Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations.

The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using 'big data' approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune's loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks.

Genomic insights from whole genome sequencing of four clonal outbreak Campylobacter jejuni assessed within the global C. jejuni population.

BACKGROUND: Whole genome sequencing (WGS) is useful for determining clusters of human cases, investigating outbreaks, and defining the population genetics of bacteria. It also provides information about other aspects of bacterial biology, including classical typing results, virulence, and adaptive strategies of the organism. Cell culture invasion and protein expression patterns of four related multilocus sequence type 21 (ST21) C. jejuni isolates from a significant Canadian water-borne outbreak were previously associated with the presence of a CJIE1 prophage. Whole genome sequencing was used to examine the genetic diversity among these isolates and confirm that previous observations could be attributed to differential prophage carriage. Moreover, we sought to determine the presence of genome sequences that could be used as surrogate markers to delineate outbreak-associated isolates. RESULTS: Differential carriage of the CJIE1 prophage was identified as the major genetic difference among the four outbreak isolates. High quality single-nucleotide variant (hqSNV) and core genome multilocus sequence typing (cgMLST) clustered these isolates within expanded datasets consisting of additional C. jejuni strains. The number and location of homopolymeric tract regions was identical in all four outbreak isolates but differed from all other C. jejuni examined. Comparative genomics and PCR amplification enabled the identification of large chromosomal inversions of approximately 93 kb and 388 kb within the outbreak isolates associated with transducer-like proteins containing long nucleotide repeat sequences. The 93-kb inversion was characteristic of the outbreak-associated isolates, and the gene content of this inverted region displayed high synteny with the reference strain. CONCLUSIONS: The four outbreak isolates were clonally derived and differed mainly in the presence of the CJIE1 prophage, validating earlier findings linking the prophage to phenotypic differences in virulence assays and protein expression. The identification of large, genetically syntenous chromosomal inversions in the genomes of outbreak-associated isolates provided a unique method for discriminating outbreak isolates from the background population. Transducer-like proteins appear to be associated with the chromosomal inversions. CgMLST and hqSNV analysis also effectively delineated the outbreak isolates within the larger C. jejuni population structure.

Pseudomonas brassicacearum strain DF41 kills Caenorhabditis elegans through biofilm-dependent and biofilm-independent mechanisms.

Pseudomonas brassicacearum DF41 is a biocontrol agent that suppresses disease caused by the fungal pathogen Sclerotinia sclerotiorum A number of exometabolites are produced by DF41 including the lipopeptide sclerosin, hydrogen cyanide (HCN) and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional level by quorum sensing (QS) and the Gac-two component regulatory system. In order to be successful, a biocontrol agent must persist in the environment at levels sufficient for pathogen control. Bacterivorous predators, including nematodes, represent a challenge to the establishment of introduced microorganisms. In the current study, DF41 was investigated for its ability to resist predation by Caenorhabditis elegans. We discovered that this bacterium is capable of killing C. elegans through two different mechanisms: the first involves exposure to toxic metabolites; and the second entails biofilm formation on the nematode head blocking the buccal cavity. Biofilm formation on nematodes, which has only been reported for Yersinia spp. and Xenorhabdus nematophila, is dependent upon the Gac system. Biofilms were not observed when bacteria were grown on NaCl-containing media, and on C. elegans biofilm-resistant mutants. Co-culturing with nematodes lead to increased expression of the pdfRI-rfiA QS genes and hcnA which is under QS control. HCN was the most nematicidal of the exometabolites, suggesting that this bacterium can respond to predator cues and upregulate expression of toxins accordingly. In summary, DF41 is able to respond to the presence of C. elegans and through two distinct mechanisms it can escape predation. IMPORTANCE: Pseudomonas brassicacearum DF41 can suppress fungal pathogens through a process known as biocontrol. To be successful, a biocontrol agent must be able to persist in the environment at levels sufficient for pathogen control. Predators including the nematode Caenorhabditis elegans represent a threat to persistence. The aim of the current study was to investigate the DF41-C. elegans interaction. We discovered that DF41 is able to escape predation through two distinct mechanisms. The first involves exposure to toxic bacterial metabolites and the second entails formation of a sticky coating on the nematode head, called a biofilm, which blocks feeding and causes starvation. This is the first report of a pseudomonad forming biofilms on the C. elegans surface. When grown with C. elegans, DF41 exhibits altered gene expression and metabolite production indicating that this bacterium can sense the presence of these predators and adjust its physiology accordingly.

