Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada.

Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher's exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 (n=22), O22:H8 (n=30), O108:H8 (n=39), O139:H19 (n=44) and O157:H7 (n=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes (P≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.

Plasmid Distribution among Escherichia coli from Livestock and Associated Wastewater: Unraveling Factors That Shape the Presence of Genes Conferring Third-Generation Cephalosporin Resistance.

A key concern with agricultural wastewater storage ponds is that they may provide an environment conducive for horizontal exchange of antibiotic resistance genes (ARGs), thereby facilitating the emergence of antibiotic resistant pathogens. Central to this exchange are mobile genetic elements like plasmids; yet, the factors shaping their presence in agricultural environments remain poorly understood. Here, using Escherichia coli as a model bacterium, we examined genetic backgrounds and plasmid profiles of generic fecal and wastewater isolates and those possessing blaCTX-M and blaCMY-2 genes (which confer resistance to third-generation cephalosporins) to delineate factors shaping the environmental persistence of plasmid-associated ARGs in beef cattle feedlots. The wastewater environment exerted minimal influence on plasmid repertoires, as the number of plasmids and distribution of different incompatibility groups did not differ between generic fecal and wastewater isolates. The blaCTX-M and blaCMY-2 genes were associated with IncF and IncA/C plasmids, respectively, and host isolates possessing these ARGs had fewer plasmids than generic isolates, suggesting ARG-bearing plasmids may associate predominantly with such hosts to compensate for the metabolic burden imposed by these plasmids. Phylogeny also appeared to be a factor for blaCTX-M genes, as their bacterial hosts were restricted to particular genetic lineages, including the environmentally adapted ET-1 clade, as noted previously for these genes. Ultimately, these findings have important implications for evaluating human health risks of agricultural wastewater with respect to environmental persistence of ARGs and may help identify options for improving wastewater treatment.

Sources of generic Escherichia coli and factors impacting guideline exceedances for food safety in an irrigation reservoir outlet and two canals.

Nearly half of all cases of foodborne illness are associated with plant-based foods such as leafy greens and raw flour. An important potential source of pathogen contamination along the food-production continuum is irrigation water, which has led to the implementation of increasingly stringent agricultural irrigation water quality requirements. To better understand factors impacting irrigation water quality, we investigated sources of generic Escherichia coli and how they varied temporally among different sampling sites. Precipitation, Campylobacter species distribution, and physicochemical water quality parameters were also investigated to substantiate microbial source tracking findings. Biweekly sampling was conducted at a reservoir outlet and two downstream canals in southern Alberta, Canada, throughout two irrigation seasons, the latter of which was notable for drought conditions. Overall, 50% of canal samples exceeded Alberta's irrigation guideline for E. coli (100 E. coli per 100 ml), whereas all reservoir samples were below guideline limits. Collectively, E. coli source apportionment, Campylobacter species distribution, and physicochemical water quality data suggest runoff from surrounding agricultural land was a contributing factor to E. coli guideline exceedances in Year 1 only. In Year 2, the majority of exceedances occurred later in the season when there was little precipitation and were largely attributed to cosmopolitan E. coli from wild birds and cattle. Similarities in E. coli host-source and Campylobacter species distributions between the reservoir and canals when the guideline was exceeded suggest the reservoir could be a primary source of E. coli during drought. Increased bacterial concentrations in canals were likely due to environmental conditions that promoted bacterial survival and in-situ proliferation. Our findings support previous accounts that many E. coli isolates possess enhanced survival capabilities, which has implications to bacterial water quality assessments and risk mitigation, particularly under drought conditions.

Multi-Year Persistence of Verotoxigenic Escherichia coli (VTEC) in a Closed Canadian Beef Herd: A Cohort Study.

In this study, fecal samples were collected from a closed beef herd in Alberta, Canada from 2012 to 2015. To limit serotype bias, which was observed in enrichment broth cultures, Verotoxigenic Escherichia coli (VTEC) were isolated directly from samples using a hydrophobic grid-membrane filter verotoxin immunoblot assay. Overall VTEC isolation rates were similar for three different cohorts of yearling heifers on both an annual (68.5 to 71.8%) and seasonal basis (67.3 to 76.0%). Across all three cohorts, O139:H19 (37.1% of VTEC-positive samples), O22:H8 (15.8%) and O?(O108):H8 (15.4%) were among the most prevalent serotypes. However, isolation rates for serotypes O139:H19, O130:H38, O6:H34, O91:H21, and O113:H21 differed significantly between cohort-years, as did isolation rates for some serotypes within a single heifer cohort. There was a high level of VTEC serotype diversity with an average of 4.3 serotypes isolated per heifer and 65.8% of the heifers classified as "persistent shedders" of VTEC based on the criteria of >50% of samples positive and ≥4 consecutive samples positive. Only 26.8% (90/336) of the VTEC isolates from yearling heifers belonged to the human disease-associated seropathotypes A (O157:H7), B (O26:H11, O111:NM), and C (O22:H8, O91:H21, O113:H21, O137:H41, O2:H6). Conversely, seropathotypes B (O26:NM, O111:NM) and C (O91:H21, O2:H29) strains were dominant (76.0%, 19/25) among VTEC isolates from month-old calves from this herd. Among VTEC from heifers, carriage rates of vt1, vt2, vt1+vt2, eae, and hlyA were 10.7, 20.8, 68.5, 3.9, and 88.7%, respectively. The adhesin gene saa was present in 82.7% of heifer strains but absent from all of 13 eae+ve strains (from serotypes/intimin types O157:H7/γ1, O26:H11/ß1, O111:NM/θ, O84:H2/ζ, and O182:H25/ζ). Phylogenetic relationships inferred from wgMLST and pan genome-derived core SNP analysis showed that strains clustered by phylotype and serotype. Further, VTEC strains of the same serotype usually shared the same suite of antibiotic resistance and virulence genes, suggesting the circulation of dominant clones within this distinct herd. This study provides insight into the diverse and dynamic nature of VTEC populations within groups of cattle and points to a broad spectrum of human health risks associated with these E. coli strains.

