Development of a Sensitive Detection Method for Stressed E. coli

Feb 22, 2011 - Office of Research and Development, National Homeland Security Research Center, U.S. Environmental Protection Agency,. Cincinnati, Ohio...
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Development of a Sensitive Detection Method for Stressed E. coli O157:H7 in Source and Finished Drinking Water by Culture-qPCR Keya Sen,*,† James L. Sinclair,† Laura Boczek,‡ and Eugene W. Rice§ †

Office of Water, Technical Support Center, ‡Office of Research and Development, National Risk Management Research Laboratories, and §Office of Research and Development, National Homeland Security Research Center, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, United States

bS Supporting Information ABSTRACT: A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 108 nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

1. INTRODUCTION E. coli O157:H7 is a bacterial pathogen that causes hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS).1,2 The organism has been implicated as the etiological agent for several waterborne disease outbreaks.3-6 The infectious dose of this pathogen is in the range of 10-100 cells.7 E. coli O157:H7 was placed on U.S. EPA’s drinking water Contaminant Candidate List in 20098 because of its potential for causing disease outbreaks from drinking water and its associated health effects. The detection of this bacterium is time-consuming and tedious. Several detection methods have been used in the last two decades. These encompass serological, biochemical, immunological as well as molecular techniques and usually a combination of strategies is employed. The target commonly used for immunological detection is the somatic O antigen that is specific to E. coli O157 serogroup9,10 or the flagellar H7 antigen.11 For molecular genetic based detection, the gene targets include the H7 flagellin (fliCH7), the lipopolysaccharide (rfbE), and the virulence factors, Shigatoxin 1 (stx1), Shigatoxin 2 (stx2), hemolysin (hly933), and intimin gene (eae).12-16 Examples of molecular -genetic techniques include PCR,16 quantitative PCR (qPCR),12,15,17 reversetranscriptase PCR (RT-PCR)13,18-20 and recently, techniques based on gene microarrays and bacteriophage reporters have been reported.21,22 Immunological methods have been used for separation and isolation of the bacteria from food or water samples, typically in an immuno-magnetic separation (IMS) format. Recently a novel method using a cantilever based immunosensor, that directly detects the pathogen in a broth or water sample, has been reported.23 For isolation of the bacterium from water traditional culture based methods typically involve membrane filtration of 0.1 to 1 L samples, followed by enrichment of the filter in a broth such as r 2011 American Chemical Society

Trypticase Soy broth (TSB), an initial IMS separation of E. coli O157 strains from the TSB cultures, that is followed by growth again on Cefixime-Tellurite MacConkey (CT -SMAC) or SMAC - 4-methylumbelliferyl-beta-D-glucuronide (MUG)24 or Rainbow Agar O157, resulting in colonies with characteristic morphology that is specific for E. coli O157:H7. Although such methods allow for the isolation of a small number of viable E. coli O157:H7 cells, often these cells are not E. coli O157:H7 when further checked by biochemical typing or molecular methods.24 Cross-reactivity of the antibody,10 growth inhibition of E. coli O157 by other E. coli strains25 in the nonselective broth, and poor recovery of stressed cells are some of the reasons cited for the false negative responses. These traditional procedures take 3-5 days. To save time, some investigators go directly to PCR/qPCR identification after IMS10 or even directly to PCR after enrichment.25,26 These direct PCR assays described in the literature can detect in addition to E. coli O157:H7, other pathogenic E. coli strains such as E. coli O55:H7,10 and nonpathogenic strains such as E. coli O157:H43.13 The reason for this is some of the targeted E. coli O157:H7 genes are also present in other E. coli strains as well as other bacteria; stx1 and stx2 are present in Shigella. In the case of an isolated colony, multiple PCRs in different tubes or multiplex PCR in a single tube is usually employed to confirm the identity.13,15,16,27,28 For public health safety, especially in the time of outbreaks, rapid screening methods are needed that detect pathogenic microbes in water. For E. coli O157:H7 such methods should Received: October 4, 2010 Accepted: January 19, 2011 Revised: January 12, 2011 Published: February 22, 2011 2250

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Environmental Science & Technology also be able to detect low numbers, since the infectious dose of the bacterium is low. Additionally, methods should be able to detect stressed cells from environmental samples and ideally be able to determine whether they are viable, so that risk assessments can be based on occurrence of viable bacteria. The goal of this project was to develop a rapid method for detection of viable E. coli O157:H7 cells in source and drinking water. Presence/ Absence medium was selected for this method as a drinking water enrichment medium that was capable of allowing the growth of stressed coliform bacteria, including E. coli O157:H7. A highly specific, probe based, multiplex qPCR detection method was combined with this enrichment process, such that stressed bacteria were recovered and detected in about 24 h from one liter of water. Several commercially available qPCR kits were tested and one multiplex PCR assay was finalized for a rapid identification of the bacterium from source and finished drinking water. To confirm the identity and also extend the strategy to include other pathogenic E. coli, a second multiplex qPCR was developed that detected stx1, stx2, and eae genes.

