Analysis of Campylobacter jejuni Subtype Distribution in the Chicken Broiler Production Continuum: a Longitudinal Examination To Identify Primary Contamination Points.

Significant knowledge gaps exist in our understanding of Campylobacter jejuni contamination of the poultry production continuum. Microbiological surveillance and genotypic characterization were undertaken on C. jejuni isolates longitudinally recovered from three poultry farms (weekly samples), the abattoir at which birds were processed, and at retail over a 542-day period in southwestern Alberta, Canada, as a model location. Subtypes were compared to concurrent isolates from diarrheic humans living in the study region. Barn outbreaks in broiler chickens occurred infrequently. Subtypes from colonized birds, including clinically relevant subtypes of C. jejuni, were recovered within barns and from subsequent production stages. When C. jejuni was detected in barns, most birds rapidly became colonized by a limited number of subtypes late in the cycle. However, the diversity of subtypes recovered from birds in the abattoir increased substantially. Moreover, birds deemed free of C. jejuni upon exit from the barn became contaminated within the abattoir environment, and a high prevalence of meat at retail was contaminated with C. jejuni, including subtypes that had not been previously observed in the barns. The observed increase in prevalence of contamination and diversity of C. jejuni subtypes along the chicken production continuum indicates that birds from a relatively small number of barns contaminate transport trucks and the abattoir with C. jejuni strains, which are collectively transferred to poultry within the abattoir and conveyed to and persist on retail products. We conclude that the abattoir was the primary contamination point of poultry by C. jejuni but only a subset of subtypes were a high risk to human beings.IMPORTANCE The longitudinal examination of Campylobacter jejuni subtypes throughout the broiler production continuum is required to determine transmission mechanisms and to identify potential reservoirs and the foodborne risk posed. We showed that a limited number of C. jejuni subtypes are responsible for infrequent outbreaks in broilers within production barns and that colonized birds from a small number of farms are introduced into the abattoir where a high prevalence of carcasses are subsequently contaminated with a diversity of subtypes, which are transferred onto poultry in retail settings. However, only a subset of strains on poultry was determined to be clinically relevant. The study findings showed that resolving C. jejuni at the subtype level is important to ascertain health risks, and the knowledge obtained in the study provides information to mitigate clinically relevant subtypes to reduce the burden of campylobacteriosis.

Rates of fluoroquinolone resistance in domestically acquired Campylobacter jejuni are increasing in people living within a model study location in Canada.

Antimicrobial resistance was evaluated in Campylobacter jejuni isolated from 1291 diarrheic people over a 15-year period (2004-2018) in southwestern Alberta, a model location in Canada with a high rate of campylobacteriosis. The prevalence of resistance to chloramphenicol, clindamycin, erythromycin, and gentamicin was low during the examination period (≤4.8%). Resistance to tetracycline remained consistently high (41.6%-65.1%), and resistance was primarily conferred by plasmid-borne tetO (96.2%). Resistance rates to ciprofloxacin and nalidixic acid increased substantially over the examination period, with a maximal fluoroquinolone resistance (FQR) prevalence of 28.9% in 2016. The majority of C. jejuni isolates resistant to ciprofloxacin (93.9%) contained a C257T single nucleotide polymorphism within the gyrA chromosomal gene. Follow up with infected people indicated that the observed increase in FQR was primarily due to domestically acquired infections. Moreover, the majority of FQ-resistant C. jejuni subtypes (82.6%) were endemic in Canada, primarily linked to cattle and chicken reservoirs; 18.4% of FQ-resistant isolates were assigned to three subtypes, predominantly associated with cattle. Study findings indicate the need to prioritize FQR monitoring in C. jejuni infections in Canada and to elucidate the dynamics of the emergence and transmission of resistant C. jejuni strains within and from cattle and chicken reservoirs.

Clinically Relevant Campylobacter jejuni Subtypes Are Readily Found and Transmitted within the Cattle Production Continuum but Present a Limited Foodborne Risk.

