2008 ES&T Aeromonas

Development and Application ofReal-Time PCR Assays forQuantifying Total and AerolysinGene-Containing Aeromonas inSource, Intermediate, and FinishedDrinking WaterC H A N G - P I N G Y U , † S A R A K . F A R R E L L , ‡

B R U C E R O B I N S O N , § A N DK U N G - H U I C H U * , †

Zachry Department of Civil Engineering, Texas A&MUniversity, College Station, TX 77843-3136, The Center forEnvironmental Biotechnology and Department of Civil andEnvironmental Engineering, University of Tennessee,Knoxville, Tennessee 37996

Received June 6, 2007. Revised manuscript receivedOctober 21, 2007. Accepted November 26, 2007.

Aeromonas spp., opportunistic pathogens, are listed as amicrobiological contaminant on the Environmental ProtectionAgency’s (EPA) Drinking Water Contaminant CandidateList. Culture-based methods for identification and quantificationof Aeromonas in drinking water are time-consuming andoften fail to differentiate pathogenic species from nonpathogenicones. This study reports successful development andapplications of two real-time PCR assays, based on 16S rRNAgene sequences and a virulence gene (aerolysin gene), forrapid and effective quantification of total and aerolysin gene-containing Aeromonas spp. The assays successfully quantifiedtotal and aerolysin gene-containing Aeromonas in source,intermediate, and finished water samples collected from sevenwater works and one pilot plant. The effectiveness ofAeromonas removal by different drinking water treatmentprocesses was examined by comparing the results obtainedfrom the EPA culture-based method and developed real-timePCR assays. Regardless of the methods, our results indicated thatconventional water treatment combination (prechlorination/coagulation/sedimentation/rapid sand filtration) and membranefiltration alone could effectively remove Aeromonas. Slowsandfiltrationalonemightnotbeeffective.Theremovalefficienciesby different disinfection treatments were not determined, dueto the lack of detectable Aeromonas. No Aeromonas wasdetected in samples with turbidity below 0.06 NTU.

IntroductionThe genus Aeromonas is a group of bacteria that areubiquitous in aquatic environments (1–3) and may also bedetected in fish, food, bottled water, and drinking water

(4–12). Since some Aeromonas spp. can produce a range ofbiotoxins (such as hemolysins, cytotoxins, aerolysins, andeven enterotoxins), they are considered as opportunisticwaterborne pathogens responsible for acute gastroenteritisand wound infections in humans (13) and animals (14). In1996, Aeromonas was listed as a microbiological contaminanton the Environmental Protection Agency’s (EPA) DrinkingWater Contaminant Candidate List. Monitoring for Aero-monas was conducted by 120 large and 180 small publicwater systems (PWS, list 2) in 2003 by using EPA approvedculture-based method, Method 1605 (Federal Register, 40CFR Part 141, March 7, 2002). Like other culture-basedmethods, this method is time-consuming. Furthermore, thismethod might not be effective for identifying pathogenicspecies from nonpathogenic ones.

Quantitative culture-independent methods, like real-timePCR, might offer a better measure for the quantification andmonitoring purposes of pathogens in drinking water. Re-cently, real-time PCR has been developed for quantifyingEscherichia coli O157:H7 and hepatitis B viruses in envi-ronmental samples (15–17). Although studies have beenreported using real-time PCR for monitoring Aeromonas spp.in stools (18) and fish tissue (19), no studies have beenreported using real-time PCR for monitoring Aeromonas spp.in drinking water, and/or other environmental samples.

Data on Aeromonas removal by water treatment facilities isessential for regulatory agencies to recommend any specifictreatment process or to impose a practical regulation onAeromonas for safe drinking water. To address the concern ofAeromonas in drinking water, we developed and appliedquantitative and sensitive molecular assays to quantify totalAeromonas spp. and virulence gene-containing Aeromonas spp.in source, intermediate, and finished water of several selectedwater utilities. The results from the EPA method and themolecular assays were compared and used to determine theeffectiveness of various water treatment processes.

Materials and MethodsDrinking Water Treatment Plants and Sampling Loca-tions. To examine the effectiveness of Aeromonas removalby various drinking water treatment processes, water sampleswere collected from different treatment units of sevendrinking water treatment plants (plants 1–7) and one pilotplant (pilot plant). Table 1 lists information of these plants,including treatment capacity, process flow diagrams of eachplant, and sampling locations. Two sampling events wereconducted between April 2004 and August 2005. For eachsampling event, seven source water, 12 intermediate, andeight finished samples were collected. Procedures for samplecollection, preservation, and storage were followed asdescribed in EPA Method 1605, an approved culture-basedmethod for Aeromonas spp. monitoring (20). Water qualityparameters, such as pH and turbidity, were measured on siteduring samples collection (for turbidity see Supporting Infor-mation Table S1). For plants 1–4, 4 L of water samples werecollected from each sample location. For plants 5–7 and pilotplant, 2 L of water samples were collected from each samplinglocation. The collected samples were packed with ice andtransported to the laboratory for analysis within 24 h.

