Environ Monit Assess (2011) 176:225238

DOI 10.1007/s10661-010-1578-1

Molecular quantification of virulence gene-containing

Aeromonas in water samples collected from different
drinking 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 produce a range of virulence factors, including

aerolysin, cytotonic enterotoxins, and serine protease, to cause acute gastroenteritis and wound
infections in humans and animals. Recognizing that not all Aeromonas strains are pathogenic, in this study, we proposed to evaluate
Aeromonas removal effectiveness based on the
presence of virulence gene-containing Aeromonas
as a proper means to assess microbial risk of
Aeromonas. We developed and applied real-time
PCR assays to quantify serine protease (ser) geneand heat-labile cytotonic enterotoxin (alt) genecontaining Aeromonas in water samples. Among
18 Aeromonas isolates from the source water,
only three isolates possessed all three genes (aer,
ser, and alt). A higher percent of isolates has
either ser gene (89%) or alt gene (72%) compared
to the percent of isolates containing aer gene
(44%). Results of this study suggested that several
different conventional and unconventional drink-

C.-P. Yu
Key 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, USA
e-mail: kchu@civil.tamu.edu

ing water treatment processes could effectively

remove Aeromonas from source water. As the
comprehensive knowledge of the distribution of
virulence factors in different Aeromonas species
is currently not available, using real-time PCR to
quantify various virulence factor genes in water
samples and/or isolates can be a practical means
for better assessment of microbial risks in water.
Keywords Virulence genes Aeromonas
Serine protease gene Heat-labile cytotonic
enterotoxin gene Drinking water

Introduction
Waterborne pathogens continue to be a major
health hazard in the United States and other
countries throughout the world. Consumption of
public drinking water is estimated to cause approximately 4.2632.80 million endemic waterborne acute gastrointestinal illnesses per year in
the United States according to two recent studies (Colford et al. 2006; Messner et al. 2006).
These infections, varying from diarrhea to respiratory disorders to heart diseases, are not caused
by classic waterborne pathogens like Vibrio
cholerae and Salmonella typhi, but by a number of emerging pathogens such as pathogenic
Escherichia coli, Cryptosporidium and Giardia,
pathogenic Yersinia, Campylobacter, Legionella,

226

Pseudomonas, and Aeromonas (Szewzyk et al.

2000). Children, the elderly, and immunocompromised individuals, which account for 2025% of
the US population, are particularly vulnerable to
these emerging pathogens. Among these emerging pathogens, Aeromonas was on the 1998 Environmental Protection Agency Drinking Water
Contaminant Candidate List 2.
Assessment of microbial risk in drinking water is challenging and commonly limited by several factors. Similar to chemical risk assessment,
prior knowledge of a doseresponse relationship
is needed for assessing microbial risk in water.
However, such information is not readily available
and limited only to a few emerging pathogens.
For example, based on available doseresponse
relationships, the calculated doses are 0.3 viruses
per 100 L for enteric viruses and 0.2 cysts per
100 L for Cryptosporidium (Rose and Gerba
1991). Such levels are below the detection limits
of conventional methods used to monitor these
microorganisms. In addition, while several pathogenic Aeromonas and mycobacterial species have
been recognized, not all environmental strains are
pathogenic (Szewzyk et al. 2000). The ability to
differentiate as well as to quantify pathogenic
species is essential for the assessment of microbial
risks in drinking water. The culture-based methods for quantifying these groups of pathogens
(such as EPA Method 1605 for Aeromonas)
cannot differentiate pathogenic from nonpathogenic species. Accordingly, the data obtained for
culture-based methods might not be appropriate
for microbial risk assessment. Additionally, many
waterborne pathogens, including V. cholerae and
Aeromonas hydrophila, have been suggested to
be present in environmental waters as viable but
nonculturable cells (VBNC) (Byrd et al. 1991;
Colwell et al. 1996; Mary et al. 2002; Halpern et al.
2007). While VBNC are not detectable by culturedependent methods, they retain their virulence
in water for a period of time and represent a
potential risk.
Real-time PCR analysis has been accepted as
a very rapid and sensitive quantitative molecular
method with applications ranging from clinical
microbiology (Bieche et al. 1999; Mercier et al.
1999) to molecular ecology (Suzuki et al. 2000;
Gruntzig et al. 2001) and environmental micro-

Environ Monit Assess (2011) 176:225238

biology (Beller et al. 2002; Dionisi et al. 2002;

