Evolution of Protein Molecules

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis.

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

During the past two decades, the technological progress of whole-genome sequencing (WGS) had changed the fields of Environmental Microbiology and Biotechnology, and, currently, is changing the underlying principles, approaches, and fundamentals of Public Health, Epidemiology, Health Economics, and national productivity. Today’s WGS technologies are able to compete with conventional techniques in cost, speed, accuracy, and resolution for day-to-day control of infectious diseases and outbreaks in clinical laboratories and in long-term epidemiological investigations. WGS gives rise to an exciting future direction for personalized Genomic Epidemiology. One of the most vital and growing public health problems is the emerging and re-emerging of multidrug-resistant (MDR) bacterial infections in the communities and healthcare settings, reinforced by a decline in antimicrobial drug discovery. In recent years, retrospective analysis provided by WGS has had a great impact on the identification and tracking of MDR microorganisms in hospitals and communities. The obtained genomic data are also important for developing novel easy-to-use diagnostic assays for clinics, as well as for antibiotic and therapeutic development at both the personal and population levels. At present, this technology has been successfully applied as an addendum to the real-time diagnostic methods currently used in clinical laboratories. However, the significance of WGS for public health may increase if: (a) unified and user-friendly bioinformatics toolsets for easy data interpretation and management are established, and (b) standards for data validation and verification are developed. Herein, we review the current and future impact of this technology on diagnosis, prevention, treatment, and control of MDR infectious bacteria in clinics and on the global scale.

/pdf/whole-genome-sequencing-targets-drug-resistant-bacterial-2g0m9gply6.pdf

Whole-genome sequencing targets drug-resistant bacterial infections.

Background
Non-typhoid Salmonella Typhimurium (S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable.

/pdf/bacteriophage-tailspike-protein-based-assay-to-monitor-phase-g9vnvaowe0.pdf

Bacteriophage tailspike protein based assay to monitor phase variable glucosylations in Salmonella O-antigens

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

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.

https://journals.asm.org/doi/pdf/10.1128/JCM.01410-14

Development of a New Molecular Subtyping Tool for Salmonella enterica Serovar Enteritidis Based on Single Nucleotide Polymorphism Genotyping Using PCR

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. 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. 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.

/pdf/high-resolution-assembly-and-characterization-of-genomes-of-4gzgnvvh8a.pdf

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

Listeria monocytogenes is the etiological agent for an often fatal foodborne illness known as listeriosis. Here, we present the complete genome sequences of 12 L. monocytogenes isolates representing the three most common serotypes of this pathogen (1/2a, 1/2b, and 4b), collected in Canada from different food products and environmental sources.

Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada.

Enteritidis based on single nucleotide polymorphism-polymerase chain reaction

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Development of a New Molecular Subtyping Tool for Salmonella enterica Serovar Enteritidis Based on Single Nucleotide Polymorphism Genotyping Using PCR

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

Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada.

Enteritidis based on single nucleotide polymorphism-polymerase chain reaction