Deletion-Based Approach for the ... - ACS Publications

Aug 28, 2014 - Department of Biology, McMaster University, Life Sciences Building, ... Sciences, McMaster University, McMaster University Medical Cent...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/est

Insertion/Deletion-Based Approach for the Detection of Escherichia coli O157:H7 in Freshwater Environments Shirley Y. Wong,† Athanasios Paschos,† Radhey S. Gupta,‡ and Herb E. Schellhorn*,† †

Department of Biology, McMaster University, Life Sciences Building, 1280 Main St. West, Hamilton, Ontario, Canada L8S 4K1 Department of Biochemistry and Biomedical Sciences, McMaster University, McMaster University Medical Centre, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5

‡

S Supporting Information *

ABSTRACT: Enterohemorrhagic Escherichia coli O157:H7 is responsible for many outbreaks of gastrointestinal illness and hemolytic uremic syndrome worldwide. Monitoring this pathogen in food and water supplies is an important public health issue. Highly conserved genetic markers, which are characteristic for specific strains, can provide direct identification of target pathogens. In this study, we examined a new detection strategy for pathogenic strains of E. coli O157:H7 serotype based on a conserved signature insertion/deletion (CSI) located in the ybiX gene using TaqMan-probe-based quantitative PCR (qPCR). The qPCR assay was linear from 1.0 × 102 to 1.0 × 107 genome copies and was specific to O157:H7 when tested against a panel of 15 non-O157:H7 E. coli. The assay also maintained detection sensitivity in the presence of competing E. coli K-12, heterologous nontarget DNA spiked in at a 1000-fold and 800-fold excess of target DNA, respectively, demonstrating the assay’s ability to detect E. coli O157:H7 in the presence of high levels of background DNA. This study thus validates the use of strain-specific CSIs as a new class of diagnostic marker for pathogen detection.

■

INTRODUCTION Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been a focus of diagnostic detection since the isolation of the E. coli O157:H7 strain EDL933 in the United States in 1975 and its association with outbreaks of hemorrhagic colitis in Michigan and Oregon in 1982.1 This facultative anaerobe is found in the gastrointestinal tract of mammals and has reservoirs primarily in cattle,2,3 sheep,4 and swine.5 Sources of contamination include contaminated beef,6 leafy green vegetables,7 and drinking water,8 which can lead to outbreaks of gastrointestinal illness and, in the most severe cases, bloody diarrhea and hemolytic uremic syndrome (HUS).9 The occurrence of E. coli O157:H7 in the food and drinking supply is thus a public health concern. DNA-based tests have been developed to detect E. coli O157:H7 and other pathogens. The first PCR tests targeted the virulence genes stx10 and eae11 in STEC (Shiga toxin-producing E. coli). Since then, multiplex PCR assays have been developed that target genes encoding O- and H-antigens in addition to virulence genes,12,13 allowing for the identification of E. coli O157:H7 strains in one PCR reaction. The use of fluorogenic TaqMan probes targeting virulence genes in quantitative PCR (qPCR) assays further increased the sensitivity and specificity of E. coli O157:H7 detection systems,14,15 achieving detection limits of 10 colony forming units (CFU) per gram with enrichment.15 However, the use of enrichment increases assay © 2014 American Chemical Society

time. Furthermore, virulence genes can be horizontally transferred among pathogenic E. coli strains, potentially confounding results of virulence-gene-based assays used to detect presumptive E. coli O157:H7 strains. This was illustrated by the 2011 outbreak of gastroenteritis and HUS in Germany that was caused by an E. coli O104:H4 strain that possesses virulence properties of both enteroaggregative and Shiga-toxinproducing E. coli.16,17 Though classified as enteroaggregative E. coli, the strain contains a mobile Stx2-phage that enables the production of Shiga toxins and is closely related to the Stx2phage from an enterohemorrhagic E. coli O111:H strain, suggesting a lateral gene transfer event that occurred in the evolutionary history of the E. coli O104:H4 outbreak strain.18 Alternative assays, employing conserved genetic markers specific to E. coli O157:H7 can potentially detect target bacteria more directly using conserved molecular markers rather than targeting laterally transferred genes. For example, the +93 mutation in the uidA gene, which is unique to E. coli O157:H7/ H- strains, renders β-glucuronidase nonfunctional and can serve as a stable genetic marker for qPCR assays.19,20 Another qPCR assay for E. coli O157:H7 detection uses open reading frame Received: Revised: Accepted: Published: 11462

June 12, 2014 August 27, 2014 August 28, 2014 August 28, 2014 dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

(ORF) Z3276, which encodes a putative fimbrial protein that is unique to E. coli O157:H7.21 Similarly, conserved signature insertions or deletions (CSIs) are also potential molecular targets for diagnostic assays. These molecular markers are the result of rare genetic changes that occurred in a common ancestor of the members within a given taxonomic clade.22 Clade-specific CSIs are unique to many bacterial taxonomic groups (see review by Bhandari et al.23)24−26 and can thus enable the elucidation of evolutionary relationships among bacterial groups. Though CSIs are most often found in higher taxonomic groups, species-specific CSIs have been identified (e.g., for the development of a pyrosequencing assay to distinguish Bacillus anthracis, the etiological agent of anthrax, from genetically similar members of the B. cereus group27). Because of their conserved nature, species- and/or strain-specific CSIs represent stable genetic markers for pathogen detection. In this study, a TaqMan-probe-based qPCR assay was developed to target an E. coli O157:H7-specific CSI located in the ybiX gene product, a member of the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. This class of oxygenases is widespread, found in both eukaryotes and bacteria. They catalyze reactions typically involving the oxidation of an organic substrate using a dioxygen molecule.28 YbiX orthologs are found in Pseudomonas aeruginosa PAO (PiuC),29 implicated in the biosynthesis of cephalosporin in Streptomyces (DAOCS),30 and the structure from Shewanella baltica OS155 Sbal_3634 ortholog is resolved (PDB ID: 3DKQ; GenBank: ABN63109.1). The specificity and dynamic range of the TaqMan-probebased assay in this work were established with several type E. coli strains and heterologous DNA. The data confirm the specificity of the assay for E. coli O157:H7 and indicate that an E. coli O157:H7-specific CSI can be used as a diagnostic marker for E. coli strains of the O157:H7 serotype, facilitating the direct and sensitive detection of targeted pathogenic E. coli. In principle, this strategy can be used for other water-borne pathogens that possess unique CSIs to improve microbiological monitoring of environmental sites of interest.