A national investigation of the prevalence and diversity of thermophilic Campylobacter species in agricultural watersheds in Canada.

The occurrence and diversity of thermophilic Campylobacter species (C. jejuni, coli, and lari) were studied in water samples from four river basins located across Canada. These basins located in Quebec (Bras d'Henri), Alberta (Oldman), Ontario (South Nation), and British Columbia (Sumas) represented some of the most intensive farming areas in Canada for hog, beef cattle, dairy cattle, and poultry, respectively. This study analyzed 769 water samples collected from 23 monitoring sites with agricultural influence, and four reference sites with limited or no agricultural influence. Water samples were collected bi-weekly over two years and analyzed for Campylobacter using a semi-quantitative minimum probable number (MPN) enrichment protocol. Putative isolates were confirmed by genus- and species-specific multiplex polymerase chain reaction (PCR) assays. A total of 377 (49%) water samples were positive for campylobacters with 355 samples having a cell density ranging from 4 to 4000 MPN L(-1). Campylobacters were more common at agricultural than reference sites in each river basin, although this difference was not significant in the Oldman and South Nation (p > 0.05). Campylobacter was significantly more common in the Bras d'Henri and Sumas (63%) compared to the South Nation (45%) and Oldman (33%) River basins (p < 0.05). C. jejuni, C. coli and C. lari were detected in each river basin, and these species occurred in 45% (n = 168), 34% (n = 128) and 19% (n = 73), of all Campylobacter positive samples, respectively. The remaining Campylobacter positive water samples without these three species (n = 67; 18%) were identified as other Campylobacter species. C. jejuni was the predominant species occurring in the Sumas, Oldman and South Nation River basins. However, in the Bras d'Henri River basin with intensive hog production, C. coli was the predominant species. This study found campylobacters to be common in some agricultural systems with intensive livestock farming activities, and different river basins could have strikingly different profiles of either C. jejuni or C. coli as the predominant waterborne thermophilic Campylobacter species.

Long-term monitoring of waterborne pathogens and microbial source tracking markers in paired agricultural watersheds under controlled and conventional tile drainage management.

Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P = 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tile-drained fields to stream could be reduced by CTD utilization.

Coherence among different microbial source tracking markers in a small agricultural stream with or without livestock exclusion practices.

Over 1,400 water samples were collected biweekly over 6 years from an intermittent stream protected and unprotected from pasturing cattle. The samples were monitored for host-specific Bacteroidales markers, Cryptosporidium species/genotypes, viruses and coliphages associated with humans or animals, and bacterial zoonotic pathogens. Ruminant Bacteroidales markers did not increase within the restricted cattle access reach of the stream, whereas the ruminant Bacteroidales marker increased significantly in the unrestricted cattle access reach. Human Bacteroidales markers significantly increased downstream of homes where septic issues were documented. Wildlife Bacteroidales markers were detected downstream of the cattle exclusion practice where stream and riparian habitat was protected, but detections decreased after the unrestricted pasture, where the stream and riparian zone was unprotected from livestock. Detection of a large number of human viruses was shown to increase downstream of homes, and similar trends were observed for the human Bacteroidales marker. There was considerable interplay among biomarkers with stream flow, season, and the cattle exclusion practices. There were no to very weak associations with Bacteroidales markers and bacterial, viral, and parasitic pathogens. Overall, discrete sample-by-sample coherence among the different microbial source tracking markers that expressed a similar microbial source was minimal, but spatial trends were physically meaningful in terms of land use (e.g., beneficial management practice) effects on sources of fecal pollution.

An enhanced technique combining pre-enrichment and passive filtration increases the isolation efficiency of Campylobacter jejuni and Campylobacter coli from water and animal fecal samples.

