observed: Enterococcus malodoratus showed a positive API result

but was variable on MicroScan panels; £ mundtii was also positive for

54

sorbitol wlien run on the API panel, but all four strains were negative on the

MicroScan panel; and £ solitarius was sorbitol negative in the API tests and

sorbitol positive in the MicroScan tests. When a conventional tube method for

sorbitol fermentation was used (9), E. malodoratus, E. mundtii, and E.

solitarius produced variable, positive, and variable results, respectively (Table

2). Thus, manual test results failed to support the validity of the results of

either panel as superior to those of the other. Two discrepancies were

observed in the inulin test (Tables 3 and 4). E. malodoratus was uniformly

inulin negative on API panels; variable results were obtained when

MicroScan panels were used. Only one discrepancy existed in the raffinose

test: variable results were obtained with Enterococcus hirae on API Rapid

Strep test strips, whereas negative results were obtained on MicroScan Pos

ID panels.

The six discrepancies seen in the carbohydrate fermentation tests for

sorbitol, inulin, and raffinose are highly variable. There was no consistent

pattern wherein one panel was positive while the other was negative. Both

systems use phenol red as the pH indicator, but there is a slight difference in

methodology. The API Rapid Strep system requires a sterile mineral oil

overlay on all the carbohydrate tests to reduce oxidative metabolism of the

carbohydrates. The MicroScan system does not utilize a mineral oil overlay.

Therefore, the discrepancies could be caused by the ability of some bacteria

to oxidatively metabolize the carbohydrates in the MicroScan system but not

in the API system. If this was the only cause of the discrepancies, one would

expect more negative results from the API system and more positive results

55

from the MicroScan system. As was pointed out above, however, this did not

occur.

Also Included In Tables 3 and 4 are two tests that were not performed

on either panel. Pigmentation is important for differentiating pigmented

Enterococcus casseliflavus and E. mundtii from nonpigmented enterococci

and fecal streptococci. Motility tests are used to distinguish motile

Enterococcus gallinarum and E. casseliflavus from nonmotile species.

Figures 1 through 3 depict selected tests that can be used to identify all

13 species of enterococci as well as S. bovis and S. equinus. The tests

shown in Tables 3 and 4 were used to construct the identification schémas,

with the exception of the sucrose test (Table 2), which is needed to distinguish

E. hirae (positive) from E. durans (negative). Also included in Figures 1

through 3 are the Lancefield group D reactions. They were included to aid in

the differentiation of E. cecorum and E. pseudoavium (group D negative) from

enterococci and fecal streptococci that produce the group D antigen.

Not all of the tests that could be used to differentiate between species

were included in these schémas. Each strain was tested several times, and

some tests yielded inconsistent results (i.e., a positive result the one time and

a negative result the next). To obtain the most consistent and reliable

Identification, only tests that produced the most consistent and reproducible

results were Included in the identification schémas. These flow charts can be

used with either the API Rapid Strep system or the MicroScan system. Some

tests depicted are only available on the API Rapid Strep strips and are noted

with an asterisk (*). Figure 2 shows that E. mundtii and E. casseliflavus are

differentiated by the sorbitol test. As stated above, E. mundtii is only sorbitol

56

positive when tested with the API Rapid Strep kit; when E, mundtiils tested

with the MicroScan system, the result is negative. Thus, when the MicroScan

system is used, differentiation between these two species must be determined

by using the motility, inulin, and raffinose results only. Figure 2 also shows

that E faecalis is p-galactosidase negative. As discussed above, this is true

only when E faecalis is tested on the API Rapid Strep panel; when tested with

the MicroScan panel or ONPG tube test, the result is positive.

The enterococci can be distinguished from other gram-positive

catalase-negative cocci. The pyrrolidonylase (pyrrolidonyl arylamidase or

pyrrolidonyl peptidase) test differentiates Enterococcus spp. from

Leuconostoc, Lactococcus and Pediococcus spp. Another test, which is

included in the API Rapid Strep panels or can be conducted as a tube test, is

for leucine aminopeptidase activity. This test differentiates Enterococcus spp.

from all other non-streptococcal isolates (11).

In conclusion, we have described key tests that can be used to

differentiate between species of enterococci and fecal streptococci. These

tests can be performed with either the API Rapid Strep panel or MicroScan

system panel. Additional tests include motility, pigmentation, and sucrose

tests. The Lancefield group D determination is optional. This test scheme is a

rapid and reproducible way to differentiate the enterococci and fecal

streptococci.

57

ACKNOWLEDGEMENTS

This work was supported by a U. S. Department of Agriculture grant to

the Food Safety Consortium. Journal paper No. J-14918 of the Iowa

Agriculture and Home Economics Experiment Station, Ames, project 2991.

We thank R. R. Facklam and M. E. Etheridge for providing cultures.

58

REFERENCES CITED

1. Collins, M. D., R. R. Facklam, J. A. E. Farrow, and R.

Williamson. 1989. Enterococcus raffinosus sp. nov., Enterococcus

solitarius sp. nov. and Enterococcus pseudoavium sp. nov. FEMS

Microbiol. Lett. 57:283-288.

2. Collins, M. D., J. A. E. Farrow, and D. Jones. 1986. Enterococcus

mundtii sp. nov. Int. J. Syst. Microbiol. 36:8-12.

3. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Bâiz, and

K. H. Schlelfer. 1984, Enterococcus avium nom. rev., comb, nov.; E.

casseliflavus nom. rev., comb, nov.; £ durans nom. rev., comb, nov.; £

gallinarum comb, nov.; and £ malodoratus sp. nov. Int. J. Syst. Microbiol.

34:220-223.

4. Collins, M. D., U. M. Rodrigues, N. E. Pigott, and R. R.

Facklam. 1991. Enterococcus dispar sp. nov. a new Enterococcus

species from human sources. Lett. Appl. Microbiol. 12:95-98.

5. Deibel, R. H. 1964. The group D streptococci. Bacteriol. Rev. 28:330-

366.

59

6. Devriese, L. A., K. Ceyssens, U. M. Rodrigues, and M. D.

Collins. 1990. Enterococcus columbae, a species from pigeon intestines.

FEMS Microbiol. Lett. 71:247-251.

7. Etheridge, M. E., R. H. Yolken, and S. L. Vonderfecht. 1988.

Enterococcus hirae implicated as a cause of diarrhea in suckling rats. J.

Clin. Microbiol. 26:1741-1744.

6. Facklam, R. R. 1972. Recognition of group D streptococcal species of

human origin by biochemical and physiological tests. J. Clin. Microbiol.

23:1131-1139.

9. Facklam, R. R., and M. D. Collins. 1989. Identification of

enterococcus species isolated from human infections by a conventional

test scheme. J. Clin. Microbiol. 27:731-734.

10. Facklam, R. R., L. G. Thacker, B. Fox, and L. Eriquez. 1982.

Presumptive identification of streptococci with a new test system, J. Clin.

Microbiol. 15:987-990.

60

11. Facklam, R. R., and J. A. Washington II. 1991. Streptococcus and

related catalase-negative gram-positive cocci, p. 238-257. In A. Balows,

W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy

(ed.). Manual of clinical microbiology, 5th ed. American Society for

Microbiology, Washington, D. C.

12. Farrow, J. A. E., and Wl. D. Collins. 1985. Enterococcus hirae, a new

species that includes amino acid assay strain NCDO 1258 and strains

causing growth depression in young chickens. Int. J. Syst. Microbiol.

35:73-75.

13. Gross, K. C., M. P. Houghton, and L. B, Senterfit. 1975.

Presumptive speciation of Streptococcus bovis and other group D

streptococci from human sources by using arginine and pyruvate tests. J.

Clin. Microbiol. 1:54-60.

14. Kusuda, R., K. Kawai, F. Salati, 0. R. Banner, and J. L. Fryer.

1991. Enterococcus seriolicida sp. nov., a fish pathogen. Int. J. Syst.

Bacteriol. 41:406-409.

15. Martinez-Murcia, A. J., and M. D. Collins. 1991. Enterococcus

sulfureus, a new yellow-pigmented Enterococcus species. FEMS

Microbiol. Lett. 80:69-74.

61

16. Mundt, J. O. 1986. Enterococci and lactic acid streptococci, p. 1063-

1066. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.),

Sergey's manual of systematic bacteriology. The Williams and Wilkins

Co., Baltimore.

17. Murray, B. E. 1990. The life and times of the Enterococcus. Clin.

Microbiol. Rev. 3:46-65.

18. Rodrigues, U., and M. D. Collins. 1990. Phylogenetic analysis of

Streptococcus saccharolyticus based on 16S rRNA sequencing. FEMS

Microbiol. Lett. 71:231-234.

19. Schlelfer, K. H., and R. Kilpper-Bâiz. 1984. Transfer of

Streptococcus faecalis and Streptococcus faecium to the genus

Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and

Enterococcus faecium comb. nov. Int. J. Syst. Bacterid. 34:31-34.

20. Sharpe, M. E., and B. G. Fewins. 1960. Serological typing of strains

of Streptococcus faecium and unclassified group D streptococci isolated

from canned hams and pig intestines. J. Gen. Microbiol. 23:621-630.

62

21. Smibert, R. M., and N. R. Krieg. 1981. General characterization, p.

409-443. In P. Gerliardt, R. G. E. Murray, R. S. Costilow, E. W. Nester, W.

A. Wood, N. R. Krieg, and G. B. Phillips (éd.). Manual of methods for

general bacteriology. American Society for Microbiology, Washington,

D.C.

63

Table 1 ; List of strains used ^

Species Strains (s)

E. avium

E. casseliflavus

E. cecorum

E. durans

E. faecalis

E. faecium

E. gallinarum

E. hirae

E. malodoratus

E. mundtii

E. pseudoavium

E. raffinosus

E. solitarius

S. bovis

S. equinus

ATCC 14025, 49462, 49463, 49465; NCDO 2366; SS -1^ ; A" ; B"

ATCC 25788; NCDO 2376; 7765F''; 443»; A8''

ATCC 43198; NCDO 2674

ATCC 19432; NCDO 498; PAH 940»; 15-20"

ATCC 4082,19433, 35038, 49477, 49478; NCDO 581; R1873"; 334-2"

ATCC 349,19434; NCDO 502; R169a»; R281a''; 2100"; 2124"

ATCC 49573; NCDO 2311, 2315, 2704

ATCC 9790; NCDO 1631,1648, 2683; ME''

ATCC 43197; NCDO 847

ATCC 43186; NCDO 582, 2374, 2377

SS-1277''; NCDO 2138

SS-1278''

SS-1279''

ATCC 9809, 33317; H-12" ; H-24"

ATCC 9812

= See tlie text for sources not listed in footnotes b through d. " From culture collection of Paul A. Hartman. ® From M. E. Etheridge. '' From R. R. Facklam.

!

Table 2: Conventional tube test results Test result®

species l l l l i l l i l l l (no. Of strains) œ œ Q. CO s X _l CO

E. avium (8) M " + (+)* + +

E. casseliflavus (5) + (+) - - + +

d E. ceœrum (2) + +

E. durans (4) +(-)--- - v

E. faecalis (8) + v'' +" +

d d

+ - • + + +

+ W + cM

+ V - + +

+ v''

(+)'' - +

b,c4

b,cjd

+

b,c4

E. faecium (7) +(-)-- v + (-)+- + +

£. gallinarum (4)

E. hirae (6)

E. malodoratus (2)

£. mundtii (4)

E. pseudoavium (2)

£. raffinosus (1)

E. solitarius (1)

+ + - + + + + + +

+

+ W + . - + V v ' ' + - + W S. bovis (4)

S.equinus (1) - - - + + + '̂'̂ - +

9+, Positive reaction (100%); -, negative reaction (100%); (+), 75% or greater siiow positive reaction; (-), 75%

or greater show negative reaction; v, variable (some strains positive, some strains negative).

b Discrepancy between Table 3 MicroScan results and Table 2 results.

c Discrepancy between published tube test results and Table 2 results.

d Discrepancy between Table 4 API results and Table 2 results.

(/) ! §•

§•

rn !Ti m m rn S 01

S I 1 I

1 i ! 13

M

I o5

m m m fn

1 I ! I m

I 13

m

I oi

m m

t 00

+ + c < + c < < < < + < < c c Crystal Violet + + + + + + + + + + + + + + Micrococcus Screen

Nitrate ' + + + + + + + + + + + + + + Voges-Proskauer

+ + + + + + + + + + + + + + + Optochin

+ • + • • Phosphatase + + + + + + + + + + + + + + + 40% Bile Escuiin

• • + + + + + + + + + + • + + L-Pyndidcnylase

• • + • • + + + + + + • + • Arginine

• • • • + + + + + + + + + + < B-Galactoacbse

• • < • • • • ' • ' • • • • • Urea

• + • + • • + • + • • • + + ' Raffinose • + • + + + + + + + 2: + + + + Lactose + < + + + + + + + + + + + + Trehatose + + + + + + + + + + + + + + + Mannose • • + + + + + + + + + + • + < 6.5% NaCi • ' + + + • < • • • • + Sorbitol • • • + • + • + + • + + Arabinose

• • • + + + + + + + + + + + + Ribose

+ < • • • • c + • • + + • Inulin

• < + + + + + + + + • + + Mannitol + + + + + + + + + + + + + + + Bacitracin

• • ' • • • • • • Pyruvate • ' • • • • • • Hemolysis

• • • ' • + + • Pigment

+ • • • + • Motility

c/>

1^' s

2 (D CO

S CO

S

I 0 % 1

8 (/)

a+, Positive reaction (100%); -, negative reaction (100%); (+), 75% or greater show positive reaction; (-), 75%

or greater show negative reaction; v, variable (some strains positive, some strains negative).