2. EXPERIMENTAL SECTION 2.1. Bacterial Strains and Growth Conditions. A total of 61 E. coli strains, 37 of which were E. coli O157, were used for the study. These strains have been previously characterized and belong to different E. coli serotypes and contain different combinations of the virulence genes [Supporting Information (SI), Table S129,30 and Table S4]. Cells were typically maintained on LB agar plates (casein peptone: 10 g/L, yeast extract: 5 g/L, sodium chloride: 10 g/L þ Agar:15 g/L) or by growth overnight in Luria Broth (LB) at 35 °C. For verification of the E. coli O157: H7 strains, Sorbitol MacConkey agar (SMAC, Oxoid, Cambridge, UK) and Rainbow Agar O157 (Biolog, Hayward, CA) were used. Seven non-E. coli species at high concentration (108 cells) were used as background to test the detection of low densities of E. coli O157: H7 strain ATCC 35150 or ATCC 43889 and details of the experiment can be found in, SI, S1). Four E. coli strains, belonging to the serogroup O111, O26, OX3:38, and O:55 were also used as background organisms for detection of low densities of E. coli O157:H7 strain 4700. These strains were chosen based on the presence of the stx1/stx2 and eae genes (SI, Tables S1 and S4). Details of the experiment can be found in SI, S2. For nutrient limitation/starvation stress experiments, E. coli O157:H7 ATCC strain 43889 was used. Details of the experiment can be found in SI, S3. After 12 and 14 days bacterial numbers had declined by log 0.57 and log 0.72, respectively, of their prestarvation level. Appropriate dilutions of starved cells were added to the water at spike levels of 1- 1000 cells. A 6X concentrated Presence-Absence broth (Remel Inc., Lenexa, KS) was added to the spiked water sample, and the sample was grown for 18-20 h at 35 °C. One mL of the sample, in duplicate, was removed before the start of the incubation and stored at -20 °C, and another mL was removed after 18 h of incubation. All samples were processed together and DNA extracted as described in section 2.2. For chlorine stress experiments, E. coli O157:H7 ATCC 43889 was also used, and details of the stressing experiment can be found in SI (S4). When buffer, tap water, well water, or creek water were to be spiked with these cells, appropriate dilutions were made shortly after chlorine stressing of the cells, which were then seeded at a concentration of 1-1000 cells into the sample. The samples were enriched and processed as described above.

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2.2. Extraction of DNA. A 1-2 mm size colony from an overnight culture was used for DNA extraction. Alternatively, one mL of a culture broth or water sample was removed and centrifuged at 10,000g for 5 min. The supernatant was removed, and the pellet was resuspended in 100 μL of Prepman Ultra Sample Preparation Reagent (catalog# 4318930, Life Technologies, Foster City, CA). The suspension was heated at 95 °C for 10 min, cooled, and centrifuged at 10,000g for 2 min. Three microliters of the supernatant was directly used in a 30 μL PCR reaction. The supernatants were stored at 4 °C if they were to be used within the week otherwise they were stored at -20 °C. Extracts stored at -20 °C performed as well as a fresh preparation in a qPCR reaction, 20 months later (data not shown). This was the final extraction procedure used for water samples as well as pure cultures. For the generation of standard curves and for positive controls, pure DNA was obtained using the WaterMaster DNA purification kit (EPICENTRE Biotechnologies, Madison, WI) as per the instructions of the manufacturer. One other DNA extraction method was evaluated: one that was used with the iQ -check kit from BioRad (Hercules, CA). Details can be found in SI, S6. 2.3. qPCR Assays. Three qPCR kits available from different vendors, and five additional primers and probes found in the literature, were tested and combined in duplex or tripex assays. All primers were made by SIGMA-Aldrich and probes by Life Technologies (Foster City, CA). The list of primers and probes used in this study, other than those that were supplied in a kit are presented in SI, Table S2. The following qPCR assays were examined. 2.3.1. Triplex qPCR 2.3.1.1. Amplification of Two Noncoding Regions from the Chromosome of E. coli O157:H7. These two regions were amplified using MicroSEQ E. coli O157:H7 detection kit that was obtained from Life Technologies (Foster City, CA). The kit became commercially available in July, 2010. The sequences of the target are proprietary. The probes are Minor groove binding (MGB) probes with 6-FAM (6-carboxyfluorescein) and VIC (Life Technologies’ proprietary dye) being the label for the probes targeted to the two respective noncoding regions and NED the label for an internal positive control (IPC). The target for the IPC, which is a synthetic piece of DNA, is also present in the master mix containing the probes. Protocols supplied with the kit were followed for setup of the qPCR reaction in an ABI Prism 7900 or StepOnePlus sequence detection system (Life Technologies, Foster City, CA). Thus PCR reactions were carried out in a final volume of 30 μL with the volume of template being 3 μL. Reactions were run as per instructions in the instruments’ manual for the Fast mode, except that an initial 2 min at 95 °C enzyme activation step was included. The Ct baseline value was set at 0.5 for FAM and VIC dyes and 0.3 for the NED dye. A sample was considered positive for E. coli O157:H7 if a Ct value lower than 35 was obtained from all three reporters (FAM, VIC, and NED). 2.3.1.2. Amplification of eae, stx1, and stx2. The primers for these targets were designed as previously described.15 The probes were similar to the TaqMan probes used by Sharma et al. except that they were modified to serve as MGB probes (SI, Table S2). Probes for eae, stx1, and stx2 were labeled with NED, FAM, and VIC, respectively. Several master mixes were evaluated including Life Technologies’ (Foster City, CA) TaqMan Universal PCR Master Mix, TaqMan Environmental Master Mix, TaqMan Fast Master Mix and 2X Environmental Fast Master 2251

