Sonja Duijn

Mycoplasma genitalium (M. genitalium) is an emerging sexually transmitted infection that has become an increasing global concern due to rising resistance to key antibiotics such as macrolides, tetracyclines, and fluoroquinolones. In the Netherlands, about 74% of M. genitalium isolates show genetic markers associated with macrolides resistance. This highlights the need for reliable culture and resistance testing and further strain characterization by means such as whole-genome sequencing (WGS). However, culturing clinical isolates of M. genitalium remains challenging due to its slow growth and host dependence. Current methods rely on Vero cell lines for co-culture, which can be unreliable in resistance testing and cause excessive contamination with host (monkey) DNA.
This study aimed to optimize in vitro culture conditions by exploring different host cell lines and media formulations to improve M. genitalium growth while minimizing host-cell contamination. We compared the effectiveness of several host cell lines (Vero, Hep-2, and Caco-2) and growth media and supplements (EMEM and low-glucose DMEM) in supporting M. genitalium growth. We monitored growth using quantitative PCR and determined the impact of these conditions on the bacterial DNA yield. Additionally, different host cell concentrations were tested for optimization bacterial growth.
The results show that the Hep-2 cell line supports the growth of M. genitalium and requires a lower initial cell load. This could result in a relatively higher Mycoplasma DNA concentration suggesting advantages for WGS. Moreover, the use of a human cell line may be useful in further reducing host DNA contamination compared to Vero cells. Although the Caco-2 cell line supported growth, its performance was inferior to both Hep-2 and Vero cell lines. EMEM medium supplemented with sodium pyruvate supported co-culture but did not improve growth, while low-glucose DMEM performed worse. Overall, Hep-2 cells appear to be a suitable alternative to Vero cells for M. genitalium co-culture.