Kikuchi et al. 2002). In addition to its sensitivity in
quantification, the real-time PCR assay can be designed for a specific strain, a phylogenetic group,
and even a group of microorganisms exhibiting
a similar function (Purkhold et al. 2000). Several
PCR-based molecular techniques have been recently developed to make rapid identification of
emerging pathogens possible (Purohit and Kapley
2002). However, most of these methods do not
provide the quantity of the emerging pathogens
in the water samplethe most essential information for risk assessment. Consequently, the
development of quantitative molecular methods
for emerging pathogens is still in its early stage
(Ibekwe and Grieve 2003).
The genus Aeromonas is a group of bacteria ubiquitous in aquatic environments, including lake and river water (Hazen et al. 1978),
seawater (Cavallo et al. 2009), bottled mineral
water (Hunter 1993), and domestic water supplies (Knochel and Jeppesen 1990; Pablos et al.
2009), even chlorinated water (LeChevallier et al.
1982; Millership and Chattopadhyay 1985). Some
Aeromonas species are considered as opportunistic waterborne pathogens responsible for acute
gastroenteritis and wound infections in humans
and animals. These species can produce many
virulence factors, such as cytotoxic enterotoxins, cytotonic enterotoxins, proteases, leukocidin,
phospholipase, endotoxin, and outer membrane
proteins (Howard et al. 1996). A. hydrophila,
Aeromonas sobria, and Aeromonas caviae are the
three most common species associated with human gastrointestinal infections (USEPA 2006).
Strains of Aeromonas trota have been shown to
carry an aerolysin gene (a toxin that forms pores
on host cells) and, therefore, are also considered
enteropathogenic (Khan et al. 1998, 1999).
Aeromonas spp. are known to be sensitive to
chlorine (Gavriel et al. 1998), although high numbers of several Aeromonas species have been isolated from biofilms in chlorinated drinking water
distribution systems (van der Kooij and Hijnen
1988; Gibbs et al. 2004; Tokajian and Hashwa
2004). Because Aeromonas can grow on small
amounts of nutrients (in the range of 1 g L1 ), regrowth occurs in most drinking water distribution
systems (Havelaar et al. 1990; Kuhn et al. 1997).

Environ Monit Assess (2011) 176:225238

227

Nevertheless, the significance of Aeromonas spp.

in drinking water to the occurrence of gastrointestinal infections is not fully understood.
The National Research Councils Committee on
Drinking Water Contaminants (Washington, DC)
has recommended a comprehensive approach
based on the virulence factor activity relationships as a means to predict potential risks from a
wide range of pathogens in drinking water. The
needs to know better about the distribution and
genetic level of virulence genes of waterborne
pathogens are supported by many previous studies
(Tourlousse et al. 2007; Miller et al. 2008). These
studies have suggested that relying on only one
single virulence gene of pathogenic strains might
possibly lead to incorrect assessment of the microbial risk of waterborne diseases because (1) many
virulence genes are unevenly distributed among
different pathogenic strains, (2) many virulence
genes are multifunctional, and (3) some virulence
can be even found in nonpathogenic strains.
To better understand the risk of Aeromonas
in drinking water, in this study, we had developed a set of real-time PCR assays to quantify
two virulence genes coded for heat-labile cytotonic enterotoxin (alt) and serine protease (ser) of
Aeromonas species in source and drinking water.
The heat-labile cytotonic enterotoxin has been extensively studied (Chopra et al. 1986; Challapalli
et al. 1988; Chopra et al. 1996). Serine protease
is a degradative enzyme which aids the invasion
of host cells, induces cell damage, and causes cell
death (Howard et al. 1996). The developed assays
were used to quantify the genes of these virulence
factors of Aeromonas in source, intermediate,
and finished drinking water from seven full-scale
water utilities and one pilot plant. Results of
this study were also compared to the total and
aerolysin gene (aer)-containing Aeromonas reported previously (Yu et al. 2008).

study. The Aeromonas bacteria include four

strains purchased from the American Type Culture Collection (ATCC; ATCC 7966T, ATCC
15468, ATCC 35993, and ATCC 49657) and 18
strains isolated from seven water treatment plants
studied 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 Bacillus
thuringiensis; Yu et al. 2005a).

Materials and methods

Real-time PCR assays for quantifying virulence

gene-containing Aeromonas spp.

