10.1007_s10661-010-1578-1

Environ Monit Assess (2011) 176:225–238DOI 10.1007/s10661-010-1578-1

Molecular quantification of virulence gene-containingAeromonas in water samples collected from differentdrinking water treatment processes

Chang-Ping Yu · Kung-Hui Chu

Received: 20 December 2009 / Accepted: 15 June 2010 / Published online: 16 July 2010© Springer Science+Business Media B.V. 2010

Abstract Pathogenic species of Aeromonas pro-duce a range of virulence factors, includingaerolysin, cytotonic enterotoxins, and serine pro-tease, to cause acute gastroenteritis and woundinfections in humans and animals. Recogniz-ing that not all Aeromonas strains are patho-genic, in this study, we proposed to evaluateAeromonas removal effectiveness based on thepresence of virulence gene-containing Aeromonasas a proper means to assess microbial risk ofAeromonas. We developed and applied real-timePCR assays to quantify serine protease (ser) gene-and heat-labile cytotonic enterotoxin (alt) gene-containing Aeromonas in water samples. Among18 Aeromonas isolates from the source water,only three isolates possessed all three genes (aer,ser, and alt). A higher percent of isolates haseither ser gene (89%) or alt gene (72%) comparedto the percent of isolates containing aer gene(44%). Results of this study suggested that severaldifferent conventional and unconventional drink-

C.-P. YuKey Laboratory of Urban Environment and Health,Institute of Urban Environment,Chinese Academy of Sciences, Xiamen, 361021, China

K.-H. Chu (B)Zachry Department of Civil Engineering,Texas A&M University, College Station,TX, 77843-3136, USAe-mail: [email protected]

ing water treatment processes could effectivelyremove Aeromonas from source water. As thecomprehensive knowledge of the distribution ofvirulence factors in different Aeromonas speciesis currently not available, using real-time PCR toquantify various virulence factor genes in watersamples and/or isolates can be a practical meansfor better assessment of microbial risks in water.

Keywords Virulence genes · Aeromonas ·Serine protease gene · Heat-labile cytotonicenterotoxin gene · Drinking water

Introduction

Waterborne pathogens continue to be a majorhealth hazard in the United States and othercountries throughout the world. Consumption ofpublic drinking water is estimated to cause ap-proximately 4.26–32.80 million endemic water-borne acute gastrointestinal illnesses per year inthe United States according to two recent stud-ies (Colford et al. 2006; Messner et al. 2006).These infections, varying from diarrhea to respi-ratory disorders to heart diseases, are not causedby classic waterborne pathogens like Vibriocholerae and Salmonella typhi, but by a num-ber of emerging pathogens such as pathogenicEscherichia coli, Cryptosporidium and Giardia,pathogenic Yersinia, Campylobacter, Legionella,

226 Environ Monit Assess (2011) 176:225–238

Pseudomonas, and Aeromonas (Szewzyk et al.2000). Children, the elderly, and immunocompro-mised individuals, which account for 20–25% ofthe US population, are particularly vulnerable tothese emerging pathogens. Among these emerg-ing pathogens, Aeromonas was on the 1998 En-vironmental Protection Agency Drinking WaterContaminant Candidate List 2.

Assessment of microbial risk in drinking wa-ter is challenging and commonly limited by sev-eral factors. Similar to chemical risk assessment,prior knowledge of a dose–response relationshipis needed for assessing microbial risk in water.However, such information is not readily availableand limited only to a few emerging pathogens.For example, based on available dose–responserelationships, the calculated doses are 0.3 virusesper 100 L for enteric viruses and 0.2 cysts per100 L for Cryptosporidium (Rose and Gerba1991). Such levels are below the detection limitsof conventional methods used to monitor thesemicroorganisms. In addition, while several patho-genic Aeromonas and mycobacterial species havebeen recognized, not all environmental strains arepathogenic (Szewzyk et al. 2000). The ability todifferentiate as well as to quantify pathogenicspecies is essential for the assessment of microbialrisks in drinking water. The culture-based meth-ods for quantifying these groups of pathogens(such as EPA Method 1605 for Aeromonas)cannot differentiate pathogenic from nonpatho-genic species. Accordingly, the data obtained forculture-based methods might not be appropriatefor microbial risk assessment. Additionally, manywaterborne pathogens, including V. cholerae andAeromonas hydrophila, have been suggested tobe present in environmental waters as viable butnonculturable cells (VBNC) (Byrd et al. 1991;Colwell et al. 1996; Mary et al. 2002; Halpern et al.2007). While VBNC are not detectable by culture-dependent methods, they retain their virulencein water for a period of time and represent apotential risk.