The utility of multiple molecular methods including whole genome sequencing as tools to differentiate Escherichia coli O157:H7 outbreaks.

A standardised method for determining Escherichia coli O157:H7 strain relatedness using whole genome sequencing or virulence gene profiling is not yet established. We sought to assess the capacity of either high-throughput polymerase chain reaction (PCR) of 49 virulence genes, core-genome single nt variants (SNVs) or k-mer clustering to discriminate between outbreak-associated and sporadic E. coli O157:H7 isolates. Three outbreaks and multiple sporadic isolates from the province of Alberta, Canada were included in the study. Two of the outbreaks occurred concurrently in 2014 and one occurred in 2012. Pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandem repeat analysis (MLVA) were employed as comparator typing methods. The virulence gene profiles of isolates from the 2012 and 2014 Alberta outbreak events and contemporary sporadic isolates were mostly identical; therefore the set of virulence genes chosen in this study were not discriminatory enough to distinguish between outbreak clusters. Concordant with PFGE and MLVA results, core genome SNV and k-mer phylogenies clustered isolates from the 2012 and 2014 outbreaks as distinct events. k-mer phylogenies demonstrated increased discriminatory power compared with core SNV phylogenies. Prior to the widespread implementation of whole genome sequencing for routine public health use, issues surrounding cost, technical expertise, software standardisation, and data sharing/comparisons must be addressed.

Chemical and biological characterization of sclerosin, an antifungal lipopeptide.

Pseudomonas sp. strain DF41 produces a lipopeptide, called sclerosin that inhibits the fungal pathogen Sclerotinia sclerotiorum . The aim of the current study was to deduce the chemical structure of this lipopeptide and further characterize its bioactivity. Mass spectrometry analysis determined the structure of sclerosin to be CH(3)-(CH(2))(6)-CH(OH)-CH(2)-CO-Dhb-Pro-Ala-Leu/Ile-Ala-Val-Val-Dhb-Thr-Val-Leu/Ile-Dhp-Ala-Ala-Ala-Val-Dhb-Dhb-Ala-Dab-Ser-Val-OH, similar to corpeptins A and B of the tolaasin group, differing by only 3 amino acids in the peptide chain. Subjecting sclerosin to various ring opening procedures revealed no new ions, suggesting that this molecule is linear. As such, sclerosin represents a new member of the tolaasin lipopeptide group. Incubation of S. sclerotinia ascospores and sclerotia in the presence of sclerosin inhibited the germination of both cell types. Sclerosin also exhibited antimicrobial activity against Bacillus species. Conversely, this lipopeptide demonstrated no zoosporicidal activity against the oomycete pathogen Phytophthora infestans . Next, we assessed the effect of DF41 and a lipopeptide-deficient mutant on the growth and development of Caenorhabditis elegans larvae. We discovered that sclerosin did not protect DF41 from ingestion by and degradation in the C. elegans digestive tract. However, another metabolite produced by this bacterium appeared to shorten the life-span of the nematode compared to C. elegans growing on Escherichia coli OP50.

Repression of the antifungal activity of Pseudomonas sp. strain DF41 by the stringent response.

