Molecula characterization of chlamdia trachomatis isolates using sequence variation in the major outer membrane protein gene (OMP1) and evaluation of their susceptibility profile.

Abstract

Chlamydia trachomatis infections are the most common bacterial sexually transmitted infections (STIs) in humans, worldwide. Due to asymptomatic nature of C. trachomatis, the need for sensitive, reliable and affordable laboratory methods for diagnosis is critical. The aim of this study was to ascertain if the genetic profiles of different C. trachomatis isolates associate with antibiotic resistance.Two hundred and sixty-five Eswab™ clinical samples were screened for Ct using Anyplex™ II STI-7 Detection. We have applied High Resolution Melting Analysis (HRMA) for the genotyping of the Ct and applied it specifically to the 14 sexually transmitted infection-related genotypes: AC, D-K and L1-L3. Based on the genotype of the OMP1 (Outer Membrane Protein) gene C. trachomatis is grouped into different serovars, which present in different clinical manifestations; with type A, B, Ba, and C causing trachoma, D-K cause urogenital infections and LI, LII & LIII associated with lymphogranuloma venereum (LGV). We confirmed the presence of the OMP1 gene with the conventional PCR. HRMA was performed to identify the C. trachomatis serovars on a Quantstudio 5 real – time PCR instrument and CDC control strains were included in the analysis. HRM analysis was done on the High-Resolution Melt Softwarev3.1. We identified the following serovars A, B, C, D, E, F, G, I, J, L3 and our prevalence for the above serovars were as follows 3.2%, 6.4%, 3.2%, 9.7%, 16.1%, 29%, 9.7%, 12.9%, 3.2% and 6.4%, respectively. None of the serovars: H, K, L1, L2 were observed. A TaqMan real time PCR assay was also performed to measure the bacterial concentration of each C. trachomatis positive sample to elucidate if there is any association with the serovar type. D-K serovars had higher bacterial load compared to A-C and L3 serovars, (p =0.0045). We also performed sanger sequencing on ribosomal proteins (L4 and L22) to determine the presence of mutations that have been previously associated with drug resistance. The ribosomal protein L4 had mutations located in 7 different positions, significant mutations associated with macrolides resistance were observed at amino acid number 109 and 151. Ribosomal protein L22 had 21 samples with mutation at amino acid number 24, that has not been associated with resistance before. Based on our study and previous studies, it is clear that macrolide resistance in C.trachomatis is multifactorial besides changes in the amino acids.