Systematic Evaluation of Whole-Genome Sequencing Based Prediction of Antimicrobial Resistance in Campylobacter jejuni and C. coli.

The increasing prevalence of antimicrobial resistance (AMR) in Campylobacter spp. is a global concern. This study evaluated the use of whole-genome sequencing (WGS) to predict AMR in Campylobacter jejuni and C. coli. A panel of 271 isolates recovered from Canadian poultry was used to compare AMR genotype to antimicrobial susceptibility testing (AST) results (azithromycin, ciprofloxacin, erythromycin, gentamicin, tetracycline, florfenicol, nalidixic acid, telithromycin, and clindamycin). The presence of antibiotic resistance genes (ARGs) was determined for each isolate using five computational approaches to evaluate the effect of: ARG screening software, input data (i.e., raw reads, draft genome assemblies), genome coverage and genome assembly software. Overall, concordance between the genotype and phenotype was influenced by the computational pipelines, level of genome coverage and the type of ARG but not by input data. For example, three of the pipelines showed a 99% agreement between detection of a tet(O) gene and tetracycline resistance, whereas agreement between the detection of tet(O) and TET resistance was 98 and 93% for two pipelines. Overall, higher levels of genome coverage were needed to reliably detect some ARGs; for example, at 15X coverage a tet(O) gene was detected in >70% of the genomes, compared to <60% of the genomes for bla(OXA). No genes associated with florfenicol or gentamicin resistance were found in the set of strains included in this study, consistent with AST results. Macrolide and fluoroquinolone resistance was associated 100% with mutations in the 23S rRNA (A2075G) and gyrA (T86I) genes, respectively. A lower association between a A2075G 23S rRNA gene mutation and resistance to clindamycin and telithromycin (92.8 and 78.6%, respectively) was found. While WGS is an effective approach to predicting AMR in Campylobacter, this study demonstrated the impact that computational pipelines, genome coverage and the genes can have on the reliable identification of an AMR genotype.

Epidemiology of Campylobacter jejuni in raccoons (Procyon lotor) on swine farms and in conservation areas in southern Ontario.

Campylobacter is a leading cause of foodborne illness in humans worldwide. Sources of infection are often difficult to identify, and are, generally, poorly understood. Recent work suggests that wildlife may represent a source of Campylobacter for human infections. Using a repeated cross-sectional study design, raccoons were trapped on five swine farms and five conservation areas in southern Ontario from 2011 to 2013. Our objectives were to: (a) assess the impact of seasonal, climatic, location, annual and raccoon demographic factors on the occurrence of Campylobacter jejuni in these animals; and (b) identify clusters of C. jejuni in space, time and space-time using spatial scan statistics. Multi-level multivariable logistic regression was used to examine the odds of isolating C. jejuni, with site and animal modelled as random intercepts. The following independent variables were examined: raccoon age and sex, year, location type, season, temperature and rainfall. A total of 1,096 samples were obtained from 627 raccoons; 46.3% were positive for C. jejuni. The following interactions and their main effects were significant (p < .05) and retained in the final model: season × temperature, year × rainfall, year × temperature. Based on the results from our multivariable model and spatial scan statistics, climatic variables (i.e. rainfall, temperature and season) were associated with the carriage of C. jejuni by raccoons, but the effects were not consistent, and varied by location and year. Although raccoons may pose a zoonotic risk due to their carriage of Campylobacter, further work is required to characterize the transmission and movement of this microorganism within the ecosystem.

"These Aren't the Strains You're Looking for": Recovery Bias of Common Campylobacter jejuni Subtypes in Mixed Cultures.

