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.

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.

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.

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.

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.

Persistence and Sexual Transmission of Filoviruses.

There is an increasing frequency of reports regarding the persistence of the Ebola virus (EBOV) in Ebola virus disease (EVD) survivors. During the 2014⁻2016 West African EVD epidemic, sporadic transmission events resulted in the initiation of new chains of human-to-human transmission. Multiple reports strongly suggest that these re-emergences were linked to persistent EBOV infections and included sexual transmission from EVD survivors. Asymptomatic infection and long-term viral persistence in EVD survivors could result in incidental introductions of the Ebola virus in new geographic regions and raise important national and local public health concerns. Alarmingly, although the persistence of filoviruses and their potential for sexual transmission have been documented since the emergence of such viruses in 1967, there is limited knowledge regarding the events that result in filovirus transmission to, and persistence within, the male reproductive tract. Asymptomatic infection and long-term viral persistence in male EVD survivors could lead to incidental transfer of EBOV to new geographic regions, thereby generating widespread outbreaks that constitute a significant threat to national and global public health. Here, we review filovirus testicular persistence and discuss the current state of knowledge regarding the rates of persistence in male survivors, and mechanisms underlying reproductive tract localization and sexual transmission.

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.

Efficacy of wastewater treatment on Arcobacter butzleri density and strain diversity.

Arcobacter butzleri is a suspected waterborne enteric pathogen that is ubiquitous in the environment, but the degree to which wastewater treatment prevents entry of A. butzleri into environmental waters and the risks posed are not well established. Untreated and treated wastewater samples (n = 260) were collected weekly from the Lethbridge and Fort Macleod wastewater treatment facilities (the two major municipal inputs in southwestern Alberta, Canada) from May 2008 to April 2009. Untreated wastewaters contained high densities of A. butzleri and fecal coliform indicators, and densities at Lethbridge were typically higher than at Fort Macleod. Data indicated that A. butzleri and fecal coliform densities in wastewater were greatest in autumn and lowest in winter. Mechanical and biological treatment of wastewaters reduced but did not eliminate fecal coliform indicators or A. butzleri. At Lethbridge, UVB irradiation of mechanically and biologically treated wastewater further reduced densities of fecal coliform indicators. There was high A. butzleri genotype diversity in all sample sources, and survival during treatment was not strain-dependent. No genotype was dominant in any sample source, but 8.9% of genotypes were recurrent over time, and 4.4% of genotypes were detected at both wastewater treatment facilities. The current study demonstrates that viable A. butzleri are able to survive wastewater treatment, including UVB irradiation, which may lead to increased density and genetic diversity of this suspected pathogen in environmental waters via wastewater effluent discharge.