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Environmental Science & Technology Mix (Master mix v 2.0). The 2X Environmental Fast Master Mix was found to be the best in obtaining signal from all three probes when all three genes were present. This master mix can be custom ordered from Life Technologies (Catalog #4401512). The final concentrations of the primers and probes for the gene targets were 0.3 μM and 100 nM respectively, for stx1 and eae and 1.5 μM and 100 nM for stx2. Reaction setup and qPCR program followed was exactly as described in 2.3.1.1, above. The baseline for FAM and VIC was set to 0.05 and NED to 0.03. Ct values up to 37, obtained from any of the three reporters, were considered positive for the presence of that gene. 2.3.2. Duplex qPCR 2.3.2.1. Amplification of a Plasmid Based Noncoding Region by the iQ-check E. coli O157:H7. This duplex qPCR kit (catalog # 357-8114, Bio-Rad) targets the junction IS91-KatP that is located upstream of the hlyA gene on the pO157 plasmid (U.S. patent WO09955908A2) present in all E. coli O157:H7 cells. A molecular beacon probe that is labeled with FAM is used as the probe for this target. A synthetic piece of DNA is included in the reaction mix and it serves as the IPC. The IPC is detected by a second probe labeled with the dye HEX. Instructions provided with the kit by the manufacturer were followed, and 5 μL of the DNA template was used. qPCR and subsequent data analysis was carried out in a MiniOpticon real-time PCR detection system (BioRad catalog # 359-1590) according to the manufacturer’s instructions specified for iQ-check E. coli O157:H7. 2.3.2.2. Amplification of the eae Gene. This gene was amplified by Life Technologies’s TaqMan pathogen detection kit (AB 4366100). This kit is a duplex qPCR kit with the eae gene and an IPC targeted by FAM and VIC labeled probes, respectively. qPCR reaction was setup according to the instructions in the kit, using 3 μL of template DNA and was performed in a ABI Prism 7900 or StepOnePlus sequence detection system (Life Technologies, Foster City, CA) in the standard mode. 2.3.2.3. Amplification of the fliCH7 Gene and rfbE Gene. The primers and probe for the fliCH7 gene were designed according to Narang et al.12 while those for rfbE gene according to Sharma et al.13 The probes were modified to MGB probes with fliCH7 gene and rfbE gene being labeled with FAM and NED dyes, respectively. Primer and probe concentrations used and conditions for amplification were exactly as described in section 2.3.1.1. 2.4. Detection of E. coli O157:H7 from Water Samples. Water samples (1 L) either from drinking water wells or from runoff from a cattle shed were obtained in sterile bottles (Table 3). Two hundred milliliters of a 6X concentrated, filter sterilized Presence-Absence broth was added to the 1 L, and samples and incubated at 35 °C for 18-20 h. One mL of each water sample was removed in duplicate prior to incubation and another mL in duplicate, after the incubation. DNA was extracted from the samples as described in section 2.2. DNA extraction from spiked water samples (100 or 1000 mL) were performed in a similar manner, after the addition of the Presence-Absence broth. An aliquot of the enriched sample was diluted and plated on Rainbow Agar O157. DNA was extracted from the black colonies.