Microbiological surveillance of the food chain plays a critical role in improving our understanding of the distribution and circulation of food-borne pathogens along the farm to fork continuum toward the development of interventions to reduce the burden of illness. The application of molecular subtyping to bacterial isolates collected through surveillance has led to the identification of strains posing the greatest risk to public health. Past evidence suggests that enrichment methods for Campylobacter jejuni, a leading bacterial foodborne pathogen worldwide, may lead to the differential recovery of subtypes, obscuring our ability to infer the composition of a mixed-strain sample and potentially biasing prevalence estimates in surveillance data. To assess the extent of potential selection bias resulting from enrichment-based isolation methods, we compared enrichment and non-enrichment isolation of mixed subtype cultures of C. jejuni, followed by subtype-specific enumeration using both colony plate-counts and digital droplet PCR. Results differed from the null hypothesis that similar proportions of C. jejuni subtypes are recovered from both methods. Our results also indicated a significant effect of subtype prevalence on isolation frequency post-recovery, with the recovery of more common subtypes being consistently favored. This bias was exacerbated when an enrichment step was included in the isolation procedure. Taken together, our results emphasize the importance of selecting multiple colonies per sample, and where possible, the use of both enrichment and non-enrichment isolation procedures to maximize the likelihood of recovering multiple subtypes present in a sample. Moreover, the effects of subtype-specific recovery bias should be considered in the interpretation of strain prevalence data toward improved risk assessment from microbiological surveillance data.

Campylobacter jejuni Strain Dynamics in a Raccoon (Procyon lotor) Population in Southern Ontario, Canada: High Prevalence and Rapid Subtype Turnover.

Free-ranging wildlife are increasingly recognized as potential reservoirs of disease-causing Campylobacter species such as C. jejuni and C. coli. Raccoons (Procyon lotor), which live at the interface of rural, urban, and more natural environments, are ideal subjects for exploring the potential role that wildlife play in the epidemiology of campylobacteriosis. We studied the prevalence and genetic diversity of Campylobacter from live-captured raccoons on five swine farms and five conservation areas in southwest Ontario. From 2011 to 2013, we collected fecal swabs (n = 1,096) from raccoons, and (n = 50) manure pit samples from the swine farm environment. We subtyped the resulting Campylobacter isolates (n = 581) using Comparative Genomic Fingerprinting (CGF) and 114 distinct subtypes were observed, including 96 and 18 subtypes among raccoon and manure pit isolates, respectively. Campylobacter prevalence in raccoons was 46.3%, with 98.7% of isolates recovered identified as C. jejuni. Novel raccoon-specific CGF subtypes (n = 40/96) accounted for 24.6% (n = 143/581) of Campylobacter isolates collected in this study. Our results also show that C. jejuni is readily acquired and lost in this wild raccoon population and that a high Campylobacter prevalence is observed despite transient carriage typically lasting 30 days or fewer. Moreover, although raccoons appeared to be colonized by species-adapted subtypes, they also harbored agriculture-associated genotypes that accounted for the majority of isolates observed (66.4%) and that are strongly associated with human infections. This suggests that raccoons may act as vectors in the transmission of clinically-relevant C. jejuni subtypes at the interface of rural, urban, and more natural environments.

A strain comparison of Campylobacter isolated from retail poultry and human clinical cases in Atlantic Canada.

Campylobacter is the leading cause of food-borne bacterial disease in Canada and many developed countries. One of the most common sources of human campylobacteriosis is considered to be the consumption or handling of raw or undercooked poultry. To date, few Canadian studies have investigated both the prevalence of Campylobacter on retail poultry and its potential impact on human clinical cases. The objective of this study was to evaluate the prevalence of Campylobacter spp. at the retail level and the correlation between subtypes recovered from chicken and those recovered from human clinical cases within the province of Nova Scotia, Canada. From this study 354 human clinical isolates were obtained from provincial hospital laboratories and a total of 480 packages of raw poultry cuts were sampled from retail outlets, yielding 312 isolates (65%), of all which were subtyped using comparative genomic fingerprinting (CGF). Of the 312 chicken isolates, the majority of isolates were C. jejuni (91.7%), followed by C. coli (7.7%) and C. lari (0.6%). Using CGF to subtype C. jejuni and C. coli isolates, 99 and 152 subtypes were recovered from chicken and clinical cases, respectively. The most prevalent human and chicken subtypes found in NS are similar to those observed nationally; indicating that the Campylobacter from this study appear to reflect of the profile of Campylobacter subtypes circulating nationally. Of the subtypes observed, only 36 subtypes were common between the two groups, however, these subtypes represented 48.3% of the clinical isolates collected. The findings from this study provides evidence that in Nova Scotia, retail poultry can act as a reservoir for Campylobacter subtypes that have been implicated in human illness.

