Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Background: Mycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.<p></p> Results: Phylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.<p></p> Conclusions: The presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.<p></p&gt

Inferring source attribution from a multi-year multi-source dataset of Salmonella in Minnesota

Salmonella enterica is a global health concern because of its widespread association with foodborne illness. Bayesian models have been developed to attribute the burden of human salmonellosis to specific sources with the ultimate objective of prioritizing intervention strategies. Important considerations of source attribution models include the evaluation of the quality of input data, assessment of whether attribution results logically reflect the data trends and identification of patterns within the data that might explain the detailed contribution of different sources to the disease burden. Here, more than 12,000 non-typhoidal Salmonella isolates from human, bovine, porcine, chicken and turkey sources that originated in Minnesota were analysed. A modified Bayesian source attribution model (available in a dedicated R package), accounting for non-sampled sources of infection, attributed 4,672 human cases to sources assessed here. Most (60%) cases were attributed to chicken, although there was a spike in cases attributed to a non-sampled source in the second half of the study period. Molecular epidemiological analysis methods were used to supplement risk modelling, and a visual attribution application was developed to facilitate data exploration and comprehension of the large multiyear data set assessed here. A large amount of within-source diversity and low similarity between sources was observed, and visual exploration of data provided clues into variations driving the attribution modelling results. Results from this pillared approach provided first attribution estimates for Salmonella in Minnesota and offer an understanding of current data gaps as well as key pathogen population features, such as serotype frequency, similarity and diversity across the sources. Results here will be used to inform policy and management strategies ultimately intended to prevent and control Salmonella infection in the state

Validation of a screening method for the detection of colistin-resistant \u3ci\u3eE. coli\u3c/i\u3e containing mcr-1 in feral swine feces

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 μg/mL) and vancomycin (8 μg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1–9.99 CFU/g (n=340), 10–49.99 CFU/g (n=170), 50–99 CFU/g (n=255), 100–149 CFU/g (n=60), and 200–2200 CFU/g (n=175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment

Gulls as Sources of Environmental Contamination by Colistin-resistant Bacteria

In 2015, the mcr-1 gene was discovered in Escherichia coli in domestic swine in China that conferred resistance to colistin, an antibiotic of last resort used in treating multi-drug resistant bacterial infections in humans. Since then, mcr-1 was found in other human and animal populations, including wild gulls. Because gulls could disseminate the mcr-1 gene, we conducted an experiment to assess whether gulls are readily colonized with mcr-1 positive E. coli, their shedding patterns, transmission among conspecifics, and environmental deposition. Shedding of mcr-1 E. coli by small gull flocks followed a lognormal curve and gulls shed one strain \u3e101 log10 CFU/g in their feces for 16.4 days, which persisted in the environment for 29.3 days. Because gulls are mobile and can shed antimicrobial-resistant bacteria for extended periods, gulls may facilitate transmission of mcr-1 positive E. coli to humans and livestock through fecal contamination of water, public areas and agricultural operations

Genome-wide diversity and phylogeography of Mycobacterium avium subsp. paratuberculosis in Canadian dairy cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease (JD) in ruminants. The control of JD in the dairy industry is challenging, but can be improved with a better understanding of the diversity and distribution of MAP subtypes. Previously established molecular typing techniques used to differentiate MAP have not been sufficiently discriminatory and/or reliable to accurately assess the population structure. In this study, the genetic diversity of 182 MAP isolates representing all Canadian provinces was compared to the known global diversity, using single nucleotide polymorphisms identified through whole genome sequencing. MAP isolates from Canada represented a subset of the known global diversity, as there were global isolates intermingled with Canadian isolates, as well as multiple global subtypes that were not found in Canada. One Type III and six “Bison type” isolates were found in Canada as well as one Type II subtype that represented 86% of all Canadian isolates. Rarefaction estimated larger subtype richness in Québec than in other Canadian provinces using a strict definition of MAP subtypes and lower subtype richness in the Atlantic region using a relaxed definition. Significant phylogeographic clustering was observed at the inter-provincial but not at the intra-provincial level, although most major clades were found in all provinces. The large number of shared subtypes among provinces suggests that cattle movement is a major driver of MAP transmission at the herd level, which is further supported by the lack of spatial clustering on an intra-provincial scale

Molecular ecology of Listeria spp., Salmonella, Escherichia coli O157:H7, and non-O157 Shiga toxin producing E. coli in northern Colorado wilderness areas

