Tracking the emergence of azithromycin resistance in multiple genotypes of typhoidal Salmonella

The rising antimicrobial resistance in Salmonella Typhi and Paratyphi A, causative agents of typhoid and paratyphoid, have led to fears of untreatable infections in South Asia. Of specific concern is the emerging resistance against azithromycin, the only remaining oral drug to treat extensively drug-resistant (XDR) typhoid. Since the first report of azithromycin resistance from Bangladesh in 2019, cases have been reported from Nepal, India, and Pakistan. The genetic basis of this resistance in all cases is a single point mutation in the efflux pump AcrB (R717Q/L). Here, we report 38 additional cases of azithromycin resistant (AzmR) S. Typhi and Paratyphi A isolated in Bangladesh between 2016 and 2018. Using genomic analysis of 56 AzmR isolates from South Asia with AcrB-R717Q/L, we confirm that this mutation has spontaneously emerged in different S. Typhi and Paratyphi A genotypes. The largest cluster of AmzR S. Typhi belonged to 4.3.1.1; Bayesian analysis predicts the mutation to have emerged ~2010. A travel-related AzmR isolate belonging to 4.3.1.1 has been reported from the UK, increasing fears of global spread. For real-time detection of AcrB-R717Q/L, we developed an extraction-free, rapid, and low-cost mismatch amplification mutation assay (MAMA). Validation of MAMA using 113 AzmR and non-AzmR isolates depicts ~100% specificity and sensitivity against phenotypic (E-test) and whole-genome sequencing assays currently used for azithromycin resistance detection. With increasing azithromycin use, AcrB-R717Q/L is likely to be acquired by XDR strains. The proposed tool for active detection and surveillance of this mutation may detect pan-oral drug resistance early, potentially giving us a window to intervene.