Changes in Escherichia coli to enteric protozoa ratios in rivers: Implications for risk-based assessment of drinking water treatment requirements.

Minimum treatment requirements are set in response to established or anticipated levels of enteric pathogens in the source water of drinking water treatment plants (DWTPs). For surface water, contamination can be determined directly by monitoring reference pathogens or indirectly by measuring fecal indicators such as Escherichia coli (E. coli). In the latter case, a quantitative interpretation of E. coli for estimating reference pathogen concentrations could be used to define treatment requirements. This study presents the statistical analysis of paired E. coli and reference protozoa (Cryptosporidium, Giardia) data collected monthly for two years in source water from 27 DWTPs supplied by rivers in Canada. E. coli/Cryptosporidium and E. coli/Giardia ratios in source water were modeled as the ratio of two correlated lognormal variables. To evaluate the potential of E. coli for defining protozoa treatment requirements, risk-based critical mean protozoa concentrations in source water were determined with a reverse quantitative microbial risk assessment (QMRA) model. Model assumptions were selected to be consistent with the World Health Organization (WHO) Guidelines for drinking-water quality. The sensitivity of mean E. coli concentration trigger levels to identify these critical concentrations in source water was then evaluated. Results showed no proportionalities between the log of mean E. coli concentrations and the log of mean protozoa concentrations. E. coli/protozoa ratios at DWTPs supplied by small rivers in agricultural and forested areas were typically 1.0 to 2.0-log lower than at DWTPs supplied by large rivers in urban areas. The seasonal variations analysis revealed that these differences were related to low mean E. coli concentrations during winter in small rivers. To achieve the WHO target of 10-6 disability-adjusted life year (DALY) per person per year, a minimum reduction of 4.0-log of Cryptosporidium would be required for 20 DWTPs, and a minimum reduction of 4.0-log of Giardia would be needed for all DWTPs. A mean E. coli trigger level of 50 CFU 100 mL-1 would be a sensitive threshold to identify critical mean concentrations for Cryptosporidium but not for Giardia. Treatment requirements higher than 3.0-log would be needed at DWTPs with mean E. coli concentrations as low as 30 CFU 100 mL-1 for Cryptosporidium and 3 CFU 100 mL-1 for Giardia. Therefore, an E. coli trigger level would have limited value for defining health-based treatment requirements for protozoa at DWTPs supplied by small rivers in rural areas.

Importance of Distributional Forms for the Assessment of Protozoan Pathogens Concentrations in Drinking-Water Sources.

The identification of appropriately conservative statistical distributions is needed to predict microbial peak events in drinking water sources explicitly. In this study, Poisson and mixed Poisson distributions with different upper tail behaviors were used for modeling source water Cryptosporidium and Giardia data from 30 drinking water treatment plants. Small differences (<0.5-log) were found between the "best" estimates of the mean Cryptosporidium and Giardia concentrations with the Poisson-gamma and Poisson-log-normal models. However, the upper bound of the 95% credibility interval on the mean Cryptosporidium concentrations of the Poisson-log-normal model was considerably higher (>0.5-log) than that of the Poisson-gamma model at four sites. The improper choice of a model may, therefore, mislead the assessment of treatment requirements and health risks associated with the water supply. Discrimination between models using the marginal deviance information criterion (mDIC) was unachievable because differences in upper tail behaviors were not well characterized with available data sets ( n<30 ). Therefore, the gamma and the log-normal distributions fit the data equally well but may predict different risk estimates when they are used as an input distribution in an exposure assessment. The collection of event-based monitoring data and the modeling of larger routine monitoring data sets are recommended to identify appropriately conservative distributions to predict microbial peak events.

Draft Genome Sequence of Romboutsia weinsteinii sp. nov. Strain CCRI-19649T Isolated from Surface Water.

Romboutsia weinsteinii sp. nov. CCRI-19649T belongs to the genus Romboutsia The strain was isolated from a water sample harvested in Québec City, Québec, Canada. The genome assembly comprised 4,134,593 bp with a 29.3% GC content. This is the first documentation that reports the genome sequence of R. weinsteinii.

Cryptosporidium hominis Is a Newly Recognized Pathogen in the Arctic Region of Nunavik, Canada: Molecular Characterization of an Outbreak.