Improved isolation techniques from environmental water and animal samples are vital to understanding Campylobacter epidemiology. In this study, the efficiency of selective enrichment in Bolton Broth (BB) followed by plating on charcoal cefoperazone deoxycholate agar (CCDA) (conventional method) was compared with an approach combining BB enrichment and passive filtration (membrane method) adapted from a method previously developed for testing of broiler meat, in the isolation of thermophilic campylobacters from surface water and animal fecal samples. The conventional method led to recoveries of Campylobacter from 36.7% of the water samples and 78.0% of the fecal samples and similar numbers, 38.3% and 76.0%, respectively, were obtained with the membrane method. To investigate the genetic diversity of Campylobacter jejuni and Campylobacter coli obtained by these two methods, isolates were analyzed using Comparative Genomic Fingerprinting, a high-resolution subtyping technique. The conventional and membrane methods yielded similar numbers of Campylobacter subtypes from water (25 and 28, respectively) and fecal (15 and 17, respectively) samples. Although there was no significant difference in recovery rates between the conventional and membrane methods, a significant improvement in isolation efficiency was obtained by using the membrane method, with a false-positive rate of 1.6% compared with 30.7% obtained using the conventional method. In conclusion, although the two methods are comparable in sensitivity, the membrane method had higher specificity, making it a cost-effective procedure for the enhanced isolation of C. jejuni and C. coli from water and animal fecal samples.

Spatial and temporal drivers of zoonotic pathogen contamination of an agricultural watershed.

In regions where animal agriculture is prominent, such as southern Alberta, higher rates of gastrointestinal illness have been reported when compared with nonagricultural regions. This difference in the rate of illness is thought to be a result of increased zoonotic pathogen exposure through environmental sources such as water. In this study, temporal and spatial factors associated with bacterial pathogen contamination of the Oldman River, which transverses this region, were analyzed using classification and regression tree analysis. Significantly higher levels of fecal indicators; more frequent isolations of Campylobacter spp., Escherichia coli O157:H7, and Salmonella enterica spp.; and higher rates of detection of pig-specific Bacteroides markers occurred at downstream sites than at upstream sites, suggesting additive stream inputs. Fecal indicator densities were also significantly higher when any one of these three bacterial pathogens was present and where there were higher total animal manure units; however, occasionally pathogens were present when fecal indicator levels were low or undetectable. Overall, Salmonella spp., Campylobacter spp., and E. coli O157:H7 presence was associated with season, animal manure units, and total rainfall on the day of sampling and 3 d in advance of sampling. Several of the environmental variables analyzed in this study appear to influence pathogen prevalence and therefore may be useful in predicting water quality and safety and in the improvement of watershed management practices in this and other agricultural regions.

Development and validation of a comparative genomic fingerprinting method for high-resolution genotyping of Campylobacter jejuni.

Campylobacter spp. are a leading cause of bacterial gastroenteritis worldwide. The need for molecular subtyping methods with enhanced discrimination in the context of surveillance- and outbreak-based epidemiologic investigations of Campylobacter spp. is critical to our understanding of sources and routes of transmission and the development of mitigation strategies to reduce the incidence of campylobacteriosis. We describe the development and validation of a rapid and high-resolution comparative genomic fingerprinting (CGF) method for C. jejuni. A total of 412 isolates from agricultural, environmental, retail, and human clinical sources obtained from the Canadian national integrated enteric pathogen surveillance program (C-EnterNet) were analyzed using a 40-gene assay (CGF40) and multilocus sequence typing (MLST). The significantly higher Simpson's index of diversity (ID) obtained with CGF40 (ID = 0.994) suggests that it has a higher discriminatory power than MLST at both the level of clonal complex (ID = 0.873) and sequence type (ID = 0.935). High Wallace coefficients obtained when CGF40 was used as the primary typing method suggest that CGF and MLST are highly concordant, and we show that isolates with identical MLST profiles are comprised of isolates with distinct but highly similar CGF profiles. The high concordance with MLST coupled with the ability to discriminate between closely related isolates suggests that CFG40 is useful in differentiating highly prevalent sequence types, such as ST21 and ST45. CGF40 is a high-resolution comparative genomics-based method for C. jejuni subtyping with high discriminatory power that is also rapid, low cost, and easily deployable for routine epidemiologic surveillance and outbreak investigations.

The occurrence and sources of Campylobacter spp., Salmonella enterica and Escherichia coli O157:H7 in the Salmon River, British Columbia, Canada.

In this study, we wished to assess the prevalence and determine the sources of three zoonotic bacterial pathogens (Salmonella, Campylobacter, and Escherichia coli O157:H7) in the Salmon River watershed in southwestern British Columbia. Surface water, sewage, and animal faecal samples were collected from the watershed. Selective bacterial culture and PCR techniques were used to isolate these three pathogens and indicator bacteria from these samples and characterize them. Campylobacter was the most prevalent pathogen in all samples, followed by Salmonella, and E. coli O157:H7. E. coli O157:H7 and Salmonella isolation rates from water, as well as faecal coliform densities correlated positively with precipitation, while Campylobacter isolation rates correlated negatively with precipitation. Analysis of DNA extracted from water samples for the presence of Bacteroides host-species markers, and comparisons of C. jejuni flaA-RFLP types and Salmonella serovars from faecal and water samples provided evidence that human sewage and specific domestic and wild animal species were sources of these pathogens; however, in most cases the source could not be determined or more than one source was possible. The frequent isolation of these zoonotic pathogens in the Salmon River highlights the risks to human health associated with intentional and unintentional consumption of untreated surface waters.