Real-time PCR analysis has been accepted asa very rapid and sensitive quantitative molecularmethod with applications ranging from clinicalmicrobiology (Bieche et al. 1999; Mercier et al.1999) to molecular ecology (Suzuki et al. 2000;Gruntzig et al. 2001) and environmental micro-

biology (Beller et al. 2002; Dionisi et al. 2002;Kikuchi et al. 2002). In addition to its sensitivity inquantification, the real-time PCR assay can be de-signed for a specific strain, a phylogenetic group,and even a group of microorganisms exhibitinga similar function (Purkhold et al. 2000). SeveralPCR-based molecular techniques have been re-cently developed to make rapid identification ofemerging pathogens possible (Purohit and Kapley2002). However, most of these methods do notprovide the quantity of the emerging pathogensin the water sample—the most essential infor-mation for risk assessment. Consequently, thedevelopment of quantitative molecular methodsfor emerging pathogens is still in its early stage(Ibekwe and Grieve 2003).

The genus Aeromonas is a group of bacte-ria ubiquitous in aquatic environments, includ-ing lake and river water (Hazen et al. 1978),seawater (Cavallo et al. 2009), bottled mineralwater (Hunter 1993), and domestic water sup-plies (Knochel and Jeppesen 1990; Pablos et al.2009), even chlorinated water (LeChevallier et al.1982; Millership and Chattopadhyay 1985). SomeAeromonas species are considered as opportunis-tic waterborne pathogens responsible for acutegastroenteritis and wound infections in humansand animals. These species can produce manyvirulence factors, such as cytotoxic enterotox-ins, cytotonic enterotoxins, proteases, leukocidin,phospholipase, endotoxin, and outer membraneproteins (Howard et al. 1996). A. hydrophila,Aeromonas sobria, and Aeromonas caviae are thethree most common species associated with hu-man gastrointestinal infections (USEPA 2006).Strains of Aeromonas trota have been shown tocarry an aerolysin gene (a toxin that forms poreson host cells) and, therefore, are also consideredenteropathogenic (Khan et al. 1998, 1999).

Aeromonas spp. are known to be sensitive tochlorine (Gavriel et al. 1998), although high num-bers of several Aeromonas species have been iso-lated from biofilms in chlorinated drinking waterdistribution systems (van der Kooij and Hijnen1988; Gibbs et al. 2004; Tokajian and Hashwa2004). Because Aeromonas can grow on smallamounts of nutrients (in the range of 1 μg L−1), re-growth occurs in most drinking water distributionsystems (Havelaar et al. 1990; Kuhn et al. 1997).

Environ Monit Assess (2011) 176:225–238 227

Nevertheless, the significance of Aeromonas spp.in drinking water to the occurrence of gastro-intestinal infections is not fully understood.The National Research Council’s Committee onDrinking Water Contaminants (Washington, DC)has recommended a comprehensive approachbased on the virulence factor activity relation-ships as a means to predict potential risks from awide range of pathogens in drinking water. Theneeds to know better about the distribution andgenetic level of virulence genes of waterbornepathogens are supported by many previous studies(Tourlousse et al. 2007; Miller et al. 2008). Thesestudies have suggested that relying on only onesingle virulence gene of pathogenic strains mightpossibly lead to incorrect assessment of the micro-bial risk of waterborne diseases because (1) manyvirulence genes are unevenly distributed amongdifferent pathogenic strains, (2) many virulencegenes are multifunctional, and (3) some virulencecan be even found in nonpathogenic strains.

To better understand the risk of Aeromonasin drinking water, in this study, we had devel-oped a set of real-time PCR assays to quantifytwo virulence genes coded for heat-labile cyto-tonic enterotoxin (alt) and serine protease (ser) ofAeromonas species in source and drinking water.The heat-labile cytotonic enterotoxin has been ex-tensively studied (Chopra et al. 1986; Challapalliet al. 1988; Chopra et al. 1996). Serine proteaseis a degradative enzyme which aids the invasionof host cells, induces cell damage, and causes celldeath (Howard et al. 1996). The developed assayswere used to quantify the genes of these virulencefactors of Aeromonas in source, intermediate,and finished drinking water from seven full-scalewater utilities and one pilot plant. Results ofthis study were also compared to the total andaerolysin gene (aer)-containing Aeromonas re-ported previously (Yu et al. 2008).

Materials and methods

Strains

Both Aeromonas (22 strains) and non-Aeromonas(18 strains) bacteria were used to validate thequantitative molecular assays developed in this

study. The Aeromonas bacteria include fourstrains purchased from the American Type Cul-ture Collection (ATCC; ATCC 7966T, ATCC15468, ATCC 35993, and ATCC 49657) and 18strains isolated from seven water treatment plantsstudied in our previous work (Yu et al. 2008).The non-Aeromonas bacteria include one E. coli(ATCC 25922), 15 estrogen-degrading bacteria(Fujii et al. 2002; Yu et al. 2007), and three strains(E. coli, Pseudomonas f luorescens, and Bacillusthuringiensis; Yu et al. 2005a).