Increasing evidence exists for the role that cattle play in the epidemiology of campylobacteriosis. In this study, the prevalence and distribution of Campylobacter jejuni were longitudinally examined at the subspecies level in the beef cattle production continuum. Animals were subdivided into two groups: those that were not administered antibiotics and those that were administered the antimicrobial growth promoter chlortetracycline and sulfamethazine (AS700). Samples were longitudinally collected throughout the confined feeding operation (CFO) period and during the slaughter process, and C. jejuni was isolated and genotyped to assess subtype richness and to elucidate transmission dynamics from farm to fork. The bacterium was frequently isolated from cattle, and the bacterial densities shed in feces increased over the CFO period. Campylobacter jejuni was also isolated from digesta, hides, the abattoir environment, and carcasses. The administration of AS700 did not conspicuously reduce the C. jejuni densities in feces or within the intestine but significantly reduced the bacterial densities and the diversity of subtypes on abattoir samples. All cattle carried multiple subtypes, including clinically relevant subtypes known to represent a risk to human health. Instances of intra-animal longitudinal transmission were observed. Although clinically relevant subtypes were transmitted to carcasses via direct contact and aerosols, the bacterium could not be isolated nor could its DNA be detected in ground beef regardless of treatment. Although the evidence indicated that beef cattle represent a significant reservoir for C. jejuni, including high-risk subtypes strongly associated with the bovine host, they do not appear to represent a significant risk for direct foodborne transmission. This implicates alternate routes of human transmission.IMPORTANCE Limited information is available on the transmission of Campylobacter jejuni subtypes in the beef production continuum and the foodborne risk posed to humans. Cattle were colonized by diverse subtypes of C. jejuni, and the densities of the bacterium shed in feces increased during the confined feeding period. Campylobacter jejuni was readily associated with the digesta, feces, and hides of cattle entering the abattoir, as well as the local environment. Moreover, C. jejuni cells were deposited on carcasses via direct contact and aerosols, but the bacterium was not detected in the ground beef generated from contaminated carcasses. We conclude that C. jejuni bacterial cells associated with beef cattle do not represent a significant risk through food consumption and suggest that clinically relevant subtypes are transmitted through alternate routes of exposure.

Enhanced microbiological surveillance reveals that temporal case clusters contribute to the high rates of campylobacteriosis in a model agroecosystem.

Infections by pathogenic Campylobacter species were determined in diarrheic (n = 2,217) and non-diarrheic control (n = 104) people in Southwestern Alberta (SWA), Canada over a 1-year period using specialized and conventional isolation, and direct PCR. Overall, 9.9% of diarrheic individuals were positive for C. jejuni (9.1%), C. upsaliensis (0.6%), and C. coli (0.5%). No C. lari was detected. Four diarrheic individuals were co-infected with C. jejuni and C. coli, and four different individuals were co-infected with C. jejuni and C. upsaliensis. Two control individuals were positive for C. jejuni. Approximately 50% of stools containing C. jejuni and/or C. coli were deemed negative by conventional isolation. Direct PCR for C. jejuni was less effective than culture-based detection. Most C. jejuni infections occurred in people living in the urban centers, but the prevalence of the bacterium was lower in females than males living in urban locations, and both males and females living in rural locations. Although C. jejuni was detected throughout the year, a trend for higher infection rates was observed in the late spring to early fall with a peak in August. Forty-six C. jejuni subtype clusters were identified, including 44 temporal case clusters attributed to 28 subtype groupings. The majority of infections (70.3%) were linked to subtypes associated with beef cattle. We conclude that many occurrences of pathogenic Campylobacter species were not detected by the conventional laboratory methodology, and temporal case clusters of C. jejuni subtypes associated with cattle contribute to the high rates of campylobacteriosis in SWA.

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.

Subtype-Specific Selection for Resistance to Fluoroquinolones but Not to Tetracyclines Is Evident in Campylobacter jejuni Isolates from Beef Cattle in Confined Feeding Operations in Southern Alberta, Canada.

