Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples

Maheux, Andrée F.; Bissonnette, Luc; Boissinot, Maurice; Bernier, Jacques; Huppé, Vicky; Bérubé, Ève; Boudreau, Dominique K.; Picard, François J.; Huletsky, Ann; Bergeron, Michel G.

doi:10.1016/j.watres.2011.01.019

Cited by 35 publications

(45 citation statements)

References 48 publications

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“…However, nonribosomal genes can be used to discriminate between different enterococcal species (21,22). However, only a handful of nonribosomal genes have been used in environmental studies to detect or identify enterococci (14,23,24). More importantly, the sequence database for the function-specific genes of environmental enterococci and other phylogenetically related genera is much more limiting than that for the 16S and 23S rRNA genes.…”

Section: Resultsmentioning

confidence: 99%

“…However, less sequencing information is available for the 23S rRNA gene than for the 16S rRNA gene, precluding robust in silico validation. As a result, validation of the Entero1 assay has relied on testing the assay against a relatively small number of environmental strains isolated from a limited number of different geographic locations (12,14). Moreover, similar to selective enterococcal media, the Entero1 assay cannot be used to determine which of the major enterococcal groups are present in a given sample.…”

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confidence: 99%

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Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

Ryu

Henson

Elk

et al. 2013

Appl Environ Microbiol

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eThe detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus-and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

Ryu

Henson

Elk

et al. 2013

Appl Environ Microbiol

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“…Quality control for each batch of mCP agar was performed, and filter, buffer, and rinse water blanks were included as sterility controls. For performing CRENAME on concentrated water samples, GN-6 filters were aseptically transferred to 3 individual 15-ml polypropylene tubes (Sarstedt, Newton, NC, USA) and processed as described by Maheux et al (27,28).…”

Section: Determination Of the Analytical Limit Of Detection (Lod) Of mentioning

confidence: 99%

“…Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME ϩ cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water (27,28).…”

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Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

Maheux

Bérubé

Boudreau

et al. 2013

Appl Environ Microbiol

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We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP ؊ /rtPCR ؉ colonies were identified as C. perfringens, whereas 3 mCP ؉ /rtPCR ؊ colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME ؉ cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME ؉ cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME ؉ cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.T he presence of Escherichia coli in drinking water indicates a recent fecal contamination, as well as a risk of waterborne gastrointestinal diseases (1). Under some circumstances, however, it has been shown that this microorganism inadequately indicates the presence of viruses and protozoan parasites of human health significance (2). For example, while chlorine in water rapidly inactivates E. coli, it leaves most disinfection-resistant pathogens almost unaffected for several hours (3). According to Payment and Franco (3), Clostridium perfringens is a suitable indicator of human enteric viruses, Giardia cysts, and Cryptosporidium oocysts in finished water and can also be used in the assessment of water treatment processes due to the resistance of Clostridium spores to chlorine. Furthermore, the presence of C. perfringens in water is also associated with fecal contamination, and it has been evaluated and utilized as an alternative indicator of fecal pollution (4-14). Thus, a simple and reliable culture-based method to isolate and enumerate C. perfringens, the membrane filtration method on mCP agar, has been elaborated and evaluated to monitor the presence of C. perfringens in water (15)(16)(17)(18)(19)(20). In the European Union, the mCP a...

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“…The HRM is simply a precise warming of the amplicon DNA [76], [80] from around 50˚C up to around 95˚C. At some point during this process, the melting temperature of the amplicon is reached and the two strands of DNA separate or "melt" apart.…”

Section: Novel Molecular Approach To Detect the Most Frequent Causatimentioning

confidence: 99%

Application of multiplex real-time PCR and Fluorescence Resonance Energy Transfer for the detection and differentiation of the most frequent causative agents of systemic infections from biological fluids

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Method for rapid and sensitive detection of Enterococcus sp. and Enterococcus faecalis/faecium cells in potable water samples

Cited by 35 publications

References 48 publications

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

Application of multiplex real-time PCR and Fluorescence Resonance Energy Transfer for the detection and differentiation of the most frequent causative agents of systemic infections from biological fluids

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