The specificity of the primer and the probe sequences to the ybiX gene in E. coli was checked with a Primer-BLAST analysis against the NCBI Nucleotide collection (nr) database. A BLASTn analysis was also performed using the target amplicon from E. coli O157:H7 str. EDL933, and the resulting Expect values (E values) for E. coli O157:H7 strains in the NCBI database were 3 × 10−34. Sequences of the primers and TaqMan probe used in this study are outlined in Table 1. Table 1. Primers and Probe Used in the Assay and the Expected Amplicon Sizes amplicon size (bp)

name

sequence (5′−3′)

O157ybiXF

CGC CAT GCT GTT TGA ACT GG (FAM)-ATT CAG AAT ATT CAG TCG CTG AAA AGC-(BHQ-1)a CAG GAT CTC TTC ATT TTC AC GGC GAA CTG GTC GTT AAT GAC TCA GAT CTC CGA CCA TTC CC

O157ybiXP

O157ybiXR ECybiXF ECybiXF

target

reference

82

E. coli O157:H7

this study

294/303b

all E. coli

this study

a

FAM = 6-carboxyfluorescein; BHQ-1 = Black Hole Quencher 1. b294 bp produced by non-O157:H7 E. coli strains; 303 bp produced by E. coli O157:H7 strains.

Bacterial Strains. E. coli strains used in this study are listed in Table 2. A total of 16 E. coli strains were tested: 14 nonO157:H7 E. coli pathogenic strains, one E. coli K-12 strain (MG1655), and one E. coli O157:H7 strain (EDL933). Table 2. E. coli Strains Used in This Study

■

MATERIALS AND METHODS Identification and Confirmation of E. coli O157:H7Specific Insertion in a Conserved Signature Indel. The conserved signature indel (CSI) with the E. coli O157:H7specific insertion was identified using previously published methods.31,32 Briefly, BLASTp searches of all E. coli K-12 ORFs were performed against the nonredundant (nr) NCBI Protein database. Protein sequences from different E. coli strains resulting from the searches were further aligned with Clustal X version 2.0,33 and alignments were then visually inspected for conserved flanking regions. Those without conserved flanking regions were not considered usable molecular markers.22 For proteins with conserved flanking regions, further BLASTp searches to the nonredundant (nr) NCBI Protein database using a shorter protein sequence surrounding the CSI as query were performed to retrieve a list of organisms with the CSI, including bacterial species in addition to E. coli. Specificity of the insertion in the CSI found in the ybiX gene to E. coli O157:H7 strains was then confirmed to be specific with a BLASTn search against E. coli strains in the nonredundant Nucleotide collection (nr/nt) NCBI database. Primer and Probe Sequences. Primers were designed for a smaller 82 bp amplicon to allow for efficient amplification.

seropathotypea

serotype

strain

pathotypeb

sourcec

A B

O157:H7 O26:H11 O26:H11 O111:NM O121:H19 O145:NM O145:NM O5:NM O113:H21 O121:NM O103:H25 O172:NM O84:NM O98:H25 O6:K2:H1 OR:H48:K-

EDL933 CL1 CL9 R82F2 CL106 N00-6496 N02-5149 N00-4067 CL3 N99-4390 N00-4859 EC6-484 EC2-044 EC3-377 CFT073 MG1655

EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC EHEC UPEC K-12

1 34 34 34 34 34 34 34 34 34 34 34 34 34 35 CGSC

C

D E − − a

Seropathotypes classified according to Karmali et al.34 bEHEC = enterohemorrhagic E. coli; UPEC = uropathogenic E. coli. cCGSC = Coli Genetic Stock Center.

Growth Conditions, DNA Extraction, and DNA Quantification of Bacterial Strains. Stocks of E. coli strains were streaked onto LB (Luria−Bertani) agar plates and grown overnight at 37 °C. Single colonies were picked and grown overnight in LB broth at 37 °C in a shaking incubator (200 rpm). DNA extractions were performed using a kit (Bacterial Genomic DNA Isolation Kit, cat. no. 17900, Norgen Biotek Corp., Ontario), and the DNA extracts were quantified with the 11463