Strains. Both Aeromonas (22 strains) and non-Aeromonas(19 strains) bacteria were used to validate the quantitativemolecular assays developed in this study (Table 3). TheAeromonas bacteria include four strains purchased from theAmerican Type Culture Collection (ATCC) (ATCC 7966T,ATCC 15468, ATCC 35993, and ATCC 49657) and eighteenstrains isolated from water treatment plants in this study.

* Corresponding author phone: (979) 845-1403; fax: (979) 862-1542; e-mail.: [email protected].

† Texas A &M University.‡ The Center for Environmental Biotechnology, University of

Tennessee.§ Department of Civil and Environmental Engineering, University

of Tennessee.

Environ. Sci. Technol. 2008, 42, 1191–1200

10.1021/es071341g CCC: $40.75 2008 American Chemical Society VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1191

Published on Web 01/17/2008

The non-Aeromonas bacteria include one E. coli. purchasedfrom ATCC (ATCC 25922), 15 estrogen-degraders isolatedfrom activated sludge (21, 22), and three strains (E. coli,Pseudomonas fluorescens, and Bacillus thuringiensis) ob-tained from the University of Tennessee, Knoxville (23).

Enumeration and Isolation of Culturable Aeromonas.Culturable Aeromonas bacteria were enumerated by usingampicillin-dextrin agar with vancomycin (ADA-V) accordingto EPA Method 1605 (20). Different volumes of water sampleswere used for filtration. For source and intermediate water,sample volumes of 100, 10, and 1 mL were used. For finishedwater, 500 mL was filtered. Duplicate samples were used.Eighteen Aeromonas strains were isolated. These isolates were

further identified according to their 16S rRNA gene sequences(∼500 bp) that were amplified with the primers Aer66f andAer613r (Table 2). These isolates were also examined for thepresence of aerolysin gene.

DNA Extraction. Water samples were filtered through 0.2µm pore-size polycarbonate membrane filters (Whatman,Clifton, NJ) to retain bacteria cells on the membranes forDNA extraction. In this study, DNA was extracted either bylysing cells eluted from the membrane or retained on themembrane. For samples collected during the first samplingevent, the eluted cells were used. The retained bacterial cellson the filter membrane were eluted by immersing themembrane in 4 mL of phosphate-buffered saline, followed

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 primer name, sequence (5′-3′) references

aerolysin gene AHCF1 5′-GAGAAGGTGACCACCAAGAACA-3′ 30AHCR1M 5′-ARCTGACATCGGCCTTGAACTC-3′ this study

Aeromonas 16S rRNA gene Aer66f 5′-GCGGCAGCGGGAAAGTAG-3′ 25Aer613r 5′-GCTTTCACATCTAACTTATCCAAC-3′ this study

1192 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 4, 2008

by vigorous agitation at the maximum velocity of a vortexmixer (Maxi MixII, Thermolyne) for 10 min. This elution stepwas repeated three times. The eluted cells were thentransferred to centrifuge tubes and pelleted by centrifugation.The supernatant was discarded, and the eluted cells werethen lysed as described by Kapley et al. (24). The derivedDNA samples were concentrated 10-fold by ethanol pre-cipitation. For samples collected during the second samplingevent, DNA was extracted directly from the cells retained onthe filter membrane. UltraClean Water DNA Kit (MoBioLaboratories, Inc.) was used for DNA extraction. The derivedDNA samples were concentrated 20 times by ethanolprecipitation. Both concentrated and unconcentrated sampleswere used as templates for SYBR Green real-time PCR assays.

Real-Time PCR Assay for Quantifying Total Aeromonasspp. The region of 16S rRNA gene of Aeromonas spp. wasused as the target region for designing real-time PCR assayfor quantifying total Aeromonas spp. A forward primer Aer66f

and a reverse primer Aer613r (Table 2), were used foramplifying 16S rRNA gene of Aeromonas spp. The forwardprimer Aer66f was previously used as a fluorescence probefor in situ hybridization (25). The reverse primer Aer613rwas modified from the primer Con607R (26) which has beenused for sequencing a portion of the 16S rRNA gene ofAeromonas (27). By aligning 132 of 16S rRNA gene sequencesof cultured Aeromonas (longer than 1000 bp that weredeposited in GenBank database during the time the assayswere designed), the forward primer (Aer66f) only has onemismatch with four sequences (X60416, X74682, AY538658,and AJ536821) and the reverse primer (Aer613r) only has onemismatch with three sequences (AY264937, AJ536820, andAJ536821). The designed primer sets were also examined fortheir uniqueness to the target gene by comparing thesequences in GenBank using the Basic Local AlignmentSearch Tool (BLAST) (28) and the Probe Match program ofthe Ribosomal Database Project (29).

