Methodologies and cell lines used for antimicrobial susceptibility testing of Chlamydia spp

Abstract

In vitro susceptibility testing was performed on strains of Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci under various conditions, including the cell line utilized, the time between infection and the addition of an antimicrobial, the concentration of inoculum, and the effect of multiple passage on the minimal chlamydicidal concentrations for the antibiotics doxycycline, azithromycin, erythromycin, ofloxacin, and tetracycline. With macrolides, the MIC varied depending upon the cell line utilized. With all antimicrobials, the MIC was related to the time at which the antimicrobial was added after infection. By an optimized cell culture passage method, all strains of chlamydia tested demonstrated survival after exposure to high levels (>100 times the MIC) of antimicrobials. Furthermore, upon retest, these surviving organisms did not demonstrate increased MICs. Thus, this phenomenon does not reflect selection of antimicrobial-resistant mutants but rather survival of some organisms in high antimicrobial concentrations (heterotypic survival). An additional 44 clinical isolates of C. trachomatis from patients with single-incident infections were tested against those from patients with recurrent or persistent infections, and heterotypic survival was seen in all isolates tested; hence, in vitro resistance did not correlate with the patient's apparent clinical outcome.

FIG. 1.

Sequential photomicrographs of MIC growth patterns of C. trachomatis serovar D (A) and C. suis strain R-19 (B) at various concentrations of doxycycline. The C. suis strain demonstrates homotypic resistance, in which most of the organisms survive at concentrations well above MIC for the C. trachomatis strain. Also shown is an example of the MIC_TP, the transition point where the concentration of antimicrobial first noticeably influences chlamydial inclusion growth (MIC_TP is 0.032 μg/ml for C. trachomatis and 0.5 μg/ml for C. suis).

FIG. 2.

Sequential photomicrographs of growth C. trachomatis serovar D at three different passage levels after initial exposure to given concentrations of doxycycline. (A) Illustration of MCC with heterotypic survival at concentrations well above the MICs after one passage in antimicrobial-free medium. (B) Illustration of MCC3 with complete growth of surviving chlamydiae after three passages in antimicrobial-free medium. (C) Illustration of the MIC pattern seen after retesting chlamydiae from the highest drug concentration in which organisms survived (arrow).