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

5 μL of 2× SsoFast EvaGreen Supermix (cat. no. 172-5201, Bio-Rad, California), 0.2 μM ECybiXF forward primer, 0.2 μM ECybiXR reverse primer, 1 μL of DNA template, and doubledistilled sterile water (to 10 μL of reaction volume). Experiments were performed on the CFX96 Touch RealTime PCR Detection System (Bio-Rad, California). Samples, positive and negative controls, and external amplification controls were performed in triplicate, while no-template controls were performed in duplicate. The qPCR program included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s (denaturation step) and 62 °C for 60 s (annealing/extension step). Amplicons were then visualized by agarose gel electrophoresis. Competition Experiments with Nontarget E. coli DNA. Competing E. coli K-12 DNA was spiked into samples with 1.0 × 104 genome copies of E. coli O157:H7 DNA. Concentrations of E. coli K-12 DNA ranged from 0 to 1.0 × 107 genome copies, representing a 0- to 1000-fold excess amount of target E. coli O157:H7 DNA. Experimental controls included plasmid (pCR4-O157 and pCR4-EC), unspiked (E. coli O157:H7 DNA only), negative (E. coli K-12 DNA only), external amplification, and no-template controls. A standard curve was performed, and reaction conditions were as outlined above under qPCR conditions. Size and specificity of amplification products were verified using agarose gels. Effect of Heterologous DNA. Fish sperm DNA (cat. no. 11467140001, Roche Applied Science, Germany) was spiked into samples with 1.0 × 104 genome copies of E. coli O157:H7 DNA, and amounts of fish sperm DNA ranged from 0 to 6.2 × 101 ng (a 1000-fold excess of target E. coli O157:H7 DNA). Experimental controls and the standard curve were performed as outlined above in spiking experiments with E. coli K-12 DNA. Size and specificity of amplification products were verified using agarose gel electrophoresis. Similar experiments were also performed using DNA isolated from lake water as competitor DNA to mimic environmental sampling conditions. qPCR Data Analysis. To assess the efficiency of the assay and determine the R2 value (coefficient of determination), a linear regression analysis was performed, with Cq (quantitative cycle) values plotted as a function of the logarithmic transformation of the starting DNA template quantity (genome copies). The signal thresholds used to determine the Cq value were established empirically, at 170 RFU (relative fluorescence units) for TaqMan probe-based reactions and at 500 RFU for EvaGreen-based reactions. qPCR experiments were performed in accordance with MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines.38 The CFX Manager Software (Bio-Rad, California) was used for qPCR data analysis. 3D Model Generation and Homology Searches. The Phyre protein fold recognition server39 was used to derive a 3D model of E. coli O157:H7 YbiX (based on the Shewanella baltica Os155 structure as a reference (PDB 3DKQ; www.ncbi. nlm.nih.gov/Structure/MMDB/mmdb.shtml)), and the model was displayed using PyMOL (The PyMOL Molecular Graphics System, Schrödinger, LLC). Protein sequence alignments were calculated with ClustalW (EMBL-EBI).40

Qubit dsDNA BR Assay Kit (cat. no. Q32850) and a Qubit fluorometer (Invitrogen, California). For DNA extraction from lake water samples (Lake Ontario), a soil DNA isolation kit (cat. no. 26500, Norgen Biotek Corp., Ontario) was used. Amplifying, Sequencing, and Aligning the Region of Interest in Bacterial Strains. Control primers ECybiXF and ECybiXR (Table 1) were used to amplify the region of interest located in the ybiX gene in E. coli strains used in this study (Table 2). PCR was performed in 50 μL reactions, consisting of 5 μL of 10× Pf u buffer with MgSO4, 1.25 U Pf u DNA polymerase (cat. no. EP0501, Thermo Fisher Scientific, Massachusetts), 0.2 M dNTPs (cat. no. R0192, Thermo Fisher Scientific, Massachusetts), 0.5 μM ECybiXF forward primer, 0.5 μM ECybiXR reverse primer, 10 ng of DNA template, and double-distilled sterile water (to 50 μL of reaction volume). Experiments were performed on the Mastercycler Gradient (Eppendorf, Germany). The PCR program was as follows: 95 °C for 3 min, followed by 30 cycles of 95 °C for 30 s, 62 °C for 30 s, 72 °C for 45 s, and then 72 °C for 5 min. Amplicons were then visualized using agarose gel electrophoresis and extracted with a kit (NucleoSpin Gel and PCR Clean-up, cat. no. 740609.250, Macherey-Nagel, Germany). Sequencing was performed on a 3730 DNA Analyzer (Applied Biosystems, California). Sequencing of PCR products were performed in duplicate using two separate PCR reactions for each strain to account for amplification artifacts. Both strands were sequenced. Sequences of the sequenced region of interest in E. coli strains were aligned using Clustal X version 2.0.33 Construction of Positive and Negative Control Plasmids. For the positive control plasmid (pCR4-O157), a 303 bp gene fragment of ybiX containing the E. coli O157:H7specific insertion was cloned into the pCR4-TOPO cloning vector using a kit (TOPO TA Cloning Kit, cat. no. 450030, Invitrogen, California). For the negative control plasmid (pCR4-EC), a 294 bp gene fragment of ybiX generated from E. coli K-12 that does not contain the E. coli O157:H7-specific insertion was cloned into the pCR4-TOPO cloning vector using the same kit. Both types of plasmids were introduced into the E. coli DH5α strain using TSS transformation36 for propagation. qPCR Conditions. For specificity testing, 0.2 ng of DNA extract (approximately 4.0 × 104 genome copies) was used as template. To establish a range of linearity and limit of detection of the assay, serial 10-fold dilutions of E. coli O157:H7 DNA extract were made, from 1.0 × 102 to 1.0 × 0 genome copies. The genome copies were calculated from the amount of DNA using the following equation:37 genome copies =

(6.02 × 1023 copy/mol) × (DNA amount in grams) (DNA length in bp) × (660g/mol/bp) (1)

qPCR experiments were performed in 10 μL reaction volumes in 96-well plates. The following components were added to reactions that used the TaqMan probe: 5 μL of 2× TaqProbe qPCR Mastermix (cat. no. Mastermix-PS, Applied Biological Materials Inc., British Columbia), 1 μM O157ybiXF forward primer, 1 μM O157ybiXR reverse primer, 0.3 μM O157ybiXP TaqMan probe (Integrated DNA Technologies, Iowa), 1 μL of DNA template, and double-distilled sterile water (to 10 μL of reaction volume). For EvaGreen dye-based external amplification controls, the 10 μL reactions consisted of

■

RESULTS Location and Specificity of the CSI in the ybiX Gene and Positions of the Primers and TaqMan Probe in ybiX. The CSI located in the ybiX gene product is 9 base pairs in size, and the insertion is found only in E. coli O157:H7 strains

11464

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

Figure 1. Protein alignment showing the 3 amino acid insertion (boxed) located in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Sequences are shown with the species name, followed by the GenBank accession number. The dashes (−) indicate amino acid identity to E. coli O157:H7 str. EDL933, and the numbers at the top indicate amino acid positions of the sequence from EDL933. Representatives of each bracketed group are shown, and the numbers in parentheses under the labels on the left indicate the number of strains and/or species belonging to the corresponding group. aEHEC = enterohemorrhagic E. coli; EPEC = enteropathogenic E. coli; ETEC = enterotoxigenic E. coli; DA-EPEC = diffuse adhering enteropathogenic E. coli; ExPEC = extraintestinal pathogenic E. coli; EAEC = enteroaggregative E. coli; STEC = Shiga toxigenic E. coli; N/A = not applicable.