( o ç c r n m m r n r n r n r n M

i I I i 8

5 5 # I 8 i i

M I M

I O)

m m m m m m m

1 i 1

S"

S' 1 1 1 1 3 S 3

S 0) i

s

3 CO

s

+ < o-

+ + + + + + + + + < »

+ + Voges-PtDSkauer

' < -i • ' ' < + < < < < ' ' Hippurate Hydrolysis

+ + + + + + + + + + + + + + Esculin

• + + + + + + + + + + ' + + Pyirolidonylase

< + + < î + 3 + < • 3 + + • a-Galactosidase

' • ' ' • • • ' • • • + • • Q-Glucuronidase

' ' • » + + + + + o- + + + < 13-Galactosidase

' ' ' ' • • • • • o- • + • ' Alkaline Phosphatase

+ + + + + + + + + + + + + + Leucine Arylamidase

• + ' ' + • + + + + + ' + ' Arginine Dehydrolase

' ' + + + + + + + + + + + + Ribose

• ' + • + ' • + + • • + + L-Arabinose < + + + + + ' + + + ' ' + + Mannitol

' o- + + + »

' • • + • ' • + Sorbitol

+ ' + + + + + + + + + + + Lactose

< + + + + + + + + + 3 + + + Trehalose

3 • • • • a- • + X • • + + • Inulin

+ ' + ' • + •«!

» + ' ' + + ' Raffinose

+ ' < ' < < + + + + < + + < Starch

+ ' ' ' • • ' < • • • • • ' Glycogen

' ' ' ' ' • ' ' ' ' • ' • • Hemolysis

' ' ' ' + ' ' • ' ' • • + ' Pigment

• ' ' ' ' ' ' + ' ' ' ' + ' Motility

if |.w CO

2 (D

ï J3

CL.

I $ c «

i

$

a+, Positive reaction (100%); -, negative reaction (100%); (+), 75% or greater show positive reaction; (-), 75%

or greater show negative reaction; v, variable (some strains positive, some strains negative).

^ Indicates where discrepancies exist between the API Rapid Strep and the MicroScan system.

Fig. 1 : Flow chart for differentiating the pyrrolidonylase-negative enterococci and fecal streptococci. The data

shown are condensed from Tables 3 and 4 . The asterisk (*) indicates that a test is available with the API

Rapid Strep but not with the MicroScan system.

GRAM-POSITIVE COCCI CATALASE- ESCULIN+ PYRROLIDONYLASE-

GROUP D + P-GALACTOSIDASE -

RIBOSE -LACTOSE -

RAFFINOSE-STARCH -*

GLYCOGEN -*

GROUP D + P-GALACTOSIDASE -

RIBOSE -LACTOSE +

RAFFINOSE + STARCH +*

GLYCOGEN +*

GROUP D-P-GALACTOSIDASE +

RIBOSE + LACTOSE +

RAFFINOSE + STARCH +*

GLYCOGEN +*

S. equinus S. bovis E. cecorum

Fig. 2: Flow chart for differentiating the pyrrolidonylase- and arabinose-positive enterococci. The data shown

are condensed from Tables 3 and 4. The asterisk (*) indicates that a test is available with the API

Rapid Strep but not with the MicroScan system. A superscript a indicates a test that has a positive result

with the API Rapid Strep and a negative result with the MicroScan system. A superscript b indicates a

test that has a negative result with the API Rapid Strep and a positive result with the MicroScan system.

GRAM-POSITIVE COCCI CATALASE- ESCULIN+ PYRROLIPONYLASE+

ARABINOSE +

PIGMENT + PIGMENT I

MOTILITY -SORBITOL INULIN -

RAFFINOSE -

E mundtii

MOTILITY + SORBITOL -INULIN +

RAFFINOSE +

E casseliflavus ARG NINE- ARG

RAFFINOSE + a-GALACTOSIDASE +*

E raffinosus

RAFFINOSE -a-GALACTOSIDASE

E avium

NINE

INULIN -MOTILITY -SORBITOL -RAFFINOSE -

P-GALACTOSIDASE +

E faecium

INULIN + MOTILITY + SORBITOL -RAFFINOSE +

P-GALACTOSIDASE +

E gallinarum

INULIN -MOTILITY -SORBITOL + RAFFINOSE -

P-GALACTOSIDASE

E faecalis

Fig. 3: Flow chart for differentiating the pyrrolidonylase-positive and arabinose-negatlve enterococci. The data

shown are condensed from Tables 2, 3, and 4. The asterisk (*) indicates that a test is available with

the API Rapid Strep but not with the MicroScan system.

GRAM-POSITIVE COCCI CATALASE- ESCULIN+ PYRROLIDONYLASE+

ARABINOSE -

MANNITOL + MANNITOL -

SUCROSE + SUCROSE - ^ oi

E. hirae E. durans

LACTOSE -RAFFINOSE ARGININE + STARCH

LACTOSE+ LACTOSE + LACTOSE+ RAFFINOSE - RAFFINOSE + RAFFINOSE ARGININE - ARGININE +/- ARGININE + STARCH -* STARCH +/-* STARCH +*

E. solitarius E. pseudoavium E. malodoratus f^^oalis

76

PAPER 2: ENTEROCOCCI IN PORK PROCESSING

77

Enterococci in Pork Processing

LINDA M. KNUDTSON AND PAUL A. HARTMAN*

Department of Microbiology, Immunology and Preventive Medicine,

Iowa State University, Ames, Iowa 50011-3211

Telephone: (515) 294-8824

FAX: (515)294-6019

^Corresponding author

78

ABSTRACT

The purpose of this study was to determine the numbers and species of

enterococci encountered on pork carcasses during different stages in the

slaughter process as well as on pork products. Three hog slaughtering plants

were surveyed, each 3 times at four processing points. Each hog was

swabbed at two sites on the carcass. Specimens were plated on two different

enterococcal media, KF Streptococcal agar and fluorescent gentamicin-

thallous-carbonate agar. Retail and spoiled pork sausage products also were

examined. Isolates were speciated by using the API Rapid Strep and Baxter

MicroScan Pes ID panels. Contamination levels varied between plants as the

carcasses progressed down the processing line; the highest counts were

obtained directly before packaging in plants A and C. The highest count for

plant B occurred at the first stage of sampling. More Enterococcus faecalis

than Enterococcus faecium were isolated from the pork carcasses. Pork

sausage results also are presented. Enterococci are useful as an indicator of

pork sanitation and to detect critical control points during processing. In some

instances, high levels of enterococci are associated with spoilage of pork

sausage.

79

INTRODUCTION

Although enterococcl are widely distributed in nature (6), the natural

habitat of these organisms is the intestinal tract of man and animals (4).

Enterococcl also have been isolated from plants and insects (14), where they

are believed to establish an epiphytic relationship (3). The importance of

enterococci as indicators of fecal pollution has been documented by several

workers (1,13). These bacteria are sometimes used as fecal indicator

organisms because they are readily isolated in large numbers from human

and animal feces. Their relative resistance to adverse conditions, such as

tolerance to extremes in temperature (5), pH, and salinity, is advantageous

when determining the sanitary history of moderately heated, frozen, salted, or

other foods and drinks in which conforms might not have survived (19).

However, because of their ability to grow in environments far removed from

the original source of fecal contamination, caution and discretion must be

exercised in attributing significance to the numbers and types of enterococci

and fecal streptococci present in foods. Once introduced into a food-

processing plant, enterococci can become established, and the subsequent

contamination of a food product does not necessarily indicate fecal pollution.

Many researchers have suggested the possibility of using enterococci as

indicators of fecal pollution by using differences in species distribution in

different hosts as a means to pinpoint the source of contamination (1, 8,15,

16). None of the enterococci can be considered absolutely "host specific," but

some species show a degree of host specificity (6). Variation in the

enterococcal flora of animals has been associated with many factors; such as

80

geographical location, diet, age, species of the animal, and even seasonal

effects (3). Despite this potential for variation, significant differences in the

distribution of enterococci and fecal streptococci have been observed in host

animals, and predominating species have been identified in some common

hosts.

In this study, numbers and species of enterococci present on pork

carcasses during fabrication and subsequent processing were examined.

Levels of enterococcal contamination were determined, and possible sources

of contamination were considered. The importance of enterococci as spoilage

organisms was investigated, as well as their importance and usefulness as

fecal indicators to determine critical control points in meat processing.

81

MATERIALS AND METHODS

Sampling location

Three pork slaughtering plants located in the Midwest were chosen for

sampling. All three plants were typical modern pork slaughtering plants of

similar design and line speeds. Each plant was visited at random on three

different occasions. All testing was performed on Tuesdays and Wednesdays,

from June through September, to reduce daily variation. On Tuesday, three

pork carcasses were selected at random at two areas of the slaughter,

immediately after singeing and polishing and after the final rinse. On

Wednesday, three carcasses from the previous day's kill were swabbed after

18-24 h in the carcass cooler. Each carcass was swabbed at two locations,

the midpoint of the loin and the outside of the ham. The side of the carcass

that was swabbed was randomly chosen. Also sampled on Wednesday were

six boneless loins. Each loin was swabbed on the lean side immediately

before packaging.

Sampling procedure

The pork carcasses were sampled on the slaughter line, so as to

disrupt normal production as little as possible. The six loins were sampled on

nearby tables in the packaging area to avoid interference with the line.

Personnel involved wore sterile gloves to avoid contamination. Sampling was

performed by using a moistened swab technique. Sterile cotton swabs were

moistened in 0.1% phosphate buffered saline at pH 7.0-7.2. The moistened

swabs were uniformly stroked 12 to 15 times across the surface of the carcass

inside a sterile 100-cm2 template. Swabs were then rotated and stroked 12

to 15 times perpendicular to the original swabbing direction. Swabs were

82

placed in 10 ml 0.1% phosphate buffered saline and stored on ice (approx.

4°C) for transport to the laboratory. Samples were plated within 4 to 7 h.

Tubes containing swabs were agitated on a vortex mixer for 20-30 seconds

before samples were diluted in 0.1% peptone water and plated.

Fresh, expired, or spoiled pork sausage samples also were examined.

These samples consisted of vacuum-packaged pork sausage chubs or

uncased sausage links furnished by processing plants or purchased at a retail

store. Five grams of pork sausage sample were aseptically transferred to a

sterile Stomacher bag containing 45 ml of 0.1% peptone water diluent. Each

sample was mixed by stomaching for 2 min in a Colworth Stomacher 400.

The samples were then decimally diluted in 9-ml 0.1% peptone water blanks

and plated on two different media selective for enterococci and fecal

streptococci.

Microbial enumeration

KF streptococcal agar (KF, Difco Laboratories, Detroit, Mich.; 7) was

prepared according to the manufacturers' instructions (Difco manual, 10th

edition, 1984). One-ml portions of appropriate dilutions were used for KF agar

pour plates. Duplicate plates were incubated at 37°C for 48 h. Colonies

exhibiting a red or pink color were counted as streptococci (7). The second

medium, fluorescent gentamicin-thallous-carbonate (fGTC) agar (12), was

prepared as specified. Appropriate dilutions of 0.1 ml were plated in

duplicate, and the plates were incubated at 37°C for 24 h. All except pinpoint

colonies were counted as enterococci. Positive fluorescence and starch

hydrolysis (zone of clearing) were also recorded. Total counts were performed

only on the processed pork sausage samples. Tryptone Glucose Extract

83

(TGE) agar (Difco Laboratories) was prepared as specified, and 1-ml portions

of the appropriate dilutions were plated in duplicate by using the pour plate

technique. The plates were incubated at 37°C for 48 h.

Statistical analysis

The method of analysis by Cochran and Cox (2) for combining

experiments was used where plants are considered to be experiments, visits

within plants to be replications within experiments, and type-stage

combinations to be treatments. Treatment by plant and treatment by visit

within plant sources of variation were pooled for treatment error. Variation

among samples for any given plant, visit, and treatment was combined for a

pooled error. An unweighted means analysis was used whenever the number

of samples differed among treatments.

Species identification

After the KF and fGTC plates had been counted, 3 colonies of each

colony type on each medium were streaked for isolation on Brain Heart

Infusion agar (Difco Laboratories). After 24 h of incubation, a gram stain and

catalase test were performed to verify gram-positive, catalase-positive

colonies. The cultures also were tested on Bile Esculin (BE) agar (Difco

Laboratories) for the ability to grow in bile and hydrolyze esculin. Cultures

positive for these tests were then identified to species by using the

classification schema developed by Knudtson and Hartman (10). This

schema was developed by using the API Rapid Strep and MicroScan Pos ID

panels.

