Chad R. Laing

Pan-genome sequence analysis using Panseq: an online tool for the rapid analysis of core and accessory genomic regions

BackgroundThe pan-genome of a bacterial species consists of a core and an accessory gene pool. The accessory genome is thought to be an important source of genetic variability in bacterial populations and is gained through lateral gene transfer, allowing subpopulations of bacteria to better adapt to specific niches. Low-cost and high-throughput sequencing platforms have created an exponential increase in genome sequence data and an opportunity to study the pan-genomes of many bacterial species. In this study, we describe a new online pan-genome sequence analysis program, Panseq.ResultsPanseq was used to identify Escherichia coli O157:H7 and E. coli K-12 genomic islands. Within a population of 60 E. coli O157:H7 strains, the existence of 65 accessory genomic regions identified by Panseq analysis was confirmed by PCR. The accessory genome and binary presence/absence data, and core genome and single nucleotide polymorphisms (SNPs) of six L. monocytogenes strains were extracted with Panseq and hierarchically clustered and visualized. The nucleotide core and binary accessory data were also used to construct maximum parsimony (MP) trees, which were compared to the MP tree generated by multi-locus sequence typing (MLST). The topology of the accessory and core trees was identical but differed from the tree produced using seven MLST loci. The Loci Selector module found the most variable and discriminatory combinations of four loci within a 100 loci set among 10 strains in 1 s, compared to the 449 s required to exhaustively search for all possible combinations; it also found the most discriminatory 20 loci from a 96 loci E. coli O157:H7 SNP dataset.ConclusionPanseq determines the core and accessory regions among a collection of genomic sequences based on user-defined parameters. It readily extracts regions unique to a genome or group of genomes, identifies SNPs within shared core genomic regions, constructs files for use in phylogeny programs based on both the presence/absence of accessory regions and SNPs within core regions and produces a graphical overview of the output. Panseq also includes a loci selector that calculates the most variable and discriminatory loci among sets of accessory loci or core gene SNPs.AvailabilityPanseq is freely available online at http://76.70.11.198/panseq. Panseq is written in Perl.

Genome evolution in major Escherichia coli O157:H7 lineages

BackgroundGenetic analysis of Escherichia coli O157:H7 strains has shown divergence into two distinct lineages, lineages I and II, that appear to have distinct ecological characteristics, with lineage I strains more commonly associated with human disease. In this study, microarray-based comparative genomic hybridization (CGH) was used to identify genomic differences among 31 E. coli O157:H7 strains that belong to various phage types (PTs) and different lineage-specific polymorphism assay (LSPA) types.ResultsA total of 4,084 out of 6,057 ORFs were detected in all E. coli O157:H7 strains and 1,751 were variably present or absent. Based on this data, E. coli O157:H7 strains were divided into three distinct clusters, which consisted of 15 lineage I (LSPA type 111111), four lineage I/II (designated in this study) (LSPA type 211111) and 12 lineage II strains (LSPA 222222, 222211, 222212, and 222221), respectively. Eleven different genomic regions that were dominant in lineage I strains (present in ≥80% of lineage I and absent from ≥ 92% of lineage II strains) spanned segments containing as few as two and up to 25 ORFs each. These regions were identified within E. coli Sakai S-loops # 14, 16, 69, 72, 78, 83, 85, 153 and 286, Sakai phage 10 (S-loops # 91, 92 and 93) and a genomic backbone region. All four lineage I/II strains were of PT 2 and possessed eight of these 11 lineage I-dominant loci. Several differences in virulence-associated loci were noted between lineage I and lineage II strains, including divergence within S-loop 69, which encodes Shiga toxin 2, and absence of the non-LEE encoded effector genes nleF and nleH1-2 and the perC homologue gene pchD in lineage II strains.ConclusionCGH data suggest the existence of two dominant lineages as well as LSPA type and PT-related subgroups within E. coli O157:H7. The genomic composition of these subgroups supports the phylogeny that has been inferred from other methods and further suggests that genomic divergence from an ancestral form and lateral gene transfer have contributed to their evolution. The genomic features identified in this study may contribute to apparent differences in the epidemiology and ecology of strains of different E. coli O157:H7 lineages.

Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River watershed, Alberta, Canada

Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from 898 faecal, 43 sewage, and 342 surface water samples from the Oldman River were characterized using bacterial subtyping methods in order to investigate potential sources of contamination of the watershed. Among these pathogens, Campylobacter spp. were the most frequently isolated from faecal, sewage, and surface water samples (266/895, 11/43, and 91/342, respectively), followed by Salmonella (67/898, 8/43, and 29/342, respectively), and E. coli O157:H7 (16/898, 2/43, and 8/342, respectively). Salmonella Rubislaw was the most common serovar isolated from water. This serovar was also isolated from two wild bird species. Most other serovars isolated from water were either not isolated from animals or were isolated from multiple species. E. coli O157:H7 was predominantly isolated from cattle. The most common phage-types of this pathogen from cattle were also the most common among water isolates, and there were exact pulsed field gel electrophoresis and comparative genomic fingerprint matches between cattle, sewage, and water isolates. Campylobacters were commonly isolated from surface waters and faeces from most animal species. Restriction fragment length polymorphism of the Campylobacter flaA gene identified several location and host species-specific (cattle, goose, pig) fingerprints. Molecular subtyping of these bacterial pathogens shows considerable promise as a tool for determining the sources of faecal pollution of water.