3. RESULTS 3.1. Specificity and Sensitivity of the Multiplex qPCR Assays. The specificity and the sensitivity of the primers and

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probes in the kits and those developed in this study were individually evaluated against a panel of bacteria obtained from different sources. The MicroSEQ kit that detected two noncoding genes in the chromosome correctly identified the 25 E. coli O157:H7 strains tested as well as 8 strains that were supplied blinded by Dr. Robert Mandrell (USDA). It also detected all 4 E. coli O157: NM strains (SI, Table S3). All other pathogenic E. coli strains tested negative by this kit including six E. coli O55 strains. Common waterborne bacteria such as A. hydrophila, P. aeruginosa, and E. faecalis were also negative. A Ct value of higher than 35 from the FAM or VIC reporters was considered negative when using this kit because some non-O157:H7 strains showed a Ct value of about 36 (e.g., Strain # 3861, Table S3) for the two targets. One mL of overnight cultures of E. coli O157:H7 usually gave a Ct value of 17.91 ((0.70) for FAM and 17.57 ( (0.88) for VIC (SI, Table S3) and values greater than 35 were far higher. The limits of detection (LOD) of this kit when using pure genomic DNA prepared from E. coli O157:H7, ATCC 35150 was 5 copies per 30 μL PCR reaction. The LOD was determined from a standard curve obtained from pure DNA concentrations of 3 to 10000 copies. This DNA was prepared by the WaterMaster DNA purification kit. The size of the E. coli genome (L) was taken to be 5.44 Mb and gene copy number (C) was determined from the equation C = M xN/LxD where M = concentration of DNA in grams, D = conversion factor from 1 Kb nucleic acid to Daltons, and N = Avogadro’s number, as described in Sen et al.32 The regression lines for the VIC detected target was y = -3.43x þ 41.32 (R2 = 0.988) and for the FAM target was y = -3.45x þ 41.21 (R2 = 0.994). When varying amount of E. coli O157:H7 cells (100-10,000 cells) were spiked in a background of 108 nontarget cells, and the DNA extracted by the Prepman Ultra Sample Preparation Reagent, the LOD estimated from three different experiments was 500 cells/mL (SI, Table S5). The actual number of E. coli O157:H7 cells in a PCR reaction of 30 μL (from 500 cells of E. coli O157:H7 spiked in a background of 108 nontarget cells/mL) was about 15 cells because the mixture of cells was first pelleted and resuspended in 100 μL of Prepman Reagent and 3 μL of this was applied to a PCR reaction. Similarly E. coli O157:H7 cells were spiked in a background of 108 cells of E. coli species (O:26, O111, O55, and OX3:H2) that contained stx1and/or stx2 or eae genes. The background gave no signal with the ABI MicroSEQ kit for the FAM and VIC dyes set to a cut off Ct of 35, when the O55 strain was absent (Background 2, SI, Table S5). When O55 was present (Background 3), a low level signal was obtained beyond Ct value of 35 for FAM but not for VIC(S1, Table S5). E. coli O157:H7 could be detected above the background levels when they were present at levels of 3000 cells or higher, estimated from 4 different experiments (SI, Table S5). The second triplex qPCR assay developed in this study detected the eae, stx1, and stx2 genes by the NED, FAM, and VIC dyes, respectively (SI, Table S4). For optimizing the reaction each of the primer and probe sets were first tested in a monoplex reaction and then combined in a triplex with DNA obtained from pure cultures. Although Ct values were different in the monoplex reaction, the LOD in the triplex reaction was 100 copies for the stx1 (y = -3.30x þ 43.66; R2 = 0.92), 50 copies for the stx2 gene (y = -3.30x þ 44.18; R2 = 0.95), and 50 copies for the eae gene (y = -3.35x þ 41.39; R2 = 0.92). The cut off Ct for the three genes were kept at 37 and the threshold was set at 0.03. This value was arrived at empirically so that stx1, stx2 genes were 2252