Drinking water treatment plants and sampling

locations
Water samples were collected from different
treatment units of seven drinking water treatment
plants (plants 17) and one pilot plant (Table 1)
as described from Yu et al. (2008). Six out of eight
plants used conventional treatment processes
(plants 13 and plants 57). Plants 13 employed
prechlorination (PreCl2 ) of the source water, followed by coagulation/flocculation/sedimentation
(C/F/S), rapid sand filtration (RSF), and chlorine
disinfection (Cl2 ). Plant 5, without using PreCl2 ,
used C/F/S, RSF, and UV and chlorine disinfection. For plants 67, source water was treated
by using slow sand filtration (SSF) and chlorine
or chloramine for disinfection. Plant 4 and the
pilot plant employed unconventional treatment
processes. For plant 4, source water is directly
treated by membrane filtration, followed by UV
disinfection and final addition of NaOH and chlorine. The pilot plant employed preozonation of
source water and SSF. The turbidity values of
source water for plants 17 were 3.20, 3.63, 6.60,
2.65, 0.50, 0.35, and 0.80, respectively. Note that
the pilot plant and plant 7 used the same source
water. Detailed information of sampling procedure, sampling locations and numbers are available in Yu et al. (2008).

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

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

2.3
0.3

Plant 6 (Vermont)
Plant 7 (Maine)

: UV disinfection

UV
SSF

: coagulation/flocculation/sedimentation

: rapid sand filtration

: chlorine disinfection

XXX

RSF

Cl2

Chloramine : chloramine disinfection

: slow sand filtration

: membrane filtration

: source water

Pilot scale

9.5

Plant 5 (Georgia)

Pilot plant (Maine)

7.9

Plant 3 (Tennessee)

2.5

5.1

Plant 2 (Tennessee)

Plant 4 (Tennessee)

1.7

Plant 1 (Tennessee)

DS

O3

Daily production
(million gallons per day)

S
SW

treatment processes

Non- conventional

Conventional treatment
processes

Plant
(Location)

A7

A4

A7

A6

A5

A3

A2

: distribution system

: ozone disinfection

: sample location

SW

SW

SW

SW

SW

SW

SW

SW

A1

Cl2

Cl2

Cl2

O3

XXX

XXX

XXX

XXX

Process flow diagrams

Table 1 Unit operation and sampling locations of drinking water treatment plants surveyed in this study (Yu et al. 2008)

B5

B3

B2

B1

SSF

SSF

SSF

RSF

RSF

RSF

RSF

C4

C7

C6

C5

C3

C2

C1

UV

Chloramine

Cl2

UV

Cl2

Cl2

Cl2

DS

DS

DS

DS

DS

DS

D8

D4a D4b

DS

DS

Cl2+NaOH

D7

D6

D5

Cl2

D3

D2

D1

228
Environ Monit Assess (2011) 176:225238

Environ Monit Assess (2011) 176:225238

229

et al. 2008). In this study, virulence genes of

serine protease (ser) and heat-labile cytotonic
enterotoxin (alt) of Aeromonas spp. were used
as biomarkers for the development of real-time
PCR assays. Table 2 lists the primer sets designed
specifically for the virulence genes.
Only published gene sequences of serine protease of Aeromonas and the genes with a sufficient
sequence length were used in the primer design. A
forward primer spF and a reverse primer spR were
designed by aligning three published serine protease gene sequences of Aeromonas (GenBank
accession numbers AF159142, Cascon et al. 2000;
X67043, Whitby et al. 1992; and AY841795, Yu
et al. 2005b; accessed on 1 December 2005). The
expected product size for the ser gene is 104 bp.
Primers altFM and altRM were modified from a
primer set (Granum et al. 1998) previously designed to target a heat-labile cytotonic enterotoxin
gene (accession number L77573; Chopra et al.
1996) of A. hydrophila. The expected product size
for the alt gene is 480 bp. The designed primer
sets were also examined for their uniqueness to
the target genes by comparing the sequences in
GenBank using the Basic Local Alignment Search
Tool (Altschul et al. 1990).

DNA extraction
Water samples were filtered through 0.2-m-poresize polycarbonate membrane filters (Whatman,
Clifton, NJ) to retain bacteria cells on the membranes for DNA extraction. The DNA was extracted directly from the filter membrane by using
the UltraClean Water DNA Kit (MoBio Laboratories, Inc.) according to the manufacturers
instructions. The derived DNA samples were con-

centrated 20-fold by ethanol precipitation. Both

concentrated and unconcentrated DNA samples
were used as templates for SYBRGreen realtime PCR assays.