Carriage of Campylobacter, Salmonella, and Antimicrobial-Resistant, Nonspecific Escherichia coli by Waterfowl Species Collected from Three Sources in Southern Ontario, Canada.

Wild birds are considered a potential source of zoonotic pathogens. We report on the occurrence of Campylobacter, Salmonella, and antimicrobial-resistant, nonspecific Escherichia coli in ducks, grebes, and swans obtained by convenience while conducting related research with Canada Geese (Branta canadensis). Samples were obtained in southern Ontario, Canada, between 2013 and 2015 from hunter-caught birds, birds submitted for postmortem diagnosis, and fresh feces from live birds in parks. A secondary objective was to characterize Campylobacter genotypes using comparative genomic fingerprinting. Salmonella and E. coli isolates were tested for susceptibility to 15 antimicrobials using the Canadian Integrated Program for Antimicrobial Resistance Surveillance test panel. A total of 71 samples were collected from 15 different waterfowl species. We detected Campylobacter, Salmonella, and E. coli in 17, 3, and 84% of samples, respectively. Ten unique Campylobacter subtypes were identified, some of which had been identified previously in water, poultry, waterfowl, and human clinical cases. Both Salmonella isolates were pansusceptible and 15% of E. coli isolates were resistant to at least one antimicrobial, including resistance to antimicrobials of highest importance to human health. Source attribution studies should examine the role of waterfowl in the dissemination of these pathogens.

Salmonella, Campylobacter, Clostridium difficile, and anti-microbial resistant Escherichia coli in the faeces of sympatric meso-mammals in southern Ontario, Canada.

The role of free-ranging wildlife in the epidemiology of enteropathogens causing clinical illness in humans and domestic animals is unclear. Salmonella enterica and anti-microbial resistant bacteria have been detected in the faeces of raccoons (Procyon lotor), but little is known about the carriage of these bacteria in other sympatric meso-mammals. Our objectives were to: (a) report the prevalence of Salmonella and associated anti-microbial resistance, Campylobacter spp, Clostridium difficile, and anti-microbial resistant Escherichia coli in the faeces of striped skunks (Mephitis mephitis) and Virginia opossums (Didelphis virginiana) in southern Ontario; and (b) compare the prevalence of these bacteria in the faeces of these meso-mammal hosts with raccoons from a previously reported study. Faecal swabs were collected from striped skunks and Virginia opossums on five swine farms and five conservation areas from 2011 to 2013. Salmonella was detected in 41% (9/22) and 5% (5/95) of faecal swabs from Virginia opossums and striped skunks, respectively. None of the Salmonella serovars carried resistance to anti-microbials. The prevalence of Campylobacter spp., C. difficile, and anti-microbial resistant E. coli ranged from 6% to 22% in striped skunk and Virginia opossums. Using exact logistic regression, Salmonella was significantly more likely to be detected in faecal swabs of Virginia opossums than skunks and significantly less likely in faecal swabs from skunks than raccoons from a previously reported study. In addition, Campylobacter spp. was significantly more likely to be detected in raccoons than opossums. Salmonella Give was detected in 8/9 (89%) of Salmonella-positive Virginia opossum faecal swabs. Our results suggest that striped skunks and Virginia opossums have the potential to carry pathogenic enteric bacteria in their faeces. The high prevalence of Salmonella Give in Virginia opossum faecal swabs in this study as well as its common occurrence in other Virginia opossum studies throughout North America suggests Virginia opossums may be reservoirs of this serovar.

A repeated cross-sectional study of the epidemiology of Campylobacter and antimicrobial resistant Enterobacteriaceae in free-living Canada geese in Guelph, Ontario, Canada.

