A custom amplicon sequencing approach to detect resistance associated mutations and sequence types in Mycoplasma genitalium

Abstract

Background: Mycoplasma genitalium resistance to antibiotic treatments is increasing, with very limited treatment alternatives on the horizon. Surveillance via sequencing of multiple M. genitalium loci would allow: monitoring of known antibiotic resistance mutations, associations between resistance/treatment failure and specific mutations, and strain typing for epidemiological purposes. In this study we assessed the performance of a custom amplicon sequencing approach, which negates the cost of library preparation for next generation sequencing.

Methods: Fifty-two M. genitalium positive samples (cervical, vaginal, anal and rectal swabs, and urine) were used. Three regions associated with M. genitalium antibiotic resistance (23S rRNA, parC and gyrA genes) were targeted, in conjunction with a locus used for differentiation of sequence types in the mgpB gene, and findings compared to Sanger sequencing.

Results: Amplicon sequencing provided adequate sequence read coverage (>30×) for the majority of samples for 23S rRNA gene (96%) and mgpB (97%), parC (78%) and gyrA (75%). Single nucleotide polymorphisms (SNPs) were characterised in samples for 23S rRNA gene (94%), parC (56%) and gyrA (4%). Unlike Sanger sequencing, mixed mutations could be identified by the amplicon sequencing method, and ratios of mutation types determined. All results, with one exception, were concordant to Sanger sequence results. Sequence diversity in the mgpB region was represented by 15 sequence types, 4 being observed in multiple samples.

Conclusions: We have demonstrated the utility of this custom amplicon sequencing approach for generating highly informative datasets with the capacity to identify and determine ratios of mixed sequences. The use of this customisable amplicon sequencing method enables cost effective, scalable amplicon sequencing of multiple target regions of interest in M. genitalium.

Keywords: 23S rRNA gene; Amplicon sequencing; Fluoroquinolone resistance; Macrolide resistance; Mycoplasma genitalium; mgpB sequence typing.