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Environ. Sci. Technol.

2008, 42, 11911200

Development and Application of


Real-Time PCR Assays for
Quantifying Total and Aerolysin
Gene-Containing Aeromonas in
Source, Intermediate, and Finished
Drinking Water
CHANG-PING YU, SARA K. FARRELL,
BRUCE ROBINSON, AND
K U N G - H U I C H U * ,
Zachry Department of Civil Engineering, Texas A&M
University, College Station, TX 77843-3136, The Center for
Environmental Biotechnology and Department of Civil and
Environmental Engineering, University of Tennessee,
Knoxville, Tennessee 37996

Received June 6, 2007. Revised manuscript received


October 21, 2007. Accepted November 26, 2007.

Aeromonas spp., opportunistic pathogens, are listed as a


microbiological contaminant on the Environmental Protection
Agencys (EPA) Drinking Water Contaminant Candidate
List. Culture-based methods for identification and quantification
of Aeromonas in drinking water are time-consuming and
often fail to differentiate pathogenic species from nonpathogenic
ones. This study reports successful development and
applications of two real-time PCR assays, based on 16S rRNA
gene sequences and a virulence gene (aerolysin gene), for
rapid and effective quantification of total and aerolysin genecontaining Aeromonas spp. The assays successfully quantified
total and aerolysin gene-containing Aeromonas in source,
intermediate, and finished water samples collected from seven
water works and one pilot plant. The effectiveness of
Aeromonas removal by different drinking water treatment
processes was examined by comparing the results obtained
from the EPA culture-based method and developed real-time
PCR assays. Regardless of the methods, our results indicated that
conventional water treatment combination (prechlorination/
coagulation/sedimentation/rapid sand filtration) and membrane
filtration alone could effectively remove Aeromonas. Slow
sandfiltrationalonemightnotbeeffective.Theremovalefficiencies
by different disinfection treatments were not determined, due
to the lack of detectable Aeromonas. No Aeromonas was
detected in samples with turbidity below 0.06 NTU.

Introduction
The genus Aeromonas is a group of bacteria that are
ubiquitous in aquatic environments (13) and may also be
detected in fish, food, bottled water, and drinking water
* Corresponding author phone: (979) 845-1403; fax: (979) 8621542; e-mail.: kchu@civil.tamu.edu.

Texas A &M University.

The Center for Environmental Biotechnology, University of


Tennessee.

Department of Civil and Environmental Engineering, University


of Tennessee.
10.1021/es071341g CCC: $40.75

Published on Web 01/17/2008

2008 American Chemical Society

(412). Since some Aeromonas spp. can produce a range of


biotoxins (such as hemolysins, cytotoxins, aerolysins, and
even enterotoxins), they are considered as opportunistic
waterborne pathogens responsible for acute gastroenteritis
and wound infections in humans (13) and animals (14). In
1996, Aeromonas was listed as a microbiological contaminant
on the Environmental Protection Agencys (EPA) Drinking
Water Contaminant Candidate List. Monitoring for Aeromonas was conducted by 120 large and 180 small public
water systems (PWS, list 2) in 2003 by using EPA approved
culture-based method, Method 1605 (Federal Register, 40
CFR Part 141, March 7, 2002). Like other culture-based
methods, this method is time-consuming. Furthermore, this
method might not be effective for identifying pathogenic
species from nonpathogenic ones.
Quantitative culture-independent methods, like real-time
PCR, might offer a better measure for the quantification and
monitoring purposes of pathogens in drinking water. Recently, real-time PCR has been developed for quantifying
Escherichia coli O157:H7 and hepatitis B viruses in environmental samples (1517). Although studies have been
reported using real-time PCR for monitoring Aeromonas spp.
in stools (18) and fish tissue (19), no studies have been
reported using real-time PCR for monitoring Aeromonas spp.
in drinking water, and/or other environmental samples.
Data on Aeromonas removal by water treatment facilities is
essential for regulatory agencies to recommend any specific
treatment process or to impose a practical regulation on
Aeromonas for safe drinking water. To address the concern of
Aeromonas in drinking water, we developed and applied
quantitative and sensitive molecular assays to quantify total
Aeromonas spp. and virulence gene-containing Aeromonas spp.
in source, intermediate, and finished water of several selected
water utilities. The results from the EPA method and the
molecular assays were compared and used to determine the
effectiveness of various water treatment processes.

Materials and Methods


Drinking Water Treatment Plants and Sampling Locations. To examine the effectiveness of Aeromonas removal
by various drinking water treatment processes, water samples
were collected from different treatment units of seven
drinking water treatment plants (plants 17) and one pilot
plant (pilot plant). Table 1 lists information of these plants,
including treatment capacity, process flow diagrams of each
plant, and sampling locations. Two sampling events were
conducted between April 2004 and August 2005. For each
sampling event, seven source water, 12 intermediate, and
eight finished samples were collected. Procedures for sample
collection, preservation, and storage were followed as
described in EPA Method 1605, an approved culture-based
method for Aeromonas spp. monitoring (20). Water quality
parameters, such as pH and turbidity, were measured on site
during samples collection (for turbidity see Supporting Information Table S1). For plants 14, 4 L of water samples were
collected from each sample location. For plants 57 and pilot
plant, 2 L of water samples were collected from each sampling
location. The collected samples were packed with ice and
transported to the laboratory for analysis within 24 h.
Strains. Both Aeromonas (22 strains) and non-Aeromonas
(19 strains) bacteria were used to validate the quantitative
molecular assays developed in this study (Table 3). The
Aeromonas bacteria include four strains purchased from the
American Type Culture Collection (ATCC) (ATCC 7966T,
ATCC 15468, ATCC 35993, and ATCC 49657) and eighteen
strains isolated from water treatment plants in this study.
VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

1191

TABLE 1. Unit Operation and Sampling Locations of Drinking Water Treatment Plants Surveyed in this Study

