Closed Genome Sequences and Antimicrobial Resistance Profiles of Eight Wild Bird Salmonella Isolates Obtained with MinION and Illumina MiSeq Sequencing.

Complete genome sequences of eight isolates of Salmonella enterica subsp. enterica from Canadian wild birds were determined by MinION and Illumina MiSeq sequencing. Assembled chromosomes had an average size of 4,833,662 bp. Salmonella enterica serovar Worthington obtained from partridge and quail carried 267-kb plasmids, which contained multiple antimicrobial resistance genes.

A New Whole Genome Culture-Independent Diagnostic Test (WG-CIDT) for Rapid Detection of Salmonella in Lettuce.

The rapid detection of foodborne microbial pathogens contaminating fresh fruits and vegetables during the intervening period between harvest and consumption could revolutionize microbial quality assurance of food usually consumed raw and those with a limited shelf life. We have developed a sensitive, shotgun whole genome sequencing protocol capable of detecting as few as 1 colony forming unit (cfu) of Salmonella enterica serovar Typhimurium spiked on 25 g of lettuce. The Ion Torrent sequencing platform was used to generate reads of globally amplified DNA from microbes recovered from the surface of lettuce followed by bioinformatic analyses of the nucleotide sequences to detect the presence of Salmonella. The test is rapid and sensitive, and appropriate for testing perishable foods, and those consumed raw, for Salmonella contamination. The test has the potential to be universally applicable to any microbial contaminant on lettuce as long as a suitable bioinformatics pipeline is available and validated. A universal test is expected to pave the way for preventive and precision food safety and the re-shaping of the entire spectrum of food safety investigations from the current disease-limiting, reactive procedure to a proactive, disease prevention process.

Genome Sequences of 13 Isolates of Salmonella enterica Serovar Typhimurium var. Copenhagen Obtained from Wild Pigeons in Canada.

Pigeon-adapted strains of Salmonella enterica serovar Typhimurium var. Copenhagen phage types 2 and 99 obtained from the provinces of Alberta, British Columbia, and Ontario, Canada, were analyzed using whole-genome sequencing. All isolates contained the Salmonella virulence plasmid despite the low pathogenicity of this lineage in their avian host.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples.

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

Development of a new molecular subtyping tool for Salmonella enterica serovar Enteritidis based on single nucleotide polymorphism genotyping using PCR.

The lack of a sufficiently discriminatory molecular subtyping tool for Salmonella enterica serovar Enteritidis has hindered source attribution efforts and impeded regulatory actions required to disrupt its food-borne transmission. The underlying biological reason for the ineffectiveness of current molecular subtyping tools such as pulsed-field gel electrophoresis (PFGE) and phage typing appears to be related to the high degree of clonality of S. Enteritidis. By interrogating the organism's genome, we previously identified single nucleotide polymorphisms (SNP) distributed throughout the chromosome and have designed a highly discriminatory PCR-based SNP typing test based on 60 polymorphic loci. The application of the SNP-PCR method to DNA samples from S. Enteritidis strains (n = 55) obtained from a variety of sources has led to the differentiation and clustering of the S. Enteritidis isolates into 12 clades made up of 2 to 9 isolates per clade. Significantly, the SNP-PCR assay was able to further differentiate predominant PFGE types (e.g., XAI.0003) and phage types (e.g., phage type 8) into smaller subsets. The SNP-PCR subtyping test proved to be an accurate, precise, and quantitative tool for evaluating the relationships among the S. Enteritidis isolates tested in this study and should prove useful for clustering related S. Enteritidis isolates involved in outbreaks.

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis.

BACKGROUND: There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome. RESULTS: The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full-sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage. CONCLUSIONS: The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.