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

conventional PCR real-time PCR (values of Ct)

strain speciesAeromonas 16S

rRNA gene aerolysinAeromonas 16S

rRNA genef aerolysinf

model strains ATCC 7966T Aeromonas hydrophila + + 10.4 16.7ATCC 15468 Aeromonas caviae + + 10.7 24.6ATCC 35993 Aeromonas sobria + +e 13.2 32.2ATCC 49657 Aeromonas trota + +e 13.1 28.6

Aeromonasisolates plant 1, no. 2 A. hydrophila (99%) + - 12.3 -

plant 2, no. 1 + + 12.7 15.9plant 3, no. 3 + - 12.0 -plant 4, no. 2 + + 11.6 15.3plant 6, no. 2 + - 10.1 -plant 2, no. 2 A. encheleia (99%) + - 12.9 -plant 7, no. 3 + - 13.4 -plant 1, no. 3 A. media (99%) + - 13.4 -plant 4, no. 3 + - 13.3 -plant 1, no. 1 A. popoffii (99%) + + 10.6 21.3plant 5, no. 2 + - 13.5 -plant 6, no. 1 + + 10.1 18.7plant 3, no. 1a Aeromonas sp. + - 13.0 -plant 3, no. 2b + + 12.6 16.0plant 4, no. 1c + + 12.9 17.0plant 5, no. 1b + + 10.8 18.4plant 7, no. 1b + - 10.5 -plant 7, no. 2d + + 11.4 16.0

non-Aeromonasstrains ATCC 25922 Escherichia coli - - - -

CEB-1 Escherichia coli - - - -CEB-2 Pseudomonas fluorescens - - - -CEB-3 Bacillus thuringiensis - - - -ARI-1 Novosphingobium tardaugens - - - -KC1 Flavobacterium sp. - - - -KC2 Chitinophaga sp. - - - -KC3 Nocardioides simplex - - - -KC4 Rhodococcus ruber - - - -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. - - - -

a Partially sequenced 16S rRNA gene showed 96% similarity to both A. media and A. veronii. b Partially sequenced 16SrRNA gene showed 96–99% similarity to both A. hydrophila, A. salmonicida, and A. bestiarum. c Partially sequenced 16SrRNA 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.

VOL. 42, NO. 4, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 1193

Real-Time PCR Assay for Quantifying Aerolysin Gene-Containing Aeromonas spp. In this study, aerolysin genewas used as a biomarker of pathogenic Aeromonas spp. Areal-time PCR assay using primers AHCF1 and AHCR1M(Table 2) was developed for quantifying aerolysin-gene-containing Aeromonas spp. These primers (AHCF1 andAHCR1M) were modified from a primer set (AHCF1 andAHCR1) designed by Kingombe et al. (30). AHCF1 and AHCR1have been used for detecting aerolysin gene in many studies(13, 18, 26, 31, 32). Compared to AHCR1 primer, the reverseprimer AHCR1M designed in this study is more specific tofive aerolysin genes of A. hydrophila that were deposited inGenBank (accession numbers M84709 (33), M16495 (34),X65043, X65044, X65045 (35)).

Real-Time PCR Analysis. The SYBR Green real-time PCRassays for quantifying aerolysin gene and 16S rRNA gene ofAeromonas were performed similarly, except using differentannealing temperatures. Each reaction was performed in atotal volume of 25 µL, with QuantiTect SYBR Green PCRMaster Mix (QIAGEN Inc., Valencia, CA), 600 nM forwardand reverse primers, and 5 µL of DNA templates. The PCRthermal cycle was 95 °C for 10 min, followed by 35 cycles of95 °C for 30 s, 61 °C (for 16S rRNA genes of Aeromonas) or57 °C (for aerolysin genes of Aeromonas) for 45 s, and 72 °Cfor 30 s. PCR amplification and detection were performed byusing a DNA Engine Opticon continuous fluorescencedetection system (MJ Research, Waltham, MA). The cyclethreshold (Ct) value was determined automatically with thecomputer software (Opticon Monitor, version 1.4, MJ Re-search). All concentrated and unconcentrated samples weremeasured twice by each assay. A subset of each sample wasspiked with 1 µL of 105 copies of standard DNA (describedbelow) as an internal control during PCR amplification. Thiswas done as a means to determine whether inhibition ofPCR had occurred. The negative controls containing onlyHPLC water were also included in each PCR run. The meltingtemperatures of amplification products were determined bymelting curves. The melting curves were obtained byoperating PCR reactions as follows: heating to 95 °C for 1min, cooling to 55 °C, and then ramping to 95 °C. Meltingcurves were checked routinely to confirm the quantificationof the desired products.