Drinking water treatment plants and samplinglocations

Water samples were collected from differenttreatment units of seven drinking water treatmentplants (plants 1–7) and one pilot plant (Table 1)as described from Yu et al. (2008). Six out of eightplants used conventional treatment processes(plants 1–3 and plants 5–7). Plants 1–3 employedprechlorination (PreCl2) of the source water, fol-lowed by coagulation/flocculation/sedimentation(C/F/S), rapid sand filtration (RSF), and chlorinedisinfection (Cl2). Plant 5, without using PreCl2,used C/F/S, RSF, and UV and chlorine disinfec-tion. For plants 6–7, source water was treatedby using slow sand filtration (SSF) and chlorineor chloramine for disinfection. Plant 4 and thepilot plant employed unconventional treatmentprocesses. For plant 4, source water is directlytreated by membrane filtration, followed by UVdisinfection and final addition of NaOH and chlo-rine. The pilot plant employed preozonation ofsource water and SSF. The turbidity values ofsource water for plants 1–7 were 3.20, 3.63, 6.60,2.65, 0.50, 0.35, and 0.80, respectively. Note thatthe pilot plant and plant 7 used the same sourcewater. Detailed information of sampling proce-dure, sampling locations and numbers are avail-able in Yu et al. (2008).

Real-time PCR assays for quantifying virulencegene-containing Aeromonas spp.

Our previous study had developed and ap-plied real-time PCR assays to quantify total andaerolysin gene-containing Aeromonas in source,intermediate, and finished drinking water (Yu


Tab

le1

Uni

tope

rati

onan

dsa

mpl

ing

loca

tion

sof

drin

king

wat

ertr

eatm

entp

lant

ssu

rvey

edin

this

stud

y(Y

uet

al.2

008)

Pla

ntD

aily

prod

ucti

onP

roce

ssfl

owdi

agra

ms

(Loc

atio

n)(m

illio

nga

llons

per

day)

Con

vent

iona

ltre

atm

ent

proc

esse

s

Pla

nt1

(Ten

ness

ee)

1.7

SW

X

XX

RS

FD

S

Cl 2

B

1A

1

C

1

D1

Cl 2

Pla

nt2

(Ten

ness

ee)

5.1

SW

X

XX

R

SF

DS

C

l 2

Cl 2

A

2

B2

C

2

D2

Pla

nt3

(Ten

ness

ee)

7.9

SW

X

XX

R

SF

DS

C

l 2

Cl 2

A

3

B3

C

3

D3

Pla

nt5

(Geo

rgia

)9.

5S

W

XX

X

RS

F D

S

UV

Cl 2

A

5

B5

C

5

D5

Pla

nt6

(Ver

mon

t)2.

3S

W

SS

F D

S

Cl 2

A

6

C6

D

6

Pla

nt7

(Mai

ne)

0.3

SW

S

SF

DS

A

7

C7

D

7

Chl

oram

ine

Non

-con

vent

iona

l

trea

tmen

tpro

cess

es

Pla

nt4

(Ten

ness

ee)

2.5

SW

D

S U

V

A4

C

4 D

4a D

4b

Cl 2

+N

aOH

Pilo

tpla

nt(M

aine

)P

ilots

cale

SW

S

SF

DS

O

3 A

7

D8

SWS

O3

SSF

XX

X

Cl 2

DS

Chl

oram

ine

UV

: sou

rce

wat

er

: coa

gula

tion/

floc

cula

tion/

sedi

men

tatio

n

: rap

id s

and

filtr

atio

n

: chl

orin

e di

sinf

ectio

n

: dis

trib

utio

n sy

stem

: mem

bran

e fi

ltrat

ion

: UV

dis

infe

ctio

n

: slo

w s

and

filtr

atio

n : c

hlor

amin

e di

sinf

ectio

n

: ozo

ne d

isin

fect

ion

: sam

ple

loca

tion

RSF


et al. 2008). In this study, virulence genes ofserine protease (ser) and heat-labile cytotonicenterotoxin (alt) of Aeromonas spp. were usedas biomarkers for the development of real-timePCR assays. Table 2 lists the primer sets designedspecifically for the virulence genes.

Only published gene sequences of serine pro-tease of Aeromonas and the genes with a sufficientsequence length were used in the primer design. Aforward primer spF and a reverse primer spR weredesigned by aligning three published serine pro-tease gene sequences of Aeromonas (GenBankaccession numbers AF159142, Cascon et al. 2000;X67043, Whitby et al. 1992; and AY841795, Yuet al. 2005b; accessed on 1 December 2005). Theexpected product size for the ser gene is 104 bp.Primers altFM and altRM were modified from aprimer set (Granum et al. 1998) previously de-signed to target a heat-labile cytotonic enterotoxingene (accession number L77573; Chopra et al.1996) of A. hydrophila. The expected product sizefor the alt gene is 480 bp. The designed primersets were also examined for their uniqueness tothe target genes by comparing the sequences inGenBank using the Basic Local Alignment SearchTool (Altschul et al. 1990).

DNA extraction

Water samples were filtered through 0.2-μm-pore-size polycarbonate membrane filters (Whatman,Clifton, NJ) to retain bacteria cells on the mem-branes for DNA extraction. The DNA was ex-tracted directly from the filter membrane by usingthe UltraClean™ Water DNA Kit (MoBio Lab-oratories, Inc.) according to the manufacturer’sinstructions. The derived DNA samples were con-

centrated 20-fold by ethanol precipitation. Bothconcentrated and unconcentrated DNA sampleswere used as templates for SYBR®Green real-time PCR assays.

