A repetitive nucleotide insertion in the rplV gene is associated with in vitro resistance to azithromycin in Rickettsia typhi

BACKGROUND: Murine typhus, caused by Rickettsia typhi, is a treatable febrile illness in Laos, where azithromycin treatment failure has been reported. Antibiotic susceptibility testing for Rickettsia spp. is challenging due to absence of resistant strains. We aimed to induce an azithromycin-resistant in R. typhi and investigate its genetic basis. METHODOLOGY: R. typhi Wilmington was cultured in azithromycin-containing media (R. typhiAZM), starting at a concentration of 0.0019 mg/L and gradually increased to 0.0625 mg/L. Resistant populations were selected up to 0.125 mg/L. MICs were determined using plaque assay and qPCR, and DNA sequencing was performed for rplD (L4), rplV (L22), and 23S rRNA domain V. Protein modeling of azithromycin-binding sites was conducted, and strain stability was assessed over 24 passages without azithromycin (R. typhi AZM (-)). RESULTS: MICs for wild type (R. typhiWT) and R. typhiAZM were 2 mg/L versus >16 mg/L (plaque assay) and 0.25 mg/L versus 8 mg/L (qPCR). A 15-nucleotides insertion (5'-AAAGGAAGAGCAACT-3') was found in the rplV of R. typhiAZM, but not other isolates. Protein modeling suggested the insertion extends the L22 loop, potentially affecting azithromycin binding site within the ribosomal exit tunnel. R. typhiAZM reverted to wild type MIC and genotype by 24 passages without azithromycin. R. typhiAZM exhibited an 8 -fold higher MIC than R. typhiWT. CONCLUSION: Repetitive insertion in rplV was associated with azithromycin resistance and may interfere with drug binding. R. typhiAZM was unstable without selective pressure. This approach may help generate resistant strains for assay validation. The role of rplV mutations in azithromycin susceptibility warrants further investigation.