Molecular typing of Salmonella enterica serovars of significance in Australia using MLVA and bacteriophage genes.
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Date
2013
Authors
Young, Chun Chun
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Heuzenroeder, Michael William
Barton, Mary Drvall
Barton, Mary Drvall
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Thesis
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Abstract
Salmonella enterica subspecies enterica is the leading causes of food-borne infections and outbreaks worldwide and are therefore routinely monitored in many countries. Both epidemiological surveillance and tracking outbreaks require typing systems to characterise bacterial isolates. Pulsed-field gel electrophoresis (PFGE) is the most widely applied molecular method for Salmonella outbreak epidemiological typing despite that the method being relatively labour-intensive, generating restriction patterns that are subjective in nature and can be difficult to analyse. Furthermore, the recent emergence of homogeneous Salmonella phage groups (e.g. S. Typhimurium DT104) has hampered the discriminatory power of PFGE in outbreak settings. The combination of these factors has led to the exploitation of alternative methods. Two of these emerging typing methods are multiple-locus variable-tandem repeat analysis (MLVA) and multiple amplification of prophage locus typing (MAPLT) that have been developed for the globally significant serovars: S. Typhimurium and S. Enteritidis. In this study, MLVA and MAPLT were being further investigated for use in other Salmonella serovars that are of significance to Australia. These serovars include S. Virchow, S. Bovismorbificans and S. Heidelberg. Overall, the developed MAPLT schemes demonstrated a comparable differentiating ability with PFGE for the serovars as a whole. However development of discriminative MLVA methods in the study was not always successful using the published MLVA loci, therefore prompting the need of examining the MLVA loci from the complete genomes of the serovars. Differentiation within predominating phage types of the studied serovars was also examined. Both MAPLT and PFGE displayed similar levels of intra-phage type differentiation suggesting the usefulness for outbreak investigations. However strains were separated optimally when using MAPLT in association with discriminative MLVA primers. The composite assay of MAPLT/MLVA for S. Bovismorbificans and S. Virchow were further examined in the retrospective outbreak studies. It was observed that outbreak isolates generally showed identical or highly similar MAPLT/MLVA profiles (one locus difference). The results suggested that MAPLT / MLVA could confirm the close genetic relationship between outbreak isolates. However result interpretation guidelines might need to be established individually for each serovar due to the differences in the relative genetic similarity displayed within each serovar. It is expected that further enhancement of the composite assays could easily be carried out even when the complete genomic data of the Salmonella serovars are lacking. It is an advantage that MAPLT could be improved through using a DOP-PCR (degenerate oligonucleotide primed-PCR) procedure to detect additional prophage loci for further strain differentiation. Overall, the study suggested that composite assays can also be developed for fine levels of differentiation of other Salmonella serovars to meet local epidemiological needs when required. Development of MAPLT involved genetic characterisation of phages residing in the studied serovars. It was noted that DNA elements shared between phages Gifsy-1 and Gifsy-2 were frequently detected from these serovars. Complete genome analysis were subsequently performed on a S. Virchow phage (PV10) and a S. Heidelberg phage (PH03), which demonstrated their high DNA similarity to Gifsy-1 and Gifsy-2 respectively. Further analysis demonstrated that Gifsys-related phages are possibly widespread within Salmonella. Since the phages were observed to carry different sets of virulence genes between serovars, this study hypothesised that Gifsy-related phages may have a significant role in shaping the epidemiology of Salmonella.
School/Discipline
School of Molecular and Biomedical Science
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2013
Provenance
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.