Date of Award

12-23-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

Nawab Alli

Second Advisor

Ashraf Khan

Abstract

Salmonella enterica is recognized worldwide as a significant cause of both human and animal disease. Recently, many random outbreaks of infections associated with the pathogens have been documented. Thus, state of the art molecular tools are necessary to conduct effective surveillance that would aid monitoring and prevention of disease. In this study, 110 Salmonella enterica serovars (Enteritidis & Javiana) strains isolated from food, environmental and clinical samples were analyzed for genetic diversity, antibiotic resistance, presence of virulence genes, plasmids, and plasmid replicon types. To assess the genetic diversity, pulsed-field gel electrophoresis (PFGE) fingerprinting, multiple-locus variable-number tandem repeat analysis (MLVA) and plasmid profiles were performed. Most of the isolates were sensitive to chloramphenicol, gentamicin, kanamycin, nalidixic acid, and sulfisoxazole, whereas some of the isolates were resistant to ampicillin and tetracycline. The isolates were screened by PCR for 19 virulence genes. Our data show that S. Javiana and S. Enteritidis isolated from foods and environmental samples carry the same virulence genes as clinical isolates. Furthermore, cdtB, pltA and pltB genes were found in all 50 S. Javiana isolates, suggesting that the CdtB toxin may contribute to its pathogenesis. In this study, some of the S. Javiana isolates carried one or more large plasmids of approximately 30, 38, 58 and 80 kb. However, all S. Enteritidis isolates carried a 58 kb plasmid, type Inc/FIIA. The PFGE fingerprinting of 50 S. Javiana indicated several foods isolates that clustered with clinical isolates. However, PFGE was not helpful in discriminating the most common S. Enteritidis. MLVA was powerful technique to genotype S. Enteritidis isolates from poultry house and clinical sources, with 18 MLVA patterns detected among the 60 isolates. This study provides data that support the potential transmission of S. Javiana and S. Enteritidis virulence factors from food and environmental sources to cause infections in humans. The presence of cdtB, pltA and pltB genes in S. Javiana suggests the importance of these genes in these serovar and it may play an important role in the virulence of the pathogens. To determine the function of cytolethal distending toxin genes in S. Javiana wild type and mutants of cytolethal distending toxin genes, cloning of these genes in expression vector, expression and purification of S. Javiana recombinant CdtB, PltA and PltB proteins were undertaken. For in-vitro studies, HeLa cells were infected with a wild type S. Javiana, its isogenic ∆,cdtB, ∆,pltA and ∆,pltB and purified recombinant proteins CdtB, PltA and PltB to determine cell cycle arrest, cytoplasmic distension, and DNase activity. These results indicated that HeLa cells infected with S. Javiana wild type strain caused G2/M cell cycle arrest and distension of cytoplasm and nuclei. However, cells infected with ∆,cdtB and ∆,pltA showed no distension.The complemented ∆,cdtB and ∆,pltA Salmonella strains showed activity like wild type. We demonstrated that purified His6-tagged CdtB alone is capable of inducing typical signs of cytolethal distension toxin such as cell cycle arrest, cytoplasmic distension and DNase activity. These results indicate that S. Javiana CdtB is likely to play an important role during host infection and disease.

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