From May through October 2016, we conducted a repeated cross-sectional study examining the effects of temporal, spatial, flock and demographic factors (i.e. juvenile vs. adult) on the prevalence of Campylobacter and antimicrobial resistant Enterobacteriaceae among 344 fresh faecal samples collected from Canada geese (Branta canadensis) from four locations where birds nested in Guelph, Ontario, Canada. The overall prevalence of Campylobacter among all fresh faecal samples was 9.3% and was greatest in the fall when these birds became more mobile following the nesting season. Based on 40 gene comparative genomic fingerprinting (CGF40), the increase in prevalence noted in the fall was matched by an increase in the number of unique CGF40 subtypes identified. Resistance to colistin was detected most commonly, in 6% of Escherichia coli isolates, and was highest in the late summer months. All colistin-resistant isolates were negative for the mcr-1 to mcr-5 genes; a chromosomal resistance mechanism (PmrB) was identified in all of these isolates. The prevalence of samples with E. coli exhibiting multi-class resistance or extended spectrum beta-lactamase was low (i.e. <2% of samples). The intra-class correlation coefficients, estimated from the variance components of multilevel logistic regression models, indicated that the shedding of Campylobacter and antimicrobial resistant E. coli among geese within a flock (i.e. birds collected from the same site on the same day) was moderately correlated. Spatial, temporal, and spatiotemporal clusters identified using the spatial scan statistic, largely supported the findings from our multi-level models. Salmonella was not isolated from any of the fresh faecal samples collected suggesting that its prevalence in this population of birds was very low.

Epidemiology of Campylobacter, Salmonella and antimicrobial resistant Escherichia coli in free-living Canada geese (Branta canadensis) from three sources in southern Ontario.

Antimicrobial resistant bacteria and zoonotic pathogens have previously been isolated from Canada geese. We examined the prevalence of three enteric bacteria (i.e. Campylobacter, Salmonella, Escherichia coli) among Canada geese from three sampling sources in southern Ontario from 2013 through 2015. Samples were obtained by convenience from hunting groups, diagnostic birds submitted for post-mortem, and fresh faeces from live birds in parks. Escherichia coli isolates were isolated and tested for susceptibility to 15 antimicrobials using the Canadian Integrated Program for Antimicrobial Resistance Surveillance test panel. The prevalences of Salmonella, Campylobacter and E. coli were 0%, 11.2% and 72.6%, respectively. Among E. coli isolates, 7.9% were resistant to ≥1 class of antimicrobials and 5.6% were resistant to ≥2 classes of antimicrobials, with some including resistance to antimicrobials of highest importance in human medicine. A significant association between season and E. coli resistance among samples from live birds was noted; summer samples had no resistant E. coli isolates, whereas spring samples demonstrated the highest prevalence of E. coli resistant to ≥1 class of antimicrobials (20.0%) among all sources. In addition, Campylobacter coli were only isolated from the spring faecal samples. Flock-level clustering was an important statistical consideration, as flock was a significant random effect in all but two of our models. Detection of Campylobacter and antimicrobial resistant E. coli from Canada geese suggests that these birds may play a role in disseminating these organisms within the environment.

Frequent Implication of Multistress-Tolerant Campylobacter jejuni in Human Infections.

Campylobacter jejuni, a major cause of bacterial foodborne illnesses, is considered highly susceptible to environmental stresses. In this study, we extensively investigated the stress tolerance of 121 clinical strains of C. jejuni against 5 stress conditions (aerobic stress, disinfectant exposure, freeze-thaw, heat treatment, and osmotic stress) that this pathogenic bacterium might encounter during foodborne transmission to humans. In contrast to our current perception about high stress sensitivity of C. jejuni, a number of clinical strains of C. jejuni were highly tolerant to multiple stresses. We performed population genetics analysis by using comparative genomic fingerprinting and showed that multistress-tolerant strains of C. jejuni constituted distinct clades. The comparative genomic fingerprinting subtypes belonging to multistress-tolerant clades were more frequently implicated in human infections than those in stress-sensitive clades. We identified unique stress-tolerant C. jejuni clones and showed the role of stress tolerance in human campylobacteriosis.

Source attribution of human campylobacteriosis at the point of exposure by combining comparative exposure assessment and subtype comparison based on comparative genomic fingerprinting.