84

RESULTS

A comparison of mean log counts of enterococci at each stage In the

slaughter process at each of three plants is shown in Fig, 1. Each point of the

figure represents 36 samples. The differences between plants and stages

were significant (P < 0.01). At stage 1, immediately after polishing, high mean

enterococcal counts were obtained from plants A and B. These results are

similar to those of Snijers et al. (18), who reported that a significant increase

in contamination on carcass surfaces occurred during polishing in a back-

scraping machine. Counts for plant C, however, were significantly lower than

for plants A and B. Different sanitation practices or newer equipment could

explain this difference, but the actual reason for these differences is not

known. At stage 2, after the final rinse, mean enterococcal counts for plants

A and B decreased significantly, approaching those of plant C. After 18-24 h

in the carcass coolers (stage 3), the mean enterococcal count from plant A

increased significantly, whereas counts at plant B actually decreased; there

was only a slight increase at plant C. Counts of samples taken immediately

before packaging (stage 4) at plants A and C were similar. At plant B,

however, counts were higher. It is evident that, although all three plants have

similar operations, significant differences in contamination existed. This

indicates that stage 3 is an important critical control point in pork processing

plant A.

Overall differences among plants are shown in Fig. 2. When mean

enterococcal counts were calculated for all samples taken at each plant, there

were significant differences between the plants (P = 0.0001). Total mean

85

counts were highest at plant A, then plant B, and finally, plant C. Further

studies are needed to determine the cause of these differences.

Fluorescent gentamicin-thallous-carbonate agar counts were used in

Fig. 1 and 2 because all enterococci and fecal streptococci grow equally well

on fGTC agar media, whereas the growth of E faecalis is favored on KF

medium. KF agar counts were performed, and these counts were lower in

almost every instance; data not shown (9).

Identifications were performed on colonies picked from both fGTC and

KF agar plates. From all samples collected at the 3 plants, 175 enterococci

were isolated. Fig. 3 represents the proportions of enterococcal species

identified: 79% of the enterococci were E faecalis, 11% E faecium, 3% E

pseudoavium, 2% each E. malodoratus and E solitarius, and 1 % each E.

casseliflavus and E durans. One percent of the enterococci isolated were not

identified (2 cultures) and may represent one or two of the 5 new species of

enterococci not included in the classification schema used to identify the

majority of isolates (10). Proportions of enterococci isolated separately from

each plant and from the two different isolation media resembled closely those

represented in Fig. 3 (9).

The processed pork sausage data (Fig. 4) includes all 3 media used,

fGTC, KF, and TGE. The differences between the fresh pork sausage counts

at plants A and B could be due to sample differences. Plant A samples

consisted of retail pork sausage chub samples, whereas plant B samples

included chub samples and uncased link samples. The uncased link samples

undergo more processing steps and had higher counts than the less

processed chubs. The lower counts found on KF medium vs. fGTC medium

86

were consistent throughout the study; almost invariably, counts were higher

on the less inhibitory fGTC medium than on KF agar. The spoiled pork

sausage samples from plant A showed signs of spoilage before reaching the

expiration date (gas was produced, bulging the packages). The expired pork

sausage from plant B showed no signs of spoilage, although the mean counts

were just as high as for the spoiled samples. Although mean enterococcal

counts (fGTC) of spoiled and expired pork sausage were similar, identities of

organisms from these 2 sources differed. Samples from the spoiled pork

sausage yielded an almost pure culture of enterococci; very few other

organisms were obtained (but not every colony was tested). On the other

hand, the expired samples contained mostly gram-positive bacilli, resembling

lactobacilli, which grew on both KF and fGTC media. Enterococcus species

were isolated, but counts were much lower than they appear in Fig. 4, About

one of every 10 colonies tested was an Enterococcus. These results indicate

that enterococci are not good shelf-life indicators.

Fig. 5 represents the proportions of enterococcal species recovered

from the pork sausage samples. (A) Represents proportions of enterococcal

species recovered from fresh pork sausage samples obtained from plants A

and B. The individual proportions from each plant were comparable, so data

were combined. As with the pork carcass results, E. faecalis was the

predominant Enterococcus species in fresh pork sausage; 83% of the isolates

were identified as E. faecalis, 11% of isolates were E. malodoratus, and 6%

were E. hirae. E. faecium was not isolated. In the spoiled pork sausage from

plant A (B, Fig. 5), E. faecium vias predominant (84%); only 14% were E.

faecalis and 2% £ durans. (C) represents proportions of enterococci from

87

expired pork sausage. In expired pork sausages, 44% of the enterococci

were £ faecalis, 23% £ pseudoavium, and 11% each were £ malodoratus,

£ rafiinosus, and £ durans.

88

DISCUSSION

The pork slaughtering plant data show that, although plants may be of

sinnilar overall design and line speed, bacterial counts on carcasses at

different points during processing can differ significantly. Undoubtedly,

differences in equipment (such as conveyer belts) and in operating protocols

(such as personnel training and hygiene, equipment sanitation and

maintenance, and plant sanitation) exist among plants.

Airborne contamination within pork slaughter and processing

establishments warrants some attention. Kotula and Emswiler-Rose (11)

reported that airborne contamination in one pork facility was 10 times higher

than in previously studied dairy facilities. During slaughter and processing,

bacteria on the surface of animals, employees, and equipment could become

airborne and cause contamination. The present work has defined some

potential critical control points. Further studies are needed to elucidate factors

contributing to high contamination levels so that appropriate remedial action

can be taken.

Several researchers have described the predominance of E faecium in

the intestinal tract of swine and other animals (1, 8,15,16) whereas E.

faecalis is believed to reside predominantly in human intestines (1, 8, 15, 16).

Therefore, the predominance of E faecalis ii. the hog carcass samples is

puzzling. Because enterococci are associated with the intestinal tracts of pigs,

enterococci isolated from a hog carcass in a slaughtering plant presumably

would arise from fecal contamination of hog carcasses by hog fecal matter;

therefore, E faecium should predominate. The overall predominance of E.

89

faecalis isolated from all 3 plants indicates that the hogs slaughtered in these

plants had an intestinal flora in which £ faecalis predominated, or that the

enterococcal contamination arose from other sources. Because E. faecalis is

the predominant species in the human intestine, human contamination of the

hog carcasses should be considered as a possible source of the carcass

contamination. The hygienic practices of employees would be of critical

importance in controlling contamination of human origin. Another explanation

would be the establishment of E. faecalis in the plant. Contamination of the

carcasses would occur when they came in contact with equipment or other

contaminated materials. The enterococcal contamination could occur at

several points in the processing line, allowing recontamination at any of

several stages in the slaughtering operation. This would explain the wide

variation in counts at different stages among plants.

From the pork slaughtering plants, the pork enters the pork processing

plant and is made into fresh pork sausage. Fresh sausage samples harbor

roughly the same species of enterococci found on the pork carcasses (Fig.

5A). Spoiled pork sausages (Fig. 5B), on the other hand, contained

predominantly E. faecium, with a smaller percentage of E. faecalis. Barnes

and Ingram (1) reported similar findings; S. faecium (E. faecium) caused

spoilage in canned hams. Enterococci predominating in hog intestines also

were identified as S. faecium (E. faecium) and unclassified group D strains

(probably new Enterococcus spp.). S. faecalis (E. faecalis) was not isolated.

When testing was performed in bacon factories, the predominant

enterococcus was S. faecalis (E. faecalis). Sharpe and Fewins (17)

serologically typed the S. faecium strains isolated from pig intestines and

90

canned hams. Two serological types were present in both the samples from

pig intestines and canned hams. But these serotypes are widespread and

have been isolated from samples from several different countries. It is

possible, however, that E. faecium arising from hog fecal contamination

causes spoilage of pork sausages if present in high numbers. Even though

fecal contamination with £ faecium was not discovered on the hog carcasses

that we examined, it is possible that this contamination occurs infrequently.

When it does occur, contamination of a single hog carcass may be sufficient to

instigate spoilage of sausage or hams made from the carcass. More

carcasses must be assayed to test this hypothesis.

In conclusion, because of the ability of enterococci to grow in

environments outside their original source, enterococcal counts alone are not

always accurate indicators of fecal pollution. But, in conjunction with species

identification, they can indicate possible sources of contamination and help to

define critical control points in need of further attention.

91

ACKNOWLEGMENTS

Supported by a USDA grant to the Food Safety Consortium. Journal

Paper No. J-14979 of the Iowa Agriculture and Home Economics Experiment

Station, Ames; project 2991. We thank C. Lynn Knipe for allowing us to be

part of this study, Anita McVey for assistance with the statistical analyses, and

Christopher Guyer for technical assistance.

92

LITERATURE CITED

1. Barnes, E. M., and M. Ingram. 1955. The identity and origin of faecal

streptococci in canned hams. Ann. Inst. Pasteur Lille. 7:115-119.

2. Cochran, W. G., and G. M. Cox. 1957. Analysis of the results of a

series of experiments, pp. 545-568. In Experimental designs, 2nd ed.

John Wiley and Sons, Inc., New York.

3. Deibel, R. H. 1964. The group D streptococci. Bacterid. Rev. 28:330-

366.

4. Garg, S. K., and B. K. Mital. 1991. Enterococci in milk and milk

products. Crit. Rev. Microbiol. 18:15-45.

5. Gordon, C. L. A. 1991. Thermal susceptibility of Streptococcus faecium

strains isolated from frankfurters. Can. J. Microbiol. 37:609-612.

6. Hartman, P. A., R. H. Deibel, and L. M. Sleverding. 1992.

Enterococci, pp. 523-531. In C. Vanderzant, D. F. Splittstoesser (eds.).

Compendium of methods for the microbiological examination of foods, 3rd

ed. American Public Health Association, Washington, D. C.

93

7. Kenner, B. A., H. F. Clark, and P. W. Kabler. 1961. Fecal

streptococci. I. Cultivation and enumeration of streptococci in surface

waters. Appl. Microbiol. 9:15-20.

8. Kjellander, J. 1960. Enteric streptococci as indicators of fecal

contamination of water. Acta Pathol. Microbiol. Scand., Suppl. 136,. 48:9-

124.

9. Knudtson, L M. 1992. Classification of enterococci and their roles in

spoilage of pork products ans as sanitary indicators in pork processing.

Ph.D. thesis. Iowa State University, Ames.

10. Knudtson, L. M., and P. A. Hartman. 1992. Routine procedures for

isolation and identification of enterococci and fecal streptococci, Appl.

Environ. Microbiol. 58; control #509-92.

11. Kotula, A. W., and B. S. Emswiler-Rose. 1988. Airborne

microorganisms in a pork processing establishment. J. Food Prot. 51:935-

937.

12. Littel, K. J., and P. A. Hartman. 1983. Fluorogenic selective and

differential medium for isolation of fecal streptococci. Appl. Environ.

Microbiol. 45:622-627.

94

13. Moussa, R. S. 1965. Species differentiation of faecal and nonfaecai

enterococci. J. Appl. Bacteriol. 28:466-472.

14. Mundt, J. O., A. H. Johnson, and R. Khatchikian. 1958. Incidence

and nature of enterococci on plant materials. Food Res. 23:186-193.

15. Fourcher, A. M., L. A. Devriese, J. F. Hernandez, and J. M.

Delattre. 1991. Enumeration by a miniaturized method of Escherichia

coii, Streptococcus bovis and enterococci as indicators of the origin of

faecal pollution of waters. J. Appl. Bacteriol. 70:525-530.

16. Ramadan, F. M., and M. S. Sabir. 1963. Differentiation studies of

fecal streptococci from farm animals. Can. J. Microbiol. 9:443-450.

17. Sharps, M. E., and B. G. Fewins. 1960. Serological typing of strains

of Streptococcus faecium and unclassified group D streptococci isolated

from canned hams and pig intestines. J. Gen. Microbiol. 23:621 -630.

18. Snijers, J. M. A., M. H. W. Janssen, G. E. Gerats, and G. P.

Cortiaensen. 1984. A comparative study of sampling techniques for

monitoring carcass contamination. Int. J. Food Microbiol. 1:229-236.

19. Thian, T. S., and P. A. Hartman. 1981. Gentamicin-thallous-

carbonate medium for isolation of fecal streptococci from foods. Appl.

Environ. Microbiol. 41:724-728.

1. Mean log counts of enterococci on fGTC agar at each stage in the slaughter process at each plant.

Stage 1 samples were taken immediately after singeing and polishing, stage 2 after the final rinse, and

stage 3 after a 24-h chill. Stage 4 samples were from loins immediately before packaging.

CM-k

4 ̂ o c

i l (0 0)

D) o

8 -

7 -

6 "

5 -

1 I I r Stage 1 Stage 2 Stage 3 Stage 4

A""

Plant A Plant B Plant C

CO o>

Fig. 2. Comparison of mean log counts for all stages at each plant. The comparison shows the overall

enterococcal counts at each plant.

Mean enterococcal count

log CFU/100 cm^

ro O) CO o

"O D) 3

>

"O Q) 3

w

"O

03 3

o

86

Fig. 3. Proportional representation of 175 enterococcal species isolated from all samples taken from pork

slaughtering plants. Complete circle represents 100% of enterococci isolated.

11%

2%

3%

E. faecalis E. faecium

E. pseudoavium I E. malodoratus I E. solitarius I E. casseliflavus ] E. durans I Ent. no ID

Fig. 4. Comparison of mean log counts of pork sausage samples from pork processing plants. Each sample

was plated on 3 different media: fluorescent gentamycin-thallous-carbonate (fGTC) agar, KF

Streptococcal (KF) agar, and Tryptone Glucose Extract (TGE) agar. Fresh A represents fresh pork

sausage produced by processing plant A. Fresh B represents fresh pork sausage from plant B. Spoiled

A indicates pork sausage samples that showed signs of spoilage before the expiration date; these were

supplied by plant A. Expired B samples are those whose expiration date was reached; these were

supplied by plant B.