TABLE 2. Primer Sets Designed for Real-Time PCR Assays


assay target
aerolysin gene
Aeromonas 16S rRNA gene

primer name, sequence (5-3)

references

AHCF1 5-GAGAAGGTGACCACCAAGAACA-3
AHCR1M 5-ARCTGACATCGGCCTTGAACTC-3
Aer66f 5-GCGGCAGCGGGAAAGTAG-3
Aer613r 5-GCTTTCACATCTAACTTATCCAAC-3

30
this study
25
this study

The non-Aeromonas bacteria include one E. coli. purchased


from ATCC (ATCC 25922), 15 estrogen-degraders isolated
from activated sludge (21, 22), and three strains (E. coli,
Pseudomonas fluorescens, and Bacillus thuringiensis) obtained from the University of Tennessee, Knoxville (23).
Enumeration and Isolation of Culturable Aeromonas.
Culturable Aeromonas bacteria were enumerated by using
ampicillin-dextrin agar with vancomycin (ADA-V) according
to EPA Method 1605 (20). Different volumes of water samples
were used for filtration. For source and intermediate water,
sample volumes of 100, 10, and 1 mL were used. For finished
water, 500 mL was filtered. Duplicate samples were used.
Eighteen Aeromonas strains were isolated. These isolates were
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008

further identified according to their 16S rRNA gene sequences


(500 bp) that were amplified with the primers Aer66f and
Aer613r (Table 2). These isolates were also examined for the
presence of aerolysin gene.
DNA Extraction. Water samples were filtered through 0.2
m pore-size polycarbonate membrane filters (Whatman,
Clifton, NJ) to retain bacteria cells on the membranes for
DNA extraction. In this study, DNA was extracted either by
lysing cells eluted from the membrane or retained on the
membrane. For samples collected during the first sampling
event, the eluted cells were used. The retained bacterial cells
on the filter membrane were eluted by immersing the
membrane in 4 mL of phosphate-buffered saline, followed

TABLE 3. Validation of Real-Time PCR Assays by Using Both Aeromonas and Non-Aeromonas Strains
conventional PCR
strain
model strains

Aeromonas
isolates

non-Aeromonas
strains

species

ATCC
ATCC
ATCC
ATCC

7966T
15468
35993
49657

Aeromonas
Aeromonas
Aeromonas
Aeromonas

plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant
plant

1,
2,
3,
4,
6,
2,
7,
1,
4,
1,
5,
6,
3,
3,
4,
5,
7,
7,

A. hydrophila (99%)

no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.
no.

2
1
3
2
2
2
3
3
3
1
2
1
1a
2b
1c
1b
1b
2d

ATCC 25922
CEB-1
CEB-2
CEB-3
ARI-1
KC1
KC2
KC3
KC4
KC5
KC6
KC7
KC8
KC9
KC10
KC11
KC12
KC13
KC14

hydrophila
caviae
sobria
trota

A. encheleia (99%)
A. media (99%)
A. popoffii (99%)
Aeromonas sp.

Escherichia coli
Escherichia coli
Pseudomonas fluorescens
Bacillus thuringiensis
Novosphingobium tardaugens
Flavobacterium sp.
Chitinophaga sp.
Nocardioides simplex
Rhodococcus ruber
Microbacterium testaceum
Aminobacter sp.
Aminobacter sp.
Sphingomonas sp.
Sphingomonas sp.
Sphingomonas sp.
Sphingomonas sp.
Brevundimonas vesicularis
Escherichia coli
Sphingomonas sp.

real-time PCR (values of Ct)

Aeromonas 16S
rRNA gene

aerolysin

Aeromonas 16S
rRNA genef

aerolysinf

+
+
+
+

+
+
+e
+e

10.4
10.7
13.2
13.1

16.7
24.6
32.2
28.6

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

+
+
+
+
+
+
+
+

12.3
12.7
12.0
11.6
10.1
12.9
13.4
13.4
13.3
10.6
13.5
10.1
13.0
12.6
12.9
10.8
10.5
11.4

15.9
15.3
21.3
18.7
16.0
17.0
18.4
16.0

a
Partially sequenced 16S rRNA gene showed 96% similarity to both A. media and A. veronii. b Partially sequenced 16S
rRNA gene showed 9699% similarity to both A. hydrophila, A. salmonicida, and A. bestiarum. c Partially sequenced 16S
rRNA gene showed 99% similarity to both A. veronii and A. culicicola. d Partially sequenced 16S rRNA gene showed 99%
similarity to both A. hydrophila and A. veronii. e Weak band. f The Ct values are the means of duplicate determinations.

by vigorous agitation at the maximum velocity of a vortex


mixer (Maxi MixII, Thermolyne) for 10 min. This elution step
was repeated three times. The eluted cells were then
transferred to centrifuge tubes and pelleted by centrifugation.
The supernatant was discarded, and the eluted cells were
then lysed as described by Kapley et al. (24). The derived
DNA samples were concentrated 10-fold by ethanol precipitation. For samples collected during the second sampling
event, DNA was extracted directly from the cells retained on
the filter membrane. UltraClean Water DNA Kit (MoBio
Laboratories, Inc.) was used for DNA extraction. The derived
DNA samples were concentrated 20 times by ethanol
precipitation. Both concentrated and unconcentrated samples
were used as templates for SYBR Green real-time PCR assays.
Real-Time PCR Assay for Quantifying Total Aeromonas
spp. The region of 16S rRNA gene of Aeromonas spp. was
used as the target region for designing real-time PCR assay
for quantifying total Aeromonas spp. A forward primer Aer66f

