Date of Award

11-2022

Degree Type

Thesis

Degree Name

MPhil in Biological and Biomedical Sciences

First Advisor

Dr. Junaid Iqbal

Second Advisor

Dr. Farah Naz Qamar

Third Advisor

Dr. Maria Fletcher

Department

Biological and Biomedical Sciences

Abstract

Enteric fever is a severe public health concern especially in LMICs with 128,000– 161,000 deaths each year caused by Salmonella Typhi and S. Paratyphi A. In 2016, a new extensively drug-resistant (XDR) S. Typhi strain started appearing in patients’ samples across the Southern Part of Pakistan. This XDR strain was resistant to the third-generation cephalosporins, the current drug of choice to treat typhoid infection. Typhoid spread via the fecal-oral route through the consumption of contaminated water and food. Isolating typhoid bacteria from environments such as sewage and drinking water samples is challenging but also important from the public health perspective. Here we are reporting several approaches which we employed to optimize the isolation of S. Typhi and S. Paratyphi A from sewage samples. Sewage samples were centrifuged and the bacteria-containing pellet was cleaned with PBS after which they were inoculated in 10 ml of Selenite F broth at 37°C overnight. After enrichment in Selenite F broth, PCR testing was performed to check the presence of S. Typhi and S. Paratyphi A, but these two bacteria were rarely isolated throughout the subsequent isolation procedures on Xylose Lysine Deoxycholate (XLD) Agar and Bismuth Sulfite medium. Different strategies were used to increase recovery, like treating enriched bacteria with 0.5% Sodium Dodecyl Sulfate and Ox bile for different durations. Serial dilutions of these treatments were then plated. Among different post-enrichment treatments Ox bile, increased the recovery of S. Typhi and S. Paratyphi A from the post enrichment broth. Based on these results we also tried pre-enrichment treatment of sewage bacteria pellet with Ox bile and found mixed results. In some cases, the recovery increased, while in others it dropped as compared to PBS. This could be explained by the variable nature of sewage and the state of bacteria present in them, 7 which may respond differently to bile. We assessed the effect of Ox bile and 0.5% SDS on 50 sewage samples collected from different sites of Karachi and were able to isolate S. Typhi from 3 samples. For confirming the antimicrobial genes and for phylogenetic analysis, 3 positive Typhi samples as well 3 unidentified samples with black colonies, underwent for whole genome sequencing. The phylogenetic analysis was performed and we were able to identify that the strains were part of the strains belonging to Pakistan, USA and Bangladesh and their similarity with the clinical strains. After bioinformatics analysis, we were able to identify that the unknown black colonies were Salmonella Melaegridis and Salmonella Kentucky.

First Page

1

Last Page

104

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