An in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection with Chlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steadystate levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations for C. trachomatis allows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.Chlamydia trachomatis is an obligate intracellular bacterial parasite whose life cycle involves alternation between the infectious extracellular, metabolically inactive elementary body (EB) form and the intracellular, metabolically active reticulate body form; the latter is the vegetative growth stage of the organism. Depending on the specific serovar involved, human infection with C. trachomatis causes a variety of ocular, pulmonary, and genital diseases. Genital infection with chlamydial serovars D to K is considered to be of major public health importance, since C. trachomatis is the most common sexually transmitted bacterium worldwide (54). Further, acute urogenital infections can progress to persistent infection, which in turn may initiate a pathogenic process leading to chronic diseases, including pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and chlamydia-induced arthritis (12, 53). Importantly, C. trachomatis has been shown to be fully viable and metabolically active in both the acute and chronic, persistent infection state. In acute infections, the bacterium can be recovered usually by standard laboratory culture. Chronic chlamydial infections are often characterized by culture negativity, although viability has...

Infections caused by the obligate intracellular bacteriumChlamydia trachomatis are among the most prevalent causes of ocular and urogenital diseases worldwide. Clinical manifestations of acute infections related to C. trachomatis serovars A to C or serovars D to K are trachoma or cervicitis and urethritis, respectively. These infections can progress to persistent infections, which may initiate a pathogenic process that leads to chronic diseases including blindness or pelvic inflammatory disease, ectopic pregnancy, tubal factor infertility, and chlamydia-induced arthritis, including Reiter's syndrome.Standard therapy for acute urogenital tract infections is a 7-day course of doxycycline or a single dose of azithomycin. Both regimens have been shown to result in satisfactory cure rates in clinical trials (20,21,32,34,40,43,49).Relapsing chlamydial infections are, however, a common problem, even though patients are often treated appropriately (6, 24, 56). Usually, recurrent infections are supposed to be a consequence of reinfection. Most of the clinical trials that have addressed relapsing chlamydial infections did not distinguish between reinfection and relapse and thus did not define the role of persistence. There are, however, recent reports of recurrent infections after appropriate antibiotic treatment which appeared to be a result of the persistence of chlamydia (15,25,38).This observation presents an apparent contradiction to results of determination of the MIC and the minimum bactericidal concentration (MBC), which clearly indicated successful suppression of chlamydial growth by clinically used antibiotics. The experimental setting involved with this kind of in vitro testing is, however, not truly reflective of the situation in vivo for chlamydial infection. In natural infections, chlamydia are usually exposed to antimicrobials long after an infection has been well established. In contrast, the conventional in vitro systems used for susceptibility testing represent a quite different condition, in that antibiotics are added usually 48 h after the infectious agent is added or are sometimes added simultaneously with the infectious agent. Recently, we could demonstrate that ciprofloxacin and ofloxacin not only failed to eradicate chlamydia from host cells but induced a persistent infection, although both antibiotics are efficient in susceptibility testing (16). Using this in vitro model, we investigated the efficacies of azithromycin, rifampin, and the combination of azithromycin and rifampin for the elimination of chlamydia from epithelial cells. MATERIALS AND METHODSCells. Cells of the HEp-2 cells line, a human laryngeal epidermoid cell line, were maintained at 37°C with 5% CO 2 in RPMI 1640 medium supplemented with 10% fetal calf serum (Seromed, Berlin, Germany), 1% L-glutamine, and 100 g of gentamicin (Seromed) per ml.Growth, purification, and titration of chlamydia. C. trachomatis serovar K/UW-31/Cx (obtained from the Washington Research Foundation, Seattle) was cultured in HEp-2 cells, as described recentl...

A 656-bp PCR fragment from rpoB was sequenced from five rifampin-resistant Chlamydia trachomatis variants selected in vitro from a wild-type parent with a surprising level of genetic variability in this region. Three variants (MIC, 4 g/ml) showed Ala5223Val in cluster I (codons 507 to 533), which harbors mutations in most rifampin-resistant bacteria. Two high-level resistance variants (MICs, 64 and 256 g/ml) showed His5263Tyr in cluster I with additional genetic variation, some of which resulted in amino acid substitutions. None of the latter was situated in clusters related to rifampin resistance in other bacteria.Rifampin exerts its antibacterial effect through binding to the ␤-subunit of bacterial RNA polymerase (RNAP). However, resistance to rifampin can develop rapidly and has been described for a number of bacteria, including Escherichia coli (11) and Mycobacterium tuberculosis (18)(19)(20). The primary mechanism of resistance is decreased binding capacity of RNAP for rifampin caused by nucleotide changes localized in the central region of the rpoB gene, specifying the ␤ subunit of the enzyme. Although there are three principal clusters harboring nucleotide changes (I to III, amino acid positions 507 to 533, 560 to 572, and 687, respectively), more than 90% of mutations are located in cluster I.The obligate intracellular bacterial pathogen Chlamydia trachomatis is a major cause of urogenital and ocular infections worldwide. This organism has been shown to be highly sensitive to rifampin in susceptibility studies (e.g., references 4, 5, 7, 17, and 24). However, there are reports of emergence of resistance in in vitro systems (12,13,21). For Chlamydia, determinations of MIC for various antibiotics traditionally have been done by adding drug along with elementary bodies to permissive cells. However, this does not represent the in vivo situation, since antibiotics are used against Chlamydia only after intracellular infection has been established. We developed an in vitro cell culture model to investigate the long-term effect of antibiotics on established infection with C. trachomatis (serovar K) in epithelial cells (8). Briefly, antibiotic-free cultured HEp-2 cells were inoculated with C. trachomatis elementary bodies at a multiplicity of infection of 0.075. As previously reported, the effect of rifampin alone was investigated, as well as its combination with azithromycin (9). Treatment, starting 2 days after infection, was done with 0.015 g of rifampin/ml, which is twofold higher than the MIC of 0.0075 g/ml. Inhibition given by antichlamydial drugs was monitored in this system over a period of 20 days. Although rifampin alone generally proved able to both inhibit chlamydial growth and suppress de novo synthesis of bacterial rRNA, recurrent infection occasionally occurred in two independent experiments. In those instances, typical chlamydial inclusions were identified and we were able to recover infectious Chlamydia from the cultures. We prepared five variants that were clearly resistant, as determined by susc...