A novel azithromycin resistance mutation in Mycoplasma genitalium induced in vitro.

BACKGROUND

Mycoplasma genitalium is a sexually transmitted bacterium of increasing concern due to issues around antimicrobial resistance. Resistance is typically mediated by SNPs; however, the difficulty of isolation and culture of M. genitalium limits the ability to analyse the impact of individual mutations.

OBJECTIVES

The aim of this study was to generate and characterize antibiotic-resistant M. genitalium mutants in vitro to understand the development of macrolide resistance in this bacterium.

METHODS

Sequential MIC assays for azithromycin were performed using the laboratory strain of M. genitalium (G37) grown in Hayflick medium. Bacteria were enumerated by droplet digital PCR (ddPCR) targeting mgpB, and a new ddPCR assay was established to detect specific mutations in the 23S rRNA gene. MICs of selected macrolide antibiotics were determined in Hayflick medium. Whole genome sequencing (WGS) was performed on the Oxford Nanopore MinION.

RESULTS

After eight passages in azithromycin, a novel 23S rRNA gene mutation, G2057A (Escherichia coli numbering), was detected. The mutant did not display a detectable growth defect and had elevated MICs to azithromycin (8-fold), josamycin (8-fold) and erythromycin (16- to 32-fold). WGS did not identify other mutations likely to contribute to reduced macrolide susceptibility.

CONCLUSIONS

A novel 23S rRNA gene mutation was identified in M. genitalium. This variation is found in Mycoplasma hominis, which is intrinsically resistant to certain macrolides. While this mutation has not been observed clinically in M. genitalium, these findings have expanded our understanding of resistance mechanisms within the Mollicutes, in particular the propensity for M. genitalium to develop resistance, even in low concentrations of antibiotic, and the interaction of azithromycin with the ribosome.