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Environmental Science & Technology detected in all previously characterized strains possessing these two genes. This triplex assay was also capable of detecting 500 cells in a background of 108 non-E. coli cells. The genes were absent in the background non-E.coli bacteria or showed slight amplifications beyond Ct values of 37 for stx2. Primers and probes were designed for the rfbE gene and fliCH7 gene, and they were combined in a duplex reaction using the same parameters for amplification as that for the two triplex reactions. All E. coli O157:H7 strains as well as those classified as E. coli O157: NM (SI, Table S4) tested positive for both genes but not any of the other strains. E. coli O55:H7 strains did not show the presence of rfbE gene. The cut off Ct for fliCH7 and rfbE was kept at 32 with the threshold being set at 0.03, because some non-H7 strains gave a signal above Ct value of 32 for fliCH7 (SI, Table S4). The LOD for fliCH7 as determined from standard curve was 10 copies (y = -3.34x þ 43.06: R2 = 0.98) and that for rfbE 100 copies (y = -3.71x þ 45.69; R2 = 0.99). The AB 4366100 kit (Life Technologies) was based on the detection of the eae gene. This kit detected all E. coli O157:H7 strains but in addition detected O55 strains as well as one O34 and one O111 strain (SI, Table S4). The target within the eae gene in this kit is probably different than the target of the eae gene in the triplex (stx1, stx2, eae) assay designed in the present study as the latter is highly specific to E. coli O157:H7 and E. coli O55: H7 strains. The LOD for this gene was 10 copies obtained from a standard curve with a regression line y = -3.3102x þ 41.589 (R2 = 0.99; amplification efficiency =100.44%). Amplification efficiency was calculated from the formula: Ex = 10(-1/slope) -1. The BioRad duplex kit correctly detected all the E. coli O157: H7 strains. The cut off Ct value had to be set to 35 because some of the non-O157 strains gave a sharp signal beyond this value. The LOD with this kit was 5 copies. This kit could detect 500 E. coli O157:H7 cells in a background of 108 nontarget cells. There was no difference in Ct values from samples prepared in the Prepman Ultra Sample Preparation Reagent (Life Technologies) or the lysis buffer provided with the iQ-check kit form BioRad. 3.2. Detection of Stressed E. coli O157:H7 in Water. Our enrichment/qPCR method was able to detect and determine viability of starved E. coli O157:H7 cells that were spiked in 1 L samples of well water at 4, 40, and 400 cells/100 mL. The water samples from the 2 wells analyzed in this study were routinely used as drinking water sources. Well-1 had 4.3  103 heterotrophic bacteria per mL, and Well-2 had 1.2  103 heterotrophic bacteria per mL. P/A broth used as a nutrient medium allowed starvation stressed spiked E. coli O157:H7 to become resuscitated and to grow in the presence of indigenous well water bacteria. When water samples were tested pre- and postenrichment after 18 h in P/A broth, for the presence of eae gene, there was a shift in Ct values from >40 to about 17-21 (þ1.6) (Table 1). Similar shift in Ct values was seen for the stx1, stx2 genes (results not shown). The 17-20 Ct value shift indicates a 5-6 log increase in genomic copies (calculated from a PCR amplification efficiency of 3.3 of the AB kit 4366100 used for the determination) which in turn indicated that the cells had increased in number. When 7 black colonies randomly picked from the Rainbow Agar O157 plates from the well water samples were tested by the eae kit, they tested negative. Black colonies isolated from spiked tap water samples, following enrichment, tested positive and the counts corresponded to a growth of 5-6 log in 18 h. This water sample lacked a coliform background and did not contain interfering bacteria that could be confused for E. coli O157:H7 on Rainbow medium. A similar increase in

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Table 1. Growth of Starved E. coli O157:H7 ATCC 43889 in Two Well Water Samples Enriched with P/A Brothb E. coli O157:H7 (CFU)a

Ct (0 h)

Ct (18 h)

difference in Ct

Well 1 uninoculated control

>40

>40

4/100 mL 40/100 mL

>40 >40

19.47 ((1.97) 18.55 ((1.71)

20.53 21.45

400/100 mL

36.92 ((2)

19.89 ((1.95)

17.03

Well 2 uninoculated control

>40

>40

4/100 mL

>40

18.65 ((1.68)

40/100 mL

>40

20.12 ((1.28)

19.88

400/100 mL

39.05 ((1.5) 18.44 ((1.77)

20.61

21.35

a CFU was determined from counts on TSB agar plates. b qPCR was performed with the AB kit # 4366100 that tests for the eae gene. Ct values are presented from PCR assays performed at least in triplicate.

