Dataset Information

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

ABSTRACT: BACKGROUND: Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates. RESULTS: We analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method. CONCLUSIONS: For S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.

SUBMITTER: Shariat N

PROVIDER: S-EPMC3840669 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

Shariat Nikki N Sandt Carol H CH DiMarzio Michael J MJ Barrangou Rodolphe R Dudley Edward G EG

BMC microbiology 20131112

<h4>Background</h4>Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed ...[more]

PMID: 24219629

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Project description:BACKGROUND:Non-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. METHODOLOGY/PRINCIPAL FINDING:The genomes of 25 S. enterica isolates covering five serovars (ten Typhimurium including three monophasic 4,[5],12:i:, four Enteritidis, three Hadar, four Heidelberg and four Kentucky) were sequenced. Most serovars were clustered in strongly supported phylogenetic clades, except for isolates of serovar Enteritidis that were scattered throughout the tree. Plasmids of varying sizes were detected in several isolates independently of serovars. Genes associated with the IncF plasmid and the IncI1 plasmid were identified in twelve and four isolates, respectively, while genes associated with the IncQ plasmid were found in one isolate. The presence of numerous genes associated with Salmonella pathogenicity islands (SPIs) was also confirmed. Components of the type III and IV secretion systems (T3SS and T4SS) varied in different isolates, which could explain in part, differences of their pathogenicity in humans and/or persistence in broilers. Conserved clusters of genes in the T3SS were detected that could be used in designing effective strategies (diagnostic, vaccination or treatments) to combat Salmonella. Antibiotic resistance genes (CMY, aadA, ampC, florR, sul1, sulI, tetAB, and srtA) and class I integrons were detected in resistant isolates while all isolates carried multidrug efflux pump systems regardless of their antibiotic susceptibility profile. CONCLUSIONS/SIGNIFICANCE:This study showed that the predominant Salmonella serovars in broiler chickens harbor genes encoding adhesins, flagellar proteins, T3SS, iron acquisition systems, and antibiotic and metal resistance genes that may explain their pathogenicity, colonization ability and persistence in chicken. The existence of mobile genetic elements indicates that isolates from a given serovar could acquire and transfer genetic material. Conserved genes in the T3SS and T4SS that we have identified are promising candidates for identification of diagnostic, antimicrobial or vaccine targets for the control of Salmonella in broiler chickens.

Project description:Salmonella enterica serovar Heidelberg is currently the 12th most common serovar of Salmonella enterica causing salmonellosis in the United States and results in twice the average incidence of blood infections caused by nontyphoidal salmonellae. Multiple outbreaks of salmonellosis caused by Salmonella Heidelberg resulted from the same poultry processor, which infected 634 people during 2013 and 2014. The hospitalization and invasive illness rates were 38% and 15%, respectively. We hypothesized that the outbreak strains of Salmonella Heidelberg had enhanced stress tolerance and virulence capabilities. We sourced nine food isolates collected during the outbreak investigation and three reference isolates to assess their tolerance to heat and sanitizers, ability to attach to abiotic surfaces, and invasiveness in vitro We performed RNA sequencing on three isolates (two outbreak-associated isolates and a reference Salmonella Heidelberg strain) with various levels of heat tolerance to gain insight into the mechanism behind the isolates' enhanced heat tolerance. We also performed genomic analyses to determine the genetic relationships among the outbreak isolates. Ultimately, we determined that (i) six Salmonella Heidelberg isolates associated with the foodborne outbreak had enhanced heat tolerance, (ii) one outbreak isolate with enhanced heat tolerance also had an enhanced biofilm-forming ability under stressful conditions, (iii) exposure to heat stress increased the expression of Salmonella Heidelberg multidrug efflux and virulence genes, and (iv) outbreak-associated isolates were likely transcriptionally primed to better survive processing stresses and, potentially, to cause illness.IMPORTANCE This study provides a deep analysis of the intrinsic stress tolerance and virulence capabilities of Salmonella Heidelberg that may have contributed to the length and severity of a recent salmonellosis outbreak. Additionally, this study provides a comprehensive analysis of the transcriptomic response of S. enterica strains to heat stress conditions and compares baseline stationary-phase gene expression among outbreak- and non-outbreak-associated Salmonella Heidelberg isolates. These data can be used in assay development to screen isolates for stress tolerance and subsequent survival. This study adds to our understanding of the strains associated with the outbreak and informs ongoing regulatory discussions on Salmonella in poultry.

Project description:BACKGROUND: Salmonella enterica subspecies I includes several closely related serovars which differ in host ranges and ability to cause disease. The basis for the diversity in host range and pathogenic potential of the serovars is not well understood, and it is not known how host-restricted variants appeared and what factors were lost or acquired during adaptations to a specific environment. Differences apparent from the genomic data do not necessarily correspond to functional proteins and more importantly differential regulation of otherwise identical gene content may play a role in the diverse phenotypes of the serovars of Salmonella. RESULTS: In this study a comparative analysis of the cytosolic proteins of serovars Typhimurium and Pullorum was performed using two-dimensional gel electrophoresis and the proteins of interest were identified using mass spectrometry. An annotated reference map was created for serovar Typhimurium containing 233 entries, which included many metabolic enzymes, ribosomal proteins, chaperones and many other proteins characteristic for the growing cell. The comparative analysis of the two serovars revealed a high degree of variation amongst isolates obtained from different sources and, in some cases, the variation was greater between isolates of the same serovar than between isolates with different sero-specificity. However, several serovar-specific proteins, including intermediates in sulphate utilisation and cysteine synthesis, were also found despite the fact that the genes encoding those proteins are present in the genomes of both serovars. CONCLUSION: Current microbial proteomics are generally based on the use of a single reference or type strain of a species. This study has shown the importance of incorporating a large number of strains of a species, as the diversity of the proteome in the microbial population appears to be significantly greater than expected. The characterisation of a diverse selection of strains revealed parts of the proteome of S. enterica that alter their expression while others remain stable and allowed for the identification of serovar-specific factors that have so far remained undetected by other methods.

Dataset Information

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

Publications

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

Similar Datasets

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