(Figure 1). None of the 463 non-O157:H7 E. coli strains and 467 representatives from other species of the Enterobacteriaceae group contain the insertion. Nor is the indel insertion present in sequences of AlkB, leprecan, EGL-9, PiuC, and other proteins defined as members of the 2-oxoglutarate and Fe (II)dependent oxygenase superfamily.28 The insertion is also present in draft genomes of nonmotile E. coli O157:NM, a Shiga-toxin-producing serotype that has been isolated from patients with hemolytic uremic syndrome.41 In E. coli O157 YbiX, the indel is located at amino acid position 194 close to the C-terminus and off the 2-oxoglutarate binding and Fe(II) coordinating site implying no interference with its catalytic activity (Figure S1, Supporting Information). The primers were designed around the CSI in the ybiX gene (Figure 2A). Amplification products produced were 82 bp for E. coli O157:H7 and 73 bp for non-O157:H7 E. coli. Control

primers produced 303 bp (O157:H7) and 294 bp (nonO157:H7) amplicons (Figure 2B). Linear Range and Sensitivity of the qPCR Assay. The qPCR assay was linear from 1.0 × 102 to 1.0 × 107 genome copies of E. coli O157:H7 (Figure 3). The assay had an amplification efficiency of 100.5% and a coefficient of determination of 0.991 (Figure 3). Specific amplification was verified by the presence of the expected 82 bp amplicon on an agarose gel (data not shown). Specificity of the Assay: E. coli Strains. To determine the specificity of the assay, 15 non-O157:H7 E. coli strains and one E. coli O157:H7 strain were used. Only E. coli O157:H7 produced amplification signals above the threshold (Figure S2, Supporting Information), while external amplification controls for all strains produced signals above the threshold. Reactions with E. coli O157:H7 DNA extract had a Cq value of 24.58 ± 0.17 (Table S1, Supporting Information). The expected 82 bp 11465

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

Figure 2. Location of primers and TaqMan probe and expected amplification products. (A) A schematic shows the E. coli O157:H7-specific insertion located in the ybiX gene. E. coli O157:H7 strains and a sample strain containing the deletion (E. coli K-12) are shown. The dashes (−) indicate a deletion. Positions of the primers and probe in the gene are indicated by the base-pair positions at the top and the labels in the alignment. Control primers are indicated by arrows in the schematic. (B) For the ybiX CSI assay, 82 bp (O157:H7; lane 2) and 73 bp (K-12; lane 3) amplicons were produced. For external amplification controls, 303 bp (O157:H7; lane 4) and 294 bp (K-12; lane 5) amplicons were produced. A 100 bp marker (M) was run in parallel (lane 1).

The insertion was present only in E. coli O157:H7 from an alignment of the sequenced region of interest in the 16 E. coli strains used in this study (Figure S3, Supporting Information). There were no differences between duplicate sequences, and sequencing of both strands produced complementary sequences (data not shown). Sensitivity of the Assay: Competition with E. coli K-12 DNA. To test the ability to specifically amplify target E. coli O157:H7 DNA in the presence of genetically similar DNA, competing E. coli K-12 DNA was spiked into qPCR reactions with E. coli O157:H7 DNA. The assay showed no decrease in sensitivity in the presence of E. coli K-12 DNA spiked in reactions at up to a 1000-fold excess amount of target DNA (a ratio of 1:1000 of O157:H7 DNA to K-12 DNA) (Figure 4A). Negative controls with 1.0 × 107 genome copies of E. coli K12 produced a signal above threshold for one technical replicate, with a Cq value of 37.82 (Table S2, Supporting Information). External amplification controls showed amplification signals above threshold (Table S3, Supporting Information). A standard curve was included and was linear from 1.0 × 103 to 1.0 × 107 genome copies (data not shown). The presence of the expected 82 bp amplification product was confirmed using agarose gel electrophoresis (data not shown).

Figure 3. Linear range of E. coli O157:H7-specific assay. A linear regression analysis of quantitative cycle (Cq) as a function of the logarithmic transformation of the starting quantity of E. coli O157:H7 is shown. 10-fold dilutions of E. coli O157:H7 genomic DNA extract used were from 1.0 × 102 to 1.0 × 107 genome copies. The efficiency (E) and the coefficient of determination (R2) are shown. Standard error was calculated for three technical replicates.

amplicon for E. coli O157:H7 was visualized using agarose gel electrophoresis (data not shown). 11466

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

external amplification controls were done for fish sperm DNA, as the control primers were designed for E. coli strains. A standard curve with a linear range of 1.0 × 103 to 1.0 × 107 genome copies (data not shown) was included. Specificities of amplification products were verified using agarose gel electrophoresis, and the expected 82 bp amplicon was seen (data not shown). Application of the Assay for Detection of E. coli O157:H7 in Freshwater Environments. To establish the selectivity and specificity of the TaqMan qPCR assay in environmental samples, tests were performed with non-EHEC environmental isolated E. coli strains and lake water samples. No cross-reactivity was observed with any of the environmental strains and water samples (Table S4, Supporting Information). To test the ability to detect E. coli O157:H7 in the presence of heterologous, environmental DNA, spiking experiments with E. coli O157:H7 (1.0 × 104 genome copies of E. coli O157:H7) were performed. No decrease in sensitivity was observed when environmental DNA (extracted from fresh water samples) was spiked into reactions at concentrations up to a 1000-fold excess amount of target DNA (Figure 4C).