9

8

7

6

5

4

3

2

1

Fresh A Fresh B Spoiled A Expired B

Processed pork sausage

Fig. 5. Proportions of enterococci isolated from processed pork sausage

samples. (A) Enterococcal spp. isolated from fresh pork sausage from

plants A and B. (B) Enterococcal spp. isolated from spoiled pork

sausage from plant A. (C) Enterococcal spp. isolated from expired

pork sausage from plant B. Complete circles represent 100% of

enterococci isolated from the respective samples.

104

11%

83%

B

84%

14%

E. faecalis E. pseudoaviun E. durans E. hirae

E. faecium E. malodoratus E. raffinosus

11%

105

PAPER 3; COMPARISON OF FLUORESCENT GENTAMICIN-THALLOUS-CARBONATEAND KF STREPTOCOCCAL AGARS TO ENUMERATE ENTEROCOCCI AND FECAL STREPTOCOCCI IN MEATS

106

Comparison of Fluorescent Gentamicin-Thallous-Carbonate and KF Streptococcal agars to Enumerate

Enterococci and Fecal Streptococci in Meats

LINDA M. KNUDTSON AND PAUL A. HARTMAN*

Department of Microbiology, Immunology and Preventive Medicine Iowa State University, Ames, Iowa 50011

Telephone: (515)294-8824 FAX: (515)294-6019

^Corresponding author

107

ABSTRACT

Two selective and differential media were compared for their abilities to

enumerate enterococci and fecal streptococci in pork, beef, and poultry

products. Counts obtained on KF Streptococcal (KF) agar were compared

with counts obtained by using Fluorescent Gentamicin-Thallous-Carbonate

(fGTC) agar. Reactions of 13 known enterococcal species also were

observed. All 13 species of enterococci as well as S. bovis and S. equinus

grew equally well on fGTC agar. KF Streptococcal medium allowed growth of

most species of enterococci, but not S. bovis and S. equinus. Comparisons

between the two media showed that counts on fGTC agar were consistently

and significantly higher than counts on KF agar for all sample sources. This

trend was not observed, however, when pure cultures of most known species

of enterococci were quantitated by using both media.

108

INTRODUCTION

EnterococcI have been considered an alternative potential indicator of

fecal pollution of foods because they have an advantage over coliforms, the

primary fecal indicator, in that they are more resistant than coliforms to most

environmental insults. This trait is shared by many potential pathogens;

therefore, enterococci can help determine the sanitary history of moderately

heated, frozen, dried or salted foods, and other foods where coliforms might

not have survived (13). Enterococci are also important in human infections

such as endocarditis and bacteremia. They may be resistant to clinically

important antibiotics (9).

Many media have been developed to isolate and enumerate

enterococci (11). In 1961, Kenner et al. (3) described new solid and liquid

media (KF streptococcal broth and agar) for enumerating enterococci. These

media contained, among other ingredients, sodium azide, bromcresol purple,

and 2,3,5-triphenyltetrazolium chloride (TTC). A new medium, gentamicin-

thallous-carbonate (GTC) agar, was reported in 1978 (1). It was superior to

KF agar for the enumeration of fecal streptococci in fecal and surface-water

samples. GTC agar was modified (fGTC agar) to make it more differential by

Littel and Hartman (8) in 1983 by incorporating a colorimetric starch substrate

plus a fluorogenic substrate, allowing differentiation of colonies on the agar

surface. Gentamicin and thallous acetate were the major selective agents.

NaHCOs, Tween 80, and KH2PO4 were added as specified earlier by

Lachica and Hartman (7) to stimulate the growth of group D streptococci. The

incorporation of amylose azure and 4-methylumbelliferyl-a-D-galactoside

109

allowed differentiation of the fecal streptococci into three phenotypic groups:

starch hydrolysis and fluorescence, no starch hydrolysis but fluorescence, and

no starch hydrolysis or fluorescence (8).

With the recent changes in classification of this group of organisms,

questions have arisen on the selectivity of the media to new members of the

genus Enterococcus. The abilities of these media to recover all species of

enterococci and fecal streptococci, as well as the differentiating characteristics

of each species on both media, are the subject of this study.

110

MATERIALS AND METHODS

Cultures

Fifty-nine strains of 13 species of enterococci and Streptococcus bovis

and Streptococcus equinus were collected from various sources (5). Cultures

also were isolated from pork carcasses during slaughter, fresh and spoiled

pork sausage, poultry, and beef products. All cultures were isolated according

to Knudtson and Hartman (6).

Media

KF Streptococcal agar (KF; Difco Laboratories, Detroit, Mich.) was

prepared according to the manufacturers' instructions. One-ml portions of

appropriate dilutions were used for KF agar pour plates. Duplicate plates

were incubated at 37°C for 48 h. Colonies exhibiting a red or pink color were

counted as streptococci (3). The second medium, fGTC agar (8) was

prepared as specified by the authors. One-tenth ml portions of appropriate

dilutions were plated in duplicate, and the plates were incubated at 37°C for

24 h. All except pinpoint colonies were counted as enterococci. Positive

fluorescence and starch hydrolysis (zones of clearing) also were recorded.

Species identification

After the KF and fGTC plates had been counted, three colonies of each

colony type on each medium were streaked for isolation on Brain Heart

Infusion agar (Difco). After 24 h of incubation, a gram stain and catalase test

were performed to verify gram-positive, catalase-positive colonies. The

cultures also were tested on Bile Esculin (BE) agar (Difco Laboratories) for

their abilities to grow in bile and hydrolyze esculin. Cultures positive for these

111

tests were identified to species by using tlie classification schema developed

by Knudtson and Hartman (5). This schema was developed by using API

Rapid Strep and MicroScan Pos ID panels. The identities of the isolates were

compared with results of known strains on both media.

112

RESULTS AND DISCUSSION

In every instance, enterococcus counts made on pork, beef, and poultry

samples by using fGTC agar were significantly higher (P < 0.01) than counts

on KF agar (Figure 1). One explanation is that a larger proportion of injured

enterococci grew on fGTC agar than on KF agar. In 1961, Kenner et al. (3)

stated that KF agar allowed the growth of S. bovis, S. equinus and the

enterococcal group consisting of S. faecalis, and its varieties S. faecalis var.

liquifaciens and zymogenes, and related S. faecalis biotypes. Significant

classification changes have occurred since that time, and many of the newly

classified species of enterococci may not have been tested for growth on KF

agar. fGTC agar allows grov^h of S. faecalis, S. faecium, S. bovis, S. equinus,

and S. avium (8), This medium was developed before the classification

changes that began in 1984 (12). Newly classified species of enterococci had

not been tested on this medium either.

When known species of enterococci were plated on each medium

(Table 1), fGTC agar allowed growth of all enterococci and fecal streptococci

tested. Strains on fGTC agar were differentiated into three groups according

to the ability to fluoresce and hydrolyze starch. E. faecalis, E. pseudoavium, E.

solitarius and S. equinus comprised a group that was both fluorescence- and

starch-negative. S. bovis was positive for both fluorescence and starch

hydrolysis. Most of the rest of the enterococcal species tested were

fluorescence-positive and starch-negative. When E. avium and £ faecium

were tested, only 38% and 71% of isolates produced fluorescence,

respectively. Littel and Hartman (8) reported similar findings; when 87 strains

of S. faecium were plated on fGTC agar, 80 (92%) showed fluorescence, and

113

7 (8%) did not. KF agar did not allow growth of known strains of E cecorum,

S. bovis or S. equinus (Tablel), other enterococci grew on this medium. This

medium contains sodium azide as a selective agent. Some streptococci, such

as S. bovis, cannot initiate growth on media that contain azide (1). Most

species reduced tetrazolium to some degree, and E. faecalis reduced it the

most strongly (Table 1). Differences in the intensity of tetrazolium reduction

were difficult to visualize and could be determined accurately only if plates

containing different species were compared side-by-side. When counts

obtained from plating diluted samples of known strains were compared (data

not shown), there were no significant differences in numbers on the two media

(except for the three species that did not grow on KF agar).

To determine if differences in counts obtained from meat samples

plated on the two media were a result of the recovery of species on fGTC agar

that did not grow on KF agar, the identities of 175 isolates collected from pork

carcasses were tabulated (Table 2). The distribution of most species of

enterococci was similar on both media. No E. cecorum, S. bovis, or S.

equinus was isolated from the pork carcasses by using either medium. Only

E. durans and E, casseliflavus were isolated on fGTC agar and not on KF

agar. These accounted for only very small percentages of isolates and could

not be sufficient to account for the differences in counts.

Another possible explanation for higher recoveries on fGTC agar than

on KF agar might be that organisms other than enterococci and fecal

streptococci grew on fGTC agar, giving false-positive results. However, more

than 95% of isolates from the colony types counted as enterococci on fGTC

agar (pinpoint colonies were not counted, although some were identified to

114

assure they were not enterococci), were identified as enterococci or fecal

streptococci (4). These results were similar to those reported by Littel and

Hartman (8), who found that 90% of isolates from sewage and fecal samples

were enterococci or fecal streptococci. Some care must be exercised,

however, when using fGTC agar. Knudtson and Hartman (6) found that when

samples (such as expired pork sausage) highly contaminated with lactobacilli

were plated on fGTC agar, enterococcal colonies could not be discerned

among a heavy background of Lactobacillus colonies.

Hartman et al. (2) stated that many, if not a large majority, of the

microorganisms in certain foods and natural environments may be in a

different physiological state than similar strains cultivated in the laboratory.

With laboratory stock cultures as test material, continuation of bacterial growth

is the concern, whereas with bacteria from the natural environment the

problem seems to be not only growth, but also growth initiation (10). The

overall effect is that a medium will usually be more inhibitory to bacteria from

natural products than to rapidly growing laboratory cultures. If this is the case,

then it is possible that the differences between counts made on fGTC and KF

agars could be a result of the inhibitory properties of KF agar (which contains

sodium azide) to enterococci and fecal streptococci present in natural

products.

In conclusion, fGTC agar is a selective and differential medium for

enterococci and fecal streptococci. It is as good as, if not better than, KF agar

to enumerate enterococci and fecal streptococci in foods. fGTC agar also

enables colony differentiation that might be useful in some circumstances. It

115

should be noted that fGTC agar can yield falsely high counts when excessive

numbers of lactobacilli are present.

116

ACKNOWLEDGMENT

Supported by a U.S.D.A. grant to the Food Safety Consortium. Journal

Paper No. J-XXXXX of the Iowa Agriculture and Home Economics Experiment

Station, Ames, project 2991.

117

REFERENCES CITED

1. Donnelly, L. S., and P. A. Hartman. 1978. Gentamicin-based medium

for the isolation of group D streptococci and application of the medium to

water analysis. Appl. Environ. Microbiol. 35:576-581.

2. Hartman, P. A., G. W. Reinbold, and D. S. Saraswat. 1966. Media

and methods for isolation and enumeration of the enterococci. Adv. Appl.

Microbiol. 8:253-289.

3. Kenner, B. A., H. F. Clark, and P. W. Kabler. 1961. Fecal

streptococci. I. Cultivation and enumeration of streptococci in surface

waters. Appl. Microbiol. 9:15-20.

4. Knudtson, L. M. 1992, Classification of enterococci and their roles in

spoilage of pork products and as sanitary indicators in pork processing.

Ph.D. thesis. Iowa State University, Ames.

5. Knudtson, L. M., and P. A. Hartman. 1992. Routine procedures for

isolation and identification of enterococci and fecal streptococci. Appl.

Environ. Microbiol. 58:3027-3031.

6. Knudtson, L. M., and P. A. Hartman. 1992. Enterococci in pork

processing. J. Food Prot. in press control # JFP-92-119.

118

7. Lachica, R. V. F., and P. A. Hartman. 1968. Two improved media for

isolating and enumerating enterococci in certain frozen foods. J. Appl.

Bacterid. 31:151-156.

8. Littel, K. J., and P. A. Hartman. 1983. Fluorogenic selective and

differential medium for isolation of fecal streptococci. Appl. Environ.

Microbiol. 45:622-627.

9. Murray, B. E. 1990. The life and times of the Enterococcus. Clin.

Microbiol. Rev. 3:46-65.

10. Ray, B. (éd.). 1989. Injured index and pathogenic bacteria: Occurrence

and detection in foods, water and feeds. CRC Press, Inc, Boca Raton,

Florida.

11. Reuter, G. 1985. Selective media for group D streptococci. Int. J. Food

Microbiol. 2:103-114.

12. Schleifer, K. H., and R. Kiipper-Bâiz. 1984. Transfer of

Streptococcus faecalis and Streptococcus faecium to the genus

Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and

Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. 34:31-34.

119

13. Thian, T. S., and P. A. Hartman. 1981. Gentamicin-thallous-

carbonate medium for isolation of fecal streptococci from foods. Appl.

Environ. Microbiol. 41:724-728.