and a reverse primer Aer613r (Table 2), were used for


amplifying 16S rRNA gene of Aeromonas spp. The forward
primer Aer66f was previously used as a fluorescence probe
for in situ hybridization (25). The reverse primer Aer613r
was modified from the primer Con607R (26) which has been
used for sequencing a portion of the 16S rRNA gene of
Aeromonas (27). By aligning 132 of 16S rRNA gene sequences
of cultured Aeromonas (longer than 1000 bp that were
deposited in GenBank database during the time the assays
were designed), the forward primer (Aer66f) only has one
mismatch with four sequences (X60416, X74682, AY538658,
and AJ536821) and the reverse primer (Aer613r) only has one
mismatch with three sequences (AY264937, AJ536820, and
AJ536821). The designed primer sets were also examined for
their uniqueness to the target gene by comparing the
sequences in GenBank using the Basic Local Alignment
Search Tool (BLAST) (28) and the Probe Match program of
the Ribosomal Database Project (29).
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Real-Time PCR Assay for Quantifying Aerolysin GeneContaining Aeromonas spp. In this study, aerolysin gene
was used as a biomarker of pathogenic Aeromonas spp. A
real-time PCR assay using primers AHCF1 and AHCR1M
(Table 2) was developed for quantifying aerolysin-genecontaining Aeromonas spp. These primers (AHCF1 and
AHCR1M) were modified from a primer set (AHCF1 and
AHCR1) designed by Kingombe et al. (30). AHCF1 and AHCR1
have been used for detecting aerolysin gene in many studies
(13, 18, 26, 31, 32). Compared to AHCR1 primer, the reverse
primer AHCR1M designed in this study is more specific to
five aerolysin genes of A. hydrophila that were deposited in
GenBank (accession numbers M84709 (33), M16495 (34),
X65043, X65044, X65045 (35)).
Real-Time PCR Analysis. The SYBR Green real-time PCR
assays for quantifying aerolysin gene and 16S rRNA gene of
Aeromonas were performed similarly, except using different
annealing temperatures. 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 was 95 C for 10 min, followed by 35 cycles of
95 C for 30 s, 61 C (for 16S rRNA genes of Aeromonas) or
57 C (for aerolysin genes of Aeromonas) for 45 s, and 72 C
for 30 s. PCR amplification and detection were performed by
using a DNA Engine Opticon continuous fluorescence
detection system (MJ Research, Waltham, MA). The cycle
threshold (Ct) value was determined automatically with the
computer software (Opticon Monitor, version 1.4, MJ Research). All concentrated and unconcentrated samples were
measured twice by each assay. A subset of each sample was
spiked with 1 L of 105 copies of standard DNA (described
below) as an internal control during PCR amplification. This
was done as a means to determine whether inhibition of
PCR had occurred. 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 melting curves were obtained by
operating PCR reactions as follows: heating to 95 C for 1
min, cooling to 55 C, and then ramping 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 232
bp-fragment of partial aerolysin gene and a 548 bp-fragment
of partial 16S rRNA gene were amplified from A. hydrophila
(ATCC 7966). These products were cloned into the vector
pCR4-TOPO (TA cloning; Invitrogen, Carlsbad, CA.). The
inserts were confirmed by sequencing using M13 primers
that flank the cloning region. The sequences of inserts were
confirmed by an Applied Biosystems 3100 DNA sequencer
(Perkin-Elmer, Foster City, CA). 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 plasmid DNA concentration
was determined using a Hoefer DyNa Quant 200 Fluorometer
(Hoefer, Pharmacia Biotech, San Francisco, CA). The copy
numbers in samples were determined as described by Yu et
al. (23).
Statistical Analysis. The experimental data were log10
transformed as described by Dionisi et al. (36). The correlation
coefficients among the results obtained from EPA method
1605 and from real-time PCR assays were calculated by using
bivariate two-tailed Pearson correlation in SPSS version 14.0.

Results
Aeromonas Strains Isolated from Water Utilities Surveyed.
Eighteen Aeromonas strains were isolated from this study.
Based on their partial 16S rRNA sequences (500 bp) (Table
3), they are A. hydorphila (five isolates), A. media (two
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008

isolates), A. encheleia (two isolates), A. popoffii (three


isolates), and Aeromonas sp. (six isolates). No A. sobria, A.
caviae, or A. trota were isolated. Our observation was
consistent with a previous study conducted by the U.S. EPA
in 20012002 (26). In the EPAs study, no A. sobria or A. trota
was identified from 205 Aeromonas strains that were isolated
from 16 water treatment utilities. Only six isolates were
identified as A. caviae (26).
Validation of Real-Time PCR Assays. The developed realtime PCR assays for quantifying total and aerolysin genecontaining Aeromonas were vigorously validated. First, the
specificity of primers for Aeromonas 16S rRNA genes was
determined by using the Probe Match Program of the
Ribosomal Database Project (http://rdp.cme.msu.edu/
probematch/, as assessed on April 8, 2007). Only sequences
that had been verified using Pintail software (37) and have
near-full-length sequences (>1200 bases) were selected for
the specificity analysis. When no base pair mismatches are
allowed, the forward primer (Aer 66f) will anneal to 858 out
of 967 Aeromonas genus 16S rRNA genes (including uncultured clones) and the reverse primers (Aer613r) will anneal
to 927 out of the 967 Aeromonas genus 16S rRNA genes. For
nontarget 16S rRNA gene sequences analysis, the forward
primer (allowed no mismatches) will anneal to only six
sequences (<0.01%) out of 128 613 of 16S rRNA gene
sequences belonging to non-Aeromonas genera within the
bacteria domain. The reverse primers (allowed no mismatches) will anneal to only two sequences (<0.01%), and
these two sequences are different from those six sequences
that can be annealed by the forward primer. The results
of the analyses indicate that these primers had a high
specificity to 16S rRNA gene sequences belonging to Aeromonas genus and a very low chance to cross-hybridize with
bacteria outside of the Aeromonas genus.
Pure cultures, both Aeromonas and non-Aeromonas
strains, were used to validate the designed real-time PCR
assays for quantifying 16S rRNA genes and aerolysin genes
of Aeromonas (Table 3). Using conventional PCR with 16S
rRNA gene primers (Table 2), Aeromonas strains used in this
study tested positive to the 16S rRNA gene-targeting PCR
assay (Table 3), while no amplification products were
observed for nontargeted bacteria. For Aeromonas 16S rRNA
gene real-time PCR assay, all tested Aeromonas strains,
including strains from ATCC and water treatment plants,
showed a Ct value of 10.113.5 for 2.5ng DNA per reaction.
The presence of aerolysin gene in each Aeromonas was
investigated by using conventional PCR with primers AHCF1
and AHCR1M (13, 26, 31, 32). No signals were detected when
the assay was challenged against non-Aeromonas bacteria.
As expected, not all Aeromonas contain an aerolysin gene.
Four Aeromonas ATCC strains and only 8 out of 18 isolates
yielded the expected fragment (aerolysin gene), indicating
the presence of the gene (Table 3). When using the developed
real-time PCR assay, all aerolysin-gene-containing strains
showed a Ct value ranging from 15.3 to 18.4 per 2.5ng of
genomic DNA, except for two isolates (A. popoffii plant 1, no.
1 and A. popoffii plant 6, no. 1) and three ATCC Aeromonas
strains (A. caviae, A. sobria, and A. trota). The higher Ct values
for the three ATCC model strains might be caused by a total
of three to six mismatches in the primers (accession numbers
AF064068, U40711, and Y00559 (38)). The results were
consistent with the observation of the weaker bands on the
gel. However, as the aerolysin gene sequences of A. popoffii
were not available in GenBank (as accessed on June fourth,
2007), it is unclear whether the slightly higher Ct value was
due to the mismatches in the primers.
Overall, these results indicated that the designed realtime PCR assay can potentially detect most Aeromonas.
Because no A. caviae, A. sobria, or A. trota were isolated in
this study, the quantities of total and aeroysin gene-