2011 Spring.Includes bibliographical references.Foodborne disease is a substantial concern in the United States and receives a great deal of attention from the government, industry, and the media. Government initiatives have alleviated some of the burden; however, without improved knowledge of the molecular epidemiology of the pathogens in a variety of environments, a comprehensive understanding of foodborne disease will remain out of reach. Listeria monocytogenes, Salmonella, Escherichia coli O157:H7 and non-O157 Shiga toxin producing E. coli (STEC) play a prominent role in the incidence of bacterial foodborne illness in the United States. Molecular subtyping methods are used extensively in foodborne disease surveillance, yet there is a knowledge gap regarding the presence, transmission and molecular ecology of these pathogens in non-food associated environments. We collected foodborne pathogen isolates from pristine wilderness environments to obtain subtyping data that may aid in the interpretation of clinical and food isolates particularly during outbreak investigations. Furthermore, the identification of subtypes present in different environments but not commonly linked to human disease may provide key information regarding the evolution of virulence in these organisms. To achieve these goals, five wilderness locations in Colorado were selected to represent pristine locations and three areas (approximately 100m2) within each location were designated; each area was sampled once during the spring, summer, and fall seasons in 2009 and 2010. A total of 450 soil, 450 water, 90 drag swab (surface soil) and 276 fecal samples were collected. Five soil samples and five water samples from each area were composited and all samples were microbiologically analyzed to detect Listeria spp. (i.e., L. monocytogenes and other Listeria spp.), Salmonella, E. coli O157:H7, and non-O157 STEC. After non-selective pre-enrichment, samples were divided and microbiologically analyzed to detect each target organism using modified versions of the United States Food and Drug Administration Bacteriological Analytical Manual. Up to four presumptive colonies for each target organism from each sample were confirmed by PCR to detect gene fragments specific to each respective organism. Overall, three samples tested positive for L. monocytogenes, including two fecal samples and one water sample. Nineteen samples contained Listeria spp. other than L. monocytogenes, 14 of which were determined to be Listeria welshimeri by sigB sequencing. The remaining five Listeria spp. were presumptively identified as Listeria rocourtiae sp. nov. by 16s rDNA sequencing; however, these isolates demonstrated notably different biochemical properties than L. rocourtiae. Salmonella was found in two samples, including one water and one fecal sample, and five non-O157 STEC were found in one fecal, one sediment, and three water samples. E. coli O157:H7 was not detected in the natural environments in Northern Colorado surveyed in this study. A molecular serotyping PCR assay revealed two L. monocytogenes isolates belonging to the 1/2 b serogroup and two isolates belonging to the 1/2a serogroup, with a single fecal sample containing two different L. monocytogenes serogroups (1/2a and 1/2b). Pulsed field gel electrophoresis typing results indicated three unique DNA fingerprints among the L. monocytogenes isolates. Two unique strains were isolated from a single fecal sample; with one strain isolated from two different fecal samples collected from the same area. All five non-O157 STEC- and both Salmonella -positive samples had unique PFGE fingerprints. All subtyping data has been deposited in PathogenTracker (www.pathogentracker.com), a publicly available WWW database. In conclusion, results from this study demonstrate a rare presence of foodborne pathogens in pristine environments and the utility of molecular subtyping from distinct environments. Further characterization of the foodborne pathogen isolates obtained from non-food associated environments in this study will expand our knowledge on the molecular ecology of foodborne pathogens in nature

The Genomic Diversity and Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis in Canadian Dairy Cattle

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative bacterium of Johne’s disease, a chronic enteritis in ruminants. Extensive collaborations with Johne’s disease researchers from across Canada provided an opportunity to build a collection of MAP isolates representing all provinces. Difficulties in interpreting the minimal genetic diversity between isolates have previously limited the power of traditional molecular epidemiological approaches in determining transmission patterns. However, the use of whole genome sequencing (WGS) now permits the diversity and relationship of MAP isolates to be assessed in much greater detail. The focus of this thesis was to investigate MAP genetic diversity and molecular epidemiology using molecular tools and phylogenetic analyses based primarily on WGS. In the following chapters the phylogeography and evolutionary dynamics of MAP in Canadian dairy cattle, explored through a combination of complementary approaches, will be presented. In Chapter 2, matrix-assisted laser desorption ionization-time of flight mass spectrometry was applied to interrogate a discriminatory short sequence repeat locus in the MAP genome, improving the discrimination of long mononucleotide repeats. In Chapter 3, WGS was used to determine the reliability of variable number tandem repeat typing (VNTR) to appropriately assess epidemiologically related and unrelated isolates. The use of WGS clarified the level of diversity and relationship between isolates and revealed that VNTR typing does not always reflect extensive within-herd MAP diversity. In Chapter 4, several thousand single nucleotide polymorphisms (SNPs) were used to infer the phylogeny of Canadian isolates in the context of the known global diversity, identifying a dominant clade that includes more than 85% of the Canadian isolates. The phylogeography of MAP at a national and provincial level was further explored, in which considerable overlap of MAP types between different provinces and within a single province was found. In Chapter 5, the impact of SNPs on the microevolution of the Canadian MAP population was investigated, identifying specific genes with an overrepresentation of nonsynonymous mutations and observing selection over time in multiple subtypes. Lastly, in Chapter 6 a novel PCR assay is described that targeted phylogenetically informative SNPs to further identify the relative importance of MAP subtypes in over 600 Canadian isolates

