John HE Nash

Public Health Agency of Canada | PHAC · National Microbiology Laboratory

Plasmids are the primary vector for horizontal transfer of antimicrobial resistance (AMR) within bacterial populations. We applied the MOB-suite, a toolset for reconstructing and typing plasmids, to 150 767 publicly available Salmonella whole-genome sequencing samples covering 1204 distinct serovars to produce a large-scale population survey of pla...

This study was to assess the gene diversity and characterize a large set of plasmids harboring extended β-lactamase (ESBL) genes from raw and digested dairy manure. A total of eighty-four plasmids that were captured in this E. coli recipient were sequenced using Illumina MiSeq sequencing technology. Twenty-four plasmids of interest were subsequentl...

Oxford Nanopore long-read sequencing offers advantages over Illumina short reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost...

Oxford Nanopore long-read sequencing offers advantages over Illumina short-reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost...

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,...

Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, vir...

Oxford Nanopore long-read sequencing offers advantages over Illumina short-reads for the identification and characterization of bacterial pathogens for outbreak detection and surveillance activities within a diagnostic public health laboratory context. Compared to Illumina, Nanopore is more cost-effective for small batches, has a lower capital cost...

Enterococcus faecium is a ubiquitous opportunistic pathogen that is exhibiting increasing levels of antimicrobial resistance (AMR). Many of the genes that confer resistance and pathogenic functions are localized on mobile genetic elements (MGEs), which facilitate their transfer between lineages. Here, features including resistance determinants, vir...

Non-typhoidal Salmonella infections represent a substantial burden of illness in humans, and the increasing prevalence of antimicrobial resistance among these infections is a growing concern. Using a combination of Salmonella isolate short-read whole-genome sequence data from select human cases, raccoons, livestock and environmental sources, and an...

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to dissemination of antibiotic resistance genes (ARGs) in the gut microbiota. The gut microbiota often suffers from various disturbances. It is not clear whether and how disturbed microbiota may affect ARG mobility under antibiotic treatments. For proof of concept, in the prese...

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serot...

Hierarchical genotyping approaches can provide insights into the source, geography and temporal distribution of bacterial pathogens. Multiple hierarchical SNP genotyping schemes have previously been developed so that new isolates can rapidly be placed within pre-computed population structures, without the need to rebuild phylogenetic trees for the...

Recreational water use is an important source of human enteric illness. Enhanced (episodic) surveillance of natural recreational waters as a supplement to beach monitoring can enrich our understanding of human health risks. From 2011 to 2013, water sampling was undertaken at recreational sites on a watershed in eastern Canada. This study compared t...

The present study investigated the impact of on-farm anaerobic digestion on the abundance of enteric bacteria, antibiotic resistance-associated gene targets and the horizontal transfer potential of extended-spectrum β-lactamase (ESBL) genes. Samples of raw and digested manure were obtained from six commercial dairy farms in Ontario, Canada. Digesti...

Monophasic Salmonella 4,[5]:12:i:- are a major public health problem because they are one of the top five Salmonella serotypes isolated from clinical cases globally and because they can carry resistance to multiple antibiotics. A total of 811 Salmonella 4,[5]:12:i:- and S . Typhimurium whole genome sequences (WGS) were generated. The various geneti...

Here, we report the complete genome sequences for 36 Canadian isolates of Salmonella enterica subsp. enterica serovar Typhimurium and its monophasic variant I 1,4,[5]:12:i:– from both clinical and animal sources. These genome sequences will provide useful references for understanding the genetic variation within this prominent serotype.

Bacterial plasmids play a large role in allowing bacteria to adapt to changing environments and can pose a significant risk to human health if they confer virulence and antimicrobial resistance (AMR). Plasmids differ significantly in the taxonomic breadth of host bacteria in which they can successfully replicate, this is commonly referred to as 'ho...

Salmonella 4,[5],12:i:- are monophasic S. Typhimurium variants incapable of producing the second-phase flagellar antigen. They have emerged since the mid-1990s to become one of the most prevalent Salmonella serotypes causing human disease world-wide. Multiple genetic events associated with different genetic elements can result in the monophasic phe...

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to the dissemination of antibiotic-resistance genes in the gut microbiota and the development of antibiotic-resistant bacterial infection, a significant threat to animal and public health. Food or water may be contaminated with multiple resistant bacteria, but animal models on...

