Ève Bérubé

The initial state of the human gut microbiome determines its reshaping by antibiotics.

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants’ microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase blaCfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.

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.

Polythiophene Biosensor for Rapid Detection of Microbial Particles in Water

Most microbial particles have a negatively charged surface and in this work, we describe a water quality monitoring application of a cationic polythiophene derivative (AH-35) for the rapid assessment of microbial contamination of water. Using E. coli as a prototype microbial particle, we demonstrate that the AH-35 polymer can provide a qualitative assessment of water if exposed to more than 500 CFU/mL, thereby paving the way to a new family of biosensors potentially useful for monitoring drinking water distribution systems.

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.

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.

Ability of three DNA-based assays to identify presumptive Escherichia coli colonies isolated from water by the culture-based mFC agar method

We tested the ability of three PCR assays, targeting uidA and tuf genes to correctly identify Escherichia coli colonies isolated from water and we compared them to two β-glucuronidase-based culture methods (Colilert(®) and Readycult(®)), in terms of specificity and sensitivity. E. coli isolates recovered on mFC agar were first tested for the presence of the uidA positive colonies were presumed to be E. coli. For further characterization, uidA-negative colonies were subsequently identified using the Vitek 2 automated system. Colilert(®) and Readycult(®) detected 436 and 442 of 468 colonies identified as E. coli on mFC corresponding to sensitivities of 93.2 and 94.4%, respectively. None of the 59 non-E. coli isolates was detected by both methods for a specificity of 100%. Two (2) uidA and 1 tuf PCR assays were also tested. The uidA PCR assays yielded positive signals for 447 (95.5%) and 434 (92.7%) of 468 E. coli isolates tested respectively, whereas the tuf PCR assay showed a sensitivity of 100%. None of the 59 non-E. coli isolates was detected by both uidA PCR assays (100% specificity), whereas tuf PCR false-positive signals were obtained with Escherichia fergusonii and Escherichia albertii. However, since these 2 species are principally found in the feces of mammals and birds, their detection indicates a fecal contamination. Consequently, using a 1-h tuf rtPCR assay to confirm the identity of E. coli colonies on mFC agar is as specific, more sensitive, and potentially more cost-efficient than culture methods based on β-glucuronidase detection.

Method for isolation of both lactose-fermenting and – non-fermenting Escherichia albertii strains from stool samples

Initially, Escherichia albertii has been described as a non-lactose fermenting bacterium and methods used to isolate it were first based on this phenotypic property. However, a recent study showed a variable lactose fermentation phenotype for E. albertii suggesting that this microorganism could have been underestimated by previous studies using isolation methods based on lactose fermentation. In this study, we present a method for the isolation and identification of both lactose fermenting and non-fermenting-E. albertii cells in stool samples, said method combining culture and isolation on mEA agar, an indole test, as well as an E. albertii-specific PCR assay for formal species identification. The ability of the procedure to detect E. albertii strains was verified using 19 E. albertii strains and 132 non-E. albertii strains representing 88 species of different origins majoritary belonging to the Enterobacteriaceae family. All indole-positive white colonies grown on mEA agar were subjected to E. albertii-specific PCR amplification; all E. albertii strains tested were detected with this assay and none of the non-E. albertii strains tested was detected. To demonstrate the ability of the procedure to directly detect E. albertii in stool samples, E. albertii-inoculated stools were tested and for all inoculated samples, E. albertii colonies were easily detected and identified. The present study provides a method enable to recover both lactose-fermenting and -non-fermenting E. albertii strains from clinical samples. This method could help to provide a better portrait of the prevalence and pathogenicity of E. albertii in clinical samples.

Rapid molecular identification of fecal origin-colonies growing on Enterococcus spp.-specific culture methods

The mEI, Chromocult® enterococci, and m-Enterococcus culture-based methods used to assess water quality by the detection of Enterococcus spp. were first compared in terms of sensitivity using (1) 41 different type strains of Enterococcus spp. and (2) environmental colonies identified by 16S rRNA sequencing. Then, two specific-rtPCR assays targeting Enterococcus spp. and Enterococcus faecalis/faecium were tested for their ability to confirm the identity of putative enterococcal colonies. The mEI, Chromocult® enterococci, and m-Enterococcus methods detected β-glucosidase activity for 28 (68.3%), 32 (78.0%), and 12 (29.3%) of the 41 reference enterococcal strains tested, respectively. Analysis with environmental colonies showed that mEI and Chromocult® enterococci media had false positive rates of 4.3% and 5.0%, respectively. Finally, the two rtPCR assays showed a specificity of 100%. Only two (2/19) colonies of E. faecium isolated from mEI agar were not detected by the Enterococcus faecium rtPCR assay, for a sensitivity of 89.5%. Our results showed that Chromocult® enterococci medium recovered more E. faecalis/faecium cells than the two other methods. Thus, the use of Chromocult® enterococci combined with the Enterococcus faecalis/faecium rtPCR assay showed the best combination to decrease the high false-positive rate obtained when the entire Enterococcus genus is targeted.

Comparison of MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods to detect Escherichia coli and total coliforms in water using 16S rRNA sequencing for colony identification

The MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods used to assess water quality by the detection of Escherichia coli and total coliforms were compared in terms of their specificity and sensitivity, using 16S rRNA sequencing for colony identification. A sewage water sample was divided in 2-μL subsamples for testing by all three culture-based methods. All growing colonies were harvested and subjected to 16S rRNA sequencing. Test results showed that all E. coli colonies were correctly identified by all three methods, for a specificity and a sensitivity of 100%. However, for the total coliform detection, the MI agar, Chromocult® coliform agar, and Compass CC agar were specific for only 69.2% (9/13), 47.2% (25/53), and 40.5% (17/42), whereas sensitive for 97.8% (45/46), 97.5% (39/40), and 85.7% (24/28), respectively. Thus, given the low level of specificity of these methods for the detection of total coliforms, confirming the identity of total coliform colonies could help to take public health decisions, in particular for cities connected to a public drinking water distribution system since the growth of few putative total coliform colonies on chromogenic agar is problematic and can lead to unnecessary and costly boiling notices from public health authorities.