■

DISCUSSION As a causative agent of outbreaks of gastrointestinal illness, enterohemorrhagic Escherichia coli O157:H7 is a major focus of molecular assay development to monitor the presence of this pathogen in food and water. Though PCR-based diagnostic assays are very sensitive, many current pathogen detection assays target virulence genes that can be horizontally transferred to other E. coli strains reducing both the specificity and utility of assays. The importance of horizontal transfer was recently exemplified in the 2011 Germany outbreak of gastroenteritis caused by an enteroaggregative E. coli O104:H4 strain that had acquired a mobile Stx2-phage,18 enabling the production of Shiga toxins that is more characteristic of Shiga-toxigenic E. coli. Identification based on more-conserved genetic targets may yield assays that are more reliable than virulence factor-targeted assays. In this study, we explored an alternative qPCR assay that uses a conserved signature indel (CSI) for direct detection of E. coli O157:H7 strains. Using competition experiments with E. coli K-12 and fish sperm DNA, we demonstrated the effectiveness of this assay for detection of E. coli O157:H7 in the presence of large amounts of nontarget DNA. CSIs present at different taxonomic levels provide a reliable means for identifying members of evolutionary clades. Most indels are vertical-inherited,42 and they have been used extensively to elucidate evolutionary histories of members within different bacterial groups, such as Gammaproteobacteria32 and Cyanobacteria.43 CSIs at a lower taxonomic level, such as species-specific CSIs, are also useful for the development of diagnostic assays,27 as they are stable molecular markers with high resolving power even at the species level. In this study, we focused on a subspecies-specific CSI that distinguishes between serotypes of E. coli. Located in the ybiX gene, this CSI translates to a 3-amino-acid insertion (amino acid sequence NIQ) found specifically in E. coli O157:H7 strains (Figure 1) and is absent from other bacterial and eukaryotic proteins defined as members of the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily (Figure S1, Supporting Information). The TaqMan-probe-based qPCR assay developed with this genetic marker offers a specific and sensitive method for

Figure 4. Detection sensitivity of E. coli O157:H7 DNA is not affected by competing E. coli K-12 DNA at tested spiking amounts and lowered by competing fish sperm DNA or environmental DNA spiked in a 1000-fold excess of target DNA. Cq values for the amplification of 1.0 × 104 genome copies of E. coli O157:H7 are plotted as a function of increasing amounts of competing (A) E. coli K-12 DNA, (B) fish sperm DNA, or (C) environmental DNA isolated from Lake Ontario that were spiked into reactions (shown as ratios of target to nontarget DNA). Standard error values calculated from technical replicates are shown as error bars.

Sensitivity of the Assay: Competition with Heterologous DNA. Fish sperm DNA was used as a source of DNA with low genetic similarity to target E. coli O157:H7 DNA to demonstrate the assay’s ability to detect E. coli O157:H7 in high levels of heterologous, nontarget DNA. At a spiking amount of a 1000-fold excess of target DNA (a ratio of 1:1000 of O157:H7 DNA to fish sperm DNA), the Cq value for the amplification of 1.0 × 104 genome copies of E. coli O157:H7 was higher than at other spiking amounts (Figure 4B). The assay showed no decrease in sensitivity when fish sperm DNA was spiked into reactions at concentrations up to an 800-fold excess amount of target DNA. External amplification controls showed amplification signals above threshold (Table S3, Supporting Information). No 11467

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

directly identifying E. coli O157:H7 strains that does not rely on virulence markers that may also be found in other strains of enterohemorrhagic E. coli. Through specificity tests using E. coli strains, the assay was able to distinguish between targeted E. coli O157:H7 and nontarget E. coli using a small 9 base-pair difference of the CSI, similar to the O157:H7-specific assay developed against the single base mismatch at the +93 position of the uidA gene.19 The assay also has a detection limit of 1.0 × 102 genome copies of E. coli O157:H7 (Figure 3), within the upper limits of the minimum infectious dose of 100 colony forming units.44 Additionally, the +93 uidA mutation is unique to E. coli O157:H7, whereas ORF Z3276 is also present in O26:H11 and O103:H2 strains, indicating that use of the CSI sequence forms a more specific measure than using ORF Z3276. External factors affect the limit of detection for pathogen detection assays when applied to environmental samples and food matrices. One factor is the use of a growth enrichment step, which increases the number of target sequences in the sample and thus improves the limit of detection of the assay. Such enrichment steps have been used in different sample types to detect E. coli O157:H7. An 18 h enrichment step can lead to a 5−6 log increase of stressed E. coli O157:H7 spiked into environmental water, lowering the detection limit to 4 CFU/ 100 mL.45 Additionally, an 8 h enrichment period, combined with propidium monoazide, which enters injured or dead cells with compromised membranes to intercalate into DNA and inhibit PCR amplification, allowed for the detection of 80 CFU of viable E. coli O157:H7 per gram of beef.21 Another factor that may reduce a given assay’s limit of detection is the presence of inhibitors, such as organic compounds, including tannic, humic, and fulvic acids. These inhibitors can be removed by activated charcoal coated with bentonite,46 Pseudomonas f luorescens,47 or the addition of bovine serum albumin48 enabling a lowered detection limit from 1.0 × 103 to 5 CFU/ g in lettuce spiked with E. coli O157:H746 and an over-4-log decrease in the limit of detection to 1.0 × 103 CFU/g in spiked oyster samples.47 Thus, a theoretical limit of detection in complex samples for the E. coli O157:H7-specific assay shown in this study can be established by taking into consideration the influences of enrichment and removal of PCR inhibitors and may be different from a limit of detection of 1.0 × 102 genome copies demonstrated in this study. Though the utility of assays are often framed in the context of extraction efficiencies of protocols,49,50 the elimination of organic inhibitors, and the addition of enrichment periods, other confounding factors may play a role in influencing an assay’s ability to sensitively detect target pathogens, such as background genetic material coextracted with samples. This aspect was examined in a PCR assay targeting the gene encoding the O-antigen of O157:H7, rf bE.51 The test was assessed by using produce washes containing 106−107 CFU of bacteria spiked with 10-fold dilutions of E. coli O157:H7 ranging from 10 to 109 CFU/mL, indicating that background flora has little effect on detection sensitivity.51 However, similar studies have not yet been done for E. coli O157:H7-specific qPCR assays. To further explore the use of the indels as diagnostics, the ybiX assay’s detection sensitivity to E. coli O157:H7 in the presence of background DNA was examined. E. coli K-12 DNA spiked into samples with target E. coli O157:H7 DNA provided a measure of the assay’s performance in samples with genetically similar, nontarget template that could potentially lead to nonspecific binding of the TaqMan