Real-time PCR analysis

The SYBRGreen real-time PCR assays for
quantifying Aeromonas-containing genes of serine protease and cytotonic enterotoxin were performed similarly, except that different annealing
temperatures were used. Each reaction was performed in a total volume of 25 L, with QuantiTect SYBR Green PCR Master Mix (QIAGEN
Inc., Valencia, CA), 600 nM forward and reverse
primers, and 5 L of DNA templates. The PCR
thermal cycle for both genes of Aeromonas was
50 C for 2 min and 95 C for 15 min, followed
by 36 cycles of 95 C for 30 s, optimum annealing
temperature (60 C for serine protease gene and
62 C for cytotonic enterotoxin gene) for 45 s, and
72 C for 30 s. PCR amplification and detection
were performed by using an iQ5 real-time PCR
detection system (BioRad). The cycle threshold
(Ct) value was determined automatically by a
computer software (iQ5 Optical System Software,
BioRad). All concentrated and unconcentrated
DNA samples were divided into three sets. To
determine the occurrence of PCR inhibition in
samples, one subset of each of the samples was
spiked with 1 L of 105 copies of plasmid DNA
(see description below for the construction of
standard curves) as internal control during PCR
amplification. The negative controls containing
only HPLC water were also included in each PCR
run. The melting temperatures of amplification
products were determined by melting curves. The

Table 2 Primer sets designed for real-time PCR assays

Assay target

Primer name, sequence (5 3 )

Aeromonas 16S rRNA gene

Aer66f
Aer613r
AHCF1
AHCR1M
spF
spR
altFM
altRM

Aerolysin gene (aer)

Serine protease gene (ser)
Cytotonic enterotoxin gene (alt)

5 -GCGGCAGCGGGAAAGTAG-3
5 -GCTTTCACATCTAACTTATCCAAC-3
5 -GAGAAGGTGACCACCAAGAACA-3
5 -ARCTGACATCGGCCTTGAACTC-3
5 -CAACCCCAACAACAGCCTG-3
5 -TAGAACTTGTGGGAGAGCATG-3
5 -ATGACCCAGTCCTGGCACG-3
5 -GCCGCTCAGGGCGAAGCC-3

References
Yu et al. (2008)
Yu et al. (2008)
This study
This study

230

melting curves were obtained by operating PCR

reaction as follows: heating to 95 C for 1 min,
cooling to 55 C, and ramping the temperature to
95 C. Melting curves were checked routinely to
confirm the quantification of the desired products.
Plasmid DNA was used as a template for
constructing standard curves. By using the designed primer sets, a 104-bp fragment of partial serine protease gene and a 480-bp fragment
of partial heat-labile cytotonic enterotoxin gene
were amplified from A. hydrophila (ATCC 7966).
These products were cloned into the vector pCR4TOPO (TA cloning; Invitrogen, Carlsbad, CA).
Clones with inserts were verified by PCR with
M13 primers that flank the cloning region. Selected clones were grown overnight in 5 ml of LB
broth with kanamycin and the plasmids were purified using Wizard Plus SV Minipreps (Promega,
Madison, WI). The sequences of inserts were
confirmed by an Applied Biosystems 3100 Genetic Analyzer (Perkin-Elmer, Foster City, CA).
The plasmid DNA concentration was determined
using a Hoefer DyNa Quant 300 Fluorometer
(Hoefer, Pharmacia Biotech, San Francisco, CA).
The copy numbers in samples were determined
by comparing the Ct of samples to the values of
standard curves (Yu et al. 2005a).

Results
Validation of real-time PCR assays
The developed real-time PCR assays for quantifying serine protease gene- and cytotonic enterotoxin gene-containing Aeromonas were vigorously
validated by using both Aeromonas and nonAeromonas strains (Table 3). The presence of serine protease gene or cytotonic enterotoxin gene
in each of Aeromonas strains was first examined
by using conventional PCR with designed primer
sets, followed by real-time PCR analysis.
By using conventional PCR, no PCR amplification products of these two genes were detected
on agarose gel when the assay was challenged
against non-Aeromonas bacteria. As expected,
not all Aeromonas contain serine protease gene
or cytotonic enterotoxin gene. Twenty out of
22 Aeromonas strains (four ATCC Aeromonas