BACKGROUND: Cryptosporidium is a leading cause of childhood diarrhea in low-resource settings, and has been repeatedly associated with impaired physical and cognitive development. In May 2013, an outbreak of diarrhea caused by Cryptosporidium hominis was identified in the Arctic region of Nunavik, Quebec. Human cryptosporidiosis transmission was previously unknown in this region, and very few previous studies have reported it elsewhere in the Arctic. We report clinical, molecular, and epidemiologic details of a multi-village Cryptosporidium outbreak in the Canadian Arctic. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the occurrence of cryptosporidiosis using a descriptive study of cases with onset between April 2013 and April 2014. Cases were defined as Nunavik inhabitants of any age presenting with diarrhea of any duration, in whom Cryptosporidium oocysts were detected by stool microscopy in a specialised reference laboratory. Cryptosporidium was identified in stool from 51 of 283 individuals. The overall annual incidence rate (IR) was 420 / 100,000 inhabitants. The IR was highest among children aged less than 5 years (1290 /100,000 persons). Genetic subtyping for stool specimens from 14/51 cases was determined by DNA sequence analysis of the 60 kDa glycoprotein (gp60) gene. Sequences aligned with C. hominis subtype Id in all cases. No common food or water source of infection was identified. CONCLUSIONS/SIGNIFICANCE: In this first observed outbreak of human cryptosporidiosis in this Arctic region, the high IR seen is cause for concern about the possible long-term effects on growth and development of children in Inuit communities, who face myriad other challenges such as overcrowding and food-insecurity. The temporal and geographic distribution of cases, as well as the identification of C. hominis subtype Id, suggest anthroponotic rather than zoonotic transmission. Barriers to timely diagnosis delayed the recognition of human cryptosporidiosis in this remote setting.

Temperature diagnostic to identify high risk areas and optimize Legionella pneumophila surveillance in hot water distribution systems.

Legionella pneumophila is frequently detected in hot water distribution systems and thermal control is a common measure implemented by health care facilities. A risk assessment based on water temperature profiling and temperature distribution within the network is proposed, to guide effective monitoring strategies and allow the identification of high risk areas. Temperature and heat loss at control points (water heater, recirculation, representative points-of-use) were monitored in various sections of five health care facilities hot water distribution systems and results used to develop a temperature-based risk assessment tool. Detailed investigations show that defective return valves in faucets can cause widespread temperature losses because of hot and cold water mixing. Systems in which water temperature coming out of the water heaters was kept consistently above 60 °C and maintained above 55 °C across the network were negative for Legionella by culture or qPCR. For systems not meeting these temperature criteria, risk areas for L. pneumophila were identified using temperature profiling and system's characterization; higher risk was confirmed by more frequent microbiological detection by culture and qPCR. Results confirmed that maintaining sufficiently high temperatures within hot water distribution systems suppressed L. pneumophila culturability. However, the risk remains as shown by the persistence of L. pneumophila by qPCR.

Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012.

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaire's disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.

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

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.

Contamination of public whirlpool spas: factors associated with the presence of Legionella spp., Pseudomonas aeruginosa and Escherichia coli.

This work explores the factors associated with contamination of public spas by Legionella spp., Pseudomonas aeruginosa and Escherichia coli. Physicochemical and microbiological parameters were measured in water samples from 95 spas inQuébec, Canada. Spa maintenance was documented by a questionnaire. Legionella spp. were detected in 23% of spas, P. aeruginosa in 41% and E. coli in 2%. Bacteria were found in concerning concentrations (Legionella spp. ≥ 500 CFU/l, P. aeruginosa ≥ 51 CFU/100 ml or E. coli ≥ 1 CFU/100 ml) in 26% ofspas. Observed physicochemical parameters frequently differed from recommended guidelines. The following factors decreased the prevalence of concerning microbial contamination: a free chlorine concentration ≥ 2 mg/l or total bromine ≥ 3 mg/l (p = 0.001), an oxidation-reduction potential (ORP) > 650 mV (p = 0.001), emptying and cleaning the spa at least monthly (p = 0.019) and a turbidity ≤ 1 NTU (p = 0.013). Proper regulations and training of spa operators are critical for better maintenance of these increasingly popular facilities.

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 ß-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.

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 ß-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 ß-glucuronidase detection.

Analytical limits of three beta-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.

[A study to assess the microbial contamination of Mya arenaria clams from the north shore of the St Lawrence River estuary, (Québec, Canada)]. / Etude de la contamination microbienne des myes (Mya arenaria) de la rive nord de l'estuaire maritime du fleuve Saint-Laurent (Québec, Canada)

The aims of the present study were to assess the microbial quality of Mya arenaria clams from the north shore of the St. Lawrence River estuary and to validate various microbial indicator microorganisms of bivalve mollusks contamination. Clams were collected from nine sites, including four harvesting sites closed by virtue of the Canadian Shellfish Sanitation Program (CSSP). Six contamination indicators (fecal coliforms, somatic coliphages, F-specific coliphages, fecal streptococci, Clostridium perfringens, and Escherichia coli) and four pathogens (Campylobacter sp., Cryptosporidium parvum, Giardia sp., and Salmonella sp.) were identified in the clams. Indicators sensibility, specificity and predictive values with respect to the presence of pathogens were calculated. Pathogenic microorganisms detection frequency in clams was important (92%). Globally, pathogens tend to be less frequently detected in opened harvesting sites (p = 0.086). Although the assessed indicators were not perfect, when F-specific coliphages are associated with E. coli or fecal coliforms, a good sensibility (62%-64%) and good positive predictive value (88%) with respect to the investigated pathogens are obtained.