probe. The detection sensitivity of the assay was not affected at E. coli K-12 spiking concentrations of up to a 1000-fold excess amount of target DNA, potentiating its use in monitoring environmental water samples to trace potential agricultural runoff or sewage contamination. Bodies of freshwater around watersheds are seeded by E. coli communities sustained in the soils, with persistent E. coli that can survive for months-long periods.52,53 The long-term persistence of indigenous E. coli leads to a constant background microbial presence in water samples collected at these sites and, depending on the bacterial load, may disrupt detection of targeted pathogenic strains. To further examine the influence of background DNA, fish sperm DNA and DNA extracted from water samples spiked into reactions with E. coli O157:H7 DNA also created a reaction environment that contained high levels of heterologous nontarget DNA that is complex in nature, thus serving as a proxy for environmental DNA. The assay’s detection of target E. coli O157:H7 DNA was unaffected by excess DNA (up to an 800-fold excess). For detection of E. coli O157:H7 in environmental samples, the assay’s effectiveness is dependent on specific detection of target DNA among background DNA. Water sample types from urban settings, such as stormwater runoff, contain a multitude of microorganisms, including Enterococcus, Campylobacter, Salmonella, and human-specific adenovirus.54 Thus, the assay’s ability to detect E. coli O157:H7 in the presence of different types of excess nontarget DNA has demonstrated its potential use on samples from environmental waters. Though E. coli O157:H7 is distributed throughout the world, it is found in environmental samples only sporadically, often due to an identifiable source of contamination.55 Bacteria, including E. coli, can survive for an extended time in water and soil, can multiply in manure and other substrates, and can be transmitted to humans through contaminated food, through drinking and surface water and, to lesser extent, by contact with animals.55 As cattle constitute a primary animal reservoir, E. coli O157:H72,3 is likely to be prevalent in the cattle farm environment and water tanks in large cattle feedlots may harbor E. coli O157:H7.56 E. coli O157:H7 can also occur in aquatic nonmammalian carriers, such as amphibians, fish, and invertebrates, and can be isolated from freshwater biofilms collected downstream of farms.55 Noteworthy, E. coli O157:H7 prevalence follows seasonal patterns, with peaks in cattle and the environment between late spring and early fall.57 In this study, we developed a qPCR assay that directly detects E. coli O157:H7 using a novel CSI-based strategy. The ability of the assay to amplify target E. coli O157:H7 DNA in the presence of excess competing background DNA and the assay’s utility in different simulated sample types were also explored. Though the applicability of this assay to the farm and environmental water samples and different food matrices has yet to be assessed comparatively and longitudinally in longterm future studies, this proof-of-principle assay represents a possible use for a conserved E. coli O157:H7-specific insertion in a CSI that enables direct identification of a clinically relevant pathogenic E. coli strain capable of causing severe human morbidity and mortality. In principle, the basic strategy employed in this study can be further used to develop CSIbased assays for other important human pathogens, including Campylobacter and Enterococcus, as well as toxigenic bacteria, such as those found in the Cyanobacteria phylum. Cladespecific indels have been identified for these important water microorganisms, and CSI-based assays can be readily developed 11468

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

(9) Karmali, M. A.; Petric, M.; Lim, C.; Fleming, P. C.; Arbus, G. S.; Lior, H. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J. Infect. Dis. 1985, 151 (5), 775−782. (10) Gannon, V. P.; King, R. K.; Kim, J. Y.; Thomas, E. J. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Appl. Environ. Microbiol. 1992, 58 (12), 3809−3815. (11) Gannon, V. P.; Rashed, M.; King, R. K.; Thomas, E. J. Detection and characterization of the eae gene of Shiga-like toxin-producing Escherichia coli using polymerase chain reaction. J. Clin. Microbiol. 1993, 31 (5), 1268−1274. (12) Nagano, I.; Kunishima, M.; Itoh, Y.; Wu, Z.; Takahashi, Y. Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction. Microbiol. Immunol. 1998, 42 (5), 371−376. (13) Hu, Y.; Zhang, Q.; Meitzler, J. C. Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR. J. Appl. Microbiol. 1999, 87 (6), 867−876. (14) Sharma, V. K.; Dean-Nystrom, E. A.; Casey, T. A. Semiautomated fluorogenic PCR assays (TaqMan) for rapid detection of Escherichia coli O157:H7 and other Shiga toxigenic E. coli. Mol. Cell. Probes 1999, 13 (4), 291−302. (15) Sharma, V. K.; Dean-Nystrom, E. A. Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Vet. Microbiol. 2003, 93 (3), 247−260. (16) Bielaszewska, M.; Mellmann, A.; Zhang, W.; Kock, R.; Fruth, A.; Bauwens, A.; Peters, G.; Karch, H. Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: A microbiological study. Lancet Infect. Dis. 2011, 11 (9), 671−676. (17) Frank, C.; Werber, D.; Cramer, J. P.; Askar, M.; Faber, M.; an der Heiden, M.; Bernard, H.; Fruth, A.; Prager, R.; Spode, A.; Wadl, M.; Zoufaly, A.; Jordan, S.; Kemper, M. J.; Follin, P.; Muller, L.; King, L. A.; Rosner, B.; Buchholz, U.; Stark, K.; Krause, G.; HUS Investigation Team. Epidemic profile of Shiga-toxin-producing Escherichia coli O104:H4 outbreak in Germany. N. Engl. J. Med. 2011, 365 (19), 1771−1780. (18) Laing, C. R.; Zhang, Y.; Gilmour, M. W.; Allen, V.; Johnson, R.; Thomas, J. E.; Gannon, V. P. A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain. PLoS One 2012, 7 (5), No. e37362. (19) Yoshitomi, K. J.; Jinneman, K. C.; Weagant, S. D. Optimization of a 3′-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR. Mol. Cell. Probes 2003, 17 (6), 275−280. (20) Yoshitomi, K. J.; Jinneman, K. C.; Weagant, S. D. Detection of Shiga toxin genes stx1, stx2, and the +93 uidA mutation of E. coli O157:H7/H-using SYBR Green I in a real-time multiplex PCR. Mol. Cell. Probes 2006, 20 (1), 31−41. (21) Li, B.; Chen, J. Q. Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker. Appl. Environ. Microbiol. 2012, 78 (15), 5297−5304. (22) Gupta, R. S. Protein phylogenies and signature sequences: A reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol. Biol. Rev. 1998, 62 (4), 1435−1491. (23) Bhandari, V.; Naushad, H. S.; Gupta, R. S. Protein based molecular markers provide reliable means to understand prokaryotic phylogeny and support Darwinian mode of evolution. Front. Cell. Infect. Microbiol. 2012, 2, 98. (24) Griffiths, E.; Gupta, R. S. Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the Deinococcus-Thermus phylum. J. Bacteriol. 2004, 186 (10), 3097− 3107.