Genotypic Characterization and Prevalence of Virulence Factors among Canadian Escherichia coli O157:H7 Strains

ABSTRACT In this study, the association between genotypic and selected phenotypic characteristics was examined in a collection of Canadian Escherichia coli O157:H7 strains isolated from humans and cattle in the provinces of Alberta, Ontario, Saskatchewan, and Quebec. In a subset of 69 strains selected on the basis of specific phage types (PTs), a strong correlation between the lineage-specific polymorphism assay (LSPA6) genotype and PT was observed with all strains of PTs 4, 14, 21, 31, 33, and 87 belonging to the LSPA6 lineage I (LSPA6-LI) genotype, while those of PTs 23, 45, 67, and 74 belonged to LSPA6 lineage II (LSPA6-LII) genotypes. This correlation was maintained when additional strains of each PT were tested. E. coli O157:H7 strains with the LSPA6-LI genotype were much more common in the collection than were the LSPA6-LII or lineage I/II (LSPA6-LI/II)-related genotypes (82.6, 11.2, and 5.8%, respectively). Of the strains tested, proportionately more LSPA6-LI than LSPA6-LII genotype strains were isolated from humans (52.7% versus 19.7%) than from cattle (47.8% versus 80.2%). In addition, 96.7% of the LSPA6-LII strains carried the stx2c variant gene, while only 50.0% of LSPA6-LI/II and 2.7% of LSPA6-LI strains carried this gene. LSPA6-LII strains were also significantly more likely to possess the colicin D gene, cda (50.8% versus 23.2%), and have combined resistance to streptomycin, sulfisoxazole, and tetracycline (72.1% versus 0.9%) than were LSPA6-LI strains. The LSPA6 genotype- and PT-related characteristics identified may be important markers of specific ecotypes of E. coli O157:H7 that have unique epidemiological and virulence characteristics.

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

ABSTRACT Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated “mobile genetic elements” such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx2 flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx2) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx2, whereas all LII strains carried variant stx2c and 4 of 14 LI/II strains had copies of both stx2 and variant stx2c. Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx2 mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies

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-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.

A comparison of Shiga-toxin 2 bacteriophage from classical enterohemorrhagic Escherichia coli serotypes and the German E. coli O104:H4 outbreak strain.

Escherichia coli O104:H4 was associated with a severe foodborne disease outbreak originating in Germany in May 2011. More than 4000 illnesses and 50 deaths were reported. The outbreak strain was a typical enteroaggregative E. coli (EAEC) that acquired an antibiotic resistance plasmid and a Shiga-toxin 2 (Stx2)-encoding bacteriophage. Based on whole-genome phylogenies, the O104:H4 strain was most closely related to other EAEC strains; however, Stx2-bacteriophage are mobile, and do not necessarily share an evolutionary history with their bacterial host. In this study, we analyzed Stx2-bacteriophage from the E. coli O104:H4 outbreak isolates and compared them to all available Stx2-bacteriophage sequences. We also compared Stx2 production by an E. coli O104:H4 outbreak-associated isolate (ON-2011) to that of E. coli O157:H7 strains EDL933 and Sakai. Among the E. coli Stx2-phage sequences studied, that from O111:H- strain JB1-95 was most closely related phylogenetically to the Stx2-phage from the O104:H4 outbreak isolates. The phylogeny of most other Stx2-phage was largely concordant with their bacterial host genomes. Finally, O104:H4 strain ON-2011 produced less Stx2 than E. coli O157:H7 strains EDL933 and Sakai in culture; however, when mitomycin C was added, ON-2011 produced significantly more toxin than the E. coli O157:H7 strains. The Stx2-phage from the E. coli O104:H4 outbreak strain and the Stx2-phage from O111:H- strain JB1-95 likely share a common ancestor. Incongruence between the phylogenies of the Stx2-phage and their host genomes suggest the recent Stx2-phage acquisition by E. coli O104:H4. The increase in Stx2-production by ON-2011 following mitomycin C treatment may or may not be related to the high rates of hemolytic uremic syndrome associated with the German outbreak strain. Further studies are required to determine whether the elevated Stx2-production levels are due to bacteriophage or E. coli O104:H4 host related factors.

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.

Rapid Determination of Escherichia coli O157:H7 Lineage Types and Molecular Subtypes by Using Comparative Genomic Fingerprinting

ABSTRACT In this study, variably absent or present (VAP) regions discovered through comparative genomics experiments were targeted for the development of a rapid, PCR-based method to subtype and fingerprint Escherichia coli O157:H7. Forty-four VAP loci were analyzed for discriminatory power among 79 E. coli O157:H7 strains of 13 phage types (PT). Twenty-three loci were found to maximize resolution among strains, generating 54 separate fingerprints, each of which contained strains of unique PT. Strains from the three previously identified major E. coli O157:H7 lineages, LSPA6-LI, LSPA6-LI/II, and LSPA6-LII, formed distinct branches on a dendrogram obtained by hierarchical clustering of comparative genomic fingerprinting (CGF) data. By contrast, pulsed-field gel electrophoresis (PFGE) typing generated 52 XbaI digestion profiles that were not unique to PT and did not cluster according to O157:H7 lineage. Our analysis identified a subpopulation comprised of 25 strains from a closed herd of cattle, all of which were of PT87 and formed a cluster distinct from all other E. coli O157:H7 strains examined. CGF found five related but unique fingerprints among the highly clonal herd strains, with two dominant subtypes characterized by a shift from the presence of locus fprn33 to its absence. CGF had equal resolution to PFGE typing but with greater specificity, generating fingerprints that were unique among phenotypically related E. coli O157:H7 lineages and PT. As a comparative genomics typing method that is amenable for use in high-throughput platforms, CGF may be a valuable tool in outbreak investigations and strain characterization.

Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O‐island 51, a 14.93 kb cryptic prophage (CP‐933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O‐island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE‐encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.