In Vitro Recombinants of Antibiotic-Resistant Chlamydia trachomatis Strains Have Statistically More Breakpoints than Clinical Recombinants for the Same Sequenced Loci and Exhibit Selection at Unexpected Loci

Abstract

Lateral gene transfer (LGT) is essential for generating between-strain genomic recombinants of Chlamydia trachomatis to facilitate the organism's evolution. Because there is no reliable laboratory-based gene transfer system for C. trachomatis, in vitro generation of recombinants from antibiotic-resistant strains is being used to study LGT. However, selection pressures imposed on in vitro recombinants likely affect statistical properties of recombination relative to naturally occurring clinical recombinants, including prevalence at particular loci. We examined multiple loci for 16 in vitro-derived recombinants of ofloxacin-and rifampin-resistant L 1 and D strains, respectively, grown with both antibiotics, and compared these with the same sequenced loci among 11 clinical recombinants. Breakpoints and recombination frequency were examined using phylogenetics, bioinformatics, and statistics. In vitro and clinical isolates clustered perfectly into two groups, without misclassification, using Ward's minimum variance based on breakpoint data. As expected, gyrA (confers ofloxacin resistance) and rpoB (confers rifampin resistance) had significantly more breakpoints among in vitro recombinants than among clinical recombinants (P < 0.0001 and P ‫؍‬ 0.02, respectively, using the Wilcoxon rank sum test). Unexpectedly, trpA also had significantly more breakpoints for in vitro recombinants (P < 0.0001). There was also significant selection at other loci. The strongest bias was for ompA in strain D (P ‫؍‬ 3.3 ؋ 10 ؊8 ). Our results indicate that the in vitro model differs statistically from natural recombination events. Additional genomic studies are needed to determine the factors responsible for the observed selection biases at unexpected loci and whether these are important for LGT to inform approaches for genetically manipulating C. trachomatis. Chlamydia trachomatis is the most prevalent sexually transmitted bacterium and the leading cause of preventable blindness in the world. More than 92 million cases are reported annually (42). Infection leads to diseases such as urethritis, cervicitis, pelvic inflammatory disease, infertility, proctitis, reactive arthritis, lymphogranuloma venereum, and trachoma. Over 70% of women and 50% of men who are infected with C. trachomatis are asymptomatic, providing a huge opportunity for unchecked transmission (23, 42). For symptomatic individuals who do seek medical care, diagnostic tests for C. trachomatis, either for initial diagnosis or test of cure, are not always performed, largely due to cost (8). Thus, empirical treatment with antibiotics is common, which may hasten the development of drug resistance. This has already occurred for Chlamydia suis, a species that is closely related to C. trachomatis in which a tetracycline transposon was acquired from other pathogens residing in the guts of pigs (13). C. trachomatis may not be far behind; a number of studies have shown increased MICs of various antibiotics for clinical C. trachomatis isolates or reference strains propagated in tissue...

Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants

“…A recent report [24] indicated a strong preference for recombination at specific positions within trpB or gyrA in several recombinant progeny originally generated by Demars and Weinfurter [4]. We used two approaches to examine selected sets of candidate hotspots identified by these authors.…”

Benzylidene Acylhydrazides Inhibit Chlamydial Growth in a Type III Secretion- and Iron Chelation-Independent Manner

“…If none of the single mutations is sufficient to cause resistance, secondary and even tertiary mutations could be created likewise. Finally, since recombinant chlamydiae can be generated from strains with resistance to different inhibitors (60,61), it should be possible to recombine the genome of MCR with another chlamydial genome to determine which SNP(s) is required and sufficient for resistance.…”

Determining the Antibiotic Resistance of Bacterial Pathogens in Sexually Transmitted Diseases

“…Significant selections were evidenced at other loci as well, results indicating that the in vitro model is statistically different from any natural recombination events. Additional genomic studies to determine the responsible factors for selection biases at unexpected loci are needed to clarify whether these are important for LGT approaches in the genetical manipulation of C. trachomatis [42].…”

“…As for C. trachomatis no reliable laboratory-based gene transfer system was available, in vitro generation of recombinants from antibiotic-resistant strains was used to study the LGT, essential for generating between-strain genomic recombinants of C. trachomatis able to facilitate the organism's evolution [42]. For 16 in vitro-derived recombinants of ofloxacin-and rifampin-resistant L1 and D strains, multiple loci were examined and compared with the same sequenced loci among 11 clinical recombinants.…”