Campylobacter jejuni was longitudinally isolated from beef cattle housed in four confined feeding operations (CFOs) in Southern Alberta, Canada, over 18 months. All of the cattle were administered a variety of antimicrobial agents (AMAs) nontherapeutically and metaphylactically during their time in the CFOs. In total, 7,966 C. jejuni isolates were recovered from cattle. More animals were colonized by the bacterium after >60 days in the CFO (interim) than were individuals upon entry at the CFO (arrival). Subtyping and resistance to seven AMAs were determined for 1,832 (23.0%) and 1,648 (20.7%) isolates, respectively. Increases in the proportion of isolates resistant to tetracycline were observed at all four CFOs between sample times and to ciprofloxacin and nalidixic acid at one or more CFOs. The vast majority of isolates resistant to tetracycline carried tetO, whereas ciprofloxacin resistance was predominantly attributed to mutations in the gyrA gene. Although considerable diversity was observed, a majority of C. jejuni isolates belonged to one of five predominant subtype clusters. There was no difference in subtype diversity by CFO, but the population structure differed between sample times. Selection for resistance to ciprofloxacin and nalidixic acid was subtype dependent, whereas selection for resistance to tetracycline was not. The findings indicate that a proportion of cattle entering CFOs carry resistant C. jejuni subtypes, and the characteristics of beef cattle CFOs facilitate transmission/proliferation of diverse subtypes, including those resistant to AMAs, which coupled with the densities of CFOs likely contribute to the high rates of cattle-associated campylobacteriosis in Southern Alberta.IMPORTANCE A small proportion of cattle entering a CFO carry Campylobacter jejuni, including subtypes resistant to AMAs. The large numbers of cattle arriving from diverse locations at the CFOs and intermingling within the CFOs over time, coupled with the high-density housing of animals, the high rates of transmission of C. jejuni subtypes among animals, and the extensive use of AMAs merge to create an ideal situation where the proliferation of diverse antimicrobial-resistant C. jejuni subtypes is facilitated. Considering that Southern Alberta reports high rates of campylobacteriosis in the human population and that many of these clinical cases are due to C. jejuni subtypes associated with cattle, it is likely that the characteristics of beef cattle CFOs favor the propagation of clinically relevant C. jejuni subtypes, including those resistant to medically important AMAs, which constitute a risk to human health.

Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni.

Enrofloxacin is registered for therapeutic use in beef cattle to treat bovine respiratory disease in Canada. A murine model was used to experimentally examine the impact of therapeutic administration of enrofloxacin on fluoroquinolone resistance development in Campylobacter jejuni. Administration of enrofloxacin to mice via subcutaneous injection or per os routes resulted in equivalent levels of bioactive enrofloxacin within the intestine, but bioactivity was short-lived (<48 h after cessation). Enrofloxacin administration did not affect densities of total bacteria, Firmicutes, or Bacteroidetes in digesta and had modest impacts on densities of Enterobacteriaceae. All mice inoculated with C. jejuni NCTC 11168 became persistently colonized by the bacterium. Enrofloxacin reduced C. jejuni cell densities within the cecal and colonic digesta for all treatments, and densities shed in feces as a function of antibiotic duration. None of the C. jejuni isolates recovered from mice after administration of enrofloxacin (n = 260) developed resistance to ciprofloxacin regardless of method or duration of administration. Furthermore, only modest shifts in the minimum inhibitory concentration of the isolates by treatment were noted. The study findings indicate that the risk posed by short-term subcutaneous administration of enrofloxacin for the development of fluoroquinolone resistance in mammals is low.

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.

Local genes for local bacteria: Evidence of allopatry in the genomes of transatlantic Campylobacter populations.

The genetic structure of bacterial populations can be related to geographical locations of isolation. In some species, there is a strong correlation between geographical distance and genetic distance, which can be caused by different evolutionary mechanisms. Patterns of ancient admixture in Helicobacter pylori can be reconstructed in concordance with past human migration, whereas in Mycobacterium tuberculosis it is the lack of recombination that causes allopatric clusters. In Campylobacter, analyses of genomic data and molecular typing have been successful in determining the reservoir host species, but not geographical origin. We investigated biogeographical variation in highly recombining genes to determine the extent of clustering between genomes from geographically distinct Campylobacter populations. Whole-genome sequences from 294 Campylobacter isolates from North America and the UK were analysed. Isolates from within the same country shared more recently recombined DNA than isolates from different countries. Using 15 UK/American closely matched pairs of isolates that shared ancestors, we identify regions that have frequently and recently recombined to test their correlation with geographical origin. The seven genes that demonstrated the greatest clustering by geography were used in an attribution model to infer geographical origin which was tested using a further 383 UK clinical isolates to detect signatures of recent foreign travel. Patient records indicated that in 46 cases, travel abroad had occurred <2 weeks prior to sampling, and genomic analysis identified that 34 (74%) of these isolates were of a non-UK origin. Identification of biogeographical markers in Campylobacter genomes will contribute to improved source attribution of clinical Campylobacter infection and inform intervention strategies to reduce campylobacteriosis.