Human campylobacteriosis is a common zoonosis with a significant burden in many countries. Its prevention is difficult because humans can be exposed to Campylobacter through various exposures: foodborne, waterborne or by contact with animals. This study aimed at attributing campylobacteriosis to sources at the point of exposure. It combined comparative exposure assessment and microbial subtype comparison with subtypes defined by comparative genomic fingerprinting (CGF). It used isolates from clinical cases and from eight potential exposure sources (chicken, cattle and pig manure, retail chicken, beef, pork and turkey meat, and surface water) collected within a single sentinel site of an integrated surveillance system for enteric pathogens in Canada. Overall, 1518 non-human isolates and 250 isolates from domestically-acquired human cases were subtyped and their subtype profiles analyzed for source attribution using two attribution models modified to include exposure. Exposure values were obtained from a concurrent comparative exposure assessment study undertaken in the same area. Based on CGF profiles, attribution was possible for 198 (79%) human cases. Both models provide comparable figures: chicken meat was the most important source (65-69% of attributable cases) whereas exposure to cattle (manure) ranked second (14-19% of attributable cases), the other sources being minor (including beef meat). In comparison with other attributions conducted at the point of production, the study highlights the fact that Campylobacter transmission from cattle to humans is rarely meat borne, calling for a closer look at local transmission from cattle to prevent campylobacteriosis, in addition to increasing safety along the chicken supply chain.

A Genome-Wide Association Study to Identify Diagnostic Markers for Human Pathogenic Campylobacter jejuni Strains.

Campylobacter jejuni is a leading human enteric pathogen worldwide and despite an improved understanding of its biology, ecology, and epidemiology, limited tools exist for identifying strains that are likely to cause disease. In the current study, we used subtyping data in a database representing over 24,000 isolates collected through various surveillance projects in Canada to identify 166 representative genomes from prevalent C. jejuni subtypes for whole genome sequencing. The sequence data was used in a genome-wide association study (GWAS) aimed at identifying accessory gene markers associated with clinically related C. jejuni subtypes. Prospective markers (n = 28) were then validated against a large number (n = 3,902) of clinically associated and non-clinically associated genomes from a variety of sources. A total of 25 genes, including six sets of genetically linked genes, were identified as robust putative diagnostic markers for clinically related C. jejuni subtypes. Although some of the genes identified in this study have been previously shown to play a role in important processes such as iron acquisition and vitamin B5 biosynthesis, others have unknown function or are unique to the current study and warrant further investigation. As few as four of these markers could be used in combination to detect up to 90% of clinically associated isolates in the validation dataset, and such markers could form the basis for a screening assay to rapidly identify strains that pose an increased risk to public health. The results of the current study are consistent with the notion that specific groups of C. jejuni strains of interest are defined by the presence of specific accessory genes.

Epidemiology of antimicrobial resistant Campylobacter spp. isolated from retail meats in Canada.

Campylobacter is an important zoonotic pathogen found in livestock and can cause illness in humans following consumption of raw and undercooked meat products. The objectives of this study were to determine the prevalence of Campylobacter spp. in retail meat (poultry, turkey, pork and beef) purchased in Alberta, Canada and to assess antimicrobial resistance and genetic relatedness of recovered Campylobacter strains with previously isolated strains from clinical and environmental sources. A Comparative Genomic Fingerprinting (CGF) method was used for assessing genetic relatedness of isolates. A total of 606 samples comprising 204, 110, 145 and 147 samples of retail chicken, turkey, ground beef and pork, respectively, were obtained. Campylobacter was isolated from 23.5% (48/204) of chicken samples and 14.2% (8/110) of turkey samples. Pork and beef samples were negative for Campylobacter. Campylobacter jejuni was the most common (94.6%) spp. found followed by C. coli (5.4%). Resistance to tetracycline was found in 48.1% of isolates, followed by resistance to ciprofloxacin (5.5%), nalidixic acid (5.5%), azithromycin (1.78%), and erythromycin (1.78%). All isolates were susceptible to clindamycin, florfenicol, gentamicin and telithromycin. Tetracycline resistance was attributable to the presence of the tetO gene. CGF analysis showed that Campylobacter isolated from poultry meat in this study were genetically related to clinical isolates recovered from human infections and to those isolated from animals and the environment.

The EpiQuant Framework for Computing Epidemiological Concordance of Microbial Subtyping Data.

