Multiple Mechanisms Confer Resistance to Azithromycin in <i>Shigella</i> in Bangladesh: a Comprehensive Whole Genome-Based Approach

Abstract

<i>Shigella</i> is the second leading cause of diarrheal deaths worldwide. Azithromycin (AZM) is a potential treatment option for <i>Shigella</i> infection; however, the recent emergence of AZM resistance in <i>Shigella</i> threatens the current treatment strategy. Therefore, we conducted a comprehensive whole genome-based approach to identify the mechanism(s) of AZM resistance in <i>Shigella</i>. We performed antimicrobial susceptibility tests, polymerase chain reaction (PCR), Sanger (amplicon) sequencing, and whole genome-based bioinformatics approaches to conduct the study. Fifty-seven (38%) of the <i>Shigella</i> isolates examined were AZM resistant; Shigella sonnei exhibited the highest rate of resistance against AZM (80%). PCR amplification for 15 macrolide resistance genes (MRGs) followed by whole-genome analysis of 13 representative <i>Shigella</i> isolates identified two AZM-modifying genes, <i>mph</i>(A) (in all <i>Shigella</i> isolates resistant to AZM) and <i>mph</i>(E) (in 2 AZM-resistant <i>Shigella</i> isolates), as well as one 23S rRNA-methylating gene, <i>erm</i>(B) (41% of AZM-resistant <i>Shigella</i> isolates) and one efflux pump mediator gene, <i>msr</i>(E) [in the same two <i>Shigella</i> isolates that harbored the <i>mph</i>(E) gene]. This is the first report of <i>msr</i>(E) and <i>mph</i>(E) genes in <i>Shigella</i>. Moreover, we found that an IncFII-type plasmid predominates and can possess all four MRGs. We also detected two plasmid-borne resistance gene clusters: IS<i>26</i>-<i>mph</i>(A)-<i>mrx</i>(A)-<i>mph</i>(R)(A)-IS<i>6100</i>, which is linked to global dissemination of MRGs, and <i>mph</i>(E)-<i>msr</i>(E)-IS<i>482</i>-IS<i>6</i>, which is reported for the first time in <i>Shigella</i>. In conclusion, this study demonstrates that MRGs in association with pathogenic IS<i>6</i> family insertion sequences generate resistance gene clusters that propagate through horizontal gene transfer (HGT) in <i>Shigella</i>. <b>IMPORTANCE</b> <i>Shigella</i> can frequently transform into a superbug due to uncontrolled and rogue administration of antibiotics and the emergence of HGT of antimicrobial resistance factors. The advent of AZM resistance in <i>Shigella</i> has become a serious concern in the treatment of shigellosis. However, there is an obvious scarcity of clinical data and research on genetic mechanisms that induce AZM resistance in <i>Shigella</i>, particularly in low- and middle-income countries. Therefore, this study is an approach to raise the alarm for the next lifeline. We show that two key MRGs [<i>mph</i>(A) and <i>erm</i>(B)] and the newly identified MRGs [<i>mph</i>(E) and <i>msr</i>(E)], with their origination in plasmid-borne pathogenic islands, are fundamental mechanisms of AZM resistance in <i>Shigella</i> in Bangladesh. Overall, this study predicts an abrupt decrease in the effectiveness of AZM against <i>Shigella</i> in the very near future and suggests prompt focus on seeking a more effective treatment alternative to AZM for shigellosis.