Application of a High-Throughput Targeted Sequence AmpliSeq Procedure to Assess the Presence and Variants of Virulence Genes in Salmonella.

We have developed a targeted, amplicon-based next-generation sequencing method to detect and analyze 227 virulence genes (VG) of Salmonella (AmpliSeqSalm_227VG) for assessing the pathogenicity potential of Salmonella. The procedure was developed using 80 reference genomes representing 75 epidemiologically-relevant serovars associated with human salmonellosis. We applied the AmpliSeqSalm_227VG assay to (a) 35 previously characterized field strains of Salmonella consisting of serovars commonly incriminated in foodborne illnesses and (b) 34 Salmonella strains with undisclosed serological or virulence attributes, and were able to divide Salmonella VGs into two groups: core VGs and variable VGs. The commonest serovars causing foodborne illnesses such as Enteritidis, Typhimurium, Heidelberg and Newport had a high number of VGs (217-227). In contrast, serovars of subspecies not commonly associated with human illnesses, such as houtenae, arizonae and salame, tended to have fewer VGs (177-195). Variable VGs were not only infrequent but, when present, displayed considerable sequence variation: safC, sseL, sseD, sseE, ssaK and stdB showed the highest variation and were linked to strain pathogenicity. In a chicken infection model, VGs belonging to rfb and sse operons showed differences and were linked with pathogenicity. The high-throughput, targeted NGS-based AmpliSeqSalm_227VG procedure provided previously unknown information about variation in select virulence genes that can now be applied to a much larger population of Salmonella for evaluating pathogenicity of various serovars of Salmonella and for risk assessment of foodborne salmonellosis.

Combined use of Oxford Nanopore and Illumina sequencing yields insights into soybean structural variation biology.

BACKGROUND: Structural variants (SVs), including deletions, insertions, duplications, and inversions, are relatively long genomic variations implicated in a diverse range of processes from human disease to ecology and evolution. Given their complex signatures, tendency to occur in repeated regions, and large size, discovering SVs based on short reads is challenging compared to single-nucleotide variants. The increasing availability of long-read technologies has greatly facilitated SV discovery; however, these technologies remain too costly to apply routinely to population-level studies. Here, we combined short-read and long-read sequencing technologies to provide a comprehensive population-scale assessment of structural variation in a panel of Canadian soybean cultivars. RESULTS: We used Oxford Nanopore long-read sequencing data (~12× mean coverage) for 17 samples to both benchmark SV calls made from Illumina short-read data and predict SVs that were subsequently genotyped in a population of 102 samples using Illumina data. Benchmarking results show that variants discovered using Oxford Nanopore can be accurately genotyped from the Illumina data. We first use the genotyped deletions and insertions for population genetics analyses and show that results are comparable to those based on single-nucleotide variants. We observe that the population frequency and distribution within the genome of deletions and insertions are constrained by the location of genes. Gene Ontology and PFAM domain enrichment analyses also confirm previous reports that genes harboring high-frequency deletions and insertions are enriched for functions in defense response. Finally, we discover polymorphic transposable elements from the deletions and insertions and report evidence of the recent activity of a Stowaway MITE. CONCLUSIONS: We show that structural variants discovered using Oxford Nanopore data can be genotyped with high accuracy from Illumina data. Our results demonstrate that long-read and short-read sequencing technologies can be efficiently combined to enhance SV analysis in large populations, providing a reusable framework for their study in a wider range of samples and non-model species.

Salmonella enterica subsp. enterica virulence potential can be linked to higher survival within a dynamic in vitro human gastrointestinal model.

Salmonella enterica subsp. enterica is one of the leading causes of human foodborne infections and several outbreaks are now associated with the consumption of fresh fruit and vegetables. This study aims at evaluating whether Salmonella virulence can be linked to an enhanced ability to survive successive digestive environments. Thirteen S. enterica strains were selected according to high and low virulence phenotypes. Lettuce inoculated separately with each S. enterica strain was used as food matrix in the TNO gastrointestinal model (TIM-1) of the human upper gastrointestinal tract. During the passage in the stomach, counts determined using PMA-qPCR were 2-5 logs higher than the cultivable counts for all strains indicating the presence of viable but non-cultivable cells. Bacterial growth was observed in the duodenum compartment after 180 min for all but one strain and growth continued into the ileal compartment. After passage through the simulated gastrointestinal tract, both virulent and avirulent S. enterica strains survived but high virulence strains had a significantly (p = 0.004) better average survival rate (1003 %-3753 %) than low virulence strains (from 25 % to 3730%). The survival rates of S. enterica strains could be linked to the presence of genes associated with acid and bile resistance and their predicted products. The presence of single nucleotide polymorphisms may also impact the function of virulence associated genes and play a role in the resulting phenotype. These data provide an understanding of the relationship between measured virulence potential and survival of S. enterica during dynamic simulated gastrointestinal transit.

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection.

The lactococcal virulent phage p2 is a model for studying the Skunavirus genus, the most prevalent group of phages causing milk fermentation failures in cheese factories worldwide. This siphophage infects Lactococcus lactis MG1363, a model strain used to study Gram-positive lactic acid bacteria. The structural proteins of phage p2 have been thoroughly described, while most of its non-structural proteins remain uncharacterized. Here, we developed an integrative approach, making use of structural biology, genomics, physiology, and proteomics to provide insights into the function of ORF47, the most conserved non-structural protein of unknown function among the Skunavirus genus. This small phage protein, which is composed of three α-helices, was found to have a major impact on the bacterial proteome during phage infection and to significantly reduce the emergence of bacteriophage-insensitive mutants.

