Determination of the inhibitory concentration 50% (IC50) of four selected drugs (chlortetracycline, doxycycline, enrofloxacin and difloxacin) that reduce in vitro the multiplication of Chlamydophila psittaci.

A total of 18 chlamydial isolates from various psittacine birds, one isolate from a domestic pigeon and one isolate from a Pekin duck were isolated in continuous Buffalo Green Monkey (BGM) kidney cell cultures. All 20 isolates were identified by nested multiplex polymerase chain reaction as Chlamydophila psittaci. These isolates were multiplied to high titres and subsequently tested for in vitro sensitivity against two tetracyclines (chlortetracycline and doxycycline) and two quinolones (enrofloxacin and difloxacin) at concentrations of 0.0, 0.25, 0.50, 1.00, and 10.00 microg/ml. Replication of chlamydia in BGM cell cultures is assayed on the basis of formation of intracytoplasmic inclusions that are visualized by Giménez staining. All isolates, although to variable degrees, are sensitive to all four drugs. The number of chlamydial inclusions decreases gradually over a broad range of increasing concentrations of the drugs. The variation in the number of inclusions between isolates is remarkably high for chlortetracycline less for doxycycline and minimal for both fluoroquinolones, the enrofloxacin and difloxacin. The decline in numbers of inclusions is highly dose-dependend and the observed reduction stretches over a wide range of drug dilutions. Therefore, it is proposed to calculate drug sensitivity values in terms of inhibitory concentration 50%, (IC5). Its calculation includes all tested drug dilutions instead of the hitherto more common minimal inhibitory concentration, MIC, which is based on results of serial dilution tests for cell-free growing bacteria. Using a logistic regression model for the calculation of the inhibitory concentration 50% of all 20 chlamydial isolates, the IC50 is 0.807 microg/ml for tetracycline, 0.497 microg/ml for doxycycline, 0.180 microg/ml for enrofloxacin and 0.168 microg/ml for difloxacin. Complete prevention of inclusion formation was already seen for enrofloxacin at a concentration of 1.0 microg/ml in 12 out of 20 and for difloxacin in 5 out of 20 isolates whereas more than 10 microg/mI chlortetracycline is needed in 15 out of 20 isolates and for doxycycline 9 out of 20 isolates yielded inclusions at 10 microg/ml.