Plasmid DNA was used as a template for constructingstandard curves. By using the designed primer sets, a 232bp-fragment of partial aerolysin gene and a 548 bp-fragmentof partial 16S rRNA gene were amplified from A. hydrophila(ATCC 7966). These products were cloned into the vectorpCR4-TOPO (TA cloning; Invitrogen, Carlsbad, CA.). Theinserts were confirmed by sequencing using M13 primersthat flank the cloning region. The sequences of inserts wereconfirmed by an Applied Biosystems 3100 DNA sequencer(Perkin-Elmer, Foster City, CA). Selected clones were grownovernight in 5 mL of LB broth with kanamycin, and theplasmids were purified using Wizard Plus SV Minipreps(Promega, Madison, WI). The plasmid DNA concentrationwas determined using a Hoefer DyNa Quant 200 Fluorometer(Hoefer, Pharmacia Biotech, San Francisco, CA). The copynumbers in samples were determined as described by Yu etal. (23).

Statistical Analysis. The experimental data were log10

transformed as described by Dionisi et al. (36). The correlationcoefficients among the results obtained from EPA method1605 and from real-time PCR assays were calculated by usingbivariate two-tailed Pearson correlation in SPSS version 14.0.

ResultsAeromonas Strains Isolated from Water Utilities Surveyed.Eighteen Aeromonas strains were isolated from this study.Based on their partial 16S rRNA sequences (∼500 bp) (Table3), they are A. hydorphila (five isolates), A. media (two

isolates), A. encheleia (two isolates), A. popoffii (threeisolates), and Aeromonas sp. (six isolates). No A. sobria, A.caviae, or A. trota were isolated. Our observation wasconsistent with a previous study conducted by the U.S. EPAin 2001–2002 (26). In the EPA’s study, no A. sobria or A. trotawas identified from 205 Aeromonas strains that were isolatedfrom 16 water treatment utilities. Only six isolates wereidentified as A. caviae (26).

Validation of Real-Time PCR Assays. The developed real-time PCR assays for quantifying total and aerolysin gene-containing Aeromonas were vigorously validated. First, thespecificity of primers for Aeromonas 16S rRNA genes wasdetermined by using the Probe Match Program of theRibosomal Database Project (http://rdp.cme.msu.edu/probematch/, as assessed on April 8, 2007). Only sequencesthat had been verified using Pintail software (37) and havenear-full-length sequences (>1200 bases) were selected forthe specificity analysis. When no base pair mismatches areallowed, the forward primer (Aer 66f) will anneal to 858 outof 967 Aeromonas genus 16S rRNA genes (including uncul-tured clones) and the reverse primers (Aer613r) will annealto 927 out of the 967 Aeromonas genus 16S rRNA genes. Fornontarget 16S rRNA gene sequences analysis, the forwardprimer (allowed no mismatches) will anneal to only sixsequences (<0.01%) out of 128 613 of 16S rRNA genesequences belonging to non-Aeromonas genera within thebacteria domain. The reverse primers (allowed no mis-matches) will anneal to only two sequences (<0.01%), andthese two sequences are different from those six sequencesthat can be annealed by the forward primer. The resultsof the analyses indicate that these primers had a highspecificity to 16S rRNA gene sequences belonging to Aero-monas genus and a very low chance to cross-hybridize withbacteria outside of the Aeromonas genus.

Pure cultures, both Aeromonas and non-Aeromonasstrains, were used to validate the designed real-time PCRassays for quantifying 16S rRNA genes and aerolysin genesof Aeromonas (Table 3). Using conventional PCR with 16SrRNA gene primers (Table 2), Aeromonas strains used in thisstudy tested positive to the 16S rRNA gene-targeting PCRassay (Table 3), while no amplification products wereobserved for nontargeted bacteria. For Aeromonas 16S rRNAgene real-time PCR assay, all tested Aeromonas strains,including strains from ATCC and water treatment plants,showed a Ct value of 10.1–13.5 for 2.5ng DNA per reaction.

The presence of aerolysin gene in each Aeromonas wasinvestigated by using conventional PCR with primers AHCF1and AHCR1M (13, 26, 31, 32). No signals were detected whenthe 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 isolatesyielded the expected fragment (aerolysin gene), indicatingthe presence of the gene (Table 3). When using the developedreal-time PCR assay, all aerolysin-gene-containing strainsshowed a Ct value ranging from 15.3 to 18.4 per 2.5ng ofgenomic DNA, except for two isolates (A. popoffii plant 1, no.1 and A. popoffii plant 6, no. 1) and three ATCC Aeromonasstrains (A. caviae, A. sobria, and A. trota). The higher Ct valuesfor the three ATCC model strains might be caused by a totalof three to six mismatches in the primers (accession numbersAF064068, U40711, and Y00559 (38)). The results wereconsistent with the observation of the weaker bands on thegel. However, as the aerolysin gene sequences of A. popoffiiwere not available in GenBank (as accessed on June fourth,2007), it is unclear whether the slightly higher Ct value wasdue to the mismatches in the primers.