BioHansel performs high-resolution genotyping of bacterial isolates by identifying phylogenetically informative single nucleotide polymorphisms (SNPs), also known as canonical SNPs, in whole genome sequencing (WGS) data. The application uses a fast k -mer matching algorithm to map pathogen WGS data to canonical SNPs contained in hierarchically stru...

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified in Salm...

Due to the public health importance of flagellar genes for typing, it is important to understand mechanisms that could alter their expression or presence. Phenotypic novelty in flagellar genes arise predominately through accumulation of mutations but horizontal transfer is known to occur. A linear plasmid termed pBSSB1 previously identified in Salm...

Previously we developed and tested the Salmonella GenoSerotyping Array (SGSA), which utilized oligonucleotide probes for O- and H- antigen biomarkers to perform accurate molecular serotyping of 57 Salmonella serotypes. Here we describe the development and validation of the ISO 17025 accredited second version of the SGSA (SGSA v. 2) with reliable an...

Reported and predicted serotypes for publicly available NCBI Illumina WGS isolates used for testing the SGSA v. 2 in silico protocol. (XLSX)

Primers and probe sequences for the SGSA v. 2. (XLSX)

Reported and predicted serotypes for isolates used for testing the SGSA v. 2 in vitro protocol. (XLSX)

In silico performance summary of the SGSA v. 2 on Illumina based on serotype. (XLSX)

We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico, modified to be RNase...

Cryptosporidium parvum is a zoonotic protozoan parasite that causes food and waterborne gastrointestinal disease and whose major animal reservoirs are cattle and small ruminants. We report here on a draft whole-genome sequence of a zoonotic isolate of C. parvum isolated from a person with cryptosporidiosis.

We report here 32 completed closed genome sequences of strains representing 30 serotypes of Salmonella . These genome sequences will provide useful references for understanding the genetic variation within Salmonella enterica serotypes, particularly as references to aid in comparative genomics studies, as well as providing information for improving...

Large-scale bacterial population genetics studies are now routine due to cost-effective Illumina short-read sequencing. However, analysing plasmid content remains difficult due to incomplete assembly of plasmids. Bacterial isolates can contain any number of plasmids and assembly remains complicated due to the presence of repetitive elements. Numero...

Bacteriophages SP-15 and ΦW-14 are members of the Myoviridae infecting Bacillus subtilis and Delftia (formerly Pseudomonas) acidovorans, respectively. What links them is that in both cases, approximately 50% of the thymine residues are replaced by hypermodified bases. The consequence of this is that the physico-chemical properties of the DNA are ra...

Here, we present the first complete genome sequence of an Escherichia coli non-O157 Shiga-toxin producing isolate, 16-9255, from serotype O121:H19. This strain is notable as a clinical case recovered from a recent Canadian flour-associated outbreak event.

We report here the completed closed genome sequences of strains representing 36 serotypes of Salmonella . These genome sequences will provide useful references for understanding the genetic variation between serotypes, particularly as references for mapping of raw reads or to create assemblies of higher quality, as well as to aid in studies of comp...

Public health and food safety institutions around the world are adopting whole genome sequencing (WGS) to replace conventional methods for characterizing Salmonella for use in surveillance and outbreak response. Falling costs and increased throughput of WGS have resulted in an explosion of data, but questions remain as to the reliability and robust...

Salmonella serotyping remains the gold-standard tool for the classification of Salmonella isolates and forms the basis of Canada’s national surveillance program for this priority foodborne pathogen. Public health officials have been increasingly looking toward whole genome sequencing (WGS) to provide a large set of data from which all the relevant...

The Salmonella Syst-OMICS consortium is sequencing 4,500 Salmonella genomes and building an analysis pipeline for the study of Salmonella genome evolution, antibiotic resistance and virulence genes. Metadata, including phenotypic as well as genomic data, for isolates of the collection are provided through the Salmonella Foodborne Syst-OMICS databas...

Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S . Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain s...

Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking and outbreak detection. In order to improve detection and reduce the burden of Salmonellosis, several rapid and high throughput molecular Salmonella serotyping methods have been developed. The a...