120

Table 1 ; Growth characteristics of known species of enterococci and fecal streptococci on fGTC and KF agars

fGTC agar KF agar No. of

SPECIES Strains Growth F s Growth Color

E avium 8 100% 38% 0 100% PINK

E. casseliflavus 5 100% 100% 0 100% PINK

E. cecorum 2 100% 100% 0 0

E. durans 4 100% 100% 0 100% PINK

E. faecalis 8 100% 0 0 100% DK RED

E. faecium 7 100% 71% 0 100% PINK

E. gallinarum 4 100% 100% 0 100% RED

E. hirae 6 100% 100% 0 100% PINK

E. malodoratus 2 100% 100% 0 100% RED

E. mundtii 4 100% 100% 0 100% RED

E. pseudoavium 2 100% 0 0 100% RED

E. raffinosus 1 100% 100% 0 100% RED

E. solitarius 1 100% 0 0 100% RED

S. bows 4 100% 100% 100% 0

S. equinus 1 100% 0 0 0

% Indicates the percentages that showed a positive result, whether it be growth, (F) fluorescence, or (S) starch hydrolysis. Color indicates the color of colonies on KF agar.

121

Table 2: Species isolated from pork carcasses on fGTC and KF agars

fGTC agar KF agar

Species identified # isolated % # isolated %

E. faecalis 49 69 87 84

E. faedum 15 19 7 6

E. durans 2 3 0 0

E. casseliflavus 1 2 0 0

E. malodoratus 1 2 2 2

E. solitarius 1 2 2 2

E. pseudoavium 1 2 4 4

Enterococcus NO ID 1 2 2 2

TOTAL 71 100 104 100

# Isolated indicates the number of each species isolated on each type of medium. % Indicates the percentage each species that was isolated.

Fig. 1. Comparison of mean log counts on fGTC and KF agars of four sample sources (pork

carcasses=/100cm2; other products =/gram). All of the differences between the two media

significant (P < 0.01).

o o c (0 0)

E O) o

ro CO

Pork carcasses Pork products Beef products Poultry products

SAMPLE SOURCE

124

PAPER 4: ANTIBIOTIC RESISTANCE AMONG ENTEROCOCCAL ISOLATES FROM CLINICAL AND ENVIRONMENTAL SOURCES

125

Antibiotic Resistance among Enterococcal Isolates from Clinical and Environmental Sources

LINDA M. KNUDTSON AND PAUL A. HARTMAN*

Department of Microbiology, Immunology and Preventive Medicine Iowa State University, Ames, Iowa 50011

Telephone: (515)294-8824 FAX: (515)294-6019

^Corresponding author

126

ABSTRACT

Antibiotic resistance among enterococci and fecal streptococci was

examined by testing 149 isolates from pork, water, and clinical material, as

well as 50 known species, for resistance to 27 different antimicrobial agents.

Tests were performed by using MicroScan Pos MIC type 6 panels. Pork

isolates exhibited less resistance than either water or clinical isolates to most

antibiotics, although a larger proportion of pork isolates than others was

resistant to tetracycline. Comparisons of antimicrobial resistance patterns

between enterococcal species revealed that Enterococcus faecium was most

resistant to p-lactam antimicrobials, especially ampicillin, whereas

Enterococcus faecalis appeared to be the most resistant to the synergistic

effects of antimicrobial combinations. Vancomycin resistance was observed

in one Enterococcus hirae isolate from water. Enterococcal isolates from any

of the sources tested did not show multiple resistance to antibiotics (such as

gentamicin, ampicillin, streptomycin, and vancomycin) used to treat serious

infections caused by gram-positive cocci.

127

INTRODUCTION

Recent attention has focused on enterococci because of their

remarkable and increasing resistance to antimicrobial agents (12). This

resistance allows them to survive in environments in which antimicrobial

agents are heavily used. Antimicrobials are given to food animals, such as

swine, to improve their growth rate and feed conversion (3). However, many

people believe that the use of antimicrobials in humans or animals is often

followed by appearance of resistant microorganisms (13). Studies by Cohen

and Tauxe (2) suggested that the antimicrobial drugs to which food animals

were exposed provided selective pressure that lead to the appearance and

persistence of drug resistant strains. Specifically, they associated the

occurrence of certain drug-resistant Salmonella sp. to antimicrobial use in

food animals. If antimicrobial use in food animals is linked to antimicrobial

resistance in Salmonella, Enterococcus species in the intestinal tract might

also be expected to develop similarly elevated resistance patterns.

Antimicrobial resistance in enterococci can be divided into two general

types, intrinsic and acquired. Intrinsic resistance is present in all or most

strains of those species, and the genes appear to reside on the chromosome.

Intrinsic resistance includes resistance to semisynthetic, penicillinase-

resistant penicillins, cephalosporins, and low levels of aminoglycosides and

clindamycin. Acquired resistance results from a mutation in cellular DNA or

acquisition of new DNA. Examples of acquired resistance include resistance

to chlorampenicol, erythromycin, tetracycline, high levels of aminoglycosides

and clindamycin, penicillin by means of penicillinase, fluoroquinolones, and

vancomycin (12). Several researchers have shown that differences in

128

antimicrobial susceptibility exist between Enterococcus faecium and

Enterococcus faecalis (4,10,11). The possible existence of similar

susceptibility differences among the more recently described species of

Enterococcus needs to be determined. An assessment of possible

correlations between a certain species and a given susceptibility pattern could

provide valuable information (14). Few data are available on antimicrobial

resistance patterns of isolates from widely divergent environmental sources,

conducted in a single laboratory by using a common procedure.

In this study, enterococci and fecal streptococci were collected from

three sources: pork, including pork carcasses and processed pork products;

water. Including samples from rivers, lakes, and well water; and clinical

material from several sources. These samples, as well as known strains, were

tested for resistance to 24 antimicrobials and 3 synergy screens with

MicroScan Pos MIC type 6 panels. The results were examined for differences

in resistance patterns between enterococci from the three different sources

(pork, water, clinical). Differences in resistance patterns between different

species of enterococci and fecal streptococci also were examined.

129

MATERIALS AND METHODS

Cultures

Fifty known strains of 13 species of enterococci and Streptococcus

bovis and S. equinus were collected from various sources and their identities

confirmed (7). Cultures also were isolated from pork carcasses during

slaughter and from fresh and spoiled pork products. All pork sample cultures

were isolated according to Knudtson and Hartman (8). Enterococci from water

samples were isolated by the membrane filter method (1). Clinical isolates

were collected from patients at one hospital over a two-year period (6).

Primary isolation on blood agar plates revealed colonies that, upon gram-

staining, were gram-positive cocci. Catalase, bile esculin, and Lancefield

grouping tests confirmed that the isolates were group D enterococci. Species

identifications were carried out as indicated by Knudtson and Hartman (7).

Antibiotic susceptibility testing

Inoculum preparation for the MicroScan Pos MIC type 6 panels (Baxter

Diagnostics, Deerfield, III.) was carried out by the log-phase technique

specified by the manufacturer. Panels were inoculated, covered, and

incubated at 37''C. After 18 to 24 hours of incubation, results were interpreted

as indicated in the manufacturer's instructions.

130

RESULTS AND DISCUSSION

The percentages of water, pork and clinical isolates that were resistant

to 27 different antimicrobial agents are shown in Table 1. Generally, smaller

proportions of the pork isolates were resistant than either the water or clinical

isolates. Only with cefazolin, imipenem, and tetracycline were larger

proportions of the pork isolates resistant than the clinical isolates. Only in

three instances, with penicillin, erythromycin, and tetracycline did higher

proportions of the pork isolates exhibit resistance than the water isolates

(Table 1). Water isolates were more resistant than either the pork or clinical

isolates to all cephalosporins, amikacin, gentamicin, imipenem and rifampin.

Only one isolate, from water, was resistant to vancomycin.

It has been suggested that animal husbandry practices have

contributed to the dissemination of antibiotic resistance among intestinal

isolates (2,3,13). Langlois et al. (9) stated that the selection for resistant

bacteria brought about by the use of antimicrobials would not be easily

reversed by partial restriction of antibiotics used in veterinary practice

because the resistant bacteria are passed on from one animal generation to

another. Resistant fecal coliforms were present in swine at the time of

slaughter, regardless of recent administration of antimicrobial agents (9). If

this were true for enterococci, isolates from pork carcasses and processed

pork should show antimicrobial resistance patterns similar to the clinical

isolates. However, the incidence of acquired resistance generally was lower

in isolates from pork than from water or clinical material. The major exception

was resistance to tetracycline, which is a common additive to swine feeds.

131

When resistance patterns of known strains and environmental isolates

were compared (Table 2), several trends could be seen. All enterococci

possessed a degree of resistance to the aminoglycosides and

cephalosporins, as would be predicted by their intrinsic resistance. Intrinsic

resistance to the semisynthetic penicillins, ticarcillin and oxacillin, also was

prevalent throughout most species of the enterococci (Table 2). Enterococcus

cecorum, however, was not resistant to any of these antimicrobials for which it

should carry intrinsic resistance. It does, however, show resistance to

clindamycin along with the other enterococci (Table 2). As stated in the

literature (4,10), Enterococcus faecium appears to carry the highest amount of

resistance to p-lactam antimicrobials, especially ampicillin. On the other

hand, it was also stated that E. faecium strains are often more refractory to the

synergistic effects of antibiotic combinations. Our results show that E. faecalis

was more often resistant to synergistic combinations of antibiotics. Grayson et

al. (5) stated that Enterococcus raffinosus showed higher levels of resistance

than E. avium to penicillin; this was supported by our findings. Resistance to

vancomycin was seen only In one strain of E. hirae, isolated from a sample of

creek water.

In conclusion, pork and pork products did not harbor enterococci with

levels of antibiotic resistance that were substantially higher than those

possessed by enterococci obtained from water or from clinical material with

the exception of resistance to tetracycline. No more resistance to antibiotics

used clinically to treat gram-positive infections in humans was observed in

any isolate from pork than was seen in water or clinical isolates. Therefore,

antibiotic resistant enterococci and fecal streptococci of pork origin do not

132

present an exceptional public health hazard. Clinical and water isolates carry

more varied antibiotic resistance patterns than isolates from pork. Also,

antibiotic-resistance patterns differ among enterococcal species, even those

isolated from environmental sources.

133

ACKNOWLEDGEMENT

Supported by a U.S.D.A. grant to the Food Safety Consortium. Journal

Paper No. J-XXXXX of the Iowa Agriculture and Home Economics Experiment

Station, Ames, project 2991.

134

LITERATURE CITED

1. American Public Healtii Association. 1989. Standard methods for the

examination of water and wastewater, 17th ed., American Public Health

Association, Washington, D.C.

2. Cohen, IW. L., and R. V. Tauxe. 1986. Drug-resistant Salmonella in the

United States: An epidemiological perspective. Science 234:964-969.

3. DuPont, H. L., and J. H. Steele. 1987. Use of antimicrobial agents in

animal feeds: Implications for human health. Rev. Infect. Dis. 9:447-460.

4. Gray, J. W., D. Stewart, and S. J. Pedler. 1991. Species

identification and antibiotic susceptibility testing of enterococci isolated

from hospitalized patients. Antimicrob. Agents Chemother. 35:1943-1945.

5. Grayson, M. L., G. M. Eliopoulos, C. B. Wennersten, K. L.

Rouff, K. Klimm, F. L. Sapico, A. S. Bayer, and R. C.

Moellering, Jr. 1991. Comparison of Enterococcus raffinosus m\\\

Enterococcus avium on the basis of penicillin susceptibility, penicillin-

binding protein analysis, and high-level aminoglycoside resistance.

Antimicrob. Agents Chemother. 35:1408-1412.

6. Knudtson, L. M. 1992. Classification of enterococci and their roles in

spoilage of pork products and as sanitary indicators in pork processing.

Ph.D. thesis. Iowa State University, Ames.

135

7. Knudtson, L. M., and P. A. Hartman. 1992. Routine procedures for

isolation and identification of enterococci and fecal streptococci. Appl.

Environ. Microbiol. 58:3027-3031.

8. Knudtson, L. M., and P. A. Hartman. 1992. Enterococci in pork

processing. J. Food Prot. in press control # JFP-92-119.

9. Langlois, B. E., K. A. Dawson, I. Leak, and D. K. Aaron. 1988.

Antimicrobial resistance of fecal colifoms from pigs in a herd not exposed

to antimicrobial agents for 126 months. Vet. Microbiol. 18:147-153.

10. Louie, M., A. E. Simor, S. Szeto, M. Patel, B. Kreiswirth, and

D. E. Low. 1992. Susceptibility testing of clinical isolates of Enterococcus

faecium and Enterococcus faecalis. J. Clin. Microbiol. 30:41-45.

11. Moeilering, R. C., Jr., O. M. Korzeniowski, M. A. Sande, and

C. B. Wennersten. 1979. Species-specific resistance to antimicrobial

synergism in Streptococcus faecium and Streptococcus faecalis. J. Infect.

Dis. 140:203-208.

12. Murray, B. E. 1990. The life and times of the Enterococcus. Clin.

Microbiol. Rev. 3:46-65.

136

13. Neu, H. C. 1992. The crisis in antibiotic resistance. Science 257:1064-

1072.

14. Rouff, K. L. 1990. Recent taxonomic changes in the genus

Enterococcus. Eur. J. Clin. Microbiol. Infect. Dis. 9:75-79.