that will help to identify these organisms in water and wastewater samples.

■

ASSOCIATED CONTENT

S Supporting Information *

Computed 3D model of E. coli YbiX with the location of the indel insertion, protein alignments of draft genomes, and Cq values for specificity testing, negative controls, and external amplification controls. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: (905) 525-9140, ext. 27316; e-mail: schell@mcmaster. ca. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Mobolaji Adeolu of the Gupta lab for identification of the ybiX indel. This study was supported with a grant from the Ontario Research Fund (Water Round) awarded by the Ontario Ministry of Research and Innovation (MRI). Support was also provided from the Natural Science and Engineering Council (Canada) NSERC discovery grants to R.S.G. and H.E.S. and by an NSERC Collaborative Research and Development grant. S.Y.W. received support from an NSERC Graduate Scholarship.

■

REFERENCES

(1) Riley, L. W.; Remis, R. S.; Helgerson, S. D.; McGee, H. B.; Wells, J. G.; Davis, B. R.; Hebert, R. J.; Olcott, E. S.; Johnson, L. M.; Hargrett, N. T.; Blake, P. A.; Cohen, M. L. Hemorrhagic colitis associated with a rare Escherichia coli serotype. N. Engl. J. Med. 1983, 308 (12), 681− 685. (2) Wells, J. G.; Shipman, L. D.; Greene, K. D.; Sowers, E. G.; Green, J. H.; Cameron, D. N.; Downes, F. P.; Martin, M. L.; Griffin, P. M.; Ostroff, S. M.; et al. Isolation of Escherichia coli serotype O157:H7 and other Shiga-like-toxin-producing E. coli from dairy cattle. J. Clin. Microbiol. 1991, 29 (5), 985−989. (3) Gannon, V. P.; Graham, T. A.; King, R.; Michel, P.; Read, S.; Ziebell, K.; Johnson, R. P. Escherichia coli O157:H7 infection in cows and calves in a beef cattle herd in Alberta, Canada. Epidemiol. Infect. 2002, 129 (1), 163−172. (4) Kudva, I. T.; Hatfield, P. G.; Hovde, C. J. Escherichia coli O157:H7 in microbial flora of sheep. J. Clin. Microbiol. 1996, 34 (2), 431−433. (5) Feder, I.; Wallace, F. M.; Gray, J. T.; Fratamico, P.; Fedorka-Cray, P. J.; Pearce, R. A.; Call, J. E.; Perrine, R.; Luchansky, J. B. Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine. Emerging Infect. Dis. 2003, 9 (3), 380−383. (6) Macdonald, C.; Drew, J.; Carlson, R.; Dzogan, S.; Tataryn, S.; Macdonald, A.; Ali, A.; Amhed, R.; Easy, R.; Clark, C.; Rodgers, F. Outbreak of Escherichia coli O157:H7 leading to the recall of retail ground beef–Winnipeg, Manitoba, May 1999. Can. Commun. Dis. Rep. 2000, 26 (13), 109−111. (7) Ackers, M. L.; Mahon, B. E.; Leahy, E.; Goode, B.; Damrow, T.; Hayes, P. S.; Bibb, W. F.; Rice, D. H.; Barrett, T. J.; Hutwagner, L.; Griffin, P. M.; Slutsker, L. An outbreak of Escherichia coli O157:H7 infections associated with leaf lettuce consumption. J. Infect. Dis. 1998, 177 (6), 1588−1593. (8) Waterborne outbreak of gastroenteritis associated with a contaminated municipal water supply, Walkerton, Ontario, May-June 2000. Can. Commun. Dis. Rep. 2000, 26, (20), 170−173. 11469