Real-time PCR analysis

The SYBR®Green real-time PCR assays forquantifying Aeromonas-containing genes of ser-ine protease and cytotonic enterotoxin were per-formed similarly, except that different annealingtemperatures were used. Each reaction was per-formed in a total volume of 25 μL, with Quanti-Tect SYBR Green PCR Master Mix (QIAGENInc., Valencia, CA), 600 nM forward and reverseprimers, and 5 μL of DNA templates. The PCRthermal cycle for both genes of Aeromonas was50◦C for 2 min and 95◦C for 15 min, followedby 36 cycles of 95◦C for 30 s, optimum annealingtemperature (60◦C for serine protease gene and62◦C for cytotonic enterotoxin gene) for 45 s, and72◦C for 30 s. PCR amplification and detectionwere performed by using an iQ5 real-time PCRdetection system (BioRad). The cycle threshold(Ct) value was determined automatically by acomputer software (iQ5 Optical System Software,BioRad). All concentrated and unconcentratedDNA samples were divided into three sets. Todetermine the occurrence of PCR inhibition insamples, one subset of each of the samples wasspiked with 1 μL of 105 copies of plasmid DNA(see description below for the construction ofstandard curves) as internal control during PCRamplification. The negative controls containingonly HPLC water were also included in each PCRrun. The melting temperatures of amplificationproducts were determined by melting curves. The

Table 2 Primer sets designed for real-time PCR assays

Assay target Primer name, sequence (5′–3′) References

Aeromonas 16S rRNA gene Aer66f 5′-GCGGCAGCGGGAAAGTAG-3′ Yu et al. (2008)Aer613r 5′-GCTTTCACATCTAACTTATCCAAC-3′

Aerolysin gene (aer) AHCF1 5′-GAGAAGGTGACCACCAAGAACA-3′ Yu et al. (2008)AHCR1M 5′-ARCTGACATCGGCCTTGAACTC-3′

Serine protease gene (ser) spF 5′-CAACCCCAACAACAGCCTG-3′ This studyspR 5′-TAGAACTTGTGGGAGAGCATG-3′

Cytotonic enterotoxin gene (alt) altFM 5′-ATGACCCAGTCCTGGCACG-3′ This studyaltRM 5′-GCCGCTCAGGGCGAAGCC-3′


melting curves were obtained by operating PCRreaction as follows: heating to 95◦C for 1 min,cooling to 55◦C, and ramping the temperature to95◦C. Melting curves were checked routinely toconfirm the quantification of the desired products.

Plasmid DNA was used as a template forconstructing standard curves. By using the de-signed primer sets, a 104-bp fragment of par-tial serine protease gene and a 480-bp fragmentof partial heat-labile cytotonic enterotoxin genewere amplified from A. hydrophila (ATCC 7966).These products were cloned into the vector pCR4-TOPO (TA cloning; Invitrogen, Carlsbad, CA).Clones with inserts were verified by PCR withM13 primers that flank the cloning region. Se-lected clones were grown overnight in 5 ml of LBbroth with kanamycin and the plasmids were pu-rified using Wizard Plus SV Minipreps (Promega,Madison, WI). The sequences of inserts wereconfirmed by an Applied Biosystems 3100 Ge-netic Analyzer (Perkin-Elmer, Foster City, CA).The plasmid DNA concentration was determinedusing a Hoefer DyNa Quant 300 Fluorometer(Hoefer, Pharmacia Biotech, San Francisco, CA).The copy numbers in samples were determinedby comparing the Ct of samples to the values ofstandard curves (Yu et al. 2005a).

Results

Validation of real-time PCR assays

The developed real-time PCR assays for quanti-fying serine protease gene- and cytotonic entero-toxin gene-containing Aeromonas were vigorouslyvalidated by using both Aeromonas and non-Aeromonas strains (Table 3). The presence of ser-ine protease gene or cytotonic enterotoxin genein each of Aeromonas strains was first examinedby using conventional PCR with designed primersets, followed by real-time PCR analysis.

By using conventional PCR, no PCR amplifi-cation products of these two genes were detectedon agarose gel when the assay was challengedagainst non-Aeromonas bacteria. As expected,not all Aeromonas contain serine protease geneor cytotonic enterotoxin gene. Twenty out of22 Aeromonas strains (four ATCC Aeromonas

strains and 16 out of 18 Aeromonas isolates)had a single and clear PCR band on agarosegel at the expected size for serine protease gene(104 bp). Similarly, 17 Aeromonas strains (fourATCC model strains and 13 of the 18 Aeromonasisolates) yielded the expected fragment (480 bp)on agarose gel, indicating the presence of cyto-tonic enterotoxin gene.