Environ Monit Assess (2011) 176:225238

strains and 16 out of 18 Aeromonas isolates)

had a single and clear PCR band on agarose
gel at the expected size for serine protease gene
(104 bp). Similarly, 17 Aeromonas strains (four
ATCC model strains and 13 of the 18 Aeromonas
isolates) yielded the expected fragment (480 bp)
on agarose gel, indicating the presence of cytotonic enterotoxin gene.
Secondly, real-time PCR assays for serine
protease gene and cytotonic enterotoxin gene
were validated by using both Aeromonas (22
strains) and non-Aeromonas (18 strains) bacteria
(Table 3). Once again, all non-Aeromonas strains
showed no amplification signals of serine protease
gene or cytotonic enterotoxin gene (i.e., Ct > 36).
Amplification of the ser gene was observed in
20 out of 22 Aeromonas strains, with a Ct value
ranging from 14.5 to 30.5 for 2.5 ng DNA per
reaction. alt gene was detected in 17 out of 22
Aereomonas strains, based on a Ct value ranging
from 13.5 to 33.6 for 2.5 ng DNA per reaction.
The wide range of Ct values observed in different
Aeromonas species is likely due to one or more
mismatches in the primers, since the primers were
designed based on a limited number of published
serine protease (ser) gene and heat-labile cytotonic enterotoxin (alt) gene sequences (of which
are mostly obtained from A. hydrophila) at the
time when these assays were developed. As new
sequences have been rapidly deposited into the
GenBank in recent years, it is not surprising to
observe one to two mismatches in the designed
primers. Nevertheless, the melting curves were
carefully examined to make sure that assays did
not generate nonspecific amplification.
The developed real-time PCR assays exhibited
a log-linear detection range across seven orders
of magnitude. High coefficients of determination
(R2 ) of the standard curves were observed: 0.999
for serine protease gene and 0.993 for cytotonic
enterotoxin gene of Aeromonas (data not shown).
The PCR amplification efficiencies for serine protease gene and cytotonic enterotoxin gene of
Aeromonas were 90% and 98%, respectively. The
efficiencies were calculated by using 10(S) 1
where S is the slope of the standard curve (Aldape
et al. 2002). Both real-time PCR assays showed
the same detection limit, 10 copies of target
gene per PCR reaction. The detection limits were

Model strains ATCC 7966

ATCC 15468
ATCC 35993
ATCC 49657
Aeromonas
Plant 1-2
isolates
Plant 2-1
Plant 3-3
Plant 4-2
Plant 6-2
Plant 2-2
Plant 7-3
Plant 13
Plant 4-3
Plant 1-1
Plant 5-2
Plant 6-1
Plant 3-1c
Plant 3-2d
Plant 4-1e
Plant 5-1d
Plant 7-1d
Plant 7-2f

Strain

Aeromonas sp.

A. popof f ii (99%)

A. media (99%)

A. encheleia (99%)

Aeromonas hydrophila
Aeromonas caviae
Aeromonas sobria
Aeromonas trota
A. hydrophila (99%)

Closest cultured
bacterial species
+
+
+b
+b

+
+
+

+
16.7
24.6
32.2
28.6

15.9

15.3

21.3

18.7

16.0
17.0
18.4

16.0

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+

+
+
16.0
29.8
28.6
24.7
16.0
15.9
18.5
18.2
26.2
15.1
14.8
14.6

21.5
15.9
22.6
20.3
18.3
14.6

22.8
30.3

+
+
+
+
+
+
+
+
+
+

+
+

+
+
+

+
+

15.3
16.5
28.8
24.6
13.5
13.7
16.0
16.1
20.1
25.3

17.1
20. 7

13.8
33.0
33.6

22.0
15.3

Aerolysin genea (aer)

Serine protease genea (ser)
Enterotoxin genea (alt)
Regular PCR Real-time PCR (Ct) Regular PCR Real-time PCR (Ct) Regular PCR Real-time PCR (Ct)

Table 3 Validation of real-time PCR assays using Aeromonas and non-Aeromonas strains

Environ Monit Assess (2011) 176:225238

231

Closest cultured
bacterial species

b Weak

Aerolysin genea (aer)

Serine protease genea (ser)
Enterotoxin genea (alt)
Regular PCR Real-time PCR (Ct) Regular PCR Real-time PCR (Ct) Regular PCR Real-time PCR (Ct)

Ct values are the means of duplicate determinations

band
c Partially sequenced 16S rRNA gene showed 96% similarity to both A. media and A. veronii
d Partially sequenced 16S rRNA gene showed 9699% similarity to both A. hydrophila, A. salmonicida, and A. bestiarum
e Partially sequenced 16S rRNA gene showed 99% similarity to both A. veronii and A. culicicola
f Partially sequenced 16S rRNA gene showed 99% similarity to both A. hydrophila and A. veronii

a The

Non-Aeromonas ATCC 25922 Escherichia coli

strains
CEB-1
Escherichia coli
CEB-2
Pseudomonas
f luorescens
CEB-3
Bacillus thuringiensis
ARI-1
Novosphingobium
tardaugens
KC1
Flavobacterium sp.
KC2
Chitinophaga sp.
KC3
Nocardioides simplex
KC4
Rhodococcus rubber
KC5
Microbacterium
testaceum
KC6
Aminobacter sp.
KC7
Aminobacter sp.
KC8
Sphingomonas sp.
KC9
Sphingomonas sp.
KC10
Sphingomonas sp.
KC11
Sphingomonas sp.
KC12
Brevundimonas
vesicularis
KC13
Escherichia coli
KC14
Sphingomonas sp.