The archaellum is the swimming organelle of the third domain, the Archaea. In the euryarchaeon Methanococcus maripaludis, genes involved in archaella formation, including the three archaellins flaB1, flaB2 and flaB3, are mainly located in the fla operon. Previous studies have shown that transcription of fla genes and expression of Fla proteins are...

We report the draft genome sequences of 25 Salmonella enterica strains representing 24 different serotypes, many of which were not available in public repositories during our selection process. These draft genomes will provide useful reference for the genetic variation between serotypes and aid in the development of molecular typing tools.

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-...

A cgMLST clustering threshold of 85% similarity balances the proportion of genomes in multi-isolate cgMLST clusters and the serovar specificity of cgMLST clusters. Analyses in this study were performed at a cgMLST clustering threshold of 85% profile similarity. This value maximized the proportion of genomes in clusters with a minimum cluster size o...

Information on genomes (n = 4,291) used to test and validate the SISTR platform. Included are error codes described in Fig 1, along with in silico predictions, and genome quality metrics. (XLSX)

Isolates of Salmonella enterica subsp. enterica serovar Heidelberg are often associated with poultry products and may cause severe human illness. Here, we report the fully assembled genome and plasmid sequences of three S . Heidelberg strains with phage types 9, 29, and 41.

Purpose: The purpose of this study was to assess safety of the traditional antidiabetic extracts of either S. purpurea or its lead active principle, morroniside at the transcriptional level. The overarching objective was to profile and validate transcriptional changes in the cytochrome P450 family of genes, in response to treatment with S. purpure...

The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic t...

Salmonella enterica subsp. enterica serovar Enteritidis is a prominent cause of human salmonellosis frequently linked to poultry products. In Canada, S. Enteritidis phage types 8, 13, and 13a predominate among both clinical and poultry isolates. Here, we report the complete genome and plasmid sequences of poultry isolates of these three phage types...

Morganella morganii is a facultative pathogen of humans, causing urinary tract and postsurgical infections. Here, we report a high-quality draft assembly of the O:1ab serotype.

Based on morphology and comparative nucleotide and protein sequence analysis, a new subfamily of the family Siphoviridae is proposed, named "Jerseyvirinae" and consisting of three genera, "Jerseylikevirus", "Sp3unalikevirus" and "K1glikevirus". To date, this subfamily consists of 18 phages for which the genomes have been sequenced. Salmonella phage...

Using a variety of genomic (BLASTN, ClustalW) and proteomic (Phage Proteomic Tree, CoreGenes) tools we have tackled the taxonomic status of members of the largest bacteriophage family, the Siphoviridae. In all over 400 phages were examined and we were able to propose 39 new genera, comprising 216 phage species, and add 62 species to two previously...

The core region of the lipopolysaccharide of Morganella morganii serotype O:1ab was obtained by hydrolysis of the LPS and studied by 2D NMR, ESI MS, and chemical methods. Its structure was highly homologous to those from the two major members of the same Proteeae tribe, Proteus mirabilis and Providencia alcalifaciens, and analysis of the M. morgani...

Volume 2, no. 2, e00348-14, 2014. Page 3: The first paragraph of the Acknowledgments should read as follows. "We sincerely thank the NCBI PGAP team for genome annotation services and Shaun Tyler at the PHAC National Microbiology Laboratory and staff at McGill University, Genome Québec, and the Centre for Applied Genomics, Toronto Hospital for Sick...

Cronobacter sakazakii is a Gram-negative pathogen found in milk-based formulae that causes infant meningitis. Bacteriophages have been proposed to control bacterial pathogens; however, comprehensive knowledge about a phage is required to ensure its safety before clinical application. We have characterized C. sakazakii phage vB_CsaM_GAP32 (GAP32), w...

The T1-like bacteriophages vB_EcoS_AHP24, AHS24, AHP42 and AKS96 of the family Siphoviridae were shown to lyse common phage types of Shiga toxin-producing Escherichia coli O157:H7 (STEC O157:H7), but not non-O157 E. coli. All contained circularly permuted genomes of 45.7-46.8 kb (43.8-44 mol% G+C) encoding 74-81 open reading frames and 1 arginyl-tR...

We report the high-quality draft genome sequences of 162 strains of Salmonella enterica subsp. enterica serovar Enteritidis representing diverse phage types and pulsed-field gel electrophoresis (PFGE) profiles. The analysis of these genomes will enable the identification of markers that are useful for differentiating strains of this highly clonal s...