137

Table 1 : Percentages of water, pork and clinical isolates resistant to 27 different antimicrobial agents

Water Pork Clinical . ... ^ . %resistant %resistant %resistant Antibiotics tested (49)3 (50) (50)

Aminoglycosides Amikacin 96 48 90 Gentamicin 88 36 74 Gentamicin synergy screen ..b — 36 Streptomycin synergy screen 6 — 32

CeohalosDorins Cephalothin 59 32 38 Cefazolin 73 30 26 Cefuroxime 94 84 84 Cefotaxime 94 60 84 Ceftriaxone 92 62 76

Penicillins Amoxicillin/K clavulanate " " 2 Ampicillin — - 4 Ampicillin/Sulbactam - — 4 Oxacillin 96 84 96 Penicillin — 2 4 Ticarcillin/K clavulanate 94 68 98

Other 6-lactams Imipenem 10 6 4

Miscellaneous Chloramphenicol — — —

Ciprofloxacin 29 6 62 Clindamycin 92 86 92 Erythromycin 35 38 62 Nitrofurantoin ~ — —

Norfloxacin 31 2 46 Rifampin 49 20 26 Sulfamethoxazole 100 98 100 Tetracycline 37 88 56 T rimethoprim/Sulfamethoxazole 22 - 36 Vancomycin 2 — -

= Numbers in parentheses indicate number of isolates tested. All isolates were susceptible.

Table 2: Percentages of resistance among enterococci and fecal streptococci.

3 Indicates number of strains tested.

^Numbers indicate the percentage of strains resistant.

PAH isolates were susceptible.

<^/S= trimethoprim/sulfamethoxazole.

Aminoglycosides

& ^ Q

0 to 5 S ^ _ o

I i f I i ! I I f I Ï I < 5 3 8 - § 4 § « g , S g ë â S 8 S S - | U j U j U j t j L j U j U j U j U j U j U j U j U j U j C o C / j u j

Amikacin 78 50 56 88 59 71 92 45 ÏÔÔ ÏÔ ÏÔÔ 50 - - ^ Gentamicin 60 67 - 56 70 65 57 75 45 100 10 100 88 06ntârnicin synsrQy 60 "• •* *" 24 — — — — — -- -- — — — — Streptomycin synergy 22 6 14 50 6

Cephalothin 20 22 — 56 42 62 71 50 18 50 20 50 — — 100 59 Cefazolin 20 22 — 56 69 68 86 58 36 83 40 50 50 -- 100 73 Cefuroxime 100 78 50 89 87 94 100 67 82 100 60 100 50 — 100 94 Cefotaxime 20 56 — 89 86 79 100 67 36 83 30 100 50 —- — 94 Ceftriaxone 20 44 — 78 82 82 100 67 45 85 20 100 50 —— — 92 Penicillins Amoxicililn/K clavulanate — -- — — — 3 — — — — -- -- " — -- —

Ampicillin — — — — — 6 —- — — -- — — — -- — —

Ampicillin/Sulbactam - - ~ — — 6 14 — — — — — — — — —

Oxacillin 100 100 — 89 93 91 100 67 100 100 80 100 100 — 100 96 Penicillin — — — — — 9 — — — — 50 — —- -- —

Ticarcillin/K clavulanate 100 100 — 67 90 79 100 67 82 100 20 100 100 — ICQ 94 Other Q-lactams Imipenem — 11 — — 1 21 14 17 9 " — -- " — - 10 Miscellaneous Chloramphenicol — — — — 2 — — — — — — — — " — —

Ciprofloxacin 80 56 — 22 40 35 29 8 27 33 — — — 75 " 29 Clindamycin 80 100 100 78 93 88 86 92 73 100 60 100 100 -- 100 92 Erythromycin 40 33 100 44 46 53 14 8 36 33 40 50 — — -- 35 Nitrofurantoin — — — — — — " — — -- — —— -- —— -- ——

Norfloxacin 60 33 — 22 33 21 29 17 27 67 -- — — 100 —» 31 Rifampin " 22 — 11 28 53 29 8 9 " -- 50 50 — 100 49 Sulfamethoxazole 100 100 100 100 95 100 100 100 100 100 70 100 100 100 100 100 Tetracycline 60 33 — 67 64 35 71 8 73 — 60 50 50 — -- 37 T/S" 40 22 50 22 24 24 100 8 -- 17 — —— 50 50 — 22 Vancomycin — — — — — — — 8

140

PAPER 5; COMPARISON OF FOUR LATEX AGGLUTINATION KITS TO RAPIDLY IDENTIFY LANCEFIELD GROUP D ENTEROCOCCI AND FECAL STREPTOCOCCI

141

Comparison of Four Latex Agglutination Kits to Rapidly Identify Lancefield Group D Enterococci and Fecal Streptococci

LINDA M. KNUDTSON AND PAUL A. HARTMAN*

Department of Microbiology, Immunology and Preventive Medicine Iowa State University, Ames, Iowa 50011

Telephone: (515)294-8824 FAX: (515)294-6019

•Corresponding author

142

ABSTRACT

Enterococci isolated from water (50 strains), clinical material (50

strains), pork products (25 strains), and other foods (25 strains) as well as 50

known strains were used to compare the abilities of four latex streptococcal

grouping kits to correctly identify group D isolates. The Streptex kit (Wellcome

Diagnostics) was 98.5% accurate and easiest to interpret. The Slidex Strepto

kit (Vitek Systems) and Strepslide kit (NCS Diagnostics) also were

acceptable; they were 96.5% and 96.0% accurate, respectively. When the

Bacto Strep Grouping kit (Difco Laboratories) was used, 99% of the group D

isolates were positive for both group D and group B, including enterococcal

strains that are group D-negative.

143

Most streptococci, enterococci, and lactococci possess group-specific

antigens, wliich are usually carbohydrate structural components of the cell

wall. Group D antigens, however, are glycerol teichoic acids. The teichoic

acids are buried in the cell wall, which makes them difficult to detect (3).

Lancefield (5) showed that these antigens could be extracted in soluble form

and identified by precipitin reactions with appropriate antisera. The

Lancefield precipitin procedure is tedious and labor intensive and has

essentially been replaced by convenient latex agglutination and

coagglutination methods. In these methods, latex beads are coated with

antisera to group-specific antigens. These latex particles agglutinate strongly

in the presence of the homologous antigen and remain in smooth suspension

if the specific antigen is absent.

Enterococci and fecal streptococci belong to Lancefield group D. Four

latex kits for the grouping of streptococci were compared for their abilities to

accurately identify 200 Lancefield group D bacteria. The four kits were the

Streptex kit (Wellcome Diagnostics, Research Triangle Park, N. C.), Slidex

Strepto-Kit (Vitek Systems, Hazelwood, Mo.), Strepslide (NCS Diagnostics,

Buffalo, N. Y.), and Bacto Strep Grouping kit (Difco, Laboratories, Detroit,

Mich.). The cultures examined included 50 enterococci and fecal streptococci

isolates from culture collections, 50 isolates from water, 50 from clinical

material, 25 from pork products, and 25 from other foods (4).

Cultures from the five sources were retrieved from frozen storage

(-70°C in 10% glycerol) and grown on Brain Heart Infusion agar (Difco) plates

to ensure culture purity. Colonies from these plates were used to perform

tests on all four kits according to the manufacturer's instructions for each kit.

144

The Streptex kit (Wellcome) employs an enzyme extraction procedure

using a proteolytic fraction from Streptomyces griseus. The extraction

procedure included a 10-minute incubation at 37°C that was simple to

perform. Of the 200 enterococci and fecal streptococci tested, only three

isolates identified as enterococci were group D-negative (1 E. faecium from

water, 1 E. faecalis from pork, and 1 E. faecium from other foods). There were

10 instances in which enterococci were positive for Lancefileld group G in

addition to group D (3 E. malodoratus an6 7 E faecalis), and 9 of these strains

were also group G-positive when tested with other kits. Some strains of group

D enterococci also possess group G antigen (1); this point is discussed by the

manufacturers.

The Slidex Strepto-Kit (Vitek) also employs an enzyme extraction, but

mutanolysin from Streptomyces globisporus is used in this kit. The extraction

procedure was essentially the same as for the Streptex procedure. Of the

same 200 enterococci and fecal streptococci tested, 193 (96.5%) were group

D-positive. Nine isolates produced strong group D agglutination and weak

agglutination with all other antisera. The manufacturer stated that strong

agglutination of one of the latex suspensions indicates the group of the test

organism and that weak reactions that occur with other latex suspensions

should be ignored. Thus, the weak false-positive reactions were an

annoyance but did not result in false identification if care was taken in

interpreting the results. Seven isolates were group D-negative with the Slidex

Strepto kit (4 E. faecium, 1 E. durans, 1 unidentified enterococcus from water,

and 1 E. durans from food). No isolates positive for group D and group G only

were observed.

145

The Strepslide (NCS Diagnostics) employs an extraction enzyme of

unspecified origin. A 10-minute incubation period at 37°C is used as with the

other two kits. When the 200 isolates were tested with this kit, 192 were group

D-positive, and 8 were group D-negative (3 E. faecium, 2 E. faecalis and 1

E.c/urans from water, and 1 E. faecium and 1 E. Wrae from food). There were

25 instances of agglutination with other group specific antisera (2 with group

B, 2 with group C, 7 with group F, and 1 with all groups). Most (but not all)

reactions were weaker than the agglutination reaction with group D. All these

isolates were accurately grouped by one of the other kits. There were 12

isolates that reacted with both group D and group G antisera (2 E.

malodoratus, 7 E. faecalis, 2 E. faecium, and 1 E. gallinarum)] the E.

malodoratus and E. faecalis were the same as those seen with the Streptex

kit.

The Bacto Strep Grouping Kit (Difco) employs an acid-dependent

extraction procedure. Antigen Extractant 1 contains a pH indicator that

changes color with the addition of Extractants 2 and 3. When combined and

allowed to react with bacteria, these extractants release antigenic material

from the bacterial cell walls. There is a 5-minute room-temperature incubation

with this kit. For suspected group B or group D isolates, the antigen extraction

procedure is replaced by a specific procedure for groups B and D. This

procedure entails direct testing of colonies from an agar plate by mixing a loop

of inoculum directly into a drop of latex suspension. When the 200 isolates

were tested by using both methods (the extraction method for groups A, C, F,

and G and the direct method for groups B and D) only two were group D-

negative (1 E. malodoratus from pork, and 1 S. bovis from other foods).

146

Agglutination was seen in 7 instances with group F, and in 13 instances with

the group G antisera (5 E. hirae, 3 E. faecalis, 2 E. malodoratus, 1 £ faecium,

1 E. casseliflavus, and 1 unidentified enterococcus). Only 3 of the group G-

positive isolates (2 E. faecalis and 1 E. malodoratus) were positive on the

Streptex or Strepslide kits. Some form of agglutination or clumping was

observed in almost every test with the group B and D latex. The agglutination

patterns were uneven and very difficult to interpret.

With the first three kits (Streptex, Slidex, and Strepslide) E.cecorum and

E. pseudoavium were negative for the group D antigen, which would be

expected because these two species of enterococci are group D-negative (2,

6). Birch et al. (1 ) stated that half of the £. faecalis isolates tested possessed

both the group D and group G antigen. In addition to 7 E. faecalis isolates (2

from water, 2 from pork, and 3 from food), 2 isolates of E. malodoratus (from

water) were consistently group D- and G-positive. The other instances of

group G positives probably were anomalous results. The Streptex kit

(Wellcome) was 98.5% accurate for the identification of group D-positive

enterococci and fecal streptococci. All procedures were straightfonmrard, easy

to perform, and required a minimum of time and attention. The Bacto Strep

Grouping kit (Difco) acid extraction procedure, on the other hand, was more

time consuming because of the number of extractants used, and the kit was

inaccurate for identifying group D isolates. Both the Strepslide (NCS) and

Slidex (Vitek) kits were acceptable in their abilities to identify group D

enterococci and fecal streptococci from clinical and environmental sources.

147

ACKNOWLEDGMENT

Supported by the U.S. Department of Agriculture grant to the Food

Safety Consortium. Journal Paper No. J-15097 of the Iowa Agriculture and

Home Economics Experiment Station, Ames; project 2991.

148

LITERATURE CITED

1. Birch, B. R., G. L. Keaney, and L. A. Ganguii. 1984. Antibiotic

susceptibility and biochemical properties of Streptococcus faecalis strains

reacting with both D and G antisera. J. Clin. Pathol. 37:1289-1292.

2. Collins, M. D., R. R. Facldam, J. A. E. Farrow, and R.

Williamson. 1989. Enterococcus raffinosus sp. nov., Enterococcus

solitarius sp. nov. and Enterococcus pseudoavium sp, nov. FEMS

Microbiol. Lett. 57:283-288.

3. Facklam, R. R., and J. A. Washington III. 1991. Streptococcus and

related catalase-negative gram-positive cocci, pp. 238-257. In A. Balows,

W. J. Hausler, K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (eds.),

Manual of Clinical Microbiology. 5th ed. American Society for

Microbiology, Washington, D. C.

4. Knudtson, L. M. 1992. Classification of enterococci and their roles in

spoilage of pork products and as sanitary indicators in pork processing.

Ph.D. thesis. Iowa State University, Ames.

5. Lancefield, R. 0. 1933. A serological differentiation of human and other

groups of hemolytic streptococci. J. Exp. Med. 57:571-595.

149

6. Williams, A. M., J. A. E. Farrow, and M. D. Collins. 1989. Reverse

transcriptase sequencing of 16S ribosomai RNA from Streptococcus

cecorum. Lett. Appl. IVIicrobiol. 8:185-189.

