Surveying Genetic Markers of Antibiotic Resistance and Genomic Background in Chlamydia trachomatis: Insights from a Multiplex NGS-Based Approach in Clinical Strains from Portugal

Objectives: To survey genetic markers of potential antimicrobial resistance (AMR) to macrolides and fluoroquinolones among C. trachomatis -positive samples from the collection of the Portuguese National Reference Laboratory for STI and explore the application of a multiplex PCR approach coupled with Next-Generation Sequencing (NGS) to provide complementary information regarding strain’s genomic backbone. Methods: 502 C. trachomatis -positive samples were subjected to PCR and sequencing of five targets, including loci potentially driving AMR ( 23S rRNA , gyrA and parC ) and loci potentially informative about strain’s genomic backbone with emphasis on LGV/non-LGV and L2/L2b differentiation (an 9bp insertion in pmpH , a 74bp insertion upstream from CT105 and the polymorphic CT442) . Results: No samples evidencing 23S rRNA mutations recognisably linked to macrolide resistant were found. Three samples harboured the Ser83Ile mutation in GyrA putatively driving fluoroquinolone resistance: two recombinant L2-L2b/D-Da (0.4%) and one L2 (0.2%). The screened regions in pmpH , upstream CT105, and CT442, were fully concordant with LGV/non-LGV differentiation. As expected, the pmpH L2b-specific genetic trait locus was detected in all L2b and recombinant L2-L2b/D-Da ompA -genotypes, but also in 96.0% of L2 specimens, which also likely possess an L2b genomic backbone. The insertion upstream CT105 exhibited full LGV-specificity, constituting a promising target for the development of rapid LGV diagnostic assays. Conclusions: This study contributes to enhance the knowledge on C. trachomatis molecular epidemiology, suggesting that the known genetic determinants of AMR are not disseminated in clinical C. trachomatis strains, and presents an exploratory approach that can be suitable for LGV/non-LGV and L2/L2b genomic background differentiation.