containing Aeromonas measured by our real-time PCR assays


are justified. However, according to the Ct values (Table 3),
our aerolysin gene assay might underestimate the gene copies
if A. caviae, A. sobria, A. trota, and A. popoffii are present as
dominant species in the samples. Also, in the cases when the
quantification of these strains are of interests or concern,
more research is needed to develop new sets of real-time
PCR assays that are specific to these strains and their aerolysin
genes.
Both assays exhibited a log-linear detection range across
7 orders of magnitude (from 5 to 5 107 copies). High
coefficients of determination (R2) of the standard curves were
observed, 0.998 for 16S rRNA genes and 0.999 for aerolysin
genes of Aeromonas , (Supporting Information Figures
S1-S2). The PCR amplification efficiencies for 16S rRNA genes
and aerolysin genes of Aeromonas were 92 and 99%,
respectively. The efficiencies were calculated by using
10(-S)-1, where S is the slope of the standard curve (39). The
detection limits for the real-time PCR assays for 16S rRNA
genes and aerolysin genes of Aeromonas were 20 copies and
10 copies per PCR reaction, respectively. These values were
comparable to reported detection limits for other bacterial
real-time PCR assays (36, 40).
Effectiveness of Aeromonas Removal by Water Treatment Process. (i) Conventional Treatment Processes. (source
water (SW) f prechlorination (PreCl2) f coagulation/
flocculation/sedimentation (C/F/S) f rapid sand filtration
(RSF) f chlorine disinfection (Cl2)): Cases of plants 13. The
concentrations of Aeromonas in water samples measured by
culture Method 1605 and by real-time PCR assays are listed
in Table 4. Removals of Aeromonas by different treatment
process ranged from >0.9 to >3.3 log reductions for plant
1, from >0.3 to >3.1 log reductions for plant 2, and from
>0.6 to 4 log reductions for plant 3 (Supporting Information
Table S2). Plant 1 and plant 2 showed similar removal
effectiveness of Aeromonas. River water was used as source
water for plants 1 and 3. Lake water was used as source water
for plant 2. The source water used by plant 3 had the highest
turbidity (NTUs were 6.57) compared to other source water
(NTUs <3) collected in this study (Supporting Information
Table S1).
Case of Plant 1. Based on two sampling events, source
water contained 1 101 to 1.9 103 CFU/100 mL culturable
Aeromonas, 5.7 102 to 1.0 104 copies/100 mL of aerolysin
gene and 1.7 104 to 9.8 104 copies/100 mL of Aeromonas
16S rRNA gene. Although the second run sample of source
water had higher turbidity, the concentrations of Aeromonas
were lower than those measured from the first run sample
with low turbidity. For all plants and samples, the real-time
PCR assays specific for Aeromonas 16S rRNA genes generally
measured higher values then that measured from culturebased method.
No Aereomonas colony, 16S rRNA, or aerolysin gene was
detected by culture and molecular methods in water samples
collected after filtration. While there were variations in
removal efficiencies based on the results of different methods,
most of the Aeromonas were removed through the preCl2
and C/F/S process. The high reduction (3.3 log reduction)
based on the culture-based assay might be due to the lesser
sensitivity of the method at low concentrations or due to the
poor culturability of injured and stressed microorganisms
due to prechlorination, especially when selective media are
used as selective agents that can have an inhibitory or toxic
effect on injured targeted bacteria (41). No virulent Aeromonas
spp. (aerolysin-gene-containing Aeromonas spp.) was detected after sedimentation. While there were no detectable
colonies in water after coagulation and sedimentation, realtime PCR assay measured 3.5 102 to 2.4 103 copies/100
mL Aeromonas 16S rRNA genes in the same water samples.