Secondly, real-time PCR assays for serineprotease gene and cytotonic enterotoxin genewere validated by using both Aeromonas (22strains) and non-Aeromonas (18 strains) bacteria(Table 3). Once again, all non-Aeromonas strainsshowed no amplification signals of serine proteasegene or cytotonic enterotoxin gene (i.e., Ct > 36).Amplification of the ser gene was observed in20 out of 22 Aeromonas strains, with a Ct valueranging from 14.5 to 30.5 for 2.5 ng DNA perreaction. alt gene was detected in 17 out of 22Aereomonas strains, based on a Ct value rangingfrom 13.5 to 33.6 for 2.5 ng DNA per reaction.The wide range of Ct values observed in differentAeromonas species is likely due to one or moremismatches in the primers, since the primers weredesigned based on a limited number of publishedserine protease (ser) gene and heat-labile cyto-tonic enterotoxin (alt) gene sequences (of whichare mostly obtained from A. hydrophila) at thetime when these assays were developed. As newsequences have been rapidly deposited into theGenBank in recent years, it is not surprising toobserve one to two mismatches in the designedprimers. Nevertheless, the melting curves werecarefully examined to make sure that assays didnot generate nonspecific amplification.

The developed real-time PCR assays exhibiteda log-linear detection range across seven ordersof magnitude. High coefficients of determination(R2) of the standard curves were observed: 0.999for serine protease gene and 0.993 for cytotonicenterotoxin gene of Aeromonas (data not shown).The PCR amplification efficiencies for serine pro-tease gene and cytotonic enterotoxin gene ofAeromonas were 90% and 98%, respectively. Theefficiencies were calculated by using 10(−S) − 1where S is the slope of the standard curve (Aldapeet al. 2002). Both real-time PCR assays showedthe same detection limit, 10 copies of targetgene per PCR reaction. The detection limits were


Tab

le3

Val

idat

ion

ofre

al-t

ime

PC

Ras

says

usin

gA

erom

onas

and

non-

Aer

omon

asst

rain

s

Stra

inC

lose

stcu

ltur

edA

erol

ysin

gene

a (aer

)Se

rine

prot

ease

gene

a (ser

)E

nter

otox

inge

nea (a

lt)ba

cter

ials

peci

esR

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)R

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)R

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)

Mod

elst

rain

sA

TC

C79

66A

erom

onas

hydr

ophi

la+

16.7

+16

.0+

15.3

AT

CC

1546

8A

erom

onas

cavi

ae+

24.6

+29

.8+

16.5

AT

CC

3599

3A

erom

onas

sobr

ia+b

32.2

+28

.6+

28.8

AT

CC

4965

7A

erom

onas

trot

a+b

28.6

+24

.7+

24.6

Aer

omon

asP

lant

1-2

A.h

ydro

phila

(99%

)−

−+

16.0

+13

.5is

olat

esP

lant

2-1

+15

.9+

15.9

+13

.7P

lant

3-3

−−

+18

.5+

16.0

Pla

nt4-

2+

15.3

+18

.2+

16.1

Pla

nt6-

2−

−+

26.2

+20

.1P

lant

2-2

A.e

nche

leia

(99%

)−

−+

15.1

+25

.3P

lant

7-3

−−

+14

.8−

−P

lant

1–3

A.m

edia

(99%

)−

−+

14.6

+17

.1P

lant

4-3

−−

−−

+20

.7P

lant

1-1

A.p

opof

fii(

99%

)+

21.3

+21

.5−

−P

lant

5-2

−−

+15

.9+

13.8

Pla

nt6-

1+

18.7

+22

.6+

33.0

Pla

nt3-

1cA

erom

onas

sp.

−−

+20

.3+

33.6

Pla

nt3-

2d+

16.0

+18

.3−

−P

lant

4-1e

+17

.0+

14.6

−−

Pla

nt5-

1d+

18.4

−−

+22

.0P

lant

7-1d

−−

+22

.8+

15.3

Pla

nt7-

2f+

16.0

+30

.3−

−


Tab

le3

(con

tinu

ed)

Stra

inC

lose

stcu

ltur

edA

erol

ysin

gene

a (aer

)Se

rine

prot

ease

gene

a (ser

)E

nter

otox

inge

nea (a

lt)ba

cter

ials

peci

esR

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)R

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)R

egul

arP

CR

Rea

l-ti

me

PC

R(C

t)

Non

-Aer

omon

asA

TC

C25

922

Esc

heri

chia

coli

−−

−−

−−

stra

ins

CE

B-1

Esc

heri

chia

coli

−−

−−

−−

CE

B-2

Pse

udom

onas

−−

−−

−−

fluo

resc

ens

CE

B-3

Bac

illus

thur

ingi

ensi

s−

−−

−−

−A

RI-

1N

ovos

phin

gobi

um−

−−

−−

−ta

rdau

gens

KC

1F

lavo

bact

eriu

msp

.−

−−

−−

−K

C2

Chi

tinop

haga

sp.