Overall, these results indicated that the designed real-time PCR assay can potentially detect most Aeromonas.Because no A. caviae, A. sobria, or A. trota were isolated inthis study, the quantities of total and aeroysin gene-


containing Aeromonas measured by our real-time PCR assaysare justified. However, according to the Ct values (Table 3),our aerolysin gene assay might underestimate the gene copiesif A. caviae, A. sobria, A. trota, and A. popoffii are present asdominant species in the samples. Also, in the cases when thequantification of these strains are of interests or concern,more research is needed to develop new sets of real-timePCR assays that are specific to these strains and their aerolysingenes.

Both assays exhibited a log-linear detection range across7 orders of magnitude (from 5 to 5 × 107 copies). Highcoefficients of determination (R2) of the standard curves wereobserved, 0.998 for 16S rRNA genes and 0.999 for aerolysingenes of Aeromonas , (Supporting Information FiguresS1-S2). The PCR amplification efficiencies for 16S rRNA genesand aerolysin genes of Aeromonas were 92 and 99%,respectively. The efficiencies were calculated by using10(-S)-1, where S is the slope of the standard curve (39). Thedetection limits for the real-time PCR assays for 16S rRNAgenes and aerolysin genes of Aeromonas were 20 copies and10 copies per PCR reaction, respectively. These values werecomparable to reported detection limits for other bacterialreal-time PCR assays (36, 40).

Effectiveness of Aeromonas Removal by Water Treat-ment Process. (i) Conventional Treatment Processes. (sourcewater (SW) f prechlorination (PreCl2) f coagulation/flocculation/sedimentation (C/F/S) f rapid sand filtration(RSF)f chlorine disinfection (Cl2)): Cases of plants 1–3. Theconcentrations of Aeromonas in water samples measured byculture Method 1605 and by real-time PCR assays are listedin Table 4. Removals of Aeromonas by different treatmentprocess ranged from >0.9 to >3.3 log reductions for plant1, from >0.3 to >3.1 log reductions for plant 2, and from>0.6 to 4 log reductions for plant 3 (Supporting InformationTable S2). Plant 1 and plant 2 showed similar removaleffectiveness of Aeromonas. River water was used as sourcewater for plants 1 and 3. Lake water was used as source waterfor plant 2. The source water used by plant 3 had the highestturbidity (NTUs were 6.5–7) compared to other source water(NTUs <3) collected in this study (Supporting InformationTable S1).

Case of Plant 1. Based on two sampling events, sourcewater contained 1 × 101 to 1.9 × 103 CFU/100 mL culturableAeromonas, 5.7 × 102 to 1.0 × 104 copies/100 mL of aerolysingene and 1.7 × 104 to 9.8 × 104 copies/100 mL of Aeromonas16S rRNA gene. Although the second run sample of sourcewater had higher turbidity, the concentrations of Aeromonaswere lower than those measured from the first run samplewith low turbidity. For all plants and samples, the real-timePCR assays specific for Aeromonas 16S rRNA genes generallymeasured higher values then that measured from culture-based method.

No Aereomonas colony, 16S rRNA, or aerolysin gene wasdetected by culture and molecular methods in water samplescollected after filtration. While there were variations inremoval 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 lessersensitivity of the method at low concentrations or due to thepoor culturability of injured and stressed microorganismsdue to prechlorination, especially when selective media areused as selective agents that can have an inhibitory or toxiceffect on injured targeted bacteria (41). No virulent Aeromonasspp. (aerolysin-gene-containing Aeromonas spp.) was de-tected after sedimentation. While there were no detectablecolonies in water after coagulation and sedimentation, real-time PCR assay measured 3.5 × 102 to 2.4 × 103 copies/100mL Aeromonas 16S rRNA genes in the same water samples.

Case of Plant 2. The culture-based method only coulddetect 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 Aero-monas 16S rRNA genes and aerolysin genes not only in sourcewater but also in samples collected after C/F/S and RSF.

Case of Plant 3. Once again, all the methods detectedAeromonas in source water: 1.1 × 103 to 1.5 × 103 CFU/100mL 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 wereno detectable Aeromonas colonies nor 16S rRNA gene ofAeromonas in all intermediate and finished samples, exceptfor the sample collected after C/F/S during the secondsampling event (83 copies of Aeromonas 16S rRNA gene/100mL). No aerolysin-gene-containing Aeromonas spp. wasdetected in samples after C/F/S.