Prevalence and diversity of waterborne Arcobacter butzleri in southwestern Alberta, Canada.

Arcobacter butzleri is a potential enteric pathogen to human beings, but its reservoirs and modes of transmission are largely unverified. Microbiological and molecular detection and subtyping techniques can facilitate surveillance of A. butzleri in hosts and environmental reservoirs. We isolated A. butzleri from 173 surface water samples (25.6%) and 81 treated wastewater samples (77.9%) collected in southwestern Alberta over a 1-year period. Arcobacter butzleri isolates (n = 500) were genotyped and compared to determine diversity of A. butzleri in southwestern Alberta. Culture methods affected the frequency of detection and genotype diversity of A. butzleri, and isolation comprehensiveness was different for surface waters and treated wastewaters. Detection of A. butzleri in the Oldman River Watershed corresponded with season, river flow rates, and fecal coliform densities. Arcobacter butzleri was detected most frequently in treated wastewater, in the Oldman River downstream from treated wastewater outfalls, and in tributaries near areas of intensive confined feeding operations. All sample sources possessed high genotype diversity, and A. butzleri isolates from treated wastewaters were genetically similar to isolates from the Oldman River downriver from treated wastewater outfall sites. In southwestern Alberta, municipal and agricultural activities contribute to the density and genotype diversity of A. butzleri in surface waters.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

BACKGROUND: Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. RESULTS: Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. CONCLUSIONS: The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Comparative Detection and Quantification of Arcobacter butzleri in Stools from Diarrheic and Nondiarrheic People in Southwestern Alberta, Canada.

Arcobacter butzleri has been linked to enteric disease in humans, but its pathogenicity and epidemiology remain poorly understood. The lack of suitable detection methods is a major limitation. Using comparative genome analysis, we developed PCR primers for direct detection and quantification ofA. butzleri DNA in microbiologically complex matrices. These primers, along with existing molecular and culture-based methods, were used to detectA. butzleri and enteric pathogens in stools of diarrheic and nondiarrheic people (n= 1,596) living in southwestern Alberta, Canada, from May to November 2008. In addition, quantitative PCR was used to compare A. butzleridensities in diarrheic and nondiarrheic stools.Arcobacter butzleriwas detected more often by PCR (59.6%) than by isolation methods (0.8%). Comparison by PCR-based detection found no difference in the prevalence ofA. butzleri between diarrheic (56.7%) and nondiarrheic (45.5%) individuals. Rates of detection in diarrheic stools peaked in June (71.1%) and October (68.7%), but there was no statistically significant correlation between the presence ofA. butzleri and patient age, sex, or place of habitation. Densities ofA. butzleriDNA in diarrheic stools (1.6 ± 0.59 log10 copies mg(-1)) were higher (P= 0.007) than in nondiarrheic stools (1.3 ± 0.63 log10copies mg(-1)). Of the 892 diarrheic samples that were positive for A. butzleri, 74.1% were not positive for other bacterial and/or viral pathogens. The current study supports previous work suggesting that A. butzleri pathogenicity is strain specific and/or dependent on other factors, such as the level of host resistance.

SuperPhy: predictive genomics for the bacterial pathogen Escherichia coli.