The stringent response (SR) enables bacteria to adapt to nutrient limitation through production of the nucleotides guanosine tetraphosphate and guanosine pentaphosphate, collectively known as (p)ppGpp. Two enzymes are responsible for the intracellular pools of (p)ppGpp: RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. We investigated how the SR affects the ability of the biological control agent Pseudomonas sp. strain DF41 to inhibit the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary. Strain DF41 relA and relA spoT mutants were generated and found to exhibit increased antifungal activity. Strain DF41 produces a lipopeptide (LP) molecule that is essential for Sclerotinia biocontrol. LP production and protease activity were both elevated in the relA and relA spoT mutants. Addition of relA but not spoT in trans restored the mutant phenotype to that of the parent. Next, we investigated whether an association exists between the SR and known regulators of biocontrol, including the Gac system and RpoS. A gacS mutant of strain DF41 produced less (p)ppGpp and exhibited a 1.7-fold decrease in relA expression compared to the wild type, suggesting that relA forms part of the Gac regulon. We discovered that rpoS transcription was reduced significantly in the SR mutants. Furthermore, rpoS provided in trans restored protease activity to wild-type levels but did not attenuate antifungal activity. Finally, relA expression was decreased in the mutants, indicating that the SR is required for maximum expression of relA.

Source Tracking Based on Core Genome SNV and CRISPR Typing of Salmonella enterica Serovar Heidelberg Isolates Involved in Foodborne Outbreaks in Québec, 2012.

Whole-genome sequencing (WGS) is the method of choice for bacterial subtyping and it is rapidly replacing the more traditional methods such as pulsed-field gel electrophoresis (PFGE). Here we used the high-resolution core genome single nucleotide variant (cgSNV) typing method to characterize clinical and food from Salmonella enterica serovar Heidelberg isolates in the context of source attribution. Additionally, clustered regularly interspaced short palindromic repeats (CRISPR) analysis was included to further support this method. Our results revealed that cgSNV was highly discriminatory and separated the outbreak isolates into distinct clusters (0-4 SNVs). CRISPR analysis was also able to distinguish outbreak strains from epidemiologically unrelated isolates. Specifically, our data clearly demonstrated the strength of these two methods to determine the probable source(s) of a 2012 epidemiologically characterized outbreak of S. Heidelberg. Using molecular cut-off of 0-10 SNVs, the cgSNV analysis of 246 clinical and food isolates of S. Heidelberg collected in Québec, in the same year of the outbreak event, revealed that retail and abattoir chicken isolates likely represent an important source of human infection to S. Heidelberg. Interestingly, the isolates genetically related by cgSNV also harbored the same CRISPR as outbreak isolates and clusters. This indicates that CRISPR profiles can be useful as a complementary approach to determine source attribution in foodborne outbreaks. Use of the genomic analysis also allowed to identify a large number of cases that were missed by PFGE, indicating that most outbreaks are probably underestimated. Although epidemiological information must still support WGS-based results, cgSNV method is a highly discriminatory method for the resolution of outbreak events and the attribution of these events to their respective sources. CRISPR typing can serve as a complimentary tool to this analysis during source tracking.

Diversity of transducer-like proteins (Tlps) in Campylobacter.

Campylobacter transducer-like proteins (Tlps), also known as methyl-accepting chemotaxis proteins (MCPs), are associated with virulence as well as niche and host adaptation. While functional attributes of these proteins are being elucidated, little has been published regarding their sequence diversity or chromosomal locations and context, although they appear to define invertible regions within Campylobacter jejuni genomes. Genome assemblies for several species of Campylobacter were obtained from the publicly available NCBI repositories. Genomes from all isolates were obtained from GenBank and assessed for Tlp content, while data from isolates with complete, finished genomes were used to determine the identity of Tlps as well as the gene content of putative invertible elements (IEs) in C. jejuni (Cj) and C. coli (Cc). Tlps from several Campylobacter species were organized into a nomenclature system and novel Tlps were defined and named for Cj and Cc. The content of Tlps appears to be species-specific, though diverse within species. Cj and Cc carried overlapping, related Tlp content, as did the three C. fetus subspecies. Tlp1 was detected in 88% of Cj isolates and approximately 43% of Cc, and was found in a different conserved chromosomal location and genetic context in each species. Tlp1 and Tlp 3 predominated in genomes from Cj whereas other Tlps were detected less frequently. Tlp13 and Tlp20 predominated in genomes from Cc while some Cj/Cc Tlps were not detected at all. Tlps 2-4 and 11-20 were less frequently detected and many showed sequence heterogeneity that could affect substrate binding, signal transduction, or both. Tlps other than Tlp1, 7, and 10 had substantial sequence identity in the C-terminal half of the protein, creating chromosomal repeats potentially capable of mediating the inversion of large chromosomal DNA. Cj and Cc Tlps were both found in association with only 14 different genes, indicating a limited genomic context. In Cj these Tlps defined IEs that were for the most part found at a single chromosomal location and comprised of a conserved set of genes. Cc IEs were situated at very different chromosomal locations, had different structures than Cj IEs, and were occasionally incomplete, therefore not capable of inversion. Tlps may have a role in Campylobacter genome structure and dynamics as well as acting as chemoreceptors mediating chemotactic responses.