150

GENERAL SUMMARY AND DISCUSSION

Over the past six years, a revised classification of the streptococci and

enterococci has emerged that is based primarily on molecular techniques

such as 16S rRNA sequencing and DNA-DNA hybridization studies. As a

result of these studies, the genus Streptococcus was divided into three

genera: Enterococcus, Lactococcus, and Streptococcus (132). The genus

Enterococcus now contains 19 species, including some species transferred

from the genus Streptococcus, new species that previously belonged to other

species, and new species not previously classified.

The first part of this dissertation involved devising classification

schemes to differentiate the species of the genus Enterococcus and

Streptococcus bovis and Streptococcus equinus. An identification system

using conventional tube tests exclusively was introduced in 1989 by Facklam

and Collins (53). However, conventional tube tests are cumbersome, costly

and labor intensive. Furthermore, our studies showed that their test results

could not be accurately reproduced. A comparison of my tube test results

with the published results of Facklam and Collins (53) revealed 17

discrepancies among 87 instances where comparisons could be made. Other

researchers (17) also have had difficulties reproducing Facklam and Collins'

results. When tube test results were compared with results obtained with the

API or MicroScan panels, 28 and 24 discrepancies, respectively, were

observed. These results indicate that Facklam and Collins' classification,

based on tube tests, should not be used to identify isolates tested with these

panels. There were also discrepancies (12) between the API Rapid Strep

151

panel and the MicroScan Pos ID panel. Some of these were possibly a result

of differences in methodologies between the two panels, but others could not

be explained.

A classification scheme was devised to identify the thirteen species of

EnterococcusXhaX belonged to the genus at the beginning of this project, as

well as S. bovis and S. equinus. Flow charts were fashioned of key tests that

could be used to differentiate these species. Three supplemental tests were

required to differentiate all of the species: pigmentation, motility and sucrose

fermentation.

The enterococci are important as potential indicators of fecal pollution

(7,108) because they are readily isolated in large numbers from human and

animal feces. The possibility of using enterococci as indicators of fecal

pollution by using differences in species distribution in different hosts as a

means to pinpoint the source of pollution has been suggested (7, 87,124,

126). Although none of the enterococci can be considered absolutely host

specific, some species do show a degree of host specificity (70). In swine,

Enterococcus faecium is the predominant organism of the intestinal tract,

whereas Enterococcus faecalis predominates in the intestinal tract of humans

(7, 87,124,126). With this in mind, the second part of my dissertation focuses

on using my newly devised classification scheme to identify enterococcal

isolates from hog carcasses during different stages in the slaughter process.

Fresh, expired and spoiled processed pork sausage also were examined.

Enterococci were enumerated and isolates were identified from these

samples to ascertain the possible fecal pollution present at different stages of

pork processing, and to pinpoint the origin(s) of that pollution. Results from

152

this study showed that enterococcal counts at different stages of the pork

process varied significantly at different plants. The polishing and back

scraping machines, as well as the scalding tank, contributed to higher

enterococcal counts in two plants. Counts from all three plants were lower

after the final rinse. In one plant counts increased significantly after a 24-hour

chill, whereas counts in the other two plants remained low. Since enterococci

are thermoduric organisms (64), even slight increases in the cooler

temperatures could have contributed to this large increase in enterococcal

counts. Since significant contamination occurred at this stage, it is a critical

control point for this plant. There are differences in many aspects of the

slaughter process at the three plants, which explains why HACCP plans must

be devised for each individual plant (146). The most interesting finding of this

study is the overall predominance of Enterococcus faecalis in all three plants

and at each processing stage as well. Because enterococci are associated

with the intestinal tracts of pigs, enterococci isolated from a hog carcass in a

slaughtering plant presumably would arise from fecal contamination of the

carcasses by hog fecal matter; therefore, £ faecium should predominate. The

predominance of E. faecalis indicates that hogs slaughtered in these three

plants during the 4-month sampling period had an intestinal flora in which E

faecalis predominated, or that the enterococcal contamination arose from

other sources. Once introduced into a slaughtering plant, enterococci can

become established, and the subsequent contamination of a food product, or

carcass, does not necessarily indicate fecal pollution. If this is the case, the E

faecalis might have been introduced by air contamination, human

contamination (E. faecalis predominates in the human intestine), or improper

153

cleaning of surfaces or equipment. Further studies are being conducted by

other investigators to pinpoint possible sources of this contamination.

Both fresh and expired pork sausage yielded low enterococcal counts

that were predominantly E. faecalis. When expired pork samples were

examined, high numbers of lactobacilli grew on the enterococcal media

resulting in falsely high counts and masking enterococcal colonies. Spoiled

pork sausage, on the other hand, yielded an almost pure culture of E. faecium.

It is possible that the contamination of hog carcasses with fecal matter occurs

infrequently: when it does occur, contamination of a single carcass may be

sufficient to instigate spoilage of sausage or hams made from the carcass.

Many media have been devised to isolate and enumerate enterococci.

KF streptococcal (KF) agar (86) contains, among other ingredients, sodium

azide, bromcresol purple, and 2, 3, 5-triphenyltetrazolium chloride (TTC). The

sodium azide selects for enterococci (72), and the reduction of tetrazolium

(TTC) differentiates E. faecalis, which strongly reduces tetrazolium, from other

enterococci which reduce tetrazolium weakly if at all. Fluorescent gentamicin-

thallous-carbonate (fGTC) agar contains gentamicin and thallous acetate (45)

as the selective agents, and NaHCOa, Tween 80 and KH2PO4 to stimulate

the growth of group D streptococci (90). The incorporation of amylose azure

and 4-methylumbelliferyl-a-D-galactoside allowed differentiation of fecal

streptococci into three categories (97). These two media were compared for

their abilities to enumerate enterococci and fecal streptococci in pork, beef,

and poultry products. In every instance, enterococcus counts made with fGTC

agar were significantly higher than counts on KF agar. Testing of known

species of enterococci on each medium revealed that all tested species of

154

enterococci and fecal streptococci grew on fGTC agar, whereas three species

(E. cecorum, S. bovis, and S. equinus) failed to grow on KF agar. Since these

species were not isolated from the meat samples with any frequency, their

inability to grow on KF agar could not account for the differences in counts

obtained by using the two agars. Identities of the enterococci isolated from the

pork, beef, and poultry samples, revealed that only Enterococcus durans and

Enterococcus casseliflavus were isolated on fGTC agar but not KF agar. The

percentages of other enterococci isolated were similar between the two

media. One explanation for the difference in counts is that a larger proportion

of injured enterococci grew on fGTC agar than on KF agar. FGTC agar is as

good as, if not better than, KF agar to enumerate enterococci and fecal

streptococci in foods. Some care must be exercised, however, when using

fGTC agar. When samples heavily contaminated with lactobacilli were plated

on fGTC agar, enterococcal colonies could not be discerned among a heavy

background of Lactobacillus colonies.

Recent attention has focused on enterococci because of their

remarkable and increasing resistance to antimicrobial agents (114). This

resistance allows them to survive in environments in which antimicrobial

agents are heavily used. Antimicrobials are given to food animals, including

swine, to improve growth rate and feed conversion. It has been suggested

that antimicrobial drugs to which food animals are exposed provide selective

pressure that leads to the appearance of antimicrobial resistant strains in food

animals (23). Enterococci and fecal streptococci collected from pork, water,

and clinical infections were tested for resistance to 27 antibiotics. Enterococci

isolated from pork samples showed less resistance than enterococci isolated

155

from water or clinical material. If food animals harbor resistant bacteria as

suggested, levels of resistance from pork isolates would be expected to be at

least as high as those from other sources. When antimicrobial resistance

patterns of different species of enterococci were compared, some trends were

seen. Almost all species of enterococci showed some degree of resistance to

the aminoglycosides, cephalosporins, the semisynthetic penicillins, and

clindamycin. Since all enterococci carry intrinsic resistance to these classes

of antimicrobials, these results were expected. Enterococcus faecium

appears to carry the highest amount of resistance to the p-lactam

antimicrobials, especially ampicillin, whereas E. faecalis strains appeared to

be more often resistant to the synergistic affects of the antibiotic combinations

tested.

The majority of streptococci, enterococci, and lactococci possess

group specific antigens which are usually carbohydrate structural components

of the cell wall. Group D antigens, however, are glycerol teichoic acids that

are buried deep in the cell wall. Because of the intracellular nature of these

teichoic acids, identification of group D isolates is difficult. Lancefield

discovered that these antigens could be extracted in soluble form and

identified by precipitin reactions with appropriate antisera. The precipitin

reaction has largely been replaced by methods in which latex beads are

coated with antisera to specific group antigens. These latex particles

agglutinate strongly in the presence of homologous antigen, and are easy to

visualize. In the final paper of this dissertation, four streptococcal grouping

kits (Streptex, Slidex, Strepslide, and Bacto) were compared for their abilities

to accurately type the group D enterococci. Isolates from culture collections.

156

water, clinical infections, pork, and other foods were tested. The Streptex

(Wellcome) kit results yielded the fewest false-negative results for group D

antigen and no false-positive results were observed. When the Slidex (Vitek)

and Strepslide (NCS) kits were used, some false-positive results were

observed. Weak agglutination reactions with antisera specific for other groups

also were observed with the latter two kits. The Bacto strep grouping kit

(Difco) employed a different procedure for identifying groups B and D which

resulted in clumps and agglutination in every group B and D test, regardless

of the group of the isolate. I recommend that the Difco kit not be used to type

presumptive group D isolates.

157

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171

AKNOWLEDGMENTS

First, I must thank my major professor, Paul A. Hartman. He allowed me

to find my own way, while never failing to support me. I would also like to

thank my committee members, Eisa Murano, Bonita Glatz, Lynn Knipe, and

Peter Pattee, for all their help and support. I thank Chris Guyers, our lab

technician, for getting up at 5 AM to swab hog carcasses so I didn't have to do

it.

Special thanks to my friends, both grad students and others, who stuck

with me even when I disappeared from the social circle for long periods of

time (like at prelims and thesis writing time): and to my family, who sounded

excited when I talked about my latest experiment, even though they had no

idea what I was talking about!

To my husband Kevin, because no matter what happens during my

professional career; I received something even more valuable than my Ph.D.

during my graduate school career, my wedding ring.

172

APPENDIX A: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM PORK

CARCASSES SAMPLED IN THREE PLANTS

173

TABLE Al : Pork carcass mean counts (of duplicate plates) from plant A

LOIN MEDIUM (LEAN) HAM SPECIES IDENTIFIED

RUN& STAGE

RUN1 STAGE 1 fGTC

KF 3.9X10^

<50 1.4X10

<50

STAGE 2 fGTC KF

7.5X10^ <50

1.4X10 <50

STAGE 3 fGTC KF

3.0X10^ 7.9X10''

1.4X10 4.6X10

STAGE 4 fGTC KF

1.3X10^ 1.5X10^

RUN 2 STAGE 1 fGTC

KF 3.1 X10^ 9.0X10^

7.3X10 9.0X10

STAGE 2 fGTC KF

<50 <50

<50 <50

STAGE 3 fGTC KF

6.6X10^ 1.4X10^

2.3X10 3.4X10

STAGE 4 fGTC KF

3.0X10^ 1.9X10^

RUN 3 STAGE 1 fGTC

KF 2.7X10^ 1.8X10^

1.5X10 7.5X10

STAGE 2 fGTC KF

<50 <50

6.3X10 6.3X10

STAGE 3 fGTC KF

<50 <50

1.0X10 4.2X10

STAGE 4 fGTC KF

1.1 XIO^ 6.2X10^

E. solitarius No enterococci

No enterococci No enterococci

E faecalis E. faecalis

E. faecalis, E. malodoratus E. faecalis

E. faecalis E. faecalis

No enterococci No enterococci

Unidentified enterococci E. faecalis

E. faecium, durans, pseudoavium E. faecalis, E. pseudoavium

E. faecalis E. faecalis

E. faecalis E. faecalis

E. faecalis E. faecalis

E. faecalis, faecium, malodoratus E. faecalis

174

TABLE A2: Pork carcass mean counts (of duplicate plates) from plant B

RUN& STAGE MEDIUM

LOIN (LEAN) HAM SPECIES IDENTIFIED

RUN1 STAGE 1 fGTC

KF 7.9X10^

<50 <50 <50

No enterococci No enterococci

STAGE 2 fGTC KF

<50 <50

1.3X10^ <50

No enterococci No enterococci

STAGE 3 fGTC KF

5.5X10^ <50

CO CM

O O

X X

T

- CO

U5

CM

E faecalis, E. faecium E. faecalis

STAGE 4 fGTC KF

2.5X10^ 1.7X10'^

E. faecalis, faecium, casseliflavus E. faecalis, E.pseudoavium

RUN 2 STAGE 1 fGTC

KF X

X

O

CO T

-' CO

<50 <50

No enterococci No enterococci

STAGE 2 fGTC KF

1.7X10^ 7.9X10^

1.4X10^ 7.9X10^

E. faecalis, E. faecium E. faecalis, E. faecium

STAGE 3 fGTC KF

7.9 Xio] 7.9X10^

6.3 Xio] 6.3X10^

E. faecalis, E. faecium E. faecalis

STAGE 4 fGTC KF

9.2 Xio] 6.3X10^

E. faecalis, E. malodoratus E. faecalis

RUNS STAGE 1 fGTC

KF 1.1 xicf

<50

1— T—

o o

X X

CO

CO CO

CO

E. faecalis No enterococci

STAGE 2 fGTC KF

<50 <50

6.3 xio] 6.3X10''