(ii) Conventional Treatment Processes with UV+ Cl2 forDisinfection. (SWf C/F/Sf RSFfUV+ Cl2): Case of plant5. 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 inSupporting Information Table S2.

Aeromonas concentrations were measured by differentmethods: 7.0 × 103 to 4.9 × 103 CFU/100 mL of culturableAeromonas, 7.2 × 103 to 1.0 × 104 copies/100 mL of aerolysingene, and 2.7 × 105 to 4.3 × 105 copies/100 mL of Aeromonas16S rRNA gene. While no Aeromonas colony was detected inwater collected after C/F/S, RSF, and UV+Cl2, Aeromonas16S rRNA genes were detected in water samples collectedafter 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, noAeromonas colonies or genes were detected in finished waterlike those collected from the other drinking water treatmentplants.

(iii) Conventional Water Treatment Processes with SlowSand Filtration (SSF). (SWfSSFfCl2 or Chloramine): Casesof plant 6–7. Measured turbidities of water samples areavailable in the (Supporting Information Table S1). Removalsof Aeromonas by different treatment process units, rangingfrom >0.6 log to >1.8 log as log reductions, are shown inSupporting Information Table S2.

Case of Plant 6 (using Cl2). The source water for plant 6is a brook, where the turbidity was below 0.5 NTU. However,detectable Aeromonas concentrations were still observed bydifferent 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). Allthe measured concentrations of Aeromonas in source waterfor plant 6 were 1 to 2 orders of magnitude lower than thoseused by plants 1–5. The finished water was free of Aeromonasbased on results from all the methods.

Case of Plant 7 (Using Chloramine). Removals of Aero-monas by different treatment process units, ranging from>0.5 to >1.0 log as log reductions (Supporting InformationTable S2).

Lake water was used as source water for plant 7. Theturbidity of the pond water was below 0.5 NTU (SupportingInformation Table S1). Low concentrations of Aeromonas inthe source water were detected: 1–10 CFU/100 mL culturableAeromonas, and 1.7 × 103 copies/100 mL of Aeromonas 16SrRNA gene (Table 4). Based on turbidities and concentrationsof Aeromonas measured by different methods, the quality ofsource water used by plant 7 was comparable to that usedby plant 6. As shown in Supporting Information Table S2,slow sand filtration alone had 0.5 log reduction of theAeromonas based on the real-time PCR assay. This result isclose to the removal efficiency of plant 6 in Vermont. Althoughthe source water quality appeared to be better than those


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used by plants 1–5, low concentrations of Aeromonas 16SrRNA gene were still detected in the water samples collectedafter slow sand filtration.

(iv) Unconventional Treatment Processes. (SW f mem-brane filtrationfUVfNaOH and Cl2): Case of plant 4. Thesource water (lake water) used by Plant 4 had low turbidity(NTU <1) (Supporting Infortmation Table S1). Removals ofAeromonas by different treatment process units in plant 4ranged from >2 to >4.4 log as log reductions (SupportingInformation Table S2).

Despite that the turbidity of source water was quite lowcompared to other plants surveyed, relatively high levels ofAeromonas 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, and1.8 × 105 to 6.3 × 105 copies/100 mL of Aeromonas 16S rRNAgene (Table 4). However, no detectable Aeromonas colony,16S rRNA gene, or aerolysin gene was present in water aftermembrane filtration. Since the pore size of the membranesis 0.1 µm, a size smaller than most sizes of bacteria, over 2.0log removal is shown by all measurement methods andgreater than 4.0 log in some cases.

(v) Unconventional Drinking Water Treatment Processes.(SWf preozonenationf slow sand filtration (SSF)): Case ofpilot plant. Removals of Aeromonas by different treatmentprocess units in pilot plant ranged from >1.0 to >1.2 as logreductions (Supporting Information Table S2).

No Aeromonas colony or genes were detected in finishedwater by all the methods (Table 4). The treatment processesof the pilot plant, preozonation followed by SSF, appearedto remove Aeromonas effectively.

Discussion

In this study, real-time PCR assays were successfully devel-oped for quantifying total and aerolysin gene-containingAeromonas spp. in water samples. Following the validationof these assays, they were applied to source, intermediateand finished water samples collected from seven drinkingwater treatment plants and one pilot plant. High correlationsbetween the results obtained from EPA culture-based methodand from real-time PCR assays for Aeromonas 16S rRNA geneand aerolysin gene were observed. The correlation coefficientbetween results of Aeromonas 16S rRNA gene and aerolysingene measured by real-time PCR assays was high (r ) 0.88).A lower correlation coefficient (r ) 0.80) between the resultsobtained from the EPA culture-based method and thoseobtained from real-time PCR assay for Aeromonas 16S rRNAgene was observed. Similarly, there was a correlation (r )0.83) between results from EPA culture-based method andaerolysin gene measured by the real-time PCR assay. All threecorrelations were significant at the 0.01 level.

