Frequency of Development and Associated Physiological Cost of Azithromycin Resistance in Chlamydia psittaci 6BC and C. trachomatis L2

Abstract

Azithromycin is a major drug used in the treatment and prophylaxis of chlamydial infections. Spontaneous azithromycin-resistant mutants of Chlamydia psittaci 6BC were isolated in vitro in the plaque assay at a frequency of about 10 ؊8 . Isogenic clonal variants with A 2058 C, A 2059 G, or A 2059 C mutations in the unique 23S rRNA gene (Escherichia coli numbering system) displayed MICs for multiple macrolides (i.e., azithromycin, erythromycin, josamycin, and spiramycin) at least 100 times higher than those of the parent strain and were also more resistant to the lincosamide clindamycin. Chlamydia trachomatis L2 variants with a Gln-to-Lys substitution in ribosomal protein L4 at position 66 (E. coli numbering system), conferring an eightfold decrease in azithromycin and erythromycin sensitivities and a fourfold decrease in josamycin and spiramycin sensitivities, were isolated following serial passage in subinhibitory concentrations of azithromycin. Each mutation was stably maintained in the absence of selection but severely affected chlamydial infectivity, as determined by monitoring the development of each isolate over 46 h in the absence of selection, in pure culture or in 1:1 competition with the isogenic parent. Data in this study support the hypothesis that the mechanisms which confer high-level macrolide resistance in chlamydiae carry a prohibitive physiological cost and may thus limit the emergence of highly resistant clones of these important pathogens in vivo.

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

“…The susceptibilities of C. psittaci 6BC to antibiotics were determined in the plaque assay as previously described (9,10). Ksm (BIOMOL) was diluted in 2ϫ DMEM (GIBCO) to a final concentration of 10 mg/mL and stored at 4°C.…”

“…PCR (data not shown) and comparative qPCR ( Table 2) analyses show that a foreign marker became protected from extracellular DNase digestion after electroporation with C. psittaci 6BC EBs. We found that the amount of DNA The frequency of mutation to drug resistance was determined by dividing the number of PFU on medium with drug by the number of PFU added to the monolayer (as measured by PFU titer in the absence of drug) (9,10). See Table S1 surviving DNase treatment increased when higher time constants were obtained, being 2 10 -fold (Ϸ1,000-fold) greater, with 2 pulses of 12.8 ms each, than in the absence of electroporation (Table 2).…”

Differences in 23S ribosomal RNA mutations between wild-type and mutant macrolide-resistant Chlamydia trachomatis isolates

“…A more recent study, however, identified no mutations in the 23S rRNA genes of resistant mutants that were selected following enrichment by serial passage in the presence of sub-inhibitory concentrations of azithromycin (14).…”

Determining the Antibiotic Resistance of Bacterial Pathogens in Sexually Transmitted Diseases

“…The LGT mechanism has not been identified, but various publications investigating possible relationships between in vitro LGT and the high frequency of LGT recombinants in clinical isolates [46] suggested that a certain percentage of the many millions of clinical infections involve more than one strain of C. trachomatis. However, a recent study failed to identify mutations in the 23S rRNA genes of resistant mutants selected following enrichment by serial passage in the presence of subinhibitory concentrations of azithromycin [47].…”