ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data.

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 serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella, and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92-97 % for O-antigens and 98-100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75-91 % for O-antigens and 62-90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

Population structure, case clusters, and genetic lesions associated with Canadian Salmonella 4,[5],12:i:- isolates.

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 genetic lesions causing the Salmonella 4,[5]:12:i:- genotype were identified and assessed with regards to their distribution in the population of 811 Salmonella 4,[5]:12:i:- and S. Typhimurium isolates, their geographical and temporal distribution, and their association with non-human sources. Several clades were identified in the population structure, and the largest two were associated almost exclusively with a short prophage insertion and insertion of a mobile element carrying loci encoding antibiotic and mercury resistance. IS26-mediated deletions and fljB point mutants appeared to spread clonally. 'Inconsistent' Salmonella 4,[5]:12:i:- isolates associated with specific, single amino acid changes in fljA and hin were found in a single clade composed of water, shellfish, and avian isolates. Inclusion of isolates from different case clusters identified previously by PFGE validated some of the clusters and invalidated others. Some wgMLST clusters of clinical isolates composed of very closely related isolates contained an isolate(s) with a different genetic lesion, suggesting continuing mobility of the implicated element responsible. Such cases may need to be left out of epidemiological investigations until sufficient numbers of isolates are included that statistical significance of association with sources is not impaired. Non-human sources were frequently found in or near clinical case clusters. Prospective surveillance and WGS of non-human sources and retrospective analysis by WGS of isolates from existing culture collections provides data critical for epidemiological investigations of food- and waterborne outbreaks.

Prevalence and phenotypic characterization of Salmonella enterica isolates from three species of wild marine turtles in Grenada, West Indies.

BACKGROUND AND AIM: Salmonella enterica causes enteric disease in mammals and may potentially be transmitted from marine turtles that shed the pathogen in the environment. Marine turtle-associated human salmonellosis is a potential public health concern in Grenada, as the island supports populations of leatherback turtles (Dermochelys coriacea), hawksbill turtles (Eretmochelys imbricata), and green turtles (Chelonia mydas) that interface with veterinarians and conservation workers, the local population, and the thousands of visitors that frequent the island yearly. To date, the prevalence of S. enterica has only been examined in a small subset of marine turtles in the Caribbean and no studies have been conducted in Grenada. The aim of this study was to quantify the prevalence of S. enterica in leatherback, hawksbill and green turtles in Grenada, characterize phenotypes and DNA profiles, and explore the potential risk to human health in the region. MATERIALS AND METHODS: A total of 102 cloacal swabs were obtained from nesting leatherback turtles and foraging hawksbill and green turtles. Samples were cultured on enrichment and selective media and isolates were phenotypically characterized using serotyping, pulsed-phase gel electrophoresis, and antibiotic susceptibility. Enrichment broths were additionally screened by polymerase chain reaction (PCR) using S. enterica-specific primers. RESULTS: S. enterica was cultured from 15/57 (26.3%) leatherback turtles, 0/28 hawksbill, and 0/17 green turtles. This included S. enterica serovars Montevideo, S. I:4,5,12:i:-, Salmonella Typhimurium, Salmonella Newport, S. I:6,7:-:-, and S. I:4,5,12:-:-. Five/15 leatherback turtles carried multiple serovars. Eight pulsotype groups were identified with multiple clustering; however, there was no clear association between pulsotype group and serotype profile. Five/71 isolates showed resistance to streptomycin or ampicillin. Twenty-one/57 leatherback turtles, 14/28 hawksbill turtles, and 8/17 green turtles tested positive for S. enterica by quantitative PCR. CONCLUSION: Nesting leatherback turtles actively shed S. enterica and poses a risk for zoonosis; however, the presence of viable pathogen in green and hawksbill species is unclear. These findings help elucidate the role of marine turtles as potential sources of zoonotic S. enterica and provide baseline data for one health research in Grenada and the wider Caribbean region.

Complete Genome Sequences for 36 Canadian Salmonella enterica Serovar Typhimurium and I 1,4,[5],12:i:- Isolates from Clinical and Animal Sources.

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.

The Salmonella enterica Plasmidome as a Reservoir of Antibiotic Resistance.

The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.

Development of a multi-locus typing scheme for an Enterobacteriaceae linear plasmid that mediates inter-species transfer of flagella.

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 Salmonella Typhi, was found to encode a flagellar operon that can mediate phase variation, which results in the rare z66 flagella phenotype. The identification and tracking of homologs of pBSSB1 is limited because it falls outside the normal replicon typing schemes for plasmids. Here we report the generation of nine new pBSSB1-family sequences using Illumina and Nanopore sequence data. Homologs of pBSSB1 were identified in 154 genomes representing 25 distinct serotypes from 67,758 Salmonella public genomes. Pangenome analysis of pBSSB1-family contigs was performed using roary and we identified three core genes amenable to a minimal pMLST scheme. Population structure analysis based on the newly developed pMLST scheme identified three major lineages representing 35 sequence types, and the distribution of these sequence types was found to span multiple serovars across the globe. This in silico pMLST scheme has shown utility in tracking and subtyping pBSSB1-family plasmids and it has been incorporated into the plasmid MLST database under the name "pBSSB1-family".