Several factors might contribute to the differences ofdetection between EPA Method 1605 (culture-based method)and real-time PCR assays. First, injured and stressed organ-isms could result in poor culturability that was not able tobe detected by the culture-based method (41). Second, theEPA culture-based method might underestimate the con-centration of Aeromonas because only yellow colonies onthe ADA-V agar plates are considered to be presumptiveAeromonas and because some Aeromonas strains are sensitiveto ampicillin, an antimicrobial agent that was used in EPAMethod 1605 to suppress the growth of background bacteria.Previous studies have reported the presence of atypical bluecolonies on ampicillin-dextrin agar plates as Aeromonas aswell as the occurrence of ampicillin-sensitive Aeromonasstrains (20, 42, 43). In addition, not all Aeromonas spp. areindole positive (44). Experiments using pure cultures sug-gested that recovery of Aeromonas was less than 100% after24 h of incubation; i.e., after prolonged incubation coloniesTA

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grew and merged, which led to lower colony counts (43). Onthe other hand, real-time PCR may overestimate the quantityof target genes due to the presence of dead cells (45). Toestimate the absolute values of target genes from viable cells,one would need to first sort the live cells from dead cells inthe sample before applying the developed real-time PCRassays. Recently, Nocker et al. (46) demonstrated that theuse of propidium monoazide (PMA) and PCR in combinationcan successfully quantify viable and dead cells over a widerange of bacteria species, including E. coli O157:H7. Theapplication of PMA also overcomes the disadvantage of usingethidium monoazide (EMA) (47–50) which can penetratesome live cells of some bacterial species. Therefore, our real-time PCR assays can be used in combination with PMA orEMA in the future to quantify viability of Aeromonas insample. Additionally, since A. hydrophila has ten 16S rRNAgene copies per genome (51), estimates from real-time PCRassays might be higher than those from culture-basedmethods. Aerolysin gene is present in A. hydrophila and otherspecies. Whereas A. hydrophila is known to have only onecopy of aerolysin gene, no information is available for otheraerloysin gene-containing Aeromonas.

One contribution of this study was to use molecular toolfor quantifying aerolysin gene-containing Aeromonas in watersamples. In this study, aerolysin genes were detected in mostsource water samples. No aerolysin gene or 16S rRNA geneof Aeromonas was detected in any finished water samples.The ratio of aerolysin genes to Aeromonas 16S rRNA genesranged from 0.023 to 0.15. The ratio measured from the sourcewater for each plant was as follows: 0.03–0.1 for plant 1; 0.15for plant 2; 0.02–0.11 for plant 3; 0.04 for plant 4; 0.023–0.027for plant 5; 0.06 for plant 6; and 0.05 for plant 7.

To our knowledge, this is the first study using both cultureand nonculture-based methods to determine removal ef-fectiveness of Aeromonas by water works. In fact, regardlessthe assessment methods used, information of the effective-ness of Aeromonas removal by drinking water is limited. Byusing ampicillin-dextrin agar (52), a study from Netherlands’water works reported that the number of culturable Aero-monas in source waters (surface water) was reduced from0.2 to 4.7 × 104 CFU/100 mL to 0-105 CFU/100 mL in treatedwaters, suggesting conventional drinking water treatmentcould remove most of Aeromonas. Results of our studyindicated that the combination of drinking water treatmentprocesses, conventional and unconventional, can effectively

remove Aeromonas from treated water. While most of theAeromonas in source water was removed by filtration process(plants 1–3, except one sample collected in plant 2), it wasstill detected in water after filtration (plants 5–7). One majordifference in the treatment processes among these plantswas prechlorination. Since the free Aeromonas cells aresusceptible to chlorine-based disinfectants (26), prechlori-nation of source water before other treatment processesmight improve the removal of Aeromonas as observed inplants 1–3. In addition, ozonation of source water asemployed by the pilot plant was also effective. Unlike thetraditional treatment processes (i.e., preCl2 + C/F/S + RSFused by Plants 1–3), using membrane filtration alone (i.e.,used by plant 4) was sufficient to remove Aeromonas. Despitethe low turbidity of the source water used by plant 6, the 16SrRNA gene of Aeromonas was detected in samples collectedafter slow sand filtration, suggesting that using slow sandfiltration alone might not be sufficient to remove Aeromonaseffectively. The removal efficiencies for Aeromonas bydifferent disinfection processes were not able to be deter-mined in this study due to the undetectable Aeromonasconcentrations in the samples collected before and afterdisinfection processes in all plants surveyed.