Shared genome analyses of notable listeriosis outbreaks, highlighting the critical importance of epidemiological evidence, input datasets and interpretation criteria.

The persuasiveness of genomic evidence has pressured scientific agencies to supplement or replace well-established methodologies to inform public health and food safety decision-making. This study of 52 epidemiologically defined Listeria monocytogenes isolates, collected between 1981 and 2011, including nine outbreaks, was undertaken (1) to characterize their phylogenetic relationship at finished genome-level resolution, (2) to elucidate the underlying genetic diversity within an endemic subtype, CC8, and (3) to re-evaluate the genetic relationship and epidemiology of a CC8-delimited outbreak in Canada in 2008. Genomes representing Canadian Listeria outbreaks between 1981 and 2010 were closed and manually annotated. Single nucleotide variants (SNVs) and horizontally acquired traits were used to generate phylogenomic models. Phylogenomic relationships were congruent with classical subtyping and epidemiology, except for CC8 outbreaks, wherein the distribution of SNV and prophages revealed multiple co-evolving lineages. Chronophyletic reconstruction of CC8 evolution indicates that prophage-related genetic changes among CC8 strains manifest as PFGE subtype reversions, obscuring the relationship between CC8 isolates, and complicating the public health interpretation of subtyping data, even at maximum genome resolution. The size of the shared genome interrogated did not change the genetic relationship measured between highly related isolates near the tips of the phylogenetic tree, illustrating the robustness of these approaches for routine public health applications where the focus is recent ancestry. The possibility exists for temporally and epidemiologically distinct events to appear related even at maximum genome resolution, highlighting the continued importance of epidemiological evidence.

Outbreak of Escherichia coli O157:H7 Infections Linked to Mechanically Tenderized Beef and the Largest Beef Recall in Canada, 2012.

Contaminated beef is a known vehicle of Escherichia coli O157:H7 infection, although more attention is given to the control of E. coli O157:H7 in ground, rather than whole-cut, beef products. In September 2012, an investigation was initiated at an Alberta, Canada, beef plant after the detection of E. coli O157:H7 in two samples of trim cut from beef originating from this plant. Later in September 2012, Alberta Health Services identified five laboratory-confirmed infections of E. coli O157:H7, and case patients reported eating needle-tenderized beef steaks purchased at a store in Edmonton, Alberta, produced with beef from the Alberta plant. In total, 18 laboratory-confirmed illnesses in Canada in September and October 2012 were linked to beef from the Alberta plant, including the five individuals who ate needle-tenderized steaks purchased at the Edmonton store. A unique strain of E. coli O157:H7, defined by molecular subtyping and whole genome sequencing, was detected in clinical isolates, four samples of leftover beef from case patient homes, and eight samples of Alberta plant beef tested by industry and food safety partners. Investigators identified several deficiencies in the control of E. coli O157:H7 at the plant; in particular, the evaluation of, and response to, the detection of E. coli O157 in beef samples during routine testing were inadequate. To control the outbreak, 4,000 tons of beef products were recalled, making it the largest beef recall in Canadian history. This outbreak, in combination with similar outbreaks in the United States and research demonstrating that mechanical tenderization can transfer foodborne pathogens present on the surface into the interior of beef cuts, prompted amendments to Canada's Food and Drug Regulations requiring mechanically tenderized beef to be labeled as such and to provide safe cooking instructions to consumers. A detailed review of this event also led to recommendations and action to improve the safety of Canada's beef supply.

