Andrée F. Maheux

Analytical comparison of nine PCR primer sets designed to detect the presence of Escherichia coli/Shigella in water samples

The analytical performance of 9 different PCR primer sets designed to detect Escherichia coli and Shigella in water has been evaluated in terms of ubiquity, specificity, and analytical detection limit. Of the 9 PCR primer sets tested, only 3 of the 5 primer sets targeting uidA gene and the primer set targeting tuf gene amplified DNA from all E. coli strains tested. However, of those 4 primer sets, only the primer set targeting the tuf gene also amplified DNA from all Shigella strains tested. For the specificity, only the primer sets targeting the uidA gene were 100% specific although the primer sets targeting 16S rRNA, phoE, and tuf genes only amplified Escherichia fergusonii as non-specific target. Finally, the primer set targeting the 16S-ITS-23S gene region, was not specific as it amplified DNA from many other Enterobacteriaceae species. In summary, only the assay targeting the tuf gene detected all E. coli/Shigella strains tested in this study. However, if it becomes important to discriminate between E. coli and E. fergusonii, assays targeting the uidA gene would represent a good choice although none of them were totally ubiquitous to detect of the presence of Shigella strains.

Analytical limits of four β-glucuronidase and β-galactosidase-based commercial culture methods used to detect Escherichia coli and total coliforms

Colilert (Colilert), Readycult Coliforms 100 (Readycult), Chromocult Coliform agar ES (Chromocult), and MI agar (MI) are beta-galactosidase and beta-glucuronidase-based commercial culture methods used to assess water quality. Their analytical performance, in terms of their respective ability to detect different strains of Escherichia coli and total coliforms, had never been systematically compared with pure cultures. Here, their ability to detect beta-glucuronidase production from E. coli isolates was evaluated by using 74 E. coli strains of different geographic origins and serotypes encountered in fecal and environmental settings. Their ability to detect beta-galactosidase production was studied by testing the 74 E. coli strains as well as 33 reference and environmental non-E. coli total coliform strains. Chromocult, MI, Readycult, and Colilert detected beta-glucuronidase production from respectively 79.9, 79.9, 81.1, and 51.4% of the 74 E. coli strains tested. These 4 methods detected beta-galactosidase production from respectively 85.1, 73.8, 84.1, and 84.1% of the total coliform strains tested. The results of the present study suggest that Colilert is the weakest method tested to detect beta-glucuronidase production and MI the weakest to detect beta-galactosidase production. Furthermore, the high level of false-negative results for E. coli recognition obtained by all four methods suggests that they may not be appropriate for identification of presumptive E. coli strains.

Rapid Concentration and Molecular Enrichment Approach for Sensitive Detection of Escherichia coli and Shigella Species in Potable Water Samples

ABSTRACT In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment]), that we coupled to an Escherichia coli/Shigella-specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli/Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non-E. coli/Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii, which was detected by the CRENAME procedure combined with the E. coli/Shigella-specific rtPCR assay (CRENAME + E. coli rtPCR). DNA from all 90 E. coli/Shigella strains tested was amplified by the CRENAME + E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME + E. coli rtPCR method detected 1.8 E. coli/Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli/Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli/Shigella-specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.

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

We have developed a rapid and robust technological solution including a membrane filtration and dissolution method followed by a molecular enrichment and a real-time PCR assay, for detecting the presence of Enterococcus sp. or Enterococcus faecalis/faecium per 100 mL of water in less than 5 h and we compared it to Method 1600 on mEI agar in terms of specificity, sensitivity, and limit of detection. The mEI and the Enterococcus sp.-specific assay detected respectively 73 (64.0%) and 114 (100%) of the 114 enterococcal strains tested. None of the 150 non-enterococcal strains tested was detected by both methods with the exception of Tetragenococcus solitarius for the Enterococcus sp. assay. The multiplexed E. faecalis/faecium assay efficiently amplified DNA from 47 of 47 (100%) E. faecalis and 27 of 27 (100%) E. faecium strains tested respectively, whereas none of the 191 non-E. faecalis/faecium strains tested was detected. By simultaneously detecting the predominant fecal enterococcal species, the E. faecalis/faecium-specific assay allows a better distinction between enterococcal strains of fecal origin and those provided by the environment than Method 1600. Our procedure allows the detection of 4.5 enterococcal colony forming units (CFU) per 100 mL in less than 5 h, whereas the mEI method detected 2.3 CFU/100 mL in 24 h (95% confidence). Thus, our innovative and highly effective method provides a rapid and easy approach to concentrate very low numbers of enterococcal cells present in a 100 mL water sample and allows a better distinction between fecal and environmental enterococcal cells than Method 1600.

