Title of Thesis

Detection, Molecular Analysis And Expressional Studies Of Local Vi Negative Strains Of Salmonella Typhi

Author(s)

Yasra Sarwar

Abstract
Typhoid is one of the most common infectious diseases in the developing countries especially in South Asian region including Pakistan. It is caused by Salmonella enterica serovar Typhi, an exclusively human pathogen.The virulence of S. Typhi is attributed to the Vi capsular antigen. The synthesis and transportation proteins of the Vi capsular polysaccharide of S.Typhi are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of S. Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative S.Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive S. Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used. The infectivity of Vi negative and Vi positive strains was tested using HeLa cell monolayers and compared the disease producing potential of the variant strains of S. Typhi. No difference was observed in ability of Vi negative and Vi positive strains to invade HeLa cell monolayer. Drug resistance pattern of Vi positive and Vi negative strains of S. Typhi was compared and no significant difference in the resistance against antibiotics of both variants of the pathogen was observed indicating that Vi negative S. Typhi strains are at par with their Vi positive counterparts as far as resistance to antimicrobials is concerned.

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1.1 Epidemiology
1.2 The Bacterium: Salmonella enterica serovar Typhi
1.3 Genome sequence of S. Typhi strains CT18 and Ty2
1.4 Genetic organization of virulence genes
1.5 Emergence of Vi negative strains of S. Typhi
1.6 Pathogenesis of Salmonella enterica serovar Typhi
1.7Antibiotic resistance
1.8 Diagnosis of typhoid
1.9 Aims and Objectives

2.1 Selection of samples
2.2 Isolation and biochemical characterization of bacteria from blood samples
2.3 Detection of typhoid antibodies in blood samples
2.4 DNA extraction from bacterial cultures
2.5 DNA extraction from blood samples
2.6 Confirmation of S.Typhi by molecular methods
2.7 Identification of Vi antigen
2.8 Detection of viaB operon by PCR
2.9 Cloning of PCR amplification product of tviD-tviE gene for sequencing
2.10 Sequencing of the cloned product
2.11 Detection of SPI-7 by PCR
2.12 Infectivity studies using HeLa cell lines
2.13 Drug sensitivity testing

3.1 Isolation and characterization of S. Typhi strains
3.2 Confirmation by molecular methods
3.3 PCR based detection of viaB operon
3.4 Transformation of tviD-tviE gene in E.coli for sequencing
3.5 Assessing the presence or absence of SPI-7
3.6 Analysis of fresh blood samples for Vi antigen
3.7 Detection of Vi-negative S. Typhi in blood samples from typhoid patients
3.8 Identification of SPI-7 by PCR
3.9 Infectivity studies by invasion Vero cell monolayer
3.10 Drug sensitivity by disc diffusion method

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