No enterococci No enterococci

STAGE 3 fGTC KF

1.4X10^ <50

<50 <50

E. faecalis, E. faecium No enterococci

STAGE 4 fGTC KF

7.1 Xio] 5.6X10^

E. faecalis

175

Table A3: Pork carcass mean counts (of duplicate plates) from plant C

RUN & LOIN STAGE MEDUIM (LEAN) HAM SPECIES IDENTIFIED

RUN 1 STAGE 1 fGTC

KF 2.7X103 3.6X1O2

6.8X10 3 8.7X10 2

No enterococci No enterococci

STAGE 2 fGTC KF

CM CM

O O

7—

T— X

X

CO CO

CO CO

1.6X1O2 6.3X10 2

No enterococci No enterococci

STAGE 3 fGTC KF

<50 6.3X102

2.4X10 2 1.2X10 2

No enterococci E. faecalis

STAGE 4 fGTC KF

2.6X102 7JX10l

E. faecalis E. faecalis

RUN 2 STAGE 1 fGTC

KF 1.3X104 1.2X10^

1.1 xio4 6.0X10 2

E. faecalis E. faecalis

STAGE 2 fGTC KF

2.9X102 7.9X10"'

7.2X10 2 1.1 X102

No enterococci No enterococci

STAGE 3 fGTC KF

<50 <50

5.1 X102 2.7X10 2

E. faecalis E. faecalis

STAGE 4 fGTC KF

5.5X102 4.2X102

E. faecium, faecalis, solitàrius E. faecalis

RUNS STAGE 1 fGTC

KF 9.3X102 1.0X102

1.2X10 3 <50

E. faecalis E. faecalis

STAGE 2 fGTC KF

8.4X102 1.0X102

1.8X1O2 <50

No enterococci No enterococci

STAGE 3 fGTC KF

<50 <50

<50 <50

E. faecalis E. faecium

STAGE 4 fGTC KF

1.1 X102 6.7X101

E. faecalis E. pseudoavium

176

APPENDIX B: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS FROM

TWO PORK PROCESSING PLANTS

177

TABLE B1 : Mean counts (of duplicate plates) from pork processing plant A

KF fGTC TOTAL STREPTOCOCCAL ENTEROCOCCAL SPECIES

SAMPLE COUNT COUNT COUNT IDENTIFIED

FRESH PORK SAUSAGE

SPOILED PORK SAUSAGE

ND 7.5X10; 1.0X10® 1.2X10®

<50 <50 <50 <50

2.0X10'=^ 1.0X10^ 1.0X10^ 1.0X10^

2.5X10® 2.5 XIO® 1.2X10®

1.7X10® 2.2X10® 3.3.x 10®

2.4X10® 2.0X10® 1.1X10®

4.0X10^ 1.4X10® 2.0X10^

2.2X10® 7.4X10^ 2.8X10; 3.0x10"

<50 <50

3.1 X104 I.OXIO'^

3.6X10® 6.1 X10^ 4.3X10® 6.4X10®

3.2X10^ 4.5X10® 8.8X10®

2.8X10^ 1.7X10® 1.4X10®

3.9X10® 2.0X10'

3.5 XIO 5 <50

2.0X10® 9.8X10®

100% E. faecalis No enterococci No enterococci No enterococci

100% E. faecium 100% E. faecium 67% E. faedum 33% E. durans 67% E.faecium 33% E. faecalis No enterococci No enterococci 100% E. faecium 50% E. faecium 50% £. facealis 80% E faecium 20% E, faecalis 83% E. faecium 17% E. faecalis 100% E faecium 50% E faecium 50% E faecalis

178

TABLE B2: Mean counts (of duplicate plates) from processing plant B

KF fGTC

SAMPLE TOTAL COUNT

STREPTOCOCCAL COUNT

ENTEROCOCCAL COUNT

SPECIES IDENTIFIED

FRESH PORK SAUSAGE

FRESH PORK SAUS. (LINK)

EXPIRED PORK SAUSAGE

EXPIRED PORK SAUS. (LINK)

1.5 X 2.0 X

1.1 X 1.2X 5.9 X 6.7 X 9.5 X

5.0 X

4.6 X 6.7 X

1.1 X 1.6 X 2.1 X 2.7 X 2.7 X 1.1 X 2.2 X 1.1 X 1.0X 9.8 X 1.0X 1.2X 1.5X 3.0 X 3.3 X

4.6 X 1.5X

Sa

S:

3.5 xio; 2.0 X 10'

2.5 x io ; 2.5X10' <50 <50 8.5X10'

4.4X10'

2.1 X 10) 3.8X10'

7.0x10; 9.0X10; 1.0X10'

<50 , 1.0X10'

<50 <50 .

8.1 X 10: 5.2X10^ 3.9X10: 3.0X10; 5.7X10'

<50 <50 <50

1.0X10 3.0X10^

2.0 X 10 q 100% E faecalis 2.0X10 6W0 E. faecalis

- 33% E hirae 1.2X10p m%E. faecalis 9.0 X 10 4 100% E faecalis 4.7 X 10 . No enterococcl 3.6 X 10 . No enterococcl 6.0 X 10 50% E faecalis

50% E malodoratus 1.7X10 100% E faecalis

5 5.8 X 10 c No enterococcl 6.5 X 10 100% E malodoratus

p 1.0 X 10 g No enterococcl 1.0 X 10 p No enterococcl 1.2X10 g m%E. faecalis 1.1 X10 g No enterococcl 1.7 XIO g m%E. faecalis 1.1 XIO g No enterococcl 1.5X 10 y No enterococcl 7.5 X 10 y No enterococcl 8.8 X 10 y No enterococcl 6.2 X 10 y No enterococcl 5.5 X 10 y No enterococcl 7.4 X 10 g No enterococcl 6.8 X 10 y No enterococcl 9.6 X 10 g No enterococcl 2.1X10 No enterococcl

Q 1.4 X 10 g No enterococcl 4.2X10 50% E. faecalis

50% E pseudoavium

PORK TRIM 1.6 X 0® 4.2X1 of 1.4X10^ 100% E faecalis (42%) 4.8 X 0^ 4.8 XIO* 1.4X10 No enterococcl

PORK TRIM 1.1 X 07 5.3X10^ 2.6X10® 100% E faecalis (72%) 6.5 X 0^ 1.6X10° 1.9X10 33% E faecalis

33% E faecium 33% E malodoratus

179

APPENDIX C: COUNTS AND ENTEROCOCCAL IDENTIFICATIONS

FROM RETAIL SAMPLES

180

Table Cl : Mean counts (of duplicate plates) from retail samples Total fGTC KF

Retail Sample Count Count Count Species ID Pork

Hormel chopped ham ND 1.5X10 5 <50 S. bovis Fresh pork sausage ND 8.9X10^ 9.6X10^ S. salivarius Hillshire farms fresh bratworst ND 7.0X10% 2.0X10^ No enterococci Mild Italian pork sausage ND 1.4X104 4.0X10^ E faecium Boneless pork loin ND 6.0X104 3.1X10^ E. faecalis Iowa pork chop ND 1.0X102 <50 No enterococci Fareway bratworst ND 3.5X10^ 5.6X10^ No enterococci JD hot pork sausage ND 2.0X10% <50 E faecalis Purnell's country pork sausage ND 2.0X10% i.oxio; No enterococci Farmland pork sausage ND 1.7X10 3 4.0X10% No enterococci JD sage pork sausage 7.5X10^ 1.0X102 <50 No enterococci JD regular pork sausage 1.0X10^ 1.0X10% <50 No enterococci Oldham's whole hog pork sausage 5.6X10^ 5.6X10% <50 E faecalis JD turkey and pork sausage 1.2X10^ 1.0X10% <50 No enterococci

Beef Signature ground beef (93% lean) 1.4X10^ 1.4X10^ 3.1X10^ E.durans/S. bovis Ground Beef (90% lean) 7.7X10; 3.1X10] 2.9X10 3 E.durans/malocloratus Ground Beef (85% lean) 2.0X10^ 9.3xio; 2.3X10^ E malodoratus Ground Beef (70% lean) 3.9X10j 4.3X10^ 2.3X10^ E.durans/faecalis Banquet Beef pot pie i.oxio; 2.5X10% <50 No enterococci

Swanson Beef pot pie 3.5X10^ 1.0X10% 1.0X10^ E faecalis Armour beef & bacon pattie <50 <50 <50 No enterococci

Poultry Chicken breast tenders 2.1X10^ <50 <50 No enterococci Whole frier (chicken) 2.0X10^ 3.0X10^ <50 No enterococci Country pride ground chicken s.zxio'^ 5.9X10^ 1.0X10^ E faecalis Banquet chicken pot pie 1.4X10^ <50 <50 No enterococci Swanson chicken pot pie 7.5X10^ <50 <50 No enterococci Banquet turkey pot pie 6.0X10^ 1.0X10% <50 No enterococci Swanson turkey pot pie 1.0X10^ <50 <50 No enterococci Longacre ground turkey 1.9X10"^ 8.6X10^ 6.0X10^ E.faecalls/faecium Armour ground turkey 1.4X104 1.3X10^ <50 No enterococci Louis Rich ground turkey 2.7X104 7.9X10^ <50 E faecalis

Cheese Hy-Vee mild Cheddar cheese 7.4X10'^ 2.3X10"^ <50 No enterococci Kraft Havarti cheese 3.0X10^ 4.4X10^ 1.4X10^ No enterococci Kraft Swiss cheese 2.2X10® 3.4X101 2.8X10 J E faecium Kraft natural gouda cheese ND 3.0X10^ 2.5X10^ E durans

181

APPENDIX D: MEAN COUNTS AND ENTEROCOCCAL IDENTIFICATIONS ' '

FROM WATER SAMPLES

182

Table D1 : Mean counts from water samples mENTERO

mENDO enterococci SOURCE coliforms/100ml /100ml SPECIES IDENTIFIED

LAKE SAMPLES Lake Laverne 56 34 E. faecium

JACUZZI SAMPLES ATFC 0 0

Gateway 0 0 Starllte 0 0

RIVER SAMPLES College Creek-1 TNTC 184 E. faecium E. faecalis College Creek-2 4520 234 E. faecium

DM River -1 200 820 E. faecium E. faecalis DM Rlver-5 10 20 E. faecium E. faecalis E. casseliflavus DM River-6 1320 520 E. faecium E. faecalis

K Creek 720 240 E. durans E. hirae Racoon River-10 470 720 E. faecium E. faecalis Skunk River -1 700 740 E. faecium E. mundtii E. casseliflavus Skunk River-2 2380 1200 E. gallinarum Squaw Creek-1 237 1000 E. faecium E. faecalis E. casseliflavus Squaw Creek-2 2150 1270 E. malodoratus

Sugar Creek 366 700 E. faedum E mundtii E. durans Worle Creek-1 810 780 E faecalis E. hirae Worle Creek-2 1370 1870 E. hirae E. gallinarum

WELL SAMPLES Mills Co.-Residence 0 0

Tama Co.-Residence 0 0 Red Rock R-87-4 0 0 Red Rock 5-RB 0 0 Red Rock 23-R 0 0 Red Rock 5-RA 0 0 Red Rock 30-0 0 0 Red Rock 29-0 0 0

Corn Field Wells -6B 6ft. 645 3 E. faecalis -6B 8ft. 0 2 E. faecalis -68 10ft. 20 5 E. faedum E. malodoratus -6B12ft. 0 11 Staphylococcus -8B 6ft. 0 38 Staphylococcus -8B 8ft. 0 10 -88 10ft. 0 1 -88 12ft. 0 1 E. casseliflavus

183

APPENDIX E; SOURCE AND IDENTIFICATIONS OF CLINICAL ISOLATES FROM

MERCY HOSPITAL MEDICAL CENTER DES MOINES, IOWA

184

TABLE E1 : Source and identification of clinical isolates

Isolate # Source

Species Identified

Isolate # Source

Species Identified

1 Urine E. faecalis 26 Genital E. faecalis

2 Genital E. faecalis 27 Genital E. faecalis

3 Urine E. faecalis 28 Urine E. faecalis

4 Urine E. faecalis 29 Urine E. faecalis

5 Urine E. faecium 30 Urine E. faecalis

6 Urine E. faecalis 31 Urine E. faecalis

7 Urine E. faecalis 32 Unknown E. faecalis

8 Urine E. faecalis 33 Urine E. faecalis

9 Urine E. faecalis 34 Unknown E. malodoratus

10 Urine E. faecalis 35 Urine E. faecalis

11 Urine E. faecium 36 Vaginal E. faecalis

12 Urine E. faecalis 37 Urine E. faecalis

13 Urine E. faecium 38 Urine E. faecalis

14 Bile E. faecium 39 Urine E. faecalis

15 Urine E. faecalis 40 Urine E. faecalis

16 Urine E. faecalis 41 Urine E. faecalis

17 Genital E. faecalis 42 Unknown E. faecalis

18 Urine E. faecalis 43 Unknown E. faecium

19 Urine E. faecalis 44 Blood E. faecalis

20 Urine E. faecalis 45 Urine E. faecalis

21 Urine E. faecalis 46 Unknown E. faecalis

22 Urine E. faecalis 47 Urine E. faecalis

23 Urine E. faecalis 48 Unknown E. faecalis

24 Urine E. faecalis 49 Urine E. faecalis

25 Urine E. faecalis 50 Urine E. faecalis