A novel azithromycin resistance mutation in Mycoplasma genitalium induced in vitro

Abstract

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.

Figure 1.

Domain V of the 23S rRNA of M. genitalium. Macrolide resistance mutations commonly found in clinical isolates are circled in red. The mutation induced in this study by exposure to azithromycin in vitro is circled in blue, with the corresponding base pair located at position 2611. The numbering used in this figure is based on E. coli sequence numbering. This figure has been drawn based on the structure presented in Pereyre et al.

Figure 2.

Detection of a novel mutation by ddPCR and Sanger sequencing. (a) Analysis of the mixed population found in passage eight. Controls are grouped on the right-hand side. Analysis with the A2058G probe (blue, top row) and WT probe (green, bottom row) is depicted. Probes targeting the other common mutations (Table 1) were also used with similar results (not shown). (b) Analysis of five passages prior to passage eight to determine when the mutation appeared. PCR was performed with the G2057A probe (blue, top row) and WT probe (green, bottom row). Respective DNA populations are indicated. (c) Sanger sequencing chromatogram of the 23S rRNA sequence of the mixed mutant and WT M. genitalium population. The red arrow corresponds to a dual peak of A/G at position 2057.

Figure 3.

Growth of the WT G37 and G2057A mutant in Hayflick medium with no azithromycin (a), 0.002 mg/L azithromycin (b), 0.008 mg/L azithromycin (c) and 0.064 mg/L azithromycin (d). Cultures were grown in triplicate and enumerated using the 23S rRNA ddPCR assay. Error bars represent 1SD. The dotted line indicates the target starting concentration for cultures.