Genomewide analysis of Salmonella Typhi and Paratyphi A, B circulating in Bangladesh to understand the population structure, transmission pattern and evolutionary dynamics using whole genome sequencing

Abstract

Enteric fever, caused by Salmonella enterica serovar Typhi (S. Typhi), Salmonella enterica Paratyphi serovars (S. Paratyphi A, B, and C), remains a major public health concern in low and middle income countries (LMIC) with poor hygiene and sanitation system. However, there is still limited understanding on the genomic epidemiology and population structure of these pathogens in low endemic settings including Bangladesh. The rapid emergence of multidrug resistance (MDR) and lack of credible systemic surveillance data representing the true disease burden emphasize the importance of genome based surveillance studies. In this thesis, a collection of whole genome sequence (WGS) data of 202 S. Typhi, 67 S. Paratyphi A and 79 S. Paratyphi B strain collected from different already existing enteric disease surveillance sites across Bangladesh was utilized with the aim to study population structure, antimicrobial resistance (AMR) dynamics and placing these sequence data in global genome context to investigate both global and regional transmission patterns. Phylogenetic analyses showed diverse S. Typhi population structure harboring nine distinct genotypes compared to S. Paratyphi A comprising only three lineages with the evidence of circulation of these Bangladeshi pathogens throughout neighboring South Asian countries. In addition, WGS analysis further classified previously serotyped Bangladeshi S. Paratyphi B strains as biotype Java, which were associated with diarrheal symptoms. Moreover, genotypic distribution and AMR patterns were observed among the S. Typhi Bangladeshi population during the study time period of 2004 to 2006. The high prevalence of reduced fluoroquinolone susceptibility with absence of MDR genes was identified among S. Typhi non-H58 genotypes 2.3.3, 3.2.2, 3.3.2 (from median 22.2% per year in 2004-2010 to 65.2% per year in 20112016)

emphasizing the shift in treatment practice towards third generation cephalosporin and azithromycin. With the rising trend of fluoroquinolone resistant S. Typhi and S. Paratyphi A strain described in this dissertation, single azithromycin resistant S. Paratyphi A strain isolated in 2018 due to acquisition of R717L mutation in acrB gene has narrowed down the overall treatment option for enteric fever. Despite of having increasing reports of antibiotic usage without prescription in Bangladesh, there was limited evidence for widespread extrinsic resistance gene and plasmid acquisition in Bangladeshi S. Java strains. Notably, a pHCM2-like cryptic plasmid carrying S. Paratyphi A strains in Bangladesh was reported first time here that has not previously detected in other global regions. A comparative Basic Local Alignment Search Tool (BLAST) analysis and pHCM2 phylogenetic analysis including plasmid sequences from the 17 S. Paratyphi A, 334 S. Typhi, and one S. Java straind from Bangladesh showed high similarity between typhoidal S. Typhi and S. Paratyphi A that were different from nontyphoidal S. Paratyphi B biotype Java. Comparative pan-genome analysis was also conducted to investigate genomic composition of core and accessory genes which revealed closed pan-genome for S. Paratyphi A but open pan-genome for S. Java. Lastly, the performance of a new WGS based diagnostic approach was evaluated by targeting unique genes for S. Typhi including globally dominant H58 lineage and S. Paratyphi A that has been considered as rapid, reliable, cost-effective compared to other currently available diagnostic methods for enteric fever detection. The findings in this dissertation will improve the understanding of genomic diversity of these pathogens to facilitate the identification of epidemiologically important new genotypes and to monitor the ongoing evolution of AMR in Bangladesh. The assimilation of WGS data will provide a framework for future genomic epidemiology studies in low-middle income countries as well as inform public health information to implement innovative diagnostic approaches, treatment regimens and enteric fever control strategies.