−−

−−

−−

KC

3N

ocar

dioi

des

sim

plex

−−

−−

−−

KC

4R

hodo

cocc

usru

bber

−−

−−

−−

KC

5M

icro

bact

eriu

m−

−−

−−

−te

stac

eum

KC

6A

min

obac

ter

sp.

−−

−−

−−

KC

7A

min

obac

ter

sp.

−−

−−

−−

KC

8Sp

hing

omon

assp

.−

−−

−−

−K

C9

Sphi

ngom

onas

sp.

−−

−−

−−

KC

10Sp

hing

omon

assp

.−

−−

−−

−K

C11

Sphi

ngom

onas

sp.

−−

−−

−−

KC

12B

revu

ndim

onas

−−

−−

−−

vesi

cula

ris

KC

13E

sche

rich

iaco

li−

−−

−−

−K

C14

Sphi

ngom

onas

sp.

−−

−−

−−

a The

Ctv

alue

sar

eth

em

eans

ofdu

plic

ate

dete

rmin

atio

nsbW

eak

band

c Par

tial

lyse

quen

ced

16S

rRN

Age

nesh

owed

96%

sim

ilari

tyto

both

A.m

edia

and

A.v

eron

iidP

arti

ally

sequ

ence

d16

SrR

NA

gene

show

ed96

–99%

sim

ilari

tyto

both

A.h

ydro

phila

,A.s

alm

onic

ida,

and

A.b

estia

rum

e Par

tial

lyse

quen

ced

16S

rRN

Age

nesh

owed

99%

sim

ilari

tyto

both

A.v

eron

iian

dA

.cul

icic

ola

f Par

tial

lyse

quen

ced

16S

rRN

Age

nesh

owed

99%

sim

ilari

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Table 4 Concentrations of Aeromonas in source, intermediate, and finished waters

Method Concentration of Aeromonas

Plant 1 A1 B1 C1 D1EPA Method 1605a 10 <1b <1b <0.2b

16S rRNA genec 1.7 × 104 (9.1 × 103)d 3.5 × 102 (1.1 × 102)d <46.2e <21.4e

Aerolysin genec 5.7 × 102 (9.1)d <27.3f <23.1f <10.7f

Serine protease genec 1.1 × 103 (4.2 × 102)d <27.3f <23.1f <10.7f

Enterotoxin genec 5.9 × 102 (2.7 × 102)d <27.3f <23.1f <10.7f

Plant 2 A2 B2 C2 D2EPA Method 1605 1.5 × 102 (50) <1 <1 <0.216S rRNA gene 7.8 × 103 (1.2 × 103) 4.3 × 102 (77.1) 66.1 (27.1) <30Aerolysin gene <1200 <30 <30 <15Serine protease gene 2.9 × 102 (50) <30 <30 <15Enterotoxin gene <60 <30 <30 <15

Plant 3 A3 B3 C3 D3EPA Method 1605 1.1 × 103 (7.0 × 102) <1 <1 <0.216S rRNA gene 8.1 × 105 (6.5 × 104) 83.3 (20) <20.7 <20Aerolysin gene 1.5 × 104 (4.0 × 103) <40 <10.3 <10Serine protease gene 5.3 × 103 (9.7 × 102) <40 <10.3 <10Enterotoxin gene 1.2 × 103 (2.4 × 102) <40 <10.3 <10

Plant 4 A4 C4 D4a D4bEPA Method 1605 6.5 × 103 (5.0 × 102) <1 <0.2 <0.216S rRNA gene 1.8 × 105 (1.4 × 104) <20 <20 <20Aerolysin gene 6.3 × 103 (1.4 × 103) <10 <10 <10Serine protease gene 1.8 × 104 (2.6 × 102) <10 <10 <10Enterotoxin gene 3.6 × 103 (2.3 × 102) <10 <10 <10

Plant 5 A5 B5 C5 D5EPA Method 1605 4.9 × 103 (4.0 × 102) <1 <1 <0.216S rRNA gene 4.3 × 105 (1.9 × 104) 2.8 × 103 (3.5 × 102) 55.5 (8.2) <40Aerolysin gene 1.0 × 104 (5.6) 2.0 × 102 (3.4) <20 <20Serine protease gene 7.9 × 103 (5.8 × 102) <30 <20 <20Enterotoxin gene 5.7 × 103 (3.4 × 103) <30 <20 <20

Plant 6 A6 C6 D6EPA Method 1605 56.5 (5.5) <1 <0.216S rRNA gene 5.4 × 103 (11.4) 96.4 (11.3) <60Aerolysin gene 3.2 × 102 (1.0 × 102) <16.7 <30Serine protease gene <522d <16.7 <30Enterotoxin gene <522e <16.7 <30

Plant 7 A7 C7 D7EPA Method 1605 10 <1 <0.216S rRNA gene 1.7 × 103 (1.6 × 102) 5.1 × 102 (50) <120Aerolysin gene <88.3d <46.1 <60Serine protease gene 6.6 × 102 (2.1 × 102) <46.1 <60Enterotoxin gene <88.3 <46.1 <60

comparable to those reported for other bacterialreal-time PCR assays (Kolb et al. 2003; Dionisiet al. 2004).