Case of Plant 2. The culture-based method only could


detect Aeromonas in source water (1.5 102 to 1.3 103
CFU/100 mL) but not in any intermediate or finished water.
However, real-time PCR assays detected measurable Aeromonas 16S rRNA genes and aerolysin genes not only in source
water but also in samples collected after C/F/S and RSF.
Case of Plant 3. Once again, all the methods detected
Aeromonas in source water: 1.1 103 to 1.5 103 CFU/100
mL culturable Aeromonas, 1.1 103 to 1.5 104 copies/
100 mL of aerolysin gene, and 1.0 104 to 8.1 105 copies/
100 mL of Aeromonas 16S rRNA gene (Table 4). There were
no detectable Aeromonas colonies nor 16S rRNA gene of
Aeromonas in all intermediate and finished samples, except
for the sample collected after C/F/S during the second
sampling event (83 copies of Aeromonas 16S rRNA gene/100
mL). No aerolysin-gene-containing Aeromonas spp. was
detected in samples after C/F/S.
(ii) Conventional Treatment Processes with UV+ Cl2 for
Disinfection. (SW f C/F/S f RSF f UV+ Cl2): Case of plant
5. No prechlorination was used for source water in plant 5.
Removals of Aeromonas by different treatment process units,
ranging from >0.04 to >3.8 log reductions, are shown in
Supporting Information Table S2.
Aeromonas concentrations were measured by different
methods: 7.0 103 to 4.9 103 CFU/100 mL of culturable
Aeromonas, 7.2 103 to 1.0 104 copies/100 mL of aerolysin
gene, and 2.7 105 to 4.3 105 copies/100 mL of Aeromonas
16S rRNA gene. While no Aeromonas colony was detected in
water collected after C/F/S, RSF, and UV+Cl2, Aeromonas
16S rRNA genes were detected in water samples collected
after C/F/S and RSF. During the second sampling event,
aerolysin gene was detected in water collected after C/F/S
(200 aerolysin gene copies/100 mL, Table 4). Similarly, no
Aeromonas colonies or genes were detected in finished water
like those collected from the other drinking water treatment
plants.
(iii) Conventional Water Treatment Processes with Slow
Sand Filtration (SSF). (SWfSSFfCl2 or Chloramine): Cases
of plant 67. Measured turbidities of water samples are
available in the (Supporting Information Table S1). Removals
of Aeromonas by different treatment process units, ranging
from >0.6 log to >1.8 log as log reductions, are shown in
Supporting Information Table S2.
Case of Plant 6 (using Cl2). The source water for plant 6
is a brook, where the turbidity was below 0.5 NTU. However,
detectable Aeromonas concentrations were still observed by
different methods: 56.5 CFU/100 mL of culturable Aeromonas,
3.2 102 copies/100 mL of aerolysin gene, and 5.4 103
copies/100 mL of Aeromonas 16S rRNA gene (Table 4). All
the measured concentrations of Aeromonas in source water
for plant 6 were 1 to 2 orders of magnitude lower than those
used by plants 15. The finished water was free of Aeromonas
based on results from all the methods.
Case of Plant 7 (Using Chloramine). Removals of Aeromonas by different treatment process units, ranging from
>0.5 to >1.0 log as log reductions (Supporting Information
Table S2).
Lake water was used as source water for plant 7. The
turbidity of the pond water was below 0.5 NTU (Supporting
Information Table S1). Low concentrations of Aeromonas in
the source water were detected: 110 CFU/100 mL culturable
Aeromonas, and 1.7 103 copies/100 mL of Aeromonas 16S
rRNA gene (Table 4). Based on turbidities and concentrations
of Aeromonas measured by different methods, the quality of
source water used by plant 7 was comparable to that used
by plant 6. As shown in Supporting Information Table S2,
slow sand filtration alone had 0.5 log reduction of the
Aeromonas based on the real-time PCR assay. This result is
close to the removal efficiency of plant 6 in Vermont. Although
the source water quality appeared to be better than those
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008

plant 7
EPA method 1605
(CFU/100 mL)

plant 6
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

plant 5
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

plant 4
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

plant 3
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

plant 2
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

plant 1
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

method

C7
<1a

4.1 102 (40)e

3.3 103 (62)e

A7
1

<50b

1.1 102 (18.3)e

<500b

8.5 103 (1.5 103)e

2.7 105 (1.2 104)e

<50b

C6
<1a

<500b

7.2 103 (1.9 103)e

C5
<1a

A6
<10a

B5
<1a

<400c

6.3 105 (6.0 104)e

A5
7.0 103 (8.5 102)e

<200b

<100c

2.2 104 (2.0 103)e

<200c

1.1 104 (3.3 103)e

<50b

C4
<1a

<100b

1.1 103 (3.6 102)e

C3
<1a

<200c

<100b

C2
<1a

<100c

<50b

C1
<1a

A4
2.5 104 (2.0 103)e

B3
<1a

1.3 103 (1.3 102)e

1.7 104 (2.1 103)e

A3
1.5 103 (6.0 102)e

<100b

5.9 103 (1.5 103)e

2.4 103 (7.3 102)e

9.8 103 (8.4 103)e


B2
<1a

<50b

1.0 103 5.0 103)e

A2
1.3 103 (3.5 102)e

B1
<1a

A1
1.9 103 (1.8 102)e

first sampling eventd

TABLE 4. Concentrations of Aeromonas in Water Samples Collected from Surveyed Plants

