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

Abstract

Shigella is the second leading cause of diarrheal deaths worldwide. Azithromycin (AZM) is a potential treatment option for Shigella infection; however, the recent emergence of AZM resistance in Shigella threatens the current treatment strategy. Therefore, we conducted a comprehensive whole genome-based approach to identify the mechanism(s) of AZM resistance in Shigella. 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 Shigella 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 Shigella isolates identified two AZM-modifying genes, mph(A) (in all Shigella isolates resistant to AZM) and mph(E) (in 2 AZM-resistant Shigella isolates), as well as one 23S rRNA-methylating gene, erm(B) (41% of AZM-resistant Shigella isolates) and one efflux pump mediator gene, msr(E) [in the same two Shigella isolates that harbored the mph(E) gene]. This is the first report of msr(E) and mph(E) genes in Shigella. Moreover, we found that an IncFII-type plasmid predominates and can possess all four MRGs. We also detected two plasmid-borne resistance gene clusters: IS26-mph(A)-mrx(A)-mph(R)(A)-IS6100, which is linked to global dissemination of MRGs, and mph(E)-msr(E)-IS482-IS6, which is reported for the first time in Shigella. In conclusion, this study demonstrates that MRGs in association with pathogenic IS6 family insertion sequences generate resistance gene clusters that propagate through horizontal gene transfer (HGT) in Shigella. IMPORTANCE Shigella 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 Shigella 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 Shigella, 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 [mph(A) and erm(B)] and the newly identified MRGs [mph(E) and msr(E)], with their origination in plasmid-borne pathogenic islands, are fundamental mechanisms of AZM resistance in Shigella in Bangladesh. Overall, this study predicts an abrupt decrease in the effectiveness of AZM against Shigella in the very near future and suggests prompt focus on seeking a more effective treatment alternative to AZM for shigellosis.

Keywords: Shigella; antimicrobial resistance mechanism; azithromycin; resistance gene cluster; whole genome.

FIG 1

Tree inferred with FastME 2.1.6.1 based on GBDP distances calculated from genome sequences. The tree was rooted at the midpoint. Values in the cluster nodes represents pseudo-bootstrap values (≥60). Species, genome size, protein count, and ribosomal sequence type are represented with multicolor blocks. Study strains are denoted with asterisks; green and red asterisks represent AZM-sensitive and -resistant Shigella isolates, respectively.

FIG 2

Comparison (BLAST atlas) of the plasmids of Shigella identified using the Gview server. The innermost multicolor ring shows the Clusters of Orthologous Groups (COG) followed by GC skews, ring backbone, pKSR100, and pZ12959_1, pZ12966_1, and pZ13032_1 in the outermost ring. Zoomed regions show AZM resistance-related gene clusters in the plasmids.