An improved DNA array-based classification method for the identification of Salmonella serotypes shows high concordance between traditional and genotypic testing.

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 and unambiguous molecular serotyping results for 112 serotypes of Salmonella which were verified both in silico and in vitro. Improvements included an expansion of the probe sets along with a new classifier tool for prediction of individual antigens and overall serotype from the array probe intensity results. The array classifier and probe sequences were validated in silico to high concordance using 36,153 draft genomes of diverse Salmonella serotypes assembled from public repositories. We obtained correct and unambiguous serotype assignments for 31,924 (88.30%) of the tested samples and a further 3,916 (10.83%) had fully concordant antigen predictions but could not be assigned to a single serotype. The SGSA v. 2 can directly use bacterial colonies with a limit of detection of 860 CFU/mL or purified DNA template at a concentration of 1.0 x 10-1 ng/µl. The SGSA v. 2 was also validated in the wet laboratory and certified using panel of 406 samples representing 185 different serotypes with correct antigen and serotype determinations for 60.89% of the panel and 18.31% correctly identified but an ambiguous overall serotype determination.

Salmonella enterica Prophage Sequence Profiles Reflect Genome Diversity and Can Be Used for High Discrimination Subtyping.

Non-typhoidal Salmonella is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of Salmonella responsible for disease outbreaks is crucial to minimize infections and eliminate ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated Salmonella strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in Salmonella. In this study, the prophage sequence diversity in different Salmonella serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of S. enterica representing 151 Salmonella serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in S. enterica genomes. Prophage sequences were highly variable among S. enterica serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of S. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating S. enterica subtypes during foodborne outbreaks.

Serovars and antimicrobial resistance of non-typhoidal Salmonella isolated from non-diarrhoeic dogs in Grenada, West Indies.

Non-typhoidal salmonellosis remains an important public health problem worldwide. Dogs may harbour Salmonella in their intestines and can easily shed Salmonella in the environment with the possibility of transmission to humans. Thus, monitoring is essential to understand the role of dogs in zoonotic transmission. The objectives of this study were to determine the shedding of Salmonella by owned, apparently healthy dogs in Grenada, West Indies, to identify the serovars, and to examine their antimicrobial susceptibility profiles. Faecal samples collected during August to October, 2016 from 144 non-diarrhoeic owned dogs were examined by enrichment and selective culture for the presence of Salmonella spp. Eight (5.6%) of the tested animals were culture positive, yielding 35 Salmonella isolates that belonged to six serovars of Salmonella enterica subspecies enterica. These were serovars Arechavaleta from two dogs, Arechavaleta and Montevideo from one dog, and Javiana, Rubislaw, Braenderup and Kiambu from one dog each. All these serovars have been reported as causes of human salmonellosis globally. Antimicrobial susceptibility tests on 35 isolates showed absence of resistance to the currently used drugs for cases of human salmonellosis, including ciprofloxacin and cefotaxime. One isolate (2.9%) was resistant to neomycin, two isolates (5.7%) showed intermediate susceptibility to neomycin, and another (2.9%) had intermediate susceptibility to tetracycline. This is the first report of isolation and antimicrobial susceptibilities of non-typhoidal Salmonella serovars from dogs in Grenada. This study shows that dogs in Grenada may be involved in the epidemiology of salmonellosis.

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.

Genetic analysis of invasive pneumococcal isolates from children in Ontario, Canada, 2007-2012.

BACKGROUND: Multilocus sequence typing (MLST) is commonly used to understand the genetic background of invasive pneumococcal disease (IPD) isolates. This study was conducted to identify serotype and genetic change among IPD isolates in Canadian children following vaccine use. METHODS: Clinical isolates collected from children ≤5 years old of Ontario, Canada with IPD during 2007-2012 were characterized with serotyping, multilocus sequence typing and antimicrobial susceptibility testing. RESULTS: One year after 13-valent pneumococcal conjugate vaccine (PCV13) implementation, a decline in 19A and 7F was observed in 2012, coincident with the rise of serogroup 15 and 22F. Clonal complex (CC) 199, CC320 and CC695 are 3 major CCs in 19A (74%). From 2007 to 2012, clonal shift was detected in the 19A population as CC320 and CC199 declined, whereas CC695 rose to a majority. Genetically, serogroup 15 was composed of 2 CCs and 7 sequence types (STs), making it more diverse than serotypes 3, 7F and 22F. Interestingly, 60% of 15C isolates were a novel ST, suggesting high single nucleotide polymorphism frequency in house-keeping genes of 15C. Several newly appeared STs found in 19A and 15 indicate the possibility of recent serotype switching events. CONCLUSION: Genetic shift because of PCV13 impact may have resulted in the decline of 19A in IPD. Recent rise of serogroup 15 infections in children could be because of its selective advantage conferred by genetic diversity, frequent recombination in the population plus drug resistance potential related to CC63 genotype. Close monitoring of serotype replacement and genetic change in IPD among children post-PCV13 is warranted.

