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.

Five years of GenoTyphi: updates to the globalSalmonellaTyphi genotyping framework

“…Isolates from Dhaka 10 have also been reported to be multi-drug resistant carrying genes conferring additional resistance to chloramphenicol, ampicillin and co-trimoxazole. Recent studies 15 has revealed that these mutations now appear to be emerging in more non-H58 genotypes in Dhaka from 2016 onwards including genotypes 2.3.3, 3.2.2, and 3.3.2.…”

Ever-Adapting RND Efflux Pumps in Gram-Negative Multidrug-Resistant Pathogens: A Race against Time

“…In recent years, other AcrB-Sa mutants have been observed, causing untreatable infections in Nepal, Bangladesh, India and Pakistan, in both Salmonella Typhi [85][86][87][88] and Salmonella Paratyphi A [85,88], summarized in Table 9. These clinical isolates are resistant to azithromycin (macrolide) by the mutations R717Q and R717L in AcrB-Sa.…”

“…Of these, 32 Salmonella Typhi and 6 Salmonella Paratyphi A isolates had a significantly high azithromycin MIC (>32 mg ml −1 ). All of these 32 highly resistant Typhi isolates had the R717 mutation (29 R717Q and 3 R717L), and five Paratyphi A isolates had the R717Q mutation [88]. It is clear that the spontaneous and divergent emergence of the "R717 mutations" in AcrB-Sa should raise great concern for the treatment of typhoid fever by macrolides.…”

The molecular basis of extensively drug-resistant Salmonella Typhi isolates from pediatric septicemia patients

“…The recent worrisome trend among some XDR S. Typhi includes a correlation between efflux pump mutations and azithromycin resistance. For instance, a point mutation(s) on the antibiotic-binding subunit AcrB of the tripartite AcrAB-TolC efflux pumps (e.g., R717Q or R717L) has recently been correlated to azithromycin-resistance among some S. Typhi and S. Paratyphi A clinical isolates (19,25,26).…”

“…They usually function as independent units in the IM to translocate antibiotics across the membrane bilayer, followed by their cooperation with RND-type efflux pumps to pump out antibiotics across the entire cell envelope (24). In typhoidal Salmonella, point mutations at amino acid position 717 (R717Q or R717L) on AcrB, the antibiotic-binding subunit of the RND-type AcrAB-TolC efflux pump, have been correlated with resistance to azithromycin in S. Typhi and S. Paratyphi A, respectively (19,25,26).…”