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. Numerous tools have been developed to analyse plasmids but the performance and functionality of the tools are variable. The MOB-suite was developed as a set of modular tools for reconstruction and typing of plasmids from draft assembly data to facilitate characterization of plasmids. Using a set of closed genomes with publicly available Illumina data, the MOB-suite identified contigs of plasmid origin with both high sensitivity and specificity (95 and 88 %, respectively). In comparison, plasmidfinder demonstrated high specificity (99 %) but limited sensitivity (50 %). Using the same dataset of 377 known plasmids, MOB-recon accurately reconstructed 207 plasmids so that they were assigned to a single grouping without other plasmid or chromosomal sequences, whereas plasmidSPAdes was only able to accurately reconstruct 102 plasmids. In general, plasmidSPAdes has a tendency to merge different plasmids together, with 208 plasmids undergoing merge events. The MOB-suite reduces the number of errors but produces more hybrid plasmids, with 84 plasmids undergoing both splits and merges. The MOB-suite also provides replicon typing similar to plasmidfinder but with the inclusion of relaxase typing and prediction of conjugation potential. The MOB-suite is written in Python 3 and is available from https://github.com/phac-nml/mob-suite.

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/.

BackgroundAdherent 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 gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype.ResultsWe sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype.ConclusionsUp to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

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 ‘host range’ and is usually described in qualitative terms of ‘narrow’ or ‘broad’. Understanding the host range potential of plasmids is of great interest due to their ability to disseminate traits such as AMR through bacterial populations and into human pathogens. We developed the MOB-suite to facilitate characterization of plasmids and introduced a whole-sequence-based classification system based on clustering complete plasmid sequences using Mash distances (https://github.com/phac-nml/mob-suite). We updated the MOB-suite database from 12 091 to 23 671 complete sequences, representing 17 779 unique plasmids. With advances in new algorithms for rapidly calculating average nucleotide identity (ANI), we compared clustering characteristics using two different distance measures – Mash and ANI – and three clustering algorithms on the unique set of plasmids. The plasmid nomenclature is designed to group highly similar plasmids together that are unlikely to have multiple representatives within a single cell. Based on our results, we determined that clusters generated using Mash and complete-linkage clustering at a Mash distance of 0.06 resulted in highly homogeneous clusters while maintaining cluster size. The taxonomic distribution of plasmid biomarker sequences for replication and relaxase typing, in combination with MOB-suite whole-sequence-based clusters have been examined in detail for all high-quality publicly available plasmid sequences. We have incorporated prediction of plasmid replication host range into the MOB-suite based on observed distributions of these sequence features in combination with known plasmid hosts from the literature. Host range is reported as the highest taxonomic rank that covers all of the plasmids which share replicon or relaxase biomarkers or belong to the same MOB-suite cluster code. Reporting host range based on these criteria allows for comparisons of host range between studies and provides information for plasmid surveillance.

eThe 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 and Remus, with double-stranded DNA (dsDNA) genomes of 131,333 bp and 134,643 bp, respectively. Despite their relatedness to Staphylococcus phages K, G1, ISP, and Twort and Listeria phages A511 and P100, Romulus and Remus can be proposed as isolates of a new species within the Twortlikevirus genus. A distinguishing feature for these phage genomes is the unique distribution of group I introns compared to that in other staphylococcal myoviruses. In addition, a hedgehog/intein domain was found within their DNA polymerase genes, and an insertion sequence-encoded transposase exhibits splicing behavior and produces a functional portal protein. From a phage therapy application perspective, Romulus and Remus infected approximately 70% of the tested S. aureus isolates and displayed promising lytic activity against these isolates. Furthermore, both phages showed a rapid initial adsorption and demonstrated biofilm-degrading capacity in a proof-of-concept experiment.

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 robustness of the data. Due to the critical importance of serovar information to public health, it is essential to have reliable serovar assignments available for all of the Salmonella records. The current study used a systematic assessment and curation of all Salmonella in the sequence read archive (SRA) to assess the state of the data and their utility. A total of 67 758 genomes were assembled de novo and quality-assessed for their assembly metrics as well as species and serovar assignments. A total of 42 400 genomes passed all of the quality criteria but 30.16 % of genomes were deposited without serotype information. These data were used to compare the concordance of reported and predicted serovars for two in silico prediction tools, multi-locus sequence typing (MLST) and the Salmonella in silico Typing Resource (SISTR), which produced predictions that were fully concordant with 87.51 and 91.91 % of the tested isolates, respectively. Concordance of in silico predictions increased when serovar variants were grouped together, 89.25 % for MLST and 94.98 % for SISTR. This study represents the first large-scale validation of serovar information in public genomes and provides a large validated set of genomes, which can be used to benchmark new bioinformatics tools.

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 defined genera (Phic3unalikevirus; L5likevirus) grouping, in total, 390 fully sequenced phage isolates. Many of the remainders are orphans which the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) chooses not to ascribe genus status at the time being.

plasmids to other Gram-negative pathogens, which might result in untreatable infections. Thus, the spread of mcr-1, especially into strains that are already resistant to carbapenems, must be stopped.