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.

Culture-Dependent Bioprospecting of Bacterial Isolates From the Canadian High Arctic Displaying Antibacterial Activity.

The goal of this study was to isolate, screen, and characterize Arctic microbial isolates from Expedition Fjord, Axel Heiberg Island, Nunavut, Canada capable of inhibiting the growth of foodborne and clinically relevant pathogens. Arctic bacteria were isolated from twelve different high Arctic habitats pertaining to active layer permafrost soil, saline spring sediments, lake sediments, and endoliths. This was achieved using (1) the cryo-iPlate, an innovative in situ cultivation device within active layer permafrost soil and (2) bulk plating of Arctic samples by undergraduate students that applied standard culturing methods. To mitigate the possibility of identifying isolates with already-known antibacterial activities, a cell-based dereplication platform was used. Ten out of the twelve Arctic habitats tested were found to yield cold-adapted isolates with antibacterial activity. Eight cold-adapted Arctic isolates were identified with the ability to inhibit the entire dereplication platform, suggesting the possibility of new mechanisms of action. Two promising isolates, initially cultured from perennial saline spring sediments and from active layer permafrost soil (Paenibacillus sp. GHS.8.NWYW.5 and Pseudomonas sp. AALPS.10.MNAAK.13, respectively), displayed antibacterial activity against foodborne and clinically relevant pathogens. Paenibacillus sp. GHS.8.NWYW.5 was capable of inhibiting methicillin resistant and susceptible Staphylococcus aureus (MRSA and MSSA), Listeria monocytogenes, Salmonella enterica and Escherichia coli O157:H7. Pseudomonas sp. AALPS.10.MNAAK.13 was observed to have antagonistic activity against MRSA, MSSA, Acinetobacter baumanii, Enterococcus faecium, and Enterococcus faecalis. After whole genome sequencing and mining, the genome of Paenibacillus sp. GHS.8.NWYW.5 was found to contain seven putative secondary metabolite biosynthetic gene clusters that displayed low homology (<50% coverage, <30% identity, and e-values > 0) to clusters identified within the genome of the type strain pertaining to the same species. These findings suggest that cold-adapted Arctic microbes may be a promising source of novel secondary metabolites for potential use in both industrial and medical settings.

Species interactions and distinct microbial communities in high Arctic permafrost affected cryosols are associated with the CH4 and CO2 gas fluxes.

Microbial metabolism of the thawing organic carbon stores in permafrost results in a positive feedback loop of greenhouse gas emissions. CO2 and CH4 fluxes and the associated microbial communities in Arctic cryosols are important in predicting future warming potential of the Arctic. We demonstrate that topography had an impact on CH4 and CO2 flux at a high Arctic ice-wedge polygon terrain site, with higher CO2 emissions and lower CH4 uptake at troughs compared to polygon interior soils. The pmoA sequencing suggested that USCα cluster of uncultured methanotrophs is likely responsible for observed methane sink. Community profiling revealed distinct assemblages across the terrain at different depths. Deeper soils contained higher abundances of Verrucomicrobia and Gemmatimonadetes, whereas the polygon interior had higher Acidobacteria and lower Betaproteobacteria and Deltaproteobacteria abundances. Genome sequencing of isolates from the terrain revealed presence of carbon cycling genes including ones involved in serine and ribulose monophosphate pathways. A novel hybrid network analysis identified key members that had positive and negative impacts on other species. Operational Taxonomic Units (OTUs) with numerous positive interactions corresponded to Proteobacteria, Candidatus Rokubacteria and Actinobacteria phyla, while Verrucomicrobia and Acidobacteria members had negative impacts on other species. Results indicate that topography and microbial interactions impact community composition.

Genomic characterisation of an international Pseudomonas aeruginosa reference panel indicates that the two major groups draw upon distinct mobile gene pools.

Pseudomonas aeruginosa is an important opportunistic pathogen, especially in the context of infections of cystic fibrosis (CF). In order to facilitate coordinated study of this pathogen, an international reference panel of P. aeruginosa isolates was assembled. Here we report the genome sequencing and analysis of 33 of these isolates and 7 reference genomes to further characterise this panel. Core genome single nucleotide variant phylogeny demonstrated that the panel strains are widely distributed amongst the P. aeruginosa population. Common loss-of-function mutations reported as adaptive during CF (such as in mucA and mexA) were identified amongst isolates from chronic respiratory infections. From the 40 strains analysed, 37 unique resistomes were predicted, based on the Resistance Gene Identifier method using the Comprehensive Antibiotic Resistance Database. Notably, hierarchical clustering and phylogenetic reconstructions based on the presence/absence of genomic islands (GIs), prophages and other regions of genome plasticity (RGPs) supported the subdivision of P. aeruginosa into two main groups. This is the largest, most diverse analysis of GIs and associated RGPs to date, and the results suggest that, at least at the largest clade grouping level (group 1 vs group 2), each group may be drawing upon distinct mobile gene pools.

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.

Whole-Genome Sequencing of Lactobacillus Species from Two Commercial Probiotic Products.

Eight Lactobacillus strains, each intrinsically resistant to an antibiotic, were isolated from two commercial probiotic products. Whole-genome sequencing identified two efflux transporters, a multidrug and extrusion protein (MATE) efflux transporter, and LmrCD, which may contribute to their intrinsic antibiotic resistance and may therefore facilitate their survival in the intestinal microbiota following antibiotic therapy.