Application of the polymerase chain reaction to the identification of Escherichia coli and coliforms in water

Fricker, E.J.; Fricker, C. R.

doi:10.1111/j.1472-765x.1994.tb00900.x

Cited by 57 publications

(32 citation statements)

References 7 publications

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“…PCR analysis for screening drinking water and environmental samples has been reported (46)(47)(48) and has been utilized to identify E. coli in primary water specimens (15,27), stool specimens (33,56), and outbreaks (23,24). In contrast, isolation of E. coli O157:H7 from water and other environmental samples is laborious.…”

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confidence: 99%

Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak

et al. 2003

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The largest reported outbreak of waterborne Escherichia coli O157:H7 in the United States occurred in upstate New York following a county fair in August 1999. Culture methods were used to isolate E. coli O157:H7 from specimens from 128 of 775 patients with suspected infections. Campylobacter jejuni was also isolated from stools of 44 persons who developed diarrheal illness after attending this fair. There was one case of a confirmed coinfection with E. coli O157:H7 and C. jejuni. Molecular detection of stx 1 and stx 2 Shiga toxin genes, immunomagnetic separation (IMS), and selective culture enrichment were utilized to detect and isolate E. coli O157:H7 from an unchlorinated well and its distribution points, a dry well, and a nearby septic tank. PCR for stx 1 and stx 2 was shown to provide a useful screen for toxin-producing E. coli O157:H7, and IMS subculture improved recovery. Pulsed-field gel electrophoresis (PFGE) was used to compare patient and environmental E. coli O157:H7 isolates. Among patient isolates, 117 of 128 (91.5%) were type 1 or 1a (three or fewer bands different). Among the water distribution system isolates, 13 of 19 (68%) were type 1 or 1a. Additionally, PFGE of C. jejuni isolates revealed that 29 of 35 (83%) had indistinguishable PFGE patterns. The PFGE results implicated the water distribution system as the main source of the E. coli O157:H7 outbreak. This investigation demonstrates the potential for outbreaks involving more than one pathogen and the importance of analyzing isolates from multiple patients and environmental samples to develop a better understanding of bacterial transmission during an outbreak.

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confidence: 99%

Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak

et al. 2003

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“…While Fricker and Fricker (15) used a PCR amplification protocol of 25 cycles and an annealing temperature of 60°C, we modified the PCR amplification protocol, using 40 cycles and an annealing temperature step of 57°C. The detection problem observed by Fricker and Fricker (15) was not observed using this optimized protocol. Among our more extended panel composed of 147 total coliform strains representing 76 enterobacterial species isolated from fecal and environmental settings (Table 1), 133 strains yielded a lacZ PCR-positive signal, for a sensitivity of 90.5%.…”

Section: Discussionmentioning

confidence: 77%

“…These primers were further used, tested, and/or validated (13)(14)(15)(16)(17)(18)(19). However, the definition of the total coliform group has changed since 1990.…”

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confidence: 99%

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

Maheux

Boudreau

Bisson

et al. 2014

Appl Environ Microbiol

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e This work demonstrates the ability of a bacterial concentration and recovery procedure combined with three different PCR assays targeting the lacZ, wecG, and 16S rRNA genes, respectively, to detect the presence of total coliforms in 100-ml samples of potable water (presence/absence test). PCR assays were first compared to the culture-based Colilert and MI agar methods to determine their ability to detect 147 coliform strains representing 76 species of Enterobacteriaceae encountered in fecal and environmental settings. Results showed that 86 (58.5%) and 109 (74.1%) strains yielded a positive signal with Colilert and MI agar methods, respectively, whereas the lacZ, wecG, and 16S rRNA PCR assays detected 133 (90.5%), 111 (75.5%), and 146 (99.3%) of the 147 total coliform strains tested. These assays were then assessed by testing 122 well water samples collected in the Québec City region of Canada. Results showed that 97 (79.5%) of the samples tested by culture-based methods and 95 (77.9%), 82 (67.2%), and 98 (80.3%) of samples tested using PCR-based methods contained total coliforms, respectively. Consequently, despite the high genetic variability of the total coliform group, this study demonstrated that it is possible to use molecular assays to detect total coliforms in potable water: the 16S rRNA molecular assay was shown to be as efficient as recommended culturebased methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.