Complete Genome Sequence of an Escherichia coli O121:H19 Strain from an Outbreak in Canada Associated with Flour.

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.

Comparison of genomes and proteomes of four whole genome-sequenced Campylobacter jejuni from different phylogenetic backgrounds.

Whole genome sequencing (WGS) has been used to assess the phylogenetic relationships, virulence and metabolic differences, and the relationship between gene carriage and host or niche differentiation among populations of C. jejuni isolates. We previously characterized the presence and expression of CJIE4 prophage proteins in four C. jejuni isolates using WGS and comparative proteomics analysis, but the isolates were not assessed further. In this study we compare the closed, finished genome sequences of these isolates to the total proteome. Genomes of the four isolates differ in phage content and location, plasmid content, capsular polysaccharide biosynthesis loci, a type VI secretion system, orientation of the ~92 kb invertible element, and allelic differences. Proteins with 99% sequence identity can be differentiated using isobaric tags for relative and absolute quantification (iTRAQ) comparative proteomic methods. GO enrichment analysis and the type of artefacts produced in comparative proteomic analysis depend on whether proteins are encoded in only one isolate or common to all isolates, whether different isolates have different alleles of the proteins analyzed, whether conserved and variable regions are both present in the protein group analyzed, and on how the analysis is done. Several proteins encoded by genes with very high levels of sequence identity in all four isolates exhibited preferentially higher protein expression in only one of the four isolates, suggesting differential regulation among the isolates. It is possible to analyze comparative protein expression in more distantly related isolates in the context of WGS data, though the results are more complex to interpret than when isolates are clonal or very closely related. Comparative proteomic analysis produced log2 fold expression data suggestive of regulatory differences among isolates, indicating that it may be useful as a hypothesis generation exercise to identify regulated proteins and regulatory pathways for more detailed analysis.

Impact of the choice of reference genome on the ability of the core genome SNV methodology to distinguish strains of Salmonella enterica serovar Heidelberg.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. The core genome single nucleotide variant pipeline (cgSNV) is one of several whole genome based sequence typing methods used for the laboratory investigation of foodborne pathogens. SNV detection using this method requires a reference genome. The purpose of this study was to investigate the impact of the choice of the reference genome on the cgSNV-informed phylogenetic clustering and inferred isolate relationships. We found that using a draft or closed genome of S. Heidelberg as reference did not impact the ability of the cgSNV methodology to differentiate among 145 S. Heidelberg isolates involved in foodborne outbreaks. We also found that using a distantly related genome such as S. Dublin as choice of reference led to a loss in resolution since some sporadic isolates were found to cluster together with outbreak isolates. In addition, the genetic distances between outbreak isolates as well as between outbreak and sporadic isolates were overall reduced when S. Dublin was used as the reference genome as opposed to S. Heidelberg.

A Syst-OMICS Approach to Ensuring Food Safety and Reducing the Economic Burden of Salmonellosis.

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 database (SalFoS), at https://salfos.ibis.ulaval.ca/. Here, we present our strategy and the analysis of the first 3,377 genomes. Our data will be used to draw potential links between strains found in fresh produce, humans, animals and the environment. The ultimate goals are to understand how Salmonella evolves over time, improve the accuracy of diagnostic methods, develop control methods in the field, and identify prognostic markers for evidence-based decisions in epidemiology and surveillance.

Draft Genome Sequence of a Necrotoxigenic Escherichia coli Isolate.

Here, we present the draft genome sequence of a necrotoxigenic Escherichia coli strain isolated from a patient following a very rapidly evolving, lethal necrotizing fasciitis.