Validation of a screening method for the detection of colistin-resistant \u3ci\u3eE. coli\u3c/i\u3e containing mcr-1 in feral swine feces

A method was developed and validated for the detection of colistin-resistant Escherichia coli containing mcr-1 in the feces of feral swine. Following optimization of an enrichment method using EC broth supplemented with colistin (1 μg/mL) and vancomycin (8 μg/mL), aliquots derived from 100 feral swine fecal samples were spiked with of one of five different mcr-1 positive E. coli strains (between 100 and 104 CFU/g), for a total of 1110 samples tested. Enrichments were then screened using a simple boil-prep and a previously developed real-time PCR assay for mcr-1 detection. The sensitivity of the method was determined in swine feces, with mcr-1 E. coli inocula of 0.1–9.99 CFU/g (n=340), 10–49.99 CFU/g (n=170), 50–99 CFU/g (n=255), 100–149 CFU/g (n=60), and 200–2200 CFU/g (n=175), which were detected with 32%, 72%, 88%, 95%, and 98% accuracy, respectively. Uninoculated controls (n = 100) were negative for mcr-1 following enrichment

Gulls as Sources of Environmental Contamination by Colistin-resistant Bacteria

In 2015, the mcr-1 gene was discovered in Escherichia coli in domestic swine in China that conferred resistance to colistin, an antibiotic of last resort used in treating multi-drug resistant bacterial infections in humans. Since then, mcr-1 was found in other human and animal populations, including wild gulls. Because gulls could disseminate the mcr-1 gene, we conducted an experiment to assess whether gulls are readily colonized with mcr-1 positive E. coli, their shedding patterns, transmission among conspecifics, and environmental deposition. Shedding of mcr-1 E. coli by small gull flocks followed a lognormal curve and gulls shed one strain \u3e101 log10 CFU/g in their feces for 16.4 days, which persisted in the environment for 29.3 days. Because gulls are mobile and can shed antimicrobial-resistant bacteria for extended periods, gulls may facilitate transmission of mcr-1 positive E. coli to humans and livestock through fecal contamination of water, public areas and agricultural operations

Environmental antimicrobial resistance gene detection from wild bird habitats using two methods: A commercially available culture-independent qPCR assay and culture of indicator bacteria followed by whole-genome sequencing

Objectives: A variety of methods have been developed to detect antimicrobial resistance (AMR) in differ-ent environments to better understand the evolution and dissemination of this public health threat. Com-parisons of results generated using different AMR detection methods, such as quantitative PCR (qPCR) and whole-genome sequencing (WGS), are often imperfect, and few studies have analysed samples in parallel to evaluate differences. In this study, we compared bacterial culture and WGS to a culture-independent commercially available qPCR assay to evaluate the concordance between methods and the utility of each in answering research questions regarding the presence and epidemiology of AMR in wild bird habitats.Methods: We first assessed AMR gene detection using qPCR in 45 bacterial isolates from which we had existing WGS data. We then analysed 52 wild bird faecal samples and 9 spatiotemporally collected water samples using culture-independent qPCR and WGS of phenotypically resistant indicator bacterial isolates.Results: Overall concordance was strong between qPCR and WGS of bacterial isolates, although concor-dance differed among antibiotic classes. Analysis of wild bird faecal and water samples revealed that more samples were determined to be positive for AMR via qPCR than via culture and WGS of bacterial isolates, although qPCR did not detect AMR genes in two samples from which phenotypically resistant isolates were found.Conclusions: Both qPCR and culture followed by sequencing may be effective approaches for characteris-ing AMR genes harboured by wild birds, although data streams produced using these different tools may have advantages and disadvantages that should be considered given the application and sample matrix.Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )Funding Agencies|Region Kalmar County, Linkoeping University; U.S. Geological Survey through the Environmental Health and Species Management Research programs of the Ecosystems Mission Area</p