The melting temperatures are 85◦C for ser-ine protease (ser) gene plasmid standards and89◦C for cytotonic enterotoxin (alt) gene plas-mid standards. The melting temperatures of the

PCR products amplified from pure strains withserine protease gene primers range from 84◦Cto 86◦C. For those amplified with cytotonic en-terotoxin gene (alt gene) primers, the meltingtemperatures are between 88◦C and 90◦C. Theslight differences in the melting temperatures ofthe PCR products reflected the heterogeneity of


Table 4 (continued)

Method Concentration of Aeromonas

Pilot plant A7 D8EPA Method 1605 10 <116S rRNA gene 1.7 × 103 (1.6 × 102) <120Aerolysin gene <88.3 <60Serine protease gene 6.6 × 102 (2.1 × 102) <60Enterotoxin gene <88.3 <60

aUnit is CFU per 100 mlbThe values refer to the method detection limits of EPA Method 1605. The calculation was based on different sample sizeused for filtrationcUnit is gene copies per 100 mldAverage concentration of duplicate samples; the number in parenthesis refers to the range of the concentrations ofduplicate sampleseThe values refer to the method detection limits of real-time PCR Aeromonas 16S rRNA gene assay (lowest detectable geneper 100 ml). The calculation was based on different sample size used for filtrationfThe values refer to the method detection limits of real-time PCR Aeromonas virulence gene assay (lowest detectable geneper 100 ml). The calculation was based on different sample size used for filtration

serine protease genes and cytotonic enterotoxingenes among different species of Aeromonas.

Application of developed assays to watersamples collected from different watertreatment processes

The validated assays were applied to quantifyserine protease gene and heat-labile enterotoxingene in water samples collected from water treat-ment plants (Table 4). The results of the watersamples were compared to the results of our pre-vious study examining the concentrations of to-tal Aeromonas (expressed as colony-forming unit[CFU] per 100 ml and as 16S rRNA gene copiesper 100 ml) and aerolysin (aer) gene-containingAeromonas (as aerolysin gene copies per 100 ml)in the same water samples (Yu et al. 2008). Serineprotease gene was detected in all source waters,except the source water for plant 6 (a brook waterwith a turbidity below 0.5 nephelometric turbid-ity units). However, heat-labile enterotoxin genewas detected only in source waters for plants 1and 3–5. No serine protease gene or heat-labileenterotoxin gene was detected in intermediate orfinished water samples. These results were con-sistent with our previous findings (1) Aeromonascolonies and 16S rRNA genes of Aeromonas weredetected in all source water, (2) no aerolysin gene

was detected in intermediate (except one fromplant 5) and finished waters, (3) 16S rRNA genesof Aeromonas were detected in some intermediatesamples of plants 1–3 and plants 5–7, and (4) noAeromonas colonies or 16S rRNA genes weredetected in finished water.

Discussion

The pathogenicity of Aeromonas sp. is likely dueto the interactions of various virulence factors(Pemberton et al. 1997). While individual viru-lence factors have been identified and character-ized, the distribution and interactions of thesevirulence factors in Aeromonas species have notbeen understood. Yet, it is believed that thevirulence genes in Aeromonas vary considerablyin different strains in the same species and de-pend on both the sources and locations whereAeromonas were isolated.

As there is a practical need for determiningclinical and environmental Aeromonas isolatesfor potential virulence, several researchers havedeveloped PCR methods by using virulence genesas biomarkers for pathogenic species (Khan etal.1999; Kingombe et al. 1999; Albert et al. 2000;Chacon et al. 2003; Sen and Rodgers 2004; Yuet al. 2008). Kingombe et al. (1999) found that


65% of 350 clinical and environmental isolatescarried act/hylA/aerA genes. Albert et al. (2000)reported that more clinical isolates (54% of115 isolates where A. caviae was the dominantstrain) contained both the alt and ast (heat-stable enterotoxin) genes than those fromenvironmental isolates (15% of 120 isolates).Only three A. hydrophila (one clinical isolate andtwo environmental isolates) possessed all threegenes, act (cytotoxic enterotoxin), alt, and ast. Senand Rodgers (2004) examined the distributionof different six virulence factors genes [elastase(ahyB), lipase (pla/lip/lipH3/alp-1), flagella Aand B (f laA and f laB), and enterotoxins (act,alt, and ast)] in 205 Aeromonas strains that wereisolated from US drinking water utilities. Intheir study, only one isolate (A. hydrophila)contained all six virulence factors genes and18 different combinations of virulence factorswere found in the different isolates. Recently,we developed real-time PCR assays to quantifytotal and aerolysin gene-containing Aeromonasin source, intermediate, and finished drinkingwaters (Yu et al. 2008). Combining results of ourprevious work (Yu et al. 2008) and this study,the distribution of the genes of three virulencefactors (aerolysin, heat-labile enterotoxin, andserine protease) in 22 Aeromonas strains wasexamined. Not surprisingly, all three virulencegenes were detected in four model Aeromonasstrains (known pathogenic strains). Only threeout of 18 isolates (two A. hydrophila and oneAeromonas popof f ii) possessed all three genes.A higher percent of isolates has either serineprotease gene (16 out of 18 isolates, 89%) or heat-labile cytotonic enterotoxin gene (13 out of 18isolates, 72%) than isolates possessing aerolysingene (eight out of 18 isolates, 44%). Our resultsare consistent with a previous study that a higherpercentage of Aeromonas isolates from waterharbored the heat-labile cytotonic enterotoxingene but not the aerolysin gene (Granum et al.1998). Serine protease is known to activate bothproaerolysin and lycerophospholipid:cholesterolacyltransferase in Aeromonas (Howard et al.1996). In our study, all the aerolysin gene-containing isolates also process serine protease,except for one isolate (from Plant 5-1 in Table 3),consistent with previous observation that