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“…1A). Although the lacZ gene has often been used in PCRs to detect the presence of TE, the sequence homology of lacZ genes among TE was not well characterized (34,(44)(45)(46)(47). By combining the findings obtained from lacZ gene sequences with those from the 16S rRNA gene, we can effectively predict clusters in Fig.…”

Section: Figmentioning

confidence: 99%

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

Zhang

Hong

LeChevallier

et al. 2015

Appl Environ Microbiol

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The current definition of coliform bacteria is method dependent, and when different culture-based methods are used, discrepancies in results can occur and affect the accuracy of identification of true coliforms. This study used an alternative approach to the identification of true coliforms by combining the phenotypic traits of the coliform isolates and the phylogenetic affiliation of 16S rRNA gene sequences with the use of lacZ and uidA genes. A collection of 1,404 isolates detected by 12 U.S. Environmental Protection Agency-approved coliform-testing methods were characterized based on their phylogenetic affiliations and responses to their original isolation media and lauryl tryptose broth, m-Endo, and MI agar media. Isolates were phylogenetically classified into 32 true-coliform, or targeted Enterobacteriaceae (TE), groups and 14 noncoliform, or nontargeted Enterobacteriaceae (NTE), groups. It was shown statistically that detecting true-positive (TP) events is more challenging than detecting true-negative (TN) events. Furthermore, most false-negative (FN) events were associated with four TE groups (i.e., Serratia group I and the Providencia, Proteus, and Morganella groups) and most false-positive (FP) events with two NTE groups, the Aeromonas and Plesiomonas groups. In Escherichia coli testing, 18 out of 145 E. coli isolates identified by enzymatic methods were validated as FN. The reasons behind the FP and FN reactions could be explained through analysis of the lacZ and uidA genes. Overall, combining the analyses of the 16S rRNA, lacZ, and uidA genes with the growth responses of TE and NTE on culture-based media is an effective way to evaluate the performance of coliform detection methods. Total coliform bacteria and Escherichia coli are considered costeffective bacterial indicators for the protection of public health. Detectable total coliforms indicate potential contamination associated with the water distribution system, while E. coli is a good indicator of fecal contamination, with a health goal (i.e., maximum contaminant level goal [MCLG]) of zero under the Revised Total Coliform Rule (RTCR) (1). When a positive result of total-coliform testing is observed, public water systems (PWSs) are required to collect and analyze three repeat samples (1). A system assessment is required when a PWS exceeds a specific frequency of total-coliform occurrence. When an E. coli maximum contaminant level (MCL) violation incurs, a PWS must have an assessment performed by the state or a state-approved entity and must correct any sanitary defects (1). False-positive (FP) results for either total-coliform or E. coli tests impose an unnecessary burden on water utilities. On the other hand, false-negative (FN) results expose consumers to potential health threats and delay response times for effective action. Therefore, accurate detection of total coliforms and E. coli in drinking water systems is crucial for both water utilities and consumers.To date, the U.S. EPA has approved 12 methods for the detection of coliform bacteria i...

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Application of the polymerase chain reaction to the identification of Escherichia coli and coliforms in water

Cited by 57 publications

References 7 publications

Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak

Detection, Isolation, and Molecular Subtyping of Escherichia coli O157:H7 and Campylobacter jejuni Associated with a Large Waterborne Outbreak

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

Phenotypic and Phylogenetic Identification of Coliform Bacteria Obtained Using 12 Coliform Methods Approved by the U.S. Environmental Protection Agency

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