Draft Genome Sequence of Bacillus megaterium Type Strain ATCC 14581.

Bacillus megaterium is a Gram-positive, rod-shaped, spore-forming bacterium of biotechnological importance. Here, we report a 5.7-Mbp draft genome sequence of B. megaterium ATCC 14581, which is the type strain of the species.

Comparative Genomic Analyses of Streptococcus pseudopneumoniae Provide Insight into Virulence and Commensalism Dynamics.

Streptococcus pseudopneumoniae (SPPN) is a recently described species of the viridans group streptococci (VGS). Although the pathogenic potential of S. pseudopneumoniae remains uncertain, it is most commonly isolated from patients with underlying medical conditions, such as chronic obstructive pulmonary disease. S. pseudopneumoniae can be distinguished from the closely related species, S. pneumoniae and S. mitis, by phenotypic characteristics, including optochin resistance in the presence of 5% CO2, bile insolubility, and the lack of the pneumococcal capsule. Previously, we reported the draft genome sequence of S. pseudopneumoniae IS7493, a clinical isolate obtained from an immunocompromised patient with documented pneumonia. Here, we use comparative genomics approaches to identify similarities and key differences between S. pseudopneumoniae IS7493, S. pneumoniae and S. mitis. The genome structure of S. pseudopneumoniae IS7493 is most closely related to that of S. pneumoniae R6, but several recombination events are evident. Analysis of gene content reveals numerous unique features that distinguish S. pseudopneumoniae from other streptococci. The presence of loci for competence, iron transport, pneumolysin production and antimicrobial resistance reinforce the phylogenetic position of S. pseudopneumoniae as an intermediate species between S. pneumoniae and S. mitis. Additionally, the presence of several virulence factors and antibiotic resistance mechanisms suggest the potential of this commensal species to become pathogenic or to contribute to increasing antibiotic resistance levels seen among the VGS.

Whole-genome sequence of Streptococcus pseudopneumoniae isolate IS7493.

Streptococcus pseudopneumoniae is a member of the viridans group streptococci (VGS) whose pathogenic significance is unclear. We announce the complete genome sequence of S. pseudopneumoniae IS7493. The genome sequence will assist in the characterization of this new organism and facilitate the development of accurate diagnostic assays to distinguish it from Streptococcus pneumoniae and Streptococcus mitis.

Acquisition of haemoglobin-bound iron by strains of the Actinobacillus minor/"porcitonsillarum" complex.

Members of the Actinobacillus minor/"porcitonsillarum" complex are common inhabitants of the swine respiratory tract. Although avirulent or of low virulence for pigs, these organisms, like pathogens, do grow in vivo and must, therefore, be able to acquire iron within the host. Here, we investigated the abilities of six members of the A. minor/"porcitonsillarum" complex to acquire iron from transferrin and various haemoglobins. Using growth assays, all six strains were shown to acquire iron from porcine, bovine and human haemoglobins but not from porcine transferrin. Analyses of whole genome sequences revealed that A. minor strains NM305(T) and 202, unlike the swine-pathogenic actinobacilli, A. pleuropneumoniae and A. suis, lack not only the transferrin-binding protein genes, tbpA and tbpB, but also the haemoglobin-binding protein gene, hgbA. Strains NM305(T) and 202, however, were found to possess other putative haemin/haemoglobin-binding protein genes that were predicted to encode mature proteins of ∼ 72 and ∼ 75 kDa, respectively. An affinity procedure based on haemin-agarose allowed the isolation of ∼ 65 and ∼ 67 kDa iron-repressible outer membrane polypeptides from membranes derived from strains NM305(T) and 202, respectively, and mass spectrometry revealed that these polypeptides were the products of the putative haemin/haemoglobin-binding protein genes. PCR approaches allowed the amplification and sequencing of homologues of both haemin/haemoglobin-binding protein genes from each of the other four strains, strains 33PN and 7ATS of the A. minor/"porcitonsillarum" complex and "A. porcitonsillarum" strains 9953L55 and 0347, suggesting that such proteins are involved in the utilization of haemoglobin-bound iron, presumably as surface receptors, by all six strains investigated.

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown.