Tracking the Emergence of Azithromycin Resistance in Multiple Genotypes of Typhoidal Salmonella

Abstract

The rising prevalence of antimicrobial resistance in Salmonella enterica serovars Typhi and Paratyphi A, causative agents of typhoid and paratyphoid, have led to fears of untreatable infections. 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 is a single point mutation in the efflux pump AcrB (R717Q/L). Here, we report 38 additional cases of azithromycin-resistant (AzmR) Salmonella 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 Salmonella Typhi and Paratyphi A genotypes. The largest cluster of AzmR Typhi belonged to genotype 4.3.1.1; Bayesian analysis predicts the mutation to have emerged sometime in 2010. A travel-related Typhi isolate with AcrB-R717Q belonging to 4.3.1.1 was isolated in the United Kingdom, 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 yielded >98% specificity and sensitivity versus phenotypic 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, giving us a window to intervene.IMPORTANCE In the early 1900s, with mortality of ∼30%, typhoid and paratyphoid ravaged parts of the world; with improved water, sanitation, and hygiene in resource-rich countries and the advent of antimicrobials, mortality dwindled to <1%. Today, the burden rests disproportionately on South Asia, where the primary means for combatting the disease is antimicrobials. However, prevalence of antimicrobial resistance is rising and, in 2016, an extensively drug resistant Typhi strain triggered an ongoing outbreak in Pakistan, leaving only one oral drug, azithromycin, to treat it. Since the description of emergence of azithromycin resistance, conferred by a point mutation in acrB (AcrB-R717Q/L) in 2019, there have been increasing numbers of reports. Using genomics and Bayesian analysis, we illustrate that this mutation emerged in approximately 2010 and has spontaneously arisen multiple times. Emergence of pan-oral drug resistant Salmonella Typhi is imminent. We developed a low-cost, rapid PCR tool to facilitate real-time detection and prevention policies.

Keywords: AMR; Bangladesh; Paratyphi; Salmonella Typhi infection; Typhi; antimicrobial drug resistance; azithromycin; paratyphoid; paratyphoid fever; typhoid.

FIG 1

Detection of azithromycin resistance and types of AcrB-717 mutation in Salmonella Typhi and Paratyphi A in Bangladesh. (A) Temporal distribution of 3043 Salmonella Typhi isolates identified in this study and by Hooda et al. (14). The total number of isolates tested is shown as the line plot from 2013 to 2018. The numbers of AzmR strains isolated each year is shown in the bar plot. (B) Azithromycin MICs among AcrB-WT and AcrB-R717Q and AcrB-R717QL mutant strains of Salmonella Typhi. (C) Temporal distribution of 587 Salmonella Paratyphi A isolates identified in this study and by Hooda et al. (14). The number of isolates is shown as the line plot from 2013 to 2018. The number of AzmR strains isolated each year is shown in the bar plot. (D) Azithromycin MICs among acrB wild-type and AcrB-R717Q and AcrB-R717QL mutant strains of Salmonella Paratyphi A. ****, P ≤ 0.0001; ***, P ≤ 0.001.

FIG 2

Spontaneous emergence of AcrB-R717Q/L mutation in five different genotypes of Salmonella Typhi. (A) A whole-genome SNP tree containing 825 Salmonella Typhi strains highlights different azithromycin-resistant Salmonella Typhi genotypes. The inner circle shows the distribution of Salmonella Typhi genotypes, and the outer circle shows the mutations in AcrB-717. (B) Temporal distribution of the acrB mutation in different genotypes between 2013 and 2018.

FIG 3

Maximum-likelihood tree based on SNP alignment of 141 Salmonella Paratyphi A strains with six azithromycin-resistant isolates. The inner circle depicts the country of isolation, the circle in the middle depicts the year of isolation, and the outer circle depicts the different mutations at AcrB-717.

FIG 4

Bayesian estimation of the maximum clade credibility tree of genotype 4.3.1.1 azithromycin-resistant and related azithromycin-sensitive Salmonella Typhi isolates. The azithromycin-resistant clade containing the AcrB-R717Q mutation is shaded in gray, and the closely related AcrB-WT strains are outside the gray box. The Salmonella Typhi strain P-stx-12 (AcrB-WT) belonging to 4.3.1.1 was used as a reference strain. The AcrB-R717Q mutation is predicted to have emerged between 2010 and 2012 in Bangladesh. The travel-related Salmonella Typhi isolate from the United Kingdom is highlighted with a red arrow. The scale bar indicates the number of substitutions per variable site per year.

FIG 5

Design and sensitivity and specificity of PCR-based mismatch amplification mutation assay (MAMA) for detecting AcrB mutations. (A) PCR cycling condition, primer sequences, and amplicon sizes generated by the AcrB MAMA and ParC control primers utilized in this study. (B) MAMA PCR primers for detecting mutation at the nucleotide position 2150 (amino acid 717) of the acrB gene. A single mismatch was incorporated at the conserved nucleotide (A to C; red, AcrB-MAMA-R) to increase allelic discrimination and chain termination in the presence of any mutation (G to A or T; red highlight). Published sequences of typhoidal Salmonella (e.g., Salmonella Typhi 5330M and Salmonella Paratyphi A 3144M [14]) with both Q (glutamine) and L (leucine) mutations were used to design the assay in silico. (C) Interpretation of the bands generated by AcrB MAMA and ParC control primers designed in this study. (D) Sensitivity and specificity of MAMA and WGS. (E) Sensitivity and specificity of MAMA and Etest compared to each other.