Table 2. Growth of Chlorine Stressed E. coli O157:H7 ATCC 43889 Spiked in Well Water, Surface Water (Mill Creek), and Tap Water Enriched with P/A Brothb E. coli O157:H7 (CFU)a

Ct (0 h)

Ct (18 h)

difference in Ct

Well 1 uninoculated control

>40

3/100 mL

39.67 ((0.24) 18.83 ((0.24)

>40

20.84

30/100 mL

36.19 ((0.75) 20.49 ((0.61)

15.7

300/100 mL

32.42 ((0.27) 19.13 ((0.078)

13.29

3000/100 mL

29.8 ((0.314) 19.58 ((0.19)

10.22

200/1000 mL

37.21 ((0.19) 21.53 ((0.24)

15.68

200/100 mL

37.7 ((1)

12.83

200/1000 mL

37.4 ((1.5)

22.67 ((1.35)

14.73

200/100 mL

>40

22.56 ((0.89)

17.44

Mill Creek Water 24.87 ((0.26)

Tap Water

a

CFU was determined from counts on TSB agar plate. b qPCR was performed with the AB kit # 4366100 that tests for the eae gene. Ct values are presented from PCR assays performed at least in triplicate.

genomic copies (Ct value shift from 13 to 21) for the eae gene was noted for chlorine stressed cells (Table 2). One water sample used in the experiment, where chlorine stressed spiked E. coli O157:H7 were resuscitated, was from a creek which contained coliform bacteria and E. coli. The presence of these bacteria did not interfere with qPCR detection of spiked E. coli O157:H7 (Table 2). Comparison of Ct values obtained from starvation stressed and chlorine stressed cells showed interesting results. Ct values obtained from 3, 30, and 300 chlorine stressed cells spiked into water from Well-1 was less than Ct values of corresponding starvation stressed cells (4, 40, 400) spiked into the same well water, pre-enrichment (Table 2 and Table 1). This was probably due to the fact that some of the chlorine stressed cells were sublethally injured and did not grow on the agar plates and thus did not contribute to the colony count on which the spike level was based. The DNA present in these injured cells, nonetheless, may have contributed to lower Ct values. 2253

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Table 3. Presence of Potential E. coli O157:H7 in Field Samples As Determined by the AB-MicroSEQ Kit (Triplex) FAM site

VIC Ct (0 h)

NED

volume of water

Ct (0 h)

Ct (18 h)

Ct (18 h)

Ct (0 h)

Ct (18 h)

Indiana

70 mL

>40

38.81 ((1.5)

>40

34. 01((1.3)

34.09 (1.2)

33.69 ((2.06)

D1, Waco, Texas

1L

>40

>40

>40

24.12 ((1.3)

34.26 ((0.8724.)

35.23 ((0.79)

IC, Waco, Texas

1L

>40

39.85 ((0.27)

>40

>40

34.56 ((1.3)

35.43 (0.17)

NF, Waco, Texas

1L

>40

>40

>40

>40

28.85 ((0.07)

29.13((0.5)

D2, Waco, Texas

1L

>40

32.49 ((0.75)

>40

30.26 ((0.64)

33.77 ((1.1)

33.75 ((0.1)

D3, Waco, Texas.

1L

>40

>40

>40

>40

32.78 ((0.45)

33.56 ((1.25)

Table 4. Presence of stx 1, stx2, eae, and E. coli O157:H7 in Field Samples Determined by MGB Probes in TaqMan Reaction (Triplex) FAM (stx1) site

volume of water

Ct (0 h)

VIC (stx2)

Ct (18 h)

Ct (0 h)

NED (eae)

Ct (18 h)

Ct (0 h)

Ct (18 h)

Indiana

70 mL

>40

>40

>40

>40

>40

>40

D1, Waco, Texas

1L

>40

24.15((0.42)

>40

25.67 ((0.59)

>40

>40

IC, Waco, Texas

1L

>40

>40

>40

>40

>40

>40

NF, Waco, Texas

1L

>40

>40

>40

31.95 ((0.62)

>40

>40

D2, Waco, Texas

1L

>40

25.03 ((0.19)

>40

25.76 ((0.62)

>40

31.5 ((0.3)

D3,Waco, Texas

1L

>40

>40

>40

22.62((0.44)

>40

>40

Table 5. Results from Different qPCR Assays for the Detection of E. coli O157:H7 in Surface Water Samplesa duplex site

a

triplex (AB, MicroSEQ)

triplex (stx1,stx2, eae)

eae (AB#436610)

fliCH7

rfbE

Bio-Rad (iQ-Check)

Indiana

-

stx1

þ

-

-

-

D1, Waco, Texas

-

stx1,stx2

þ

þ

-

-

IC, Waco, Texas

-

-

þ

þ

-

-

NF, Waco, Texas D2, Waco,Texas

þ

stx2 stx1,stx2,eae

þ þ

þ þ

þ

þ

D3,Waco,Texas

-

stx2

þ

þ

-

-

A positive signal was obtained only in postenrichment (18 h) samples.

