Treatment failures of chlamydial infections are still a matter of concern, despite the in vitro sensibility of the organisms by MIC testing (8
). Persistent developmental forms have been described in vitro and associated with antimicrobial refractoriness (1
). Our model instead focuses on the active developmental cycle and studies differential susceptibility of its various developmental forms. This issue requires consideration, as clinical treatment is usually directed against a fully established infection and a mixture of multiple developmental forms. In the present in vitro model, we divided the cycle into three phases and the extracellular EBs and described the pharmacodynamic characteristics of the antimicrobials used most often by time-killing at various serum concentrations. Since chlamydiae differ in the length of the growth cycle, we compared a fast-replicating and a slow-replicating species.
We have demonstrated a reduced antimicrobial susceptibility in the extracellular phase and the very late and early states of the cycle. These phases can be correlated to the presence of elementary bodies that have been shown to have a reduced metabolism (2
). One remarkable exception is the immediate activity of rifampin against early stages of both C. trachomatis
and C. pneumoniae
. Especially the latter one was killed before the replicative phase commenced. Microarray analyses have identified the immediate-early genes of C. trachomatis
), of which none was described to interact with rifampin. The RNA polymerases as a major target of rifampin may be present in early chlamydiae as a preformed protein, but the superior activity of rifampin in comparison to other drugs requires further studies. In mid phase, differences between the antibiotics were more distinct. By comparing the two species, killing was faster for ciprofloxacin in C. trachomatis
and for erythromycin in C. pneumoniae
. Rifampin was again most effective. In late phase, all tested antibiotics act with the same kinetic. The time p.i. before drugs have to be applied in order to achieve sufficient killing corresponds to 60% of the cycle in fast- and slow-replicating species. Recondensation into EBs was associated with refractoriness to treatment. As antibiotics are classically divided into concentration-dependent and concentration-independent agents, we tested the influence of three physiologically relevant doses on killing of C. trachomatis
. To our knowledge, this is the first study that reports the increased susceptibility of chlamydiae to higher concentrations of antimicrobials and justifies the application of the highest tolerable dose.
The pharmacodynamics of antichlamydial antibiotics are most often characterized by MICs. They correspond to the drug concentration at the site of infection that is supposed to be exceeded in order to mediate activity. In addition, MIC is a useful tool to monitor the development of resistance. As resistance is not a matter of serious concern in chlamydiae and serum as well as intracellular concentrations of all tested antibiotics exceed MICs, comparison of MICs in the case of chlamydiae is of little value for the comparison of antibiotics. Additionally, MIC determination often neglects the fact that the in vivo treatment is directed against an established infection (17
). Our model of time-killing resembled the in vivo situation more closely. Physiologically relevant doses of drugs were used, and different stages of the cycle were distinguished. Drug interactions like synergism could be easily monitored. The results provided a rationale for improved antimicrobial treatment and gave information on the biology of the pathogen during the cycle. The model did not account for developmental forms resembling persistent infections, as they could be induced in vitro under various conditions (1
). Killing was detected over 3 logarithmic steps; regrowth at lower levels that has been reported in some studied was not monitored (4
Chlamydial infections are usually treated with macrolides or tetracyclines. Nevertheless, therapeutic failures and reinfections often occur, resulting in serious sequelae like pelvic inflammatory disease or tubal infertility (7
). Our in vitro study demonstrated the superior activity of rifampin over other often-used antibiotics. In addition, rifampin is well tolerated and cheap. The drawback of rifampin is the fast occurrence of resistance by a point mutation in the rpoB
). Failure due to resistance has been described in C. trachomatis
) but interestingly not in C. pneumoniae
. A combination of rifampin with other drugs has been proven successful in preventing resistance (11
). Macrolides seem to be a good combinational partner as they not only are the second most active antimicrobial class but also show synergistic activity with rifampin. The combination of azithromycin and rifampin was already shown to be effective in an animal model of C. pneumoniae
mouse pneumonitis (18
In summary, our model of chlamydial time-killing provided further evidence for rifampin as a highly active antichlamydial drug. Clinical studies are needed to demonstrate its clinical effectiveness in therapy-refractory infections.