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/.
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.
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.
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".
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-tRNA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the structural proteins were identical among the four phages. Further proteomic analysis identified seven structural proteins responsible for tail fiber, tail tape measure protein, major capsid, portal protein as well as major and minor tail proteins. Bioinformatic analyses on the proteins revealed that genomes of AHP24, AHS24, AHP42 and AKS96 did not encode for bacterial virulence factors, integration-related proteins or antibiotic resistance determinants. All four phages were highly lytic to STEC O157:H7 with considerable potential as biocontrol agents. Comparative genomic, proteomic and phylogenetic analysis suggested that the four phages along with 17 T1-like phage genomes from database of National Center for Biotechnology Information (NCBI) can be assigned into a proposed subfamily "Tunavirinae" with further classification into five genera, namely "Tlslikevirus" (TLS, FSL SP-126), "Kp36likevirus" (KP36, F20), Tunalikevirus (T1, ADB-2 and Shf1), "Rtplikevirus" (RTP, vB_EcoS_ACG-M12) and "Jk06likevirus" (JK06, vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96, phiJLA23, phiKP26, phiEB49). The fact that the viruses related to JK06 have been isolated independently in Israel (JK06) (GenBank Assession #, NC_007291), Canada (vB_EcoS_Rogue1, AHP24, AHS24, AHP42, AKS96) and Mexico (phiKP26, phiJLA23) (between 2005 and 2011) indicates that these similar phages are widely distributed, and that horizontal gene transfer does not always prevent the characterization of bacteriophage evolution. With this new scheme, any new discovered phages with same type can be more properly identified. Genomic- and proteomic-based taxonomic classification of phages would facilitate better understanding phages diversity and genetic traits involved in phage evolution.
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 tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: “G7cvirus” (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), “Lit1virus” (LIT1, PA26 and vB_PaeP_C2-10_Ab09), “Sp58virus” (SP058 and SP076), and “Dss3virus” (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of “G7cvirus”, Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the “Enquartavirinae”.
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. Digestion significantly abated populations of viable coliforms in all six farms. Conjugative transfer of plasmids carrying β-lactamase genes from manure bacteria enriched overnight with buffered peptone containing 4 mg/liter cefotaxime into a β-lactam-sensitive green fluorescent protein (GFP)-labeled Escherichia coli recipient strain was evaluated in patch matings. Digestion significantly decreased the frequency of the horizontal transfer of ESBL genes. Twenty-five transconjugants were sequenced, revealing six distinct plasmids, ranging in size from 40 to 180 kb. A variety of ESBL genes were identified:
,
,
,
,
, and
.
was the most prevalent ESBL gene detected on plasmids harbored by transconjugants. Various mobile genetic elements were found located proximal to resistance genes. Ten gene targets, including
,
(A),
(B),
(B),
(F),
,
,
,
, and
, were quantified by quantitative PCR on a subset of 18 raw and 18 digested samples. Most targets were significantly more abundant in raw manure; however,
(B) and
(F) targets were more abundant in digested samples. Overall, on-farm digestion of dairy manure abated coliform bacteria, a number of antibiotic resistance-associated gene targets, and the potential for
conjugation of plasmids conferring resistance to extended-spectrum β-lactams and other classes of antibiotics into E. coli CV601.
Using livestock manure for fertilization can entrain antibiotic-resistant bacteria into soil. Manure on some dairy farms is anaerobically digested before being land applied. Recommending the widespread implementation of the practice should be founded on understanding the impact of this treatment on various endpoints of human health concern. Although lab-scale anaerobic treatments have shown potential for reducing the abundance of antibiotic resistance genes, there are very few data from commercial farms. Anaerobic digestion of manure on six dairy farms efficiently abated coliform bacteria, E. coli, and a majority of antibiotic resistance-associated gene targets. In addition, the conjugation potential of plasmids carrying ESBL genes into introduced E. coli strain CV601 was reduced. Overall, anaerobic digestion abated coliform bacteria, the genes that they carry, and the potential for ESBL-carrying plasmid transfer.
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.
Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking, and outbreak detection. To improve ...detection and reduce the burden of salmonellosis, several rapid and high-throughput molecular Salmonella serotyping methods have been developed.The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAP Salmonella serotyping assay), to the Salmonella genoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically important Salmonella serovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host range Salmonella serovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity of Salmonella serovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively.It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping of Salmonella.
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