The iQ-Check TM E. coli O157:H7 detection kit also demonstrated a decrease in Ct value of the spiked chlorine or starvation stressed cells in well water, postenrichment (Ct value of FAM signal decreased by 15-20). The seven black colonies isolated from the well water samples tested negative with this kit. 3.3. Detection of E. coli O157:H7 from Surface Water. Six surface water samples were tested for E. coli O157:H7. These were obtained from a river in northern Indiana and a creek near a dairy farm near Waco, Texas. When tested by the MicroSEQ E. coli O157:H7 kit, as well as for eae, stx1, and stx 2 in the triplex assay, only one sample, D2 tested positive, with decreased Ct values for all three fluorescent reporters, postenrichment (Table 3 and Table 4). This sample also tested positive by the iQ-check E. coli O157:H7 kit from Bio-Rad, postenrichment (Table 5). There appears to be minimal inhibition of the IPC in the surface water samples prepared in the PrepMan Ultra buffer, and a Ct value of around 34.17 ((0.89) was usually obtained from the NED dye (Table 3). The Ct value was similar to that obtained with PrepMan Ultra buffer preparations of pure cultures (Ct= 33.20 ( 0.52).

When tested for the eae gene using the AB TaqMan pathogen detection kit, which has a broader specificity than the in-house triplex assay that also tests for eae, the duplex PCR assay showed a decrease in Ct value postenrichment in all 6 samples for eae (SI, Table S6 and Table 5). Again the IPC showed minimal inhibition with well water and surface water sample tested, and a Ct value of 28.6 ((1.28) was usually obtained from the VIC dye.

4. DISCUSSION In the proposed method genes specific to E. coli O157:H7 were directly detected in a water sample following a short enrichment of the target organisms. The enrichment is necessary for increasing the sensitivity of the method as well as for viability determinations. Membrane filtration was avoided to exclude addition of further stress to the cells and instead the water sample was directly enriched by addition of a 6X P/A broth. The suitability of this nonselective broth for recovery of stressed E. coli O157:H7, both starvation and chlorine induced, has been well established in our laboratory. Overnight enrichment was 2254

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Environmental Science & Technology thought to be sufficient to get a good concentration of the target cells (without concentrating them on a membrane filter first). One liter sample volume was chosen because it has been shown that E. coli O157:H7 is more often isolated from 1 L samples than 0.1 L.24 Our results showed similar levels of growth from small numbers of stressed E. coli O157:H7 that were spiked in either 1 L samples or 100 mL samples as determined by qPCR (Table 2). Rainbow Agar or CT-SMAC did not prove to be selective enough for the isolation of E. coli O157:H7 from the surface waters, because background bacteria also gave similar colony morphology as the E. coli O157:H7. This result was not unexpected because other investigators have observed this and were often unable to recover E. coli O157:H7 from environmental water samples by IMS-cultivation methods.24,25 Commercially available qPCR detection kits, including a fourth kit from Shanghai ZJ biotech Co. Ltd., China (results not shown), and previously published probe based assays for E. coli O157:H7 were evaluated. This was done so that previously developed assays were not unnecessarily duplicated, thus simplifying the method development process. Several combinations of the five genes stx1, stx2, eae, rfbE, and fliCH7 were examined so that signals from the targets were not lost when combining them in multiplex assays. Two triplex assays were optimized to be used together in the same PCR run: one was the new MicroSEQ kit and the other was a triplex assay, developed in this study, that detects the virulence genes stx1, stx2, and eae. The new MicroSEQ kit from Life Technologies detects all E. coli O157 strains including some E. coli O157:NM strains; it did not detect O55: H7 and 0111 strains. The stx1, stx2, and eae probes were similar to those developed by Sharma et al. 15 but were modified into MGB probes, that uses a nonfluorescent quencher, to make the assay compatible with the MicroSEQ kit assay so that the reactions could be carried out in the same assay plate. The MGB probes show increased specificity, and the eae gene was only detected in E. coli O157 and E. coli O55:H7 strains unlike Ibekwe et al.’s probe that detected eae from several other serotypes.33 A strain was considered E. coli O157:H7 if it tested positive by the MicroSEQ kit, had the eae gene and in addition had stx1 and/or stx 2. The iQ Check kit from BioRad was also suitable and all E. coli O157 strains tested positive. However, the instrument setting for automatic analysis for positives and negatives cannot be used, because the cut off Ct value for the iQ Check E. coli O157:H7 program, is around 48 at which value some of the non-E. coli O157:H7 would give a signal. The triplex assay developed in this study for stx1, stx2, and eae was not tested in the Bio-Rad platform. However, with the use of suitable dyes that are compatible with the sequence detection systems of BioRad, such as Hex (instead of VIC) and Cy5/Texas Red (instead of NED), in addition to FAM, the triplex assay can be easily carried out in the instrument. The primers and probes of rfbE and fliCH7 proved to be specific for E. coli O157:H7 in this study, but are capable of detecting other H7 strains (fliCH7) as well as other nonpathogenic E. coli O157 (rfbE).12,13 Thus this duplex assay will need to be further evaluated using strains presented in Sharma et al. (2006) and Narang et al. (2009).12,13 The assay can potentially be used for further confirmation of E. coli O157: H7. While the MicroSEQ kit already informs about the presence of E. coli O157 strains, knowing what virulence genes are present is also an important consideration. The detection of stx1, stx2, and eae also allows for the detection of E. coli strains that carry these genes and are often the cause of severe diarrhea34 (SI, Table S4). The eae gene target as specified by the new MGB probe