BACKGROUND: Predictive genomics is the translation of raw genome sequence data into a phenotypic assessment of the organism. For bacterial pathogens, these phenotypes can range from environmental survivability, to the severity of human disease. Significant progress has been made in the development of generic tools for genomic analyses that are broadly applicable to all microorganisms; however, a fundamental missing component is the ability to analyze genomic data in the context of organism-specific phenotypic knowledge, which has been accumulated from decades of research and can provide a meaningful interpretation of genome sequence data. RESULTS: In this study, we present SuperPhy, an online predictive genomics platform ( http://lfz.corefacility.ca/superphy/ ) for Escherichia coli. The platform integrates the analytical tools and genome sequence data for all publicly available E. coli genomes and facilitates the upload of new genome sequences from users under public or private settings. SuperPhy provides real-time analyses of thousands of genome sequences with results that are understandable and useful to a wide community, including those in the fields of clinical medicine, epidemiology, ecology, and evolution. SuperPhy includes identification of: 1) virulence and antimicrobial resistance determinants 2) statistical associations between genotypes, biomarkers, geospatial distribution, host, source, and phylogenetic clade; 3) the identification of biomarkers for groups of genomes on the based presence/absence of specific genomic regions and single-nucleotide polymorphisms and 4) in silico Shiga-toxin subtype. CONCLUSIONS: SuperPhy is a predictive genomics platform that attempts to provide an essential link between the vast amounts of genome information currently being generated and phenotypic knowledge in an organism-specific context.

Extensive characterization of Campylobacter jejuni chicken isolates to uncover genes involved in the ability to compete for gut colonization.

BACKGROUND: Campylobacter jejuni is responsible for human foodborne enteritis. This bacterium is a remarkable colonizer of the chicken gut, with some strains outcompeting others for colonization. To better understand this phenomenon, the objective of this study was to extensively characterize the phenotypic performance of C. jejuni chicken strains and associate their gut colonizing ability with specific genes. RESULTS: C. jejuni isolates (n = 45) previously analyzed for the presence of chicken colonization associated genes were further characterized for phenotypic properties influencing colonization: autoagglutination and chemotaxis as well as adhesion to and invasion of primary chicken caecal cells. This allowed strains to be ranked according to their in vitro performance. After their in vitro capacity to outcompete was demonstrated in vivo, strains were then typed by comparative genomic fingerprinting (CGF). In vitro phenotypical properties displayed a linear variability among the tested strains. Strains possessing higher scores for phenotypical properties were able to outcompete others during chicken colonization trials. When the gene content of strains was compared, some were associated with different phenotypical scores and thus with different outcompeting capacities. Use of CGF profiles showed an extensive genetic variability among the studied strains and suggested that the outcompeting capacity is not predictable by CGF profile. CONCLUSION: This study revealed a wide array of phenotypes present in C. jejuni strains, even though they were all recovered from chicken caecum. Each strain was classified according to its in vitro competitive potential and its capacity to compete for chicken gut colonization was associated with specific genes. This study also exposed the disparity existing between genetic typing and phenotypical behavior of C. jejuni strains.

Development of a comparative genomic fingerprinting assay for rapid and high resolution genotyping of Arcobacter butzleri.

BACKGROUND: Molecular typing methods are critical for epidemiological investigations, facilitating disease outbreak detection and source identification. Study of the epidemiology of the emerging human pathogen Arcobacter butzleri is currently hampered by the lack of a subtyping method that is easily deployable in the context of routine epidemiological surveillance. In this study we describe a comparative genomic fingerprinting (CGF) method for high-resolution and high-throughput subtyping of A. butzleri. Comparative analysis of the genome sequences of eleven A. butzleri strains, including eight strains newly sequenced as part of this project, was employed to identify accessory genes suitable for generating unique genetic fingerprints for high-resolution subtyping based on gene presence or absence within a strain. RESULTS: A set of eighty-three accessory genes was used to examine the population structure of a dataset comprised of isolates from various sources, including human and non-human animals, sewage, and river water (n=156). A streamlined assay (CGF40) based on a subset of 40 genes was subsequently developed through marker optimization. High levels of profile diversity (121 distinct profiles) were observed among the 156 isolates in the dataset, and a high Simpson's Index of Diversity (ID) observed (ID > 0.969) indicate that the CGF40 assay possesses high discriminatory power. At the same time, our observation that 115 isolates in this dataset could be assigned to 29 clades with a profile similarity of 90% or greater indicates that the method can be used to identify clades comprised of genetically similar isolates. CONCLUSIONS: The CGF40 assay described herein combines high resolution and repeatability with high throughput for the rapid characterization of A. butzleri strains. This assay will facilitate the study of the population structure and epidemiology of A. butzleri.