Strain

Table 3 (continued)

232
Environ Monit Assess (2011) 176:225238

Environ Monit Assess (2011) 176:225238

233

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

Method
Plant 1
EPA Method 1605a
16S rRNA genec
Aerolysin genec
Serine protease genec
Enterotoxin genec
Plant 2
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene
Plant 3
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene
Plant 4
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene
Plant 5
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene
Plant 6
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene
Plant 7
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene

Concentration of Aeromonas
A1
10
1.7 104 (9.1 103 )d
5.7 102 (9.1)d
1.1 103 (4.2 102 )d
5.9 102 (2.7 102 )d
A2
1.5 102 (50)
7.8 103 (1.2 103 )
<1200
2.9 102 (50)
<60
A3
1.1 103 (7.0 102 )
8.1 105 (6.5 104 )
1.5 104 (4.0 103 )
5.3 103 (9.7 102 )
1.2 103 (2.4 102 )
A4
6.5 103 (5.0 102 )
1.8 105 (1.4 104 )
6.3 103 (1.4 103 )
1.8 104 (2.6 102 )
3.6 103 (2.3 102 )
A5
4.9 103 (4.0 102 )
4.3 105 (1.9 104 )
1.0 104 (5.6)
7.9 103 (5.8 102 )
5.7 103 (3.4 103 )
A6
56.5 (5.5)
5.4 103 (11.4)
3.2 102 (1.0 102 )
<522d
<522e
A7
10
1.7 103 (1.6 102 )
<88.3d
6.6 102 (2.1 102 )
<88.3

B1
<1b
3.5 102 (1.1 102 )d
<27.3f
<27.3f
<27.3f
B2
<1
4.3 102 (77.1)
<30
<30
<30
B3
<1
83.3 (20)
<40
<40
<40

B5
<1
2.8 103 (3.5 102 )
2.0 102 (3.4)
<30
<30

comparable to those reported for other bacterial

real-time PCR assays (Kolb et al. 2003; Dionisi
et al. 2004).
The melting temperatures are 85 C for serine protease (ser) gene plasmid standards and
89 C for cytotonic enterotoxin (alt) gene plasmid standards. The melting temperatures of the

C1
<1b
<46.2e
<23.1f
<23.1f
<23.1f
C2
<1
66.1 (27.1)
<30
<30
<30
C3
<1
<20.7
<10.3
<10.3
<10.3
C4
<1
<20
<10
<10
<10
C5
<1
55.5 (8.2)
<20
<20
<20
C6
<1
96.4 (11.3)
<16.7
<16.7
<16.7
C7
<1
5.1 102 (50)
<46.1
<46.1
<46.1

D1
<0.2b
<21.4e
<10.7f
<10.7f
<10.7f
D2
<0.2
<30
<15
<15
<15
D3
<0.2
<20
<10
<10
<10
D4a
<0.2
<20
<10
<10
<10
D5
<0.2
<40
<20
<20
<20
D6
<0.2
<60
<30
<30
<30
D7
<0.2
<120
<60
<60
<60

D4b
<0.2
<20
<10
<10
<10

PCR products amplified from pure strains with

serine protease gene primers range from 84 C
to 86 C. For those amplified with cytotonic enterotoxin gene (alt gene) primers, the melting
temperatures are between 88 C and 90 C. The
slight differences in the melting temperatures of
the PCR products reflected the heterogeneity of

234

Environ Monit Assess (2011) 176:225238

Table 4 (continued)
Method
Pilot plant
EPA Method 1605
16S rRNA gene
Aerolysin gene
Serine protease gene
Enterotoxin gene

Concentration of Aeromonas
A7
10
1.7 103 (1.6 102 )
<88.3
6.6 102 (2.1 102 )
<88.3