Enterococcus ureasiticus sp. nov. and Enterococcus quebecensis sp. nov., isolated from water

Three enterococcal isolates, CCRI-16620, CCRI-16986(T) and CCRI-16985(T), originating from water were characterized using morphological, biochemical and molecular taxonomic methods. 16S rRNA gene sequence analysis classified all three strains in the Enterococcus faecalis species group. The phylogenetic tree of 16S rRNA gene sequences showed that the three isolates form two separate branches. The first branch is represented by strains CCRI-16620 and CCRI-16986(T) and the second branch by strain CCRI-16985(T). Further sequence analysis of the housekeeping genes rpoA (encoding RNA polymerase α subunit), pheS (phenylalanyl-tRNA synthase), tufA (elongation factor Tu) and atpD (ATP synthase β-subunit) as well as the results of amplified fragment length polymorphism (AFLP) DNA fingerprinting and DNA-DNA hybridization experiments confirmed the distinct status of these strains. Moreover, biochemical tests allowed phenotypic differentiation of the strains from the other species of the E. faecalis species group. On the basis of the results obtained, the names Enterococcus ureasiticus sp. nov. (type strain CCRI-16986(T) = CCUG 59304(T) = DSM 23328(T) = LMG 26304(T)) and Enterococcus quebecensis sp. nov. (type strain CCRI-16985(T) = CCUG 59306(T) = DSM 23327(T) = LMG 26306(T)) are proposed for the two hitherto undescribed species.

Analytical limits of three β-glucosidase-based commercial culture methods used in environmental microbiology, to detect enterococci

The enzyme-based test methods Enterolert, Chromocult Enterococci agar, and mEI agar, used to assess water quality through the detection Enterococcus spp., have been compared in terms of their analytical specificity and their ability to detect various enterococcal strains. To achieve this goal, we have tested 110 different non-enterococcal bacterial strains and 101 strains of Enterococcus spp. isolated from diverse origins. The results obtained showed that 69 (68.3%), 84 (83.2%), and 89 (88.1%) of the 101 enterococcal strains tested respectively yielded a positive signal with Enterolert, mEI, and Chromocult Enterococci. Regarding the specificity, none of the non-Enterococcus spp. strains tested were detectable by any of the three culture methods, except for Granulicatella adiacens which turned out positive on Chromocult Enterococci. The results of this study showed that, based on our collection of strains, the Enterolert test method detected less enterococcal strains than the two other methods.

Abilities of the mCP Agar method and CRENAME alpha toxin-specific real-time PCR assay to detect Clostridium perfringens spores in drinking water.

ABSTRACT 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.

Molecular Method for Detection of Total Coliforms in Drinking Water Samples

ABSTRACT 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 culture-based methods. This assay might be used in combination with an Escherichia coli molecular assay to assess drinking water quality.

Comparative analysis of classical and molecular microbiology methods for the detection of Escherichia coli and Enterococcus spp. in well water

The microbiological quality of 165 1 litre well water samples collected in the Québec City region was assessed by culture-based methods (mFC agar, Chromocult coliform agar, Colilert(®), MI agar, Chromocult enterococci, Enterolert™, and mEI agar) and by a molecular microbiology strategy, dubbed CRENAME-rtPCR, developed for the detection of Escherichia coli, Enterococcus spp., Enterococcus faecalis/faecium, and Bacillus atrophaeus subsp. globigii. In these drinking water samples, approved culture-based methods detected E. coli at rates varying from 1.8 to 3.6% and Enterococcus spp. at rates varying from 3.0 to 11.5%, while the molecular microbiology approach for E. coli was found to be as efficient, detecting contamination in 3.0% of samples. In contrast, CRENAME-rtPCR detected Enterococcus spp. in 27.9% of samples while the E. faecalis/faecium molecular assay did not uncover a single contaminated sample, thereby revealing a discrepancy in the coverage of waterborne enterococcal species detected by classical and molecular microbiology methods. The validation of the CRENAME-E. coli rtPCR test as a new tool to assess the quality of drinking water will require larger scale studies elaborated to demonstrate its equivalence to approved methods.

Characterization of Escherichia fergusonii and Escherichia albertii isolated from water

The aim of this study was to characterize Escherichia fergusonii and Escherichia albertii isolated from water.