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TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum.

ABSTRACT: Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N6(GTG)4, N6(CAC)4, N6(CGG)4, N6(CCG)4 and N6(CTG)4, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N6(GTG)4 primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.

SUBMITTER: Majchrzak M

PROVIDER: S-EPMC4204001 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:BACKGROUND: Salmonella enterica subsp. enterica serovar Virchow has been recognized as a significant health burden in Asia, Australia and Europe. In addition to its global distribution, S. Virchow is clinically significant due to the frequency at which it causes invasive infections and its association with outbreaks arising from food-borne transmission. Here, we examine the genome of an invasive isolate of S. Virchow SVQ1 (phage type 8) from an outbreak in southeast Queensland, Australia. In addition to identifying new potential genotyping targets that could be used for discriminating between S. Virchow strains in outbreak scenarios, we also aimed to carry out a comprehensive comparative analysis of the S. Virchow genomes. RESULTS: Genome comparisons between S. Virchow SVQ1 and S. Virchow SL491, a previously published strain, identified a high degree of genomic similarity between the two strains with fewer than 200 single nucleotide differences. Clustered Regularly Interspaced Palindromic Repeats (CRISPR) regions were identified as a highly variable region that could be used to discriminate between S. Virchow isolates. We amplified and sequenced the CRISPR regions of fifteen S. Virchow isolates collected from seven different outbreaks across Australia. We observed three allelic types of the CRISPR region from these isolates based on the presence/absence of the spacers and were able to discriminate S. Virchow phage type 8 isolates originating from different outbreaks. A comparison with 27 published Salmonella genomes found that the S. Virchow SVQ1 genome encodes 11 previously described Salmonella Pathogenicity Islands (SPI), as well as additional genomic islands including a remnant integrative conjugative element that is distinct from SPI-7. In addition, the S. Virchow genome possesses a novel prophage that encodes the Type III secretion system effector protein SopE, a key Salmonella virulence factor. The prophage shares very little similarity to the SopE prophages found in other Salmonella serovars suggesting an independent acquisition of sopE. CONCLUSIONS: The availability of this genome will serve as a genome template and facilitate further studies on understanding the virulence and global distribution of the S. Virchow serovar, as well as the development of genotyping methods for outbreak investigations.

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TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum.

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