D8
<1
<120
<60
<60
<60

a Unit

is CFU per 100 ml

values refer to the method detection limits of EPA Method 1605. The calculation was based on different sample size
used for filtration
c Unit is gene copies per 100 ml
d Average concentration of duplicate samples; the number in parenthesis refers to the range of the concentrations of
duplicate samples
e The values refer to the method detection limits of real-time PCR Aeromonas 16S rRNA gene assay (lowest detectable gene
per 100 ml). The calculation was based on different sample size used for filtration
f The values refer to the method detection limits of real-time PCR Aeromonas virulence gene assay (lowest detectable gene
per 100 ml). The calculation was based on different sample size used for filtration
b The

serine protease genes and cytotonic enterotoxin

genes among different species of Aeromonas.
Application of developed assays to water
samples collected from different water
treatment processes
The validated assays were applied to quantify
serine protease gene and heat-labile enterotoxin
gene in water samples collected from water treatment plants (Table 4). The results of the water
samples were compared to the results of our previous study examining the concentrations of total Aeromonas (expressed as colony-forming unit
[CFU] per 100 ml and as 16S rRNA gene copies
per 100 ml) and aerolysin (aer) gene-containing
Aeromonas (as aerolysin gene copies per 100 ml)
in the same water samples (Yu et al. 2008). Serine
protease gene was detected in all source waters,
except the source water for plant 6 (a brook water
with a turbidity below 0.5 nephelometric turbidity units). However, heat-labile enterotoxin gene
was detected only in source waters for plants 1
and 35. No serine protease gene or heat-labile
enterotoxin gene was detected in intermediate or
finished water samples. These results were consistent with our previous findings (1) Aeromonas
colonies and 16S rRNA genes of Aeromonas were
detected in all source water, (2) no aerolysin gene

was detected in intermediate (except one from

plant 5) and finished waters, (3) 16S rRNA genes
of Aeromonas were detected in some intermediate
samples of plants 13 and plants 57, and (4) no
Aeromonas colonies or 16S rRNA genes were
detected in finished water.

Discussion
The pathogenicity of Aeromonas sp. is likely due
to the interactions of various virulence factors
(Pemberton et al. 1997). While individual virulence factors have been identified and characterized, the distribution and interactions of these
virulence factors in Aeromonas species have not
been understood. Yet, it is believed that the
virulence genes in Aeromonas vary considerably
in different strains in the same species and depend on both the sources and locations where
Aeromonas were isolated.
As there is a practical need for determining
clinical and environmental Aeromonas isolates
for potential virulence, several researchers have
developed PCR methods by using virulence genes
as biomarkers for pathogenic species (Khan et
al.1999; Kingombe et al. 1999; Albert et al. 2000;
Chacon et al. 2003; Sen and Rodgers 2004; Yu
et al. 2008). Kingombe et al. (1999) found that

Environ Monit Assess (2011) 176:225238

65% of 350 clinical and environmental isolates

carried act/hylA/aerA genes. Albert et al. (2000)
reported that more clinical isolates (54% of
115 isolates where A. caviae was the dominant
strain) contained both the alt and ast (heatstable enterotoxin) genes than those from
environmental isolates (15% of 120 isolates).
Only three A. hydrophila (one clinical isolate and
two environmental isolates) possessed all three
genes, act (cytotoxic enterotoxin), alt, and ast. Sen
and Rodgers (2004) examined the distribution
of different six virulence factors genes [elastase
(ahyB), lipase (pla/lip/lipH3/alp-1), flagella A
and B (f laA and f laB), and enterotoxins (act,
alt, and ast)] in 205 Aeromonas strains that were
isolated from US drinking water utilities. In
their study, only one isolate (A. hydrophila)
contained all six virulence factors genes and
18 different combinations of virulence factors
were found in the different isolates. Recently,
we developed real-time PCR assays to quantify
total and aerolysin gene-containing Aeromonas
in source, intermediate, and finished drinking
waters (Yu et al. 2008). Combining results of our
previous work (Yu et al. 2008) and this study,
the distribution of the genes of three virulence
factors (aerolysin, heat-labile enterotoxin, and
serine protease) in 22 Aeromonas strains was
examined. Not surprisingly, all three virulence
genes were detected in four model Aeromonas
strains (known pathogenic strains). Only three
out of 18 isolates (two A. hydrophila and one
Aeromonas popof f ii) possessed all three genes.
A higher percent of isolates has either serine
protease gene (16 out of 18 isolates, 89%) or heatlabile cytotonic enterotoxin gene (13 out of 18
isolates, 72%) than isolates possessing aerolysin
gene (eight out of 18 isolates, 44%). Our results
are consistent with a previous study that a higher
percentage of Aeromonas isolates from water
harbored the heat-labile cytotonic enterotoxin
gene but not the aerolysin gene (Granum et al.
1998). Serine protease is known to activate both
proaerolysin and lycerophospholipid:cholesterol
acyltransferase in Aeromonas (Howard et al.
1996). In our study, all the aerolysin genecontaining isolates also process serine protease,
except for one isolate (from Plant 5-1 in Table 3),
consistent with previous observation that