A fundamental assumption in the use and interpretation of microbial subtyping results for public health investigations is that isolates that appear to be related based on molecular subtyping data are expected to share commonalities with respect to their origin, history, and distribution. Critically, there is currently no approach for systematically assessing the underlying epidemiology of subtyping results. Our aim was to develop a method for directly quantifying the similarity between bacterial isolates using basic sampling metadata and to develop a framework for computing the epidemiological concordance of microbial typing results. We have developed an analytical model that summarizes the similarity of bacterial isolates using basic parameters typically provided in sampling records, using a novel framework (EpiQuant) developed in the R environment for statistical computing. We have applied the EpiQuant framework to a data set comprising 654 isolates of the enteric pathogen Campylobacter jejuni from Canadian surveillance data in order to examine the epidemiological concordance of clusters obtained by using two leading C. jejuni subtyping methods. The EpiQuant framework can be used to directly quantify the similarity of bacterial isolates based on basic sample metadata. These results can then be used to assess the concordance between microbial epidemiological and molecular data, facilitating the objective assessment of subtyping method performance and paving the way for the improved application of molecular subtyping data in investigations of infectious disease.

Molecular epidemiology of Campylobacter jejuni human and chicken isolates from two health units.

A study was conducted over a 2-year period in the Perth District and Wellington-Dufferin-Guelph health units in Ontario, with an objective of using comparative genomic fingerprinting (CGF) with a 40-gene assay (CGF40) to investigate the association between human cases of campylobacteriosis and spatially and temporally related Campylobacter isolates from retail chicken. CGF results were available for isolates from 115 human cases and 718 retail chicken samples. These data were combined with CGF results from a large reference database of Campylobacter isolates. Isolates were categorized into types based on >90% CGF40 fingerprint similarity (CGF-90%). CGF-90% types were categorized as chicken associated (CA90) when the proportion of animal isolates in the given type that originated from chicken was at least 80% and was statistically significant. Risk factor data were collected from cases by questionnaire. Urban cases were significantly more likely than rural cases to be CA90 and there were significantly fewer CA90 cases in the second year of the study. Due to the population distribution in Canada and most industrialized countries, the majority of campylobacteriosis cases are urban dwellers. Therefore, the association between urban cases and chicken-associated types of Campylobacter emphasizes the importance of educational and food safety efforts to reduce the impact of Campylobacter from retail chicken on public health. Sources other than chicken may be more important for rural dwellers.

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.

A framework for assessing the concordance of molecular typing methods and the true strain phylogeny of Campylobacter jejuni and C. coli using draft genome sequence data.

Tracking of sources of sporadic cases of campylobacteriosis remains challenging, as commonly used molecular typing methods have limited ability to unambiguously link genetically related strains. Genomics has become increasingly prominent in the public health response to enteric pathogens as methods enable characterization of pathogens at an unprecedented level of resolution. However, the cost of sequencing and expertise required for bioinformatic analyses remains prohibitive, and these comprehensive analyses are limited to a few priority strains. Although several molecular typing methods are currently widely used for epidemiological analysis of campylobacters, it is not clear how accurately these methods reflect true strain relationships. To address this, we have developed a framework and associated computational tools to rapidly analyze draft genome sequence data for the assessment of molecular typing methods against a "gold standard" based on the phylogenetic analysis of highly conserved core (HCC) genes with high sequence quality. We analyzed 104 publicly available whole genome sequences (WGS) of C. jejuni and C. coli. In addition to in silico determination of multi-locus sequence typing (MLST), flaA, and porA type, as well as comparative genomic fingerprinting (CGF) type, we inferred a "reference" phylogeny based on 389 HCC genes. Molecular typing data were compared to the reference phylogeny for concordance using the adjusted Wallace coefficient (AWC) with confidence intervals. Although MLST targets the sequence variability in core genes and CGF targets insertions/deletions of accessory genes, both methods are based on multi-locus analysis and provided better estimates of true phylogeny than methods based on single loci (porA, flaA). A more comprehensive WGS dataset including additional genetically related strains, both epidemiologically linked and unlinked, will be necessary to more comprehensively assess the performance of subtyping methods for outbreak investigations and surveillance activities. Analyses of the strengths and weaknesses of widely used typing methodologies in inferring true strain relationships will provide guidance in the interpretation of this data for epidemiological purposes.

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.