22Motivation: The recent widespread application of whole-genome sequencing (WGS) for microbial 23

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.

23Whole-genome sequencing (WGS) of microbial pathogens has become an essential 24 part of modern epidemiological investigations. Although WGS data can be analyzed 25 using a number of different approaches, such as traditional phylogenetic methods, a 26 critical requirement for global systems for pathogen surveillance is the development of 27 approaches for transforming sequence data into WGS-based subtypes, which creates a 28 nomenclature that describes their higher-order relationships to one another. To this end, 29 subtype similarity thresholds are needed to define clusters of subtypes representing 30 lineages of interest. WGS-based subtyping presents a challenge since both the addition 31 of novel genome sequences and small adjustments in similarity thresholds can have a 32 dramatic impact on cluster composition and stability. We present the Neighbourhood 33 Adjusted Wallace Coefficient (nAWC), a method for evaluating cluster stability based on 34 computing cluster concordance between neighbouring similarity thresholds. The nAWC 35 can be used to identify areas in in which distance thresholds produce robust clusters. 36Using datasets from Salmonella enterica and Campylobacter jejuni, representing 37 strongly and weakly clonal bacterial species respectively, we show that clusters 38 generated using such thresholds are both stable and reflect basic units in their overall 39 population structure. Our results suggest that the nAWC could be useful for defining 40 robust clusters compatible with nomenclatures for global WGS-based surveillance 41 networks, which require stable clusters to be defined that both harness the 42 discriminatory power of WGS data while allowing for long-term tracking of strains of 43 interest. 44 45 46 Molecular subtyping of microbial pathogens has become integral to modern 47 epidemiological investigations of infectious disease, playing a key role in public health 48 efforts worldwide (Foxman and Riley, 2001). Subtyping is used to discriminate isolates 49 from the same species and to identify clusters of similar isolates based on their genetic 50 relatedness. In the context of molecular surveillance networks such as PulseNet, this 51 information has made it possible to confirm possible disease outbreaks, to identify 52 outbreak cases that may not have been possible to distinguish among a background of 53 sporadic cases, and to further link cases to potential sources of infection even in the 54 absence of clear epidemiological linkage (Boxrud et al., 2010; Tauxe et al., 2010). 55 Over the last several decades, pathogen subtyping approaches have evolved from 56 methods based on assessing phenotypic characteristics to methods based on genetic 57 variation detectable through gel electrophoresis and sequencing. More recently, 58 advances in a new generation of high-throughput sequencing methods has led to the 59 increasing adoption of whole-genome sequencing (WGS) as a primary method for the 60 characterization of microbial pathogens (Gilmour et al., 2013; Köser et al., 2012). As 61 tradi...

Antimicrobial resistance was evaluated in Campylobacter jejuni isolated from 1,291 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 mutation 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 FQR C. jejuni subtypes (82.6%) were endemic in Canada, primarily linked to cattle and chicken reservoirs; 18.4% of FQR 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.

The decreased costs of genome sequencing have increased capability to apply wholegenome sequence on epidemiological surveillance of zoonotic Campylobacter jejuni.However, knowledge about how genetically similar epidemiologically linked isolates can be is vital for correct application of this methodology. To address this issue in C. jejuni we investigated the spatial and temporal signals in the genomes of a major clonal complex and generalist lineage, ST-45 CC, by exploiting the population structure and genealogy and applying genome-wide association analysis of 340 isolates from across Europe collected over a wide time-range. The occurrence and strength of the geographical signal varied between sublineages and followed the clonal frame when present, while no evidence of a temporal signal was found. Certain sublineages of ST-45 CC formed discrete and genetically isolated clades to which geography and time had left only negligible traces in the genomes. We hypothesize that these ST-45 CC clades form globally expanded monomorphic clones possibly spread across Europe by migratory birds. In addition, we observed an incongruence between the genealogy of the strains and MLST typing, thereby challenging the existing clonal complex definition and use of a common MLST-based nomenclature for the ST-45 CC of C. jejuni.