aerolysin/hemolysin and serine protease geneswere present in all the β-hemolytic strains(Chacon et al. 2003).

The ability to quantify known virulence genesin water samples is critical to assess and man-age risks associated with Aeromonas in drinkingwater. In this study, real-time PCR assays target-ing two Aeromonas virulence genes (serine pro-tease gene and cytotonic enterotoxin gene) havebeen developed and applied to water samples.These results complement very nicely the resultsof our previous study reporting quantitative dataof Aeromonas concentrations in water samplesin units of CFU per milliliter, 16S rRNA genecopies per milliliter, and aerolysin gene copiesper milliliter (Yu et al. 2008). When comparingthe results of this study and our previous study,high correlations between the results obtainedfrom EPA culture-based method and from real-time PCR assays for Aeromonas serine proteasegenes and cytotonic enterotoxin genes were ob-served. The correlation coefficient (r) betweenresults from EPA culture-based method andreal-time PCR assay for serine protease gene was0.82 (P = 0.048). The correlation coefficient be-tween results from EPA culture-based methodand real-time PCR assay for Aeromonas cyto-tonic enterotoxin (alt) gene was 0.90 (P = 0.1).The correlation coefficient between results fromAeromonas serine protease and cytotonic entero-toxin gene measured by real-time PCR assays was0.84 (P = 0.16). However, due to the limited datapoints for these two genes (ser and alt genes), thecorrelations were not significant at the 0.01 levelas reported in our previous study (Yu et al. 2008).

Sequence variability of a virulence gene in var-ious pathogens poses a great challenge for thedevelopment of real-time PCR and biochip assaysto be used for accurate identification and quan-tification of pathogens in environmental samples(Tourlousse et al. 2007). In this study, we observedhigh Ct values when using Aeromonas isolatesto validate the developed real-time PCR assays.The high Ct values might have been causedby the sequence variability in these virulencegenes, resulting in the mismatches in the primersdesigned from the limited sequences in theGenBank. As the virulence genes in the GenBankincreases, one would be able to design improved


sets of real-time PCR assays for virulence gene-containing Areomonas.

Waterborne diseases not only cause humansuffering, but also result in tremendous economicloss. By using virulence genes as biomarkers inthe design of real-time PCR, this study presentsa new approach for better surveillance and con-trol of Aeromonas for safe drinking water. Morestudies are needed to fill the knowledge gap in thedistribution of virulence factors in environmen-tal Aeromonas isolates, to survey water qualityin terms of concentrations of different virulencegenes, to assess the potential cost of using mole-cular tools, and to standardize the applications ofdeveloped molecular tools for water quality mon-itoring. Nevertheless, this study can be viewedas a model approach for (1) applying molecu-lar techniques to be used as routine monitoringmethods for other emerging pathogens and (2)collecting quantitative data that is necessary forassessing potential health threats due to a widerange of pathogens in potable and recreationalwaters. One can also envision that a differentform of dose–response relationship, expressed as“copies of virulence genes” rather than as “num-ber of pathogens,” might be developed for betterassessment of microbial risk.

Conclusions

This study applied the newly developed real-timePCR assays to quantify serine protease (ser) gene-containing and heat-labile cytotonic enterotoxin(alt) gene-containing Aeromonas in water sam-ples. Several conclusions can be drawn from theresults of this work:

1. A higher percentage of isolates has either theser gene (89%) or alt gene (72%) comparedto the percentage of isolates containing theaer gene (44%). Only three out of our 18Aeromonas isolates possessed all three genes(aer, ser, and alt).

2. By applying real-time PCR assays andthe culture-based method, we have shownthat both conventional and unconventionaldrinking water treatment processes could

effectively remove Aeromonas from treatedwater.

3. Using real-time PCR to quantify various viru-lence factor genes in water samples, as demon-strated in this study, can be a model approachfor collecting quantitative data that is neces-sary for assessing potential microbial risks inwater.

Acknowledgement This work was supported in partby the National Science Foundation, Award No. BES-0439389. We also thank the support by the CAS/SAFEAInternational Partnership Program for Creative ResearchTeams (KZCX2-YW-T08).

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