Typhoid fever is a life-threatening infectious disease that requires antibiotic treatment to avoid deaths and complications. However, the global spread of extensively drug-resistant (XDR) S. Typhi threatens antibiotic therapy, leaving azithromycin the only remaining oral drug effective against XDR typhoid. Azithromycin resistance has recently appeared in South Asia, posing an imminent threat of untreatable typhoid.

This study will decode the genetics of S. Typhi samples from South Asia obtained from the ACT-South Asia trial to investigate the epidemiological and evolutionary pathways of azithromycin-resistant S. Typhi. Experiments will also identify factors driving the emergence of azithromycin-resistant S. Typhi and determine the effects of resistance-conferring mutations on the organisms’ survival and fitness.

Further, this study will investigate the correlation between azithromycin treatment responses and azithromycin susceptibility of S. Typhi organisms inside and outside human cells. This project will generate critical data to guide public health control measures and improve the treatment of typhoid fever.

We will longitudinally expose an azithromycin-susceptible (AzithS) S. Typhi isolates to sub-MICs of azithromycin or azithromycin+cefixime/ciprofloxacin to investigate the effects of single versus combination drug on the evolutionary rate of azithromycin resistance in S. Typhi. Serially transferred cultures (evolving populations) will be genome-sequenced to identify azithromycin resistance mutations.

We will also generate azithromycin-resistant (AzithR) isogenic mutants by introducing known azithromycin resistance mutations (R717Q/R717L, acrB gene) into susceptible isolates using Lambda-red homologous recombination. The growth dynamics, antimicrobial tolerance and fitness of isogenic mutants will be measured in comparison with parental isolates. Transcriptional profiling in response to sub-MIC of azithromycin will also be studied in a parental AzithS isolate and its AzithR isogenic mutant using RNA-sequencing. These experiments will identify factors that may accelerate or decelerate the development of AzithR S.Typhi, and determine the fitness effect of azithromycin resistance mutations and bacterial responses to azithromycin exposure.