Salmonella enterica subsp. enterica serovar Enteritidis is an important zoonotic food-borne pathogen causing serious human illnesses frequently linked to poultry products. Here, we report fully assembled genome sequences of 16 S. Enteritidis strains with common pulsed-field gel electrophoresis (PFGE) and phage types (8, 13, 13a, and 14b) that predo...

The renewed interest in controlling Staphylococcus aureus infections using their natural enemies, bacteriophages, has led to the isolation of a limited number of virulent phages so far. These phages are all members of the Twortlikevirus, displaying little variance. We present two novel closely related (95.9% DNA homology) lytic myoviruses, Romulus...

Here, we report the first complete genome sequence of Actinobacillus suis, an important opportunistic pathogen of swine. By comparing the genome sequence of A. suis with those of other members of the family Pasteurellaceae, we hope to better understand the role of these organisms in health and disease in swine.

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 mitig...

Accession numbers and gene coordinates used for in silico MLST analysis.

Alignment of NcoI optical map of NRC857c with the in silico-generated map of LF82. The vertical lines are alignment marks identifying similar restriction fragments between two aligned contigs. The region highlighted in red is a region of DNA that is translocated in LF82.

Genes unique to NRG857c and/or LF82.

Iron transport in AIEC NRG857c and aerobactin uptake mutant.

Alignment of NcoI optical map of NRC857c with nine super-contigs generated from shotgun sequencing. The NcoI optical restriction map of NRG857c was aligned with the in silico-generated NcoI restriction maps of nine super-contigs arising from the shotgun sequencing and assembly of the genome. The vertical lines are alignment marks identifying simila...

Table S2: Predicted Genomic Islands in NRG857c.

List of E. coli genomes used for comparative genomics analyses.

Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the...

Oligonucleotide primers used for microarray result validation by qRT-PCR.

A. pleuropneumoniaegenes which are differentially expressed in infected pig lungs (150 genes). Individual genes and their corresponding locus tag are sorted according to their functional class and fold change. q-values as calculated by SAM are indicated in %.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease which causes great economic losses worldwide. Many virulence factors are involved in the pathogenesis, namely capsular polysaccharides, RTX toxins, LPS and many iron acquisition systems. In order to identify genes that are expressed in vivo du...

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-β-1,6-N-acetyl-d-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plate...

Biofilm formation is an important virulence trait of many bacterial pathogens. It has been reported in the literature that only two of the reference strains of the swine pathogen Actinobacillus pleuropneumoniae, representing serotypes 5b and 11, were able to form biofilm in vitro. In this study, we compared biofilm formation by the serotype 1 refer...

Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components f...

IntroductionAquaculture-Relevant Microarray Research on SalmonidsMicroarray Research on CatfishAquaculture-Relevant Microarray Research on FlatfishAquaculture-Relevant Microarray Research on ZebrafishThe Use of DNA Microarrays to Investigate Aquatic Animal PathogensExamples of Novel Microarray Platforms for Aquaculture-Related ResearchOur First Gli...

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The Gram-negative rod Actinobacillus pleuropneumoniae is a facultative anaerobic pathogen of the porcine respiratory tract, and HlyX, the A. pleuropneumoniae homologue of fumarate and nitrate reduction regulator (FNR), has been shown to be important for persistence. An A. pleuropneumoniae hlyX deletion mutant has a decreased generation time but hig...

Host-pathogen interactions are of great importance in understanding the pathogenesis of infectious microorganisms. We developed in vitro models to study the host-pathogen interactions of porcine respiratory tract pathogens using two immortalized epithelial cell lines, namely, the newborn pig trachea (NPTr) and St. Jude porcine lung (SJPL) cell line...

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Mi...

Mean Log2Ratio for genes that are divergent or highly divergent/absent in at least one reference strain of A. pleuropneumoniae. A table listing all the genes that are either divergent or highly divergent/absent in at least one of the 15 reference strains. The number of strains for which a particular gene is identified as divergent or highly diverge...

Actinobacillus pleuropneumoniae, the causative agent of porcine contagious pleuropneumonia, is an important pathogen of swine throughout the world. It must rapidly overcome the innate pulmonary immune defenses of the pig to cause disease. To better understand this process, the objective of this study was to identify genes that are differentially ex...