<40c

<20b

D4b
<0.2a

D7
<0.2a

<100c

<50b

D6
<0.2a

<100c

<50b

D5
<0.2a

<400c

<200b

D4a
<1a

<100c

<50b

D3
<0.2a

<57.1c

<28.6b

D2
<0.2a

<100c

<50b

D1
<0.2a

A7
10

5.4 103 (11.4)e

3.2 102 (1.0 102)e

A6
56.5 (5.5)e

4.3 105 (1.9 104)e

1.0 104 (5.6)e

A5
4.9 103 (4.0 102)e

1.8 105 (1.4 104)e

6.3 103 (1.4 103)e

A4
6.5 103 (5.0 x102)e

8.1 105 (6.5 104)e

1.5 104 (4.0 103)e

A3
1.1 103 (7.0 102)e

7.8 103 (1.2 103)e

<1200

A2
1.5 102 (50)e

1.7 104 (9.1 103)e

5.7 102(9.1)e

A1
10

concentration of Aeromonas

2.8 103 .5 102)e

2.0 102 (3.4)e

B5
<1a

83.3 (20)e

<40b

B3
<1a

4.3 103 77.1)e

<30b

B2
<1a

3.5 102 (1.1 102)e

<27.3b

B1
<1a

C7
<1a

96.4 (11.3)e

<16.7b

C6
<1a

55.5 (8.2)e

<20b

C5
<1a

<20c

<10b

C4
<1a

<20.7c

<10.3b

C3
<1a

66.1 (27.1)e

<30b

C2
<1a

<46.2c

<23.1b

C1
<1a

second sampling eventd

<20c

<10b

D4b
<0.2a

D7
<0.2a

<60c

<30b

D6
<0.2a

<40c

<20b

D5
<0.2a

<20c

<10b

D4a
<1a

<20c

<10b

D3
<0.2a

<30c

<15b

D2
<0.2a

<21.4c

<10.7b

D1
<0.2a

<60b

<120c

<88.3b

1.7 103 (1.6 102)e

<100b

<200c

<2000b

<4000c

D8
<1a
A7
1

pilot plant
EPA method 1605
(CFU/100 mL)
aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

a
The values refer to the detection limits per 100 mL of EPA Method 1605 (lowest detectable CFU per 100 mL). The calculation was based on different sample size used for
filtration. b The values refer to the detection limits per 100 mL of real-time PCR aerolysin gene assay (lowest detectable aerolysin gene per 100 mL). The calculation was based on
different sample size used for filtration. c The values refer to the detection limits per 100 mL of real-time PCR Aeromonas 16S rRNA gene assay (lowest detectable Aeromonas 16S
rRNA gene per 100 mL). The calculation was based on different sample size used for filtration. d A1-A7, B1-B5, C1-C7, and D1-D8 represent sampling locations as shown in Table 1.
e
Average concentration of duplicate samples; the number in parenthesis refers to the range of the concentrations of duplicate samples.

D8
<1a
A7
10

<120c
5.1 102(50)e
1.7 103 (1.6 102)e
<200c
<4000c

<200c

<60b
<100b

aerolysin gene
(copies/ 100 mL)
16S rRNA gene
(copies/100 mL)

<2000b

<100b

<88.3b

<46.1b

second sampling eventd


concentration of Aeromonas
first sampling eventd
method

TABLE 4. Continued

used by plants 15, low concentrations of Aeromonas 16S


rRNA gene were still detected in the water samples collected
after slow sand filtration.
(iv) Unconventional Treatment Processes. (SW f membrane filtration f UV f NaOH and Cl2): Case of plant 4. The
source water (lake water) used by Plant 4 had low turbidity
(NTU <1) (Supporting Infortmation Table S1). Removals of
Aeromonas by different treatment process units in plant 4
ranged from >2 to >4.4 log as log reductions (Supporting
Information Table S2).
Despite that the turbidity of source water was quite low
compared to other plants surveyed, relatively high levels of
Aeromonas were measured by different methods: 2.5 104
to 6.5 103 CFU/100 mL of culturable Aeromonas, 6.3 103
to 2.2 104 copies/100 mL of aerolysin gene, and
1.8 105 to 6.3 105 copies/100 mL of Aeromonas 16S rRNA
gene (Table 4). However, no detectable Aeromonas colony,
16S rRNA gene, or aerolysin gene was present in water after
membrane filtration. Since the pore size of the membranes
is 0.1 m, a size smaller than most sizes of bacteria, over 2.0
log removal is shown by all measurement methods and
greater than 4.0 log in some cases.
(v) Unconventional Drinking Water Treatment Processes.
(SWf preozonenation f slow sand filtration (SSF)): Case of
pilot plant. Removals of Aeromonas by different treatment
process units in pilot plant ranged from >1.0 to >1.2 as log
reductions (Supporting Information Table S2).
No Aeromonas colony or genes were detected in finished
water by all the methods (Table 4). The treatment processes
of the pilot plant, preozonation followed by SSF, appeared
to remove Aeromonas effectively.

Discussion
In this study, real-time PCR assays were successfully developed for quantifying total and aerolysin gene-containing
Aeromonas spp. in water samples. Following the validation
of these assays, they were applied to source, intermediate
and finished water samples collected from seven drinking
water treatment plants and one pilot plant. High correlations
between the results obtained from EPA culture-based method
and from real-time PCR assays for Aeromonas 16S rRNA gene
and aerolysin gene were observed. The correlation coefficient
between results of Aeromonas 16S rRNA gene and aerolysin
gene measured by real-time PCR assays was high (r ) 0.88).
A lower correlation coefficient (r ) 0.80) between the results
obtained from the EPA culture-based method and those
obtained from real-time PCR assay for Aeromonas 16S rRNA
gene was observed. Similarly, there was a correlation (r )
0.83) between results from EPA culture-based method and
aerolysin gene measured by the real-time PCR assay. All three
correlations were significant at the 0.01 level.
Several factors might contribute to the differences of
detection between EPA Method 1605 (culture-based method)
and real-time PCR assays. First, injured and stressed organisms could result in poor culturability that was not able to
be detected by the culture-based method (41). Second, the
EPA culture-based method might underestimate the concentration of Aeromonas because only yellow colonies on
the ADA-V agar plates are considered to be presumptive
Aeromonas and because some Aeromonas strains are sensitive
to ampicillin, an antimicrobial agent that was used in EPA
Method 1605 to suppress the growth of background bacteria.
Previous studies have reported the presence of atypical blue
colonies on ampicillin-dextrin agar plates as Aeromonas as
well as the occurrence of ampicillin-sensitive Aeromonas
strains (20, 42, 43). In addition, not all Aeromonas spp. are
indole positive (44). Experiments using pure cultures suggested that recovery of Aeromonas was less than 100% after
24 h of incubation; i.e., after prolonged incubation colonies
VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