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designed in this study appears to be specific for E. coli O157:H7 and O55:H7 strains and does not recognize O34 or O111 strains that are recognized by the AB 4366100 kit (which is also based on the eae gene target). Four of five E. coli O55:H7 strains tested, were recognized by the eae gene target. This is not unexpected since the eae gene of O55:H7 and E. coli O157:H7 are virtually identical.35 If a sample tests positive for stx1 and/or stx2 genes, but not for the targets in the MicroSEQ kit, then it can be inferred that a pathogenic strain of E. coli other than E. coli O157:H7 is present. Indeed in the six samples from 4 locations that were tested, only one sample, D2, proved positive by all tests and thus had E. coli O157:H7. It would appear three other samples had Shiga toxin producing strains. The AB 4366100 kit that is specific for eae can be included in the procedure because it has a broad specificity and thus will indicate other pathogenic strains of E. coli, in addition to E. coli O157:H7. Thus, a rapid and sensitive method for the detection of viable E. coli O157:H7 cells in water, which takes about 24 h from the time of collection of water to detection, has been developed. The method involves a brief period of enrichment of the water sample followed by direct detection of E. coli O157:H7 by two triplex qPCR assays, one that is commercially available and the other that can be designed to detect the virulence genes. The procedure can be conducted on a single PCR plate and can be used for source as well as finished drinking waters.

’ ASSOCIATED CONTENT

bS

Supporting Information. The bacterial strains and the sources they were obtained from, the primers and probes used in the study, the specificity and sensitivity of the different qPCR kits and assays, chlorine stressing procedures, and DNA extraction protocols have been included. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (513)569-7026. Fax: (513)569-7191. E-mail: sen.keya@ epa.gov.

’ ACKNOWLEDGMENT We thank Dr. Robert Mandrell and Dr. V. Sharma, USDA, Dr. P. Feng, FDA, CFSAN, and Dr. Dennis Lye of US EPA for the kind gift of E. coli and other bacterial strains and Dr. Stacy Pfaller of US EPA for suggestions and careful review of the manuscript. ’ REFERENCES (1) Griffin, P. M.; Tauxe, R. V. The Epidemiology of Infections Caused by Escherichia coli O157:H7, Other Enterohemorrhagic E. coli, and the Associated Hemolytic Uremic Syndrome. Epidemiol. Rev. 1991, 13, 60–98. (2) Remis, R. S.; MacDonald, K. L.; Riley, L. W.; Puhr, N. D.; Wells, J. G.; Davis, B. R.; Blake, P. A.; Cohen, M. L. Sporadic Cases of Hemorrhagic Colitis Associated With Escherichia coli O157:H7. Ann. Intern. Med. 1984, 101, 624–626. (3) Swerdlow, D. L.; Woodruff, B. A.; Brady, R. C.; Griffin, P. M.; Tippen, S.; Donnell, H. D., Jr.; Geldreich, E.; Payne, B. J.; Meyer, A., Jr.; Wells, J. G. A Waterborne Outbreak in Missouri of Escherichia coli O157: H7 Associated With Bloody Diarrhea and Death. Ann. Intern. Med. 1992, 117, 812–819. (4) Licence, K.; Oates, K. R.; Synge, B. A.; Reid, T. M. An Outbreak of E. coli O157 Infection With Evidence of Spread From Animals to Man 2255

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