235

aerolysin/hemolysin and serine protease genes

were present in all the -hemolytic strains
(Chacon et al. 2003).
The ability to quantify known virulence genes
in water samples is critical to assess and manage risks associated with Aeromonas in drinking
water. In this study, real-time PCR assays targeting two Aeromonas virulence genes (serine protease gene and cytotonic enterotoxin gene) have
been developed and applied to water samples.
These results complement very nicely the results
of our previous study reporting quantitative data
of Aeromonas concentrations in water samples
in units of CFU per milliliter, 16S rRNA gene
copies per milliliter, and aerolysin gene copies
per milliliter (Yu et al. 2008). When comparing
the results of this study and our previous study,
high correlations between the results obtained
from EPA culture-based method and from realtime PCR assays for Aeromonas serine protease
genes and cytotonic enterotoxin genes were observed. The correlation coefficient (r) between
results from EPA culture-based method and
real-time PCR assay for serine protease gene was
0.82 (P = 0.048). The correlation coefficient between results from EPA culture-based method
and real-time PCR assay for Aeromonas cytotonic enterotoxin (alt) gene was 0.90 (P = 0.1).
The correlation coefficient between results from
Aeromonas serine protease and cytotonic enterotoxin gene measured by real-time PCR assays was
0.84 (P = 0.16). However, due to the limited data
points for these two genes (ser and alt genes), the
correlations were not significant at the 0.01 level
as reported in our previous study (Yu et al. 2008).
Sequence variability of a virulence gene in various pathogens poses a great challenge for the
development of real-time PCR and biochip assays
to be used for accurate identification and quantification of pathogens in environmental samples
(Tourlousse et al. 2007). In this study, we observed
high Ct values when using Aeromonas isolates
to validate the developed real-time PCR assays.
The high Ct values might have been caused
by the sequence variability in these virulence
genes, resulting in the mismatches in the primers
designed from the limited sequences in the
GenBank. As the virulence genes in the GenBank
increases, one would be able to design improved

236

sets of real-time PCR assays for virulence genecontaining Areomonas.

Waterborne diseases not only cause human
suffering, but also result in tremendous economic
loss. By using virulence genes as biomarkers in
the design of real-time PCR, this study presents
a new approach for better surveillance and control of Aeromonas for safe drinking water. More
studies are needed to fill the knowledge gap in the
distribution of virulence factors in environmental Aeromonas isolates, to survey water quality
in terms of concentrations of different virulence
genes, to assess the potential cost of using molecular tools, and to standardize the applications of
developed molecular tools for water quality monitoring. Nevertheless, this study can be viewed
as a model approach for (1) applying molecular techniques to be used as routine monitoring
methods for other emerging pathogens and (2)
collecting quantitative data that is necessary for
assessing potential health threats due to a wide
range of pathogens in potable and recreational
waters. One can also envision that a different
form of doseresponse relationship, expressed as
copies of virulence genes rather than as number of pathogens, might be developed for better
assessment of microbial risk.

Conclusions
This study applied the newly developed real-time
PCR assays to quantify serine protease (ser) genecontaining and heat-labile cytotonic enterotoxin
(alt) gene-containing Aeromonas in water samples. Several conclusions can be drawn from the
results of this work:
1. A higher percentage of isolates has either the
ser gene (89%) or alt gene (72%) compared
to the percentage of isolates containing the
aer gene (44%). Only three out of our 18
Aeromonas isolates possessed all three genes
(aer, ser, and alt).
2. By applying real-time PCR assays and
the culture-based method, we have shown
that both conventional and unconventional
drinking water treatment processes could

Environ Monit Assess (2011) 176:225238

effectively remove Aeromonas from treated

water.
3. Using real-time PCR to quantify various virulence factor genes in water samples, as demonstrated in this study, can be a model approach
for collecting quantitative data that is necessary for assessing potential microbial risks in
water.
Acknowledgement This work was supported in part
by the National Science Foundation, Award No. BES0439389. We also thank the support by the CAS/SAFEA
International Partnership Program for Creative Research
Teams (KZCX2-YW-T08).

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