Novel features of Mycoplasma genitalium genomes identified through Oxford Nanopore sequence analysis of isolates from Australia

Mycoplasma genitalium is a fastidious human pathogen with increasing antimicrobial resistance, yet its genomic landscape remains poorly characterized due to difficulties with culture, including prolonged incubation periods and low DNA yields from both clinical and cultured samples. Consequently, there are few publicly available genome sequences. In this study, a Vero cell culture protocol was optimized to increase M. genitalium DNA yield and integrated with Oxford Nanopore technology. As a result, 22 complete genome sequences were generated with a mean sequencing depth of 51.01×. Comparative genomics revealed that 59% of isolates contained a translocated rRNA operon, with junction flanks showing ~90% identity to the MgPar repetitive regions known to be associated with genomic rearrangement. Phylogenetic analysis revealed multiple groups encompassing both recent and deeply branching lineages. High rates of macrolide (90.9%) and fluoroquinolone (45.5%) resistance were observed. All isolates with quinolone resistance mutations also carried macrolide resistance mutations. Notably, all three isolates with mgpB 161 allele had the same resistance profile: A2059G, H69R, S83I and M95I at 23S, L4, parC and gyrA, respectively. This work provides the first complete M. genitalium genome generated using Oxford Nanopore sequencing from Vero cell-propagated isolates, underscoring the novelty and technical advancement of this approach.