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470

Environmental Science & Technology

Article

(25) Griffiths, E.; Gupta, R. S. Molecular signatures in protein sequences that are characteristics of the phylum Aquificae. Int. J. Syst. Evol. Microbiol. 2006, 56 (Pt 1), 99−107. (26) Griffiths, E.; Petrich, A. K.; Gupta, R. S. Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. Microbiology 2005, 151 (Pt 8), 2647−2657. (27) Ahmod, N. Z.; Gupta, R. S.; Shah, H. N. Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group. J. Microbiol. Methods 2011, 87 (3), 278−285. (28) Aravind, L.; Koonin, E. V. The DNA-repair protein AlkB, EGL9, and leprecan define new families of 2-oxoglutarate- and irondependent dioxygenases. Genome Biol. 2001, 2 (3), research0007.1− research0007.8. (29) van Delden, C.; Page, M. G.; Kohler, T. Involvement of Fe uptake systems and AmpC beta-lactamase in susceptibility to the siderophore monosulfactam BAL30072 in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2013, 57 (5), 2095−2102. (30) Kovacevic, S.; Weigel, B. J.; Tobin, M. B.; Ingolia, T. D.; Miller, J. R. Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase. J. Bacteriol. 1989, 171 (2), 754−760. (31) Bhandari, V.; Gupta, R. S. Molecular signatures for the phylum Synergistetes and some of its subclades. Antonie Van Leeuwenhoek 2012, 102 (4), 517−540. (32) Gao, B.; Mohan, R.; Gupta, R. S. Phylogenomics and protein signatures elucidating the evolutionary relationships among the Gammaproteobacteria. Int. J. Syst. Evol. Microbiol. 2009, 59 (Pt 2), 234−247. (33) Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.; McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.; Gibson, T. J.; Higgins, D. G. Clustal W and Clustal X version 2.0. Bioinformatics 2007, 23 (21), 2947− 2948. (34) Karmali, M. A.; Mascarenhas, M.; Shen, S.; Ziebell, K.; Johnson, S.; Reid-Smith, R.; Isaac-Renton, J.; Clark, C.; Rahn, K.; Kaper, J. B. Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. J. Clin. Microbiol. 2003, 41 (11), 4930−4940. (35) Culham, D. E.; Lu, A.; Jishage, M.; Krogfelt, K. A.; Ishihama, A.; Wood, J. M. The osmotic stress response and virulence in pyelonephritis isolates of Escherichia coli: Contributions of RpoS, ProP, ProU and other systems. Microbiology 2001, 147 (Pt 6), 1657− 1670. (36) Chung, C. T.; Niemela, S. L.; Miller, R. H. One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. U. S. A. 1989, 86 (7), 2172−2175. (37) Whelan, J. A.; Russell, N. B.; Whelan, M. A. A method for the absolute quantification of cDNA using real-time PCR. J. Immunol. Methods 2003, 278 (1−2), 261−269. (38) Bustin, S. A.; Benes, V.; Garson, J. A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M. W.; Shipley, G. L.; Vandesompele, J.; Wittwer, C. T. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009, 55 (4), 611−622. (39) Kelley, L. A.; Sternberg, M. J. Protein structure prediction on the Web: A case study using the Phyre server. Nat. Protoc. 2009, 4 (3), 363−371. (40) Chenna, R.; Sugawara, H.; Koike, T.; Lopez, R.; Gibson, T. J.; Higgins, D. G.; Thompson, J. D. Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 2003, 31 (13), 3497− 3500. (41) Gunzer, F.; Bohm, H.; Russmann, H.; Bitzan, M.; Aleksic, S.; Karch, H. Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. J. Clin. Microbiol. 1992, 30 (7), 1807−1810.

(42) Vali, U.; Brandstrom, M.; Johansson, M.; Ellegren, H. Insertiondeletion polymorphisms (indels) as genetic markers in natural populations. BMC Genet. 2008, 9, 8. (43) Gupta, R. S.; Mathews, D. W. Signature proteins for the major clades of Cyanobacteria. BMC Evol. Biol. 2010, 10, 24. (44) Paton, J. C.; Paton, A. W. Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 1998, 11 (3), 450−479. (45) Sen, K.; Sinclair, J. L.; Boczek, L.; Rice, E. W. Development of a sensitive detection method for stressed E. coli O157:H7 in source and finished drinking water by culture-qPCR. Environ. Sci. Technol. 2011, 45 (6), 2250−2256. (46) Lee, J. L.; Levin, R. E. Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment. Food Microbiol. 2011, 28 (3), 562−567. (47) Abolmaaty, A.; Gu, W.; Witkowsky, R.; Levin, R. E. The use of activated charcoal for the removal of PCR inhibitors from oyster samples. J. Microbiol. Methods 2007, 68 (2), 349−352. (48) Farell, E. M.; Alexandre, G. Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates. BMC Res. Notes 2012, 5, 257. (49) Mull, B.; Hill, V. R. Recovery and detection of Escherichia coli O157:H7 in surface water, using ultrafiltration and real-time PCR. Appl. Environ. Microbiol. 2009, 75 (11), 3593−3597. (50) Sanchez, G.; Elizaquivel, P.; Aznar, R. A single method for recovery and concentration of enteric viruses and bacteria from freshcut vegetables. Int. J. Food Microbiol. 2012, 152 (1−2), 9−13. (51) Timmons, C.; Dobhal, S.; Fletcher, J.; Ma, L. M. Primers with 5′ flaps improve the efficiency and sensitivity of multiplex PCR assays for the detection of Salmonella and Escherichia coli O157:H7. J. Food Prot. 2013, 76 (4), 668−673. (52) Whitman, R. L.; Nevers, M. B.; Byappanahalli, M. N. Examination of the watershed-wide distribution of Escherichia coli along Southern Lake Michigan: An integrated approach. Appl. Environ. Microbiol. 2006, 72 (11), 7301−7310. (53) Byappanahalli, M. N.; Whitman, R. L.; Shively, D. A.; Sadowsky, M. J.; Ishii, S. Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lakes watershed. Environ. Microbiol 2006, 8 (3), 504−513. (54) Sidhu, J. P.; Hodgers, L.; Ahmed, W.; Chong, M. N.; Toze, S. Prevalence of human pathogens and indicators in stormwater runoff in Brisbane, Australia. Water Res. 2012, 46 (20), 6652−6660. (55) Ferens, W. A.; Hovde, C. J. Escherichia coli O157:H7: Animal reservoir and sources of human infection. Foodborne Pathog. Dis. 2011, 8 (4), 465−487. (56) Sargeant, J. M.; Sanderson, M. W.; Smith, R. A.; Griffin, D. D. Escherichia coli O157 in feedlot cattle feces and water in four major feeder-cattle states in the USA. Prev. Vet. Med. 2003, 61 (2), 127−135. (57) Edrington, T. S.; Callaway, T. R.; Ives, S. E.; Engler, M. J.; Looper, M. L.; Anderson, R. C.; Nisbet, D. J. Seasonal shedding of Escherichia coli O157:H7 in ruminants: A new hypothesis. Foodborne Pathog. Dis. 2006, 3 (4), 413−421.

11470

dx.doi.org/10.1021/es502794h | Environ. Sci. Technol. 2014, 48, 11462−11470