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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.
1191
TABLE 1. Unit Operation and Sampling Locations of Drinking Water Treatment Plants Surveyed in this Study
references
AHCF1 5-GAGAAGGTGACCACCAAGAACA-3
AHCR1M 5-ARCTGACATCGGCCTTGAACTC-3
Aer66f 5-GCGGCAGCGGGAAAGTAG-3
Aer613r 5-GCTTTCACATCTAACTTATCCAAC-3
30
this study
25
this study
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.
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.
1193
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
1194
1195
1196
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
A7
1
<50b
<500b
<50b
C6
<1a
<500b
C5
<1a
A6
<10a
B5
<1a
<400c
A5
7.0 103 (8.5 102)e
<200b
<100c
<200c
<50b
C4
<1a
<100b
C3
<1a
<200c
<100b
C2
<1a
<100c
<50b
C1
<1a
A4
2.5 104 (2.0 103)e
B3
<1a
A3
1.5 103 (6.0 102)e
<100b
<50b
A2
1.3 103 (3.5 102)e
B1
<1a
A1
1.9 103 (1.8 102)e
<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
A6
56.5 (5.5)e
A5
4.9 103 (4.0 102)e
A4
6.5 103 (5.0 x102)e
A3
1.1 103 (7.0 102)e
<1200
A2
1.5 102 (50)e
5.7 102(9.1)e
A1
10
concentration of Aeromonas
B5
<1a
83.3 (20)e
<40b
B3
<1a
<30b
B2
<1a
<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
<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
<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
TABLE 4. Continued
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
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-
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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
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
Supporting materials
Supporting materials
ii
Supporting materials
iii
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)
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)
Plant 1
Coagulation/
sedimentation
Filtration
>3.3 loga
Aerolysin gene
1.6 log
Plant 2
Coagulation/
sedimentation
>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
>1.7 log
4.0 log
Plant 4
Membrane
UV
Disinfection
Membrane
UV
>3.8 log
Aerolysin gene
>2.0 log
>2.8 log
>3.2 loga
>4.0 loga
Plant 5
Coagulation/
sedimentation
Filtration
Disinfection
Coagulation/
sedimentation
Filtration
>3.8 log
>3.7 log
>1.2 log
1.7 log
>1.0 log
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
0.9 log
>0.6 loga
1.7 log
Plant 7
Filtration
Disinfection
Filtration
>1.0 log
Aerolysin gene
0.5 log
Disinfection
>0.2loga
Disinfection
>0.6 loga
Pilot Plant
>1.0 loga
Aerolysin gene
>1.2 loga
Supporting materials
>0.1 loga
Disinfection
Aerolysin gene
Plant 6
Disinfection
Disinfection
>1.0 log
>4.4 log
Aerolysin gene
Filtration
>0.9 log
Disinfection
Filtration
Filtration
>3.1 log
Aerolysin gene
Disinfection
>2.3 log