Turbidity is an easy measure of water quality. Everydrinking water treatment plant measures turbidity routinely.To examine whether there is a relationship between turbidityand Aeromonas concentrations, all the quantitative datapoints and turbidity values were plotted together. As shownin Figure 1, no relationship can be established betweenturbidity and Aeromonas colonies, or between turbidity andconcentration of Aeromonas 16S rRNA genes, or betweenturbidity and concentrations of aerolysin genes of Aeromonas.However, a common trend was observed: no Aeromonasconcentrations can be detected by any of the methods whenturbidities were below 0.06 NTU (11 out of 54 samples werewith turbidities below 0.06 NTU). Potential use of NTU <0.06 as the first step screening criteria to indicate the presenceof Aeromonas in water samples will be beneficial andpractical. However, more field samples are needed tostatistically validate such an approach.

AcknowledgmentsThis work was supported in part by the EnvironmentalProtection Agency, the Water Treatment Technology As-sistance Center at the University of New Hampshire (Sub-

FIGURE 1. Turbidity (NTU) and quantification of Aeromonas in all water samples collected in this study.


award no. 04-866), and by the National Science Foundation,award no. BES-0439389.

Supporting Information AvailableStandard curves for quantifying 16S rRNA genes and aerolysingenes of Aeromonas, turbidity of collected samples from eachplant, and average removal by different treatment processesin each plant. This material is available free of charge via theInternet at http://pubs.acs.org.

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ES071341G


Supporting materials i

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 *

1Zachry Department of Civil Engineering, Texas A &M University, College Station, TX77843-3136 2The 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 ii

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.

A B

C

Supporting materials iii

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.

A B

C

Supporting materials vi

Table S1 Turbidity of samples collected from each plant. Plant # Sample Location

(turbidity 1*, turbidity2**) Plant1

A1 (1.25, 3.20)

B1 (0.36, 1.00)

C1 (0.033, 0.1)

D1 (0.033, 0.06)

Plant 2

A2 (1.00, 3.63)

B2 (0.90, 0.89)

C2 (0.04, 0.08)

D2 (0.09, 0.07)

Plant 3

A3 (7.00, 6.60)

B3 (0.70, 0.70)

C3 (0.11, 0.10)

D3 (0.04, 0.03)

Plant 5

A5 (1.60, 2.65)

B5 (0.35, 0.19)

C5 (0.07, 0.08)

D5 (0.25, 0.30)

Plant 6

A6 (0.31, 0.50) C6

(0.06, 0.06) D6

(0.06, 0.06) Plant 7

A7 (0.56, 0.35) C7

(0.10, 0.08) D7

(0.14, 0.14) Plant 4

A4 (0.60, 0.80) C4

(0.01, 0.03) D4a

(0.01, 0.03) D4b

(0.03, 0.03) Pilot Plant

A7 (0.56, 0.35) D8

(0.10, 0.09) *form 1st sampling event. ** from 2nd sampling event.

Supporting materials v

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

a Calculations were based on method detection limits.

Log reduction Method First Sampling Event Second Sampling Event

Plant 1 Coagulation/ sedimentation Filtration Disinfection Coagulation/

sedimentation Filtration Disinfection

EPA Method 1605 >3.3 loga - - >1.0 loga - -

Aerolysin gene >2.3 loga - - >1.3 loga - -

16S rRNA gene 1.6 log >1.4 loga - 1.7 log >0.9 loga -




Aerolysin gene >1.8 loga - - - - -

16S rRNA gene 1.1 log >0.8 loga - 1.3 log 0.8 log >0.3 loga





16S rRNA gene >1.7 log - - 4.0 log >0.6 log -

Plant 4 Membrane UV Disinfection Membrane UV Disinfection



16S rRNA gene >3.2 loga - - >4.0 loga - -




Aerolysin gene >1.2 loga - - 1.7 log >1.0 loga -

16S rRNA gene 1.5 log 1.9 log >0.04 loga 2.2 loga 1.7 loga >0.1 loga

Plant 6 Filtration Disinfection Filtration Disinfection

EPA Method 1605 - - >1.8 loga -

Aerolysin gene - - >1.3 loga -

16S rRNA gene 0.9 log >0.6 loga 1.7 log >0.2loga

Plant 7 Filtration Disinfection Filtration Disinfection

EPA Method 1605 - - >1.0 loga -

Aerolysin gene - - - -

16S rRNA gene - - 0.5 log >0.6 loga

Pilot Plant Preozonation and slow sand filtration Preozonation and slow sand filtration

EPA Method 1605 - >1.0 loga

Aerolysin gene - -

16S rRNA gene - >1.2 loga

Documents

2008 ES&T Aeromonas