Commensal Escherichia coli Strains of Bovine Origin Competitively Mitigated Escherichia coli O157:H7 in a Gnotobiotic Murine Intestinal Colonization Model with or without Physiological Stress.

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7. Currently, there are no effective methods of eliminating this important zoonotic pathogen from cattle, and colonization resistance in relation to EHEC O157:H7 in cattle is poorly understood. We developed a gnotobiotic EHEC O157:H7 murine model to examine aspects of the cattle pathogen-microbiota interaction, and to investigate competitive suppression of EHEC O157:H7 by 18 phylogenetically distinct commensal E. coli strains of bovine origin. As stress has been suggested to influence enteric colonization by EHEC O157:H7 in cattle, corticosterone administration (±) to incite a physiological stress response was included as an experimental variable. Colonization of the intestinal tract (IT) of mice by the bovine EHEC O157:H7 strain, FRIK-2001, mimicked characteristics of bovine IT colonization. In this regard, FRIK-2001 successfully colonized the IT and temporally incited minimal impacts on the host relative to other EHEC O157:H7 strains, including on the renal metabolome. The presence of the commensal E. coli strains decreased EHEC O157:H7 densities in the cecum, proximal colon, and distal colon. Moreover, histopathologic changes and inflammation markers were reduced in the distal colon of mice inoculated with commensal E. coli strains (both propagated separately and communally). Although stress induction affected the behavior of mice, it did not influence EHEC O157:H7 densities or disease. These findings support the use of a gnotobiotic murine model of enteric bovine EHEC O157:H7 colonization to better understand pathogen-host-microbiota interactions toward the development of effective on-farm mitigations for EHEC O157:H7 in cattle, including the identification of bacteria capable of competitively colonizing the IT.

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.

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.

Molecular Epidemiological Evidence Implicates Cattle as a Primary Reservoir of Campylobacter jejuni Infecting People via Contaminated Chickens.

The study aimed to determine the relative contribution of cattle to the burden of illness in a model agroecosystem with high rates of human campylobacteriosis (≥ 115 cases/100 K), and high densities of cattle, including large numbers of cattle housed in confined feeding operations (i.e., in southwestern Alberta, Canada). To accomplish this, a large-scale molecular epidemiological analysis of Campylobacter jejuni circulating within the study location was completed. In excess of 8000 isolates of C. jejuni from people (n = 2548 isolates), chickens (n = 1849 isolates), cattle (n = 2921 isolates), and water (n = 771 isolates) were subtyped. In contrast to previous studies, the source attribution estimates of clinical cases attributable to cattle vastly exceeded those attributed to chicken (i.e., three- to six-fold). Moreover, cattle were often colonized by C. jejuni (51%) and shed the bacterium in their feces. A large proportion of study isolates were found in subtypes primarily associated with cattle (46%), including subtypes infecting people and those associated with chickens (19%). The implication of cattle as a primary amplifying reservoir of C. jejuni subtypes in circulation in the study location is supported by the strong cattle association with subtypes that were found in chickens and in people, a lack of evidence indicating the foodborne transmission of C. jejuni from beef and dairy, and the large number of cattle and the substantial quantities of untreated manure containing C. jejuni cells. Importantly, the evidence implicated cattle as a source of C. jejuni infecting people through a transmission pathway from cattle to people via the consumption of chicken. This has implications for reducing the burden of campylobacteriosis in the study location and elsewhere.

Combining analytical epidemiology and genomic surveillance to identify risk factors associated with the spread of antimicrobial resistance in Salmonella enterica subsp. enterica serovar Heidelberg.

Antimicrobial resistance (AMR) has become a critical threat to public health worldwide. The use of antimicrobials in food and livestock agriculture, including the production of poultry, is thought to contribute to the dissemination of antibiotic resistant bacteria (ARB) and the genes and plasmids that confer the resistant phenotype (ARG). However, the relative contribution of each of these processes to the emergence of resistant pathogens in poultry production and their potential role in the transmission of resistant pathogens in human infections, requires a deeper understanding of the dynamics of ARB and ARG in food production and the factors involved in the increased risk of transmission.