1197

FIGURE 1. Turbidity (NTU) and quantification of Aeromonas in all water samples collected in this study.
grew and merged, which led to lower colony counts (43). On
the other hand, real-time PCR may overestimate the quantity
of target genes due to the presence of dead cells (45). To
estimate the absolute values of target genes from viable cells,
one would need to first sort the live cells from dead cells in
the sample before applying the developed real-time PCR
assays. Recently, Nocker et al. (46) demonstrated that the
use of propidium monoazide (PMA) and PCR in combination
can successfully quantify viable and dead cells over a wide
range of bacteria species, including E. coli O157:H7. The
application of PMA also overcomes the disadvantage of using
ethidium monoazide (EMA) (4750) which can penetrate
some live cells of some bacterial species. Therefore, our realtime PCR assays can be used in combination with PMA or
EMA in the future to quantify viability of Aeromonas in
sample. Additionally, since A. hydrophila has ten 16S rRNA
gene copies per genome (51), estimates from real-time PCR
assays might be higher than those from culture-based
methods. Aerolysin gene is present in A. hydrophila and other
species. Whereas A. hydrophila is known to have only one
copy of aerolysin gene, no information is available for other
aerloysin gene-containing Aeromonas.
One contribution of this study was to use molecular tool
for quantifying aerolysin gene-containing Aeromonas in water
samples. In this study, aerolysin genes were detected in most
source water samples. No aerolysin gene or 16S rRNA gene
of Aeromonas was detected in any finished water samples.
The ratio of aerolysin genes to Aeromonas 16S rRNA genes
ranged from 0.023 to 0.15. The ratio measured from the source
water for each plant was as follows: 0.030.1 for plant 1; 0.15
for plant 2; 0.020.11 for plant 3; 0.04 for plant 4; 0.0230.027
for plant 5; 0.06 for plant 6; and 0.05 for plant 7.
To our knowledge, this is the first study using both culture
and nonculture-based methods to determine removal effectiveness of Aeromonas by water works. In fact, regardless
the assessment methods used, information of the effectiveness of Aeromonas removal by drinking water is limited. By
using ampicillin-dextrin agar (52), a study from Netherlands
water works reported that the number of culturable Aeromonas in source waters (surface water) was reduced from
0.2 to 4.7 104 CFU/100 mL to 0-105 CFU/100 mL in treated
waters, suggesting conventional drinking water treatment
could remove most of Aeromonas. Results of our study
indicated that the combination of drinking water treatment
processes, conventional and unconventional, can effectively
1198

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008

remove Aeromonas from treated water. While most of the


Aeromonas in source water was removed by filtration process
(plants 13, except one sample collected in plant 2), it was
still detected in water after filtration (plants 57). One major
difference in the treatment processes among these plants
was prechlorination. Since the free Aeromonas cells are
susceptible to chlorine-based disinfectants (26), prechlorination of source water before other treatment processes
might improve the removal of Aeromonas as observed in
plants 13. In addition, ozonation of source water as
employed by the pilot plant was also effective. Unlike the
traditional treatment processes (i.e., preCl2 + C/F/S + RSF
used by Plants 13), using membrane filtration alone (i.e.,
used by plant 4) was sufficient to remove Aeromonas. Despite
the low turbidity of the source water used by plant 6, the 16S
rRNA gene of Aeromonas was detected in samples collected
after slow sand filtration, suggesting that using slow sand
filtration alone might not be sufficient to remove Aeromonas
effectively. The removal efficiencies for Aeromonas by
different disinfection processes were not able to be determined in this study due to the undetectable Aeromonas
concentrations in the samples collected before and after
disinfection processes in all plants surveyed.
Turbidity is an easy measure of water quality. Every
drinking water treatment plant measures turbidity routinely.
To examine whether there is a relationship between turbidity
and Aeromonas concentrations, all the quantitative data
points and turbidity values were plotted together. As shown
in Figure 1, no relationship can be established between
turbidity and Aeromonas colonies, or between turbidity and
concentration of Aeromonas 16S rRNA genes, or between
turbidity and concentrations of aerolysin genes of Aeromonas.
However, a common trend was observed: no Aeromonas
concentrations can be detected by any of the methods when
turbidities were below 0.06 NTU (11 out of 54 samples were
with turbidities below 0.06 NTU). Potential use of NTU <
0.06 as the first step screening criteria to indicate the presence
of Aeromonas in water samples will be beneficial and
practical. However, more field samples are needed to
statistically validate such an approach.

Acknowledgments
This work was supported in part by the Environmental
Protection Agency, the Water Treatment Technology Assistance Center at the University of New Hampshire (Sub-

award no. 04-866), and by the National Science Foundation,


award no. BES-0439389.

Supporting Information Available


Standard curves for quantifying 16S rRNA genes and aerolysin
genes of Aeromonas, turbidity of collected samples from each
plant, and average removal by different treatment processes
in each plant. This material is available free of charge via the
Internet at http://pubs.acs.org.

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ES071341G

Supporting Materials
for
Development and application of real-time PCR assays for
quantifying total and aerolysin gene-containing Aeromonas in
source, intermediate, and finished drinking water
Chang-Ping Yu1, Sara K. Farrell2, Bruce Robinson3, and Kung-Hui Chu1 *
1

Zachry Department of Civil Engineering, Texas A &M University, College Station,


TX77843-3136

The Center for Environmental Biotechnology, 3Department of Civil and Environmental


Engineering, University of Tennessee, Knoxville, TN 37996

Content
Figure S1 Construction of standard curves for quantifying Aeromonas 16S rRNA genes.
(A)10-fold serial dilutions of plasmid DNA (carrying a partial 16S rRNA gene
of A. hydrophila) ranging from 5 to 5 107 copies were used as templates for
real-time PCR. The dash line indicates the threshold fluorescence, a level at
which the threshold cycle (Ct) was determined. (B) A standard curve was
generated by plotting log quantity (starting copy numbers) vs. Ct (threshold
cycle). The coefficient of determination of the straight line, R2, was 0.998. (C)
PCR products were verified by taking first derivative of the melting curves.
Figure S2 Construction of standard curves for quantifying aerolysin genes of Aeromonas. (A)
10-fold serial dilutions of plasmid DNA (carrying a partial 16S rRNA gene of A.
hydrophila) ranging from 5 to 5 107 copies were used as templates for
real-time PCR. The dash line indicates the threshold fluorescence, a level at
which the threshold cycle (Ct) was determined. (B) A standard curve was
generated by plotting log quantity (starting copy numbers) vs. Ct (threshold
cycle). The coefficient of determination of the straight line, R2, was 0.999. (C)
PCR products were verified by taking first derivative of the melting curves.
Table S1 Turbidity of collected samples from each plant.
Table S2

Average removal by different treatment processes in each plant.

