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A novel azithromycin resistance mutation in Mycoplasma genitalium induced in vitro
Teck-Phui Chua1,2,3, Jennifer Danielewski2,3, Catriona S Bradshaw4,5,6
1Department of Obstetrics, Gynaecology, and Newborn Health, University of Melbourne, Parkville, Victoria, Australia.
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.
Insights
A novel mutation in the 23S rRNA gene was identified in Mycoplasma genitalium, conferring resistance to macrolide antibiotics like azithromycin. This finding enhances understanding of antimicrobial resistance in this sexually transmitted bacterium.
Area of Science:
- Microbiology
- Genetics
- Antimicrobial Resistance
Background:
- Mycoplasma genitalium is a growing concern due to antimicrobial resistance.
- Difficulty in culturing M. genitalium hinders mutation impact analysis.
Purpose of the Study:
- Generate and characterize in vitro antibiotic-resistant M. genitalium mutants.
- Understand macrolide resistance development in M. genitalium.
Main Methods:
- Sequential MIC assays for azithromycin resistance.
- Droplet digital PCR (ddPCR) for bacterial enumeration and mutation detection.
- Whole genome sequencing (WGS) for comprehensive genetic analysis.
Main Results:
- A novel 23S rRNA gene mutation (G2057A) was detected after azithromycin exposure.
- The mutant showed elevated MICs to azithromycin, josamycin, and erythromycin.
- No other significant mutations affecting macrolide susceptibility were identified.
Conclusions:
- A novel 23S rRNA gene mutation linked to macrolide resistance in M. genitalium was identified.
- This mutation, also found in Mycoplasma hominis, expands knowledge of resistance mechanisms in Mollicutes.
- M. genitalium can develop resistance even at low antibiotic concentrations, impacting azithromycin-ribosome interactions.
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