Supporting materials

Figure S1 Construction of standard curves for quantifying Aeromonas 16S rRNA


genes. (A)10-fold serial dilutions of plasmid DNA (carrying a partial 16S rRNA gene
of A. hydrophila) ranging from 5 to 5 107 copies were used as templates for
real-time PCR. The dash line indicates the threshold fluorescence, a level at which
the threshold cycle (Ct) was determined. (B) A standard curve was generated by
plotting log quantity (starting copy numbers) vs. Ct (threshold cycle). The coefficient
of determination of the straight line, R2, was 0.998. (C) PCR products were verified
by taking first derivative of the melting curves.

Supporting materials

ii

Figure S2 Construction of standard curves for quantifying aerolysin genes of


Aeromonas. (A) 10-fold serial dilutions of plasmid DNA (carrying a partial 16S
rRNA gene of A. hydrophila) ranging from 5 to 5 107 copies were used as templates
for real-time PCR. The dash line indicates the threshold fluorescence, a level at
which the threshold cycle (Ct) was determined. (B) A standard curve was generated
by plotting log quantity (starting copy numbers) vs. Ct (threshold cycle). The
coefficient of determination of the straight line, R2, was 0.999. (C) PCR products
were verified by taking first derivative of the melting curves.

Supporting materials

iii

Table S1 Turbidity of samples collected from each plant.

Plant #
Plant1
Plant 2
Plant 3
Plant 5
Plant 6
Plant 7
Plant 4
Pilot
Plant

A1
(1.25, 3.20)
A2
(1.00, 3.63)
A3
(7.00, 6.60)
A5
(1.60, 2.65)
A6
(0.31, 0.50)
A7
(0.56, 0.35)
A4
(0.60, 0.80)
A7
(0.56, 0.35)

Sample Location
(turbidity 1*, turbidity2**)
B1
C1
(0.36, 1.00)
(0.033, 0.1)
B2
C2
(0.90, 0.89)
(0.04, 0.08)
B3
C3
(0.70, 0.70)
(0.11, 0.10)
B5
C5
(0.35, 0.19)
(0.07, 0.08)
C6
(0.06, 0.06)
C7
(0.10, 0.08)
C4
(0.01, 0.03)

*form 1st sampling event.


** from 2nd sampling event.

Supporting materials

vi

D1
(0.033, 0.06)
D2
(0.09, 0.07)
D3
(0.04, 0.03)
D5
(0.25, 0.30)
D6
(0.06, 0.06)
D7
(0.14, 0.14)
D4a
D4b
(0.01, 0.03) (0.03, 0.03)
D8
(0.10, 0.09)

Table S2 Average removal by different treatment processes in each plant.


Log reduction
Method

First Sampling Event

Plant 1

Coagulation/
sedimentation

Filtration

EPA Method 1605

>3.3 loga

Aerolysin gene

1.6 log

Plant 2

Coagulation/
sedimentation

EPA Method 1605

>1.4 log

>1.8 log

>1.0 loga

1.1 log

>0.8 log

Plant 3

Coagulation/
sedimentation

Filtration

>3.2 log

>1.3 log

1.7 log

Disinfection

Coagulation/
sedimentation
>2.2 log

1.3 log

0.8 log

>0.3 loga

Disinfection

Coagulation/
sedimentation

Filtration

Disinfection

>3.0 log

>0.6 log

>2.6 log

16S rRNA gene

>1.7 log

4.0 log

Plant 4

Membrane

UV

Disinfection

Membrane

UV

>3.8 log

Aerolysin gene

>2.0 log

>2.8 log

16S rRNA gene

>3.2 loga

>4.0 loga

Plant 5

Coagulation/
sedimentation

Filtration

Disinfection

Coagulation/
sedimentation

Filtration

EPA Method 1605

>3.8 log

>3.7 log

>1.2 log

1.7 log

>1.0 log

16S rRNA gene

1.5 log

1.9 log

>0.04 loga

2.2 loga

1.7 loga

Filtration

Disinfection

Filtration
>1.8 log

Aerolysin gene

>1.3 loga

16S rRNA gene

0.9 log

>0.6 loga

1.7 log

Plant 7

Filtration

Disinfection

Filtration
>1.0 log

Aerolysin gene

16S rRNA gene

0.5 log

Disinfection

>0.2loga
Disinfection

>0.6 loga

Pilot Plant

Preozonation and slow sand filtration

EPA Method 1605

>1.0 loga

Aerolysin gene

16S rRNA gene

>1.2 loga

Calculations were based on method detection limits.

Supporting materials

>0.1 loga

EPA Method 1605

EPA Method 1605

Disinfection

Aerolysin gene

Plant 6

Disinfection

Disinfection

>1.0 log

>4.4 log

Aerolysin gene

EPA Method 1605

Filtration

>0.9 log

Disinfection

16S rRNA gene

EPA Method 1605

Filtration

Filtration

>3.1 log

Aerolysin gene

Disinfection

>2.3 log

16S rRNA gene

Second Sampling Event


Coagulation/
sedimentation

Preozonation and slow sand filtration

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