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Genital Chlamydia trachomatis infections is the most common sexually transmitted bacteria worldwide and is one of the aetiological microorganisms of chronic prostatitis (CP) and pelvic inflammatory diseases (PID). Antibiotics is finding the most suitable treatment and play a crucial role in the treatment of infection caused by C. trachomatis. Nevertheless, little is known about the in vitro effects of interactions between the antibiotics used against C. trachomatis. In this study, we investigate the in vitro effects of five antimicrobial agents, which are commonly used in the treatment of C. trachomatis infection, alone or in combination with each other by a microdilution checkerboard assay. Our aim was to determine whether there is evidence of in vitro interaction between various antibiotics that could account for its admirable clinical success.
2. Materials and Methods
2.1. Cells and Organisms
Cells of the McCoy cells line, mouse fibroblasts monolayers, were maintained at 37°C with 5% CO2 in antimicrobial-free designed minimal essential medium containing 10% fetal bovine serum. Laboratory reference strains for C. trachomatis (serovar D, E, G) were maintained and routinely used in our laboratory for research. Clinical strains were obtained from twenty-six males and fifteen females excluded pregnant woman who had a culture-documented C. trachomatis genital infection from September 2005 through to June 2009 at Tianjin institute of sexually transmitted diseases, China. All the patients did not use antibiotics two weeks before they came to the clinics. Urethral and prostatic specimens which were positive by detection of direct immunofluorescence staining were collected in transport media (DMEM medium, 5% calf serum, glucose, amphotericin B, gentamicin). The absence of other organisms such as gonococci, Trichomonas vaginalis and genital mycoplasmas was proved before cell culture.
2.2. Growth, purification, and titration of Chlamydia
The reference C. trachomatis strains, and the clinical isolates of C. trachomatis were cultured in McCoy cells by multiple passage, as described before . Briefly, at 48hs postinfection the Chlamydia were harvested in transport media and stored at -80°C. The infectivity of the Chlamydia was expressed as the number of inclusion forming units (IFU) per milliliter. The inoculum size of infectious Chlamydial forms for all MIC comparisons was 104 inclusion-forming units (IFU) in each well, resulting in the infection of approximately 90% of the cells in the monolayer.
2.3. Serotyping of isolates
All the forty-one clinical isolates from patients were typed by detection of restriction fragment length polymorphism in the gene encoding the major outer membrance protein of C. trachomatis .
2.4. Antibacterial compounds
Azithromycin, minocyline, doxycycline and rifampin were supplied as powders from the National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China, and solubilized according to the manufacturers' instructions. Moxifloxacin was obtained from Bayer Healthcare Co. Ltd. (Moxifloxacin hydrochloride, sodium chloride injection 250mlï¼š0.4g moxifloxacin, 2.25g NaCl).
2.5. Determination of MICs
Determination of the MICs was performed by inoculating Chlamydia strains onto monolayers of the McCoy cell line on 96-well microtiter plates as described recently . Appropriate controls were included. Briefly, McCoy cells that had been seeded into 96-well plates and incubated for 24hs at 37°C with 5% CO2 were pretreated with 30 µg of DEAE-dextranperml for 30 mins. Each well was inoculated with 0.1 ml of the microorganism diluted to yield 104 inclusion-forming units per ml. The plates were centrifuged at 1,200-g for 1 h. The wells were then aspirated and overlaid with 0.1 ml of medium containing 1 µg of cycloheximide per ml and serial twofold dilutions of azithromycin, moxifloxacin, doxycycline, minocyline and rifampin. After incubation at 37°C with 5% CO2 for 44-48hs, the cultures were fixed with methanol and stained with iodine dye. The MIC was deï¬ned as the lowest antibiotic concentration at which no inclusion was seen.
2.6. Determination of ∑FICs
A microtitration checkerboard assay was used to determine the fractionary inhibitory concentration (FIC) and ∑FIC index of the paired combinations of antibacterial agents against the C. trachomatis Stains. The FIC value was calculated from the MIC of antibiotic A alone and the MIC of antibiotic A in combination: FIC of antibiotic A = MIC of antibiotic A in combination/MIC of antibiotic A alone. The FIC of antibiotic B was calculated in the same manner and the sum of the two FIC agents combined to give the ∑FIC index: ∑FIC index= FIC of antibiotic A +FIC of antibiotic B. The value of the ∑FIC index was then used to determine whether synergism, additivity, indifference or antagonism occurred between the antibiotic agents. The following values, which were supported by other publication of the American Society of Microbiology [5, 6], were used to interpret the nature of the interactions: synergism ≤0.5, additivity 0.5-1, indifference 1-2, antagonism >2.
2.7. Statistical analysis
Statistical analyses were performed with SPSS software, version 11.5. One-Way ANOVA (analysis of variance) was used to compare analysis of multiple samples which had synergistic and additivity activity in the clinical isolates. And if they had statistically important differences, LSDï¼ˆleast-significant differentï¼‰was used to compare analysis of every two samples to identify which combination was the best. P<0.05 considered statistically important difference.
3.1. Antimicrobial susceptibility alone results
The minimal inhibitory concentrations (MICs) of five antibiotics used against C. trachomatis reference strains were in sensitive range. All the forty-one C. trachomatis strains from patients with recent STDs were serotyped. Thirty-eight are serovar E, two were serovar G and one is serovar J. Table 1 gives the MIC ranges of five antibiotics used for these Chlamydia clinical isolates. All the C. trachomatis strains did not exhibit homotypic resistance, a pattern in which the majority of organisms survive antimicrobial concentrations were well above the MIC range.
3.2. The ∑FIC index of six antibiotics combinations
The ∑FIC index of six antibiotics combinations used against C. trachomatis reference strains are presented in Table 2. Compared with the effect of drugs, additivity effects were observed when azithromycin was combined with moxifloxacin, doxycycline and rifampin, and drug combinations seemed to be more suitable to kill C. trachomatis serovar D and E. Neverless, antagonism effects were observed when minocyline was combined with azithromycin, moxifloxacin and rifampin.
The ∑FIC index of six antibiotics combinations used against C. trachomatis clinical isolates are presented in Table 3. Slightly more than one half of all test results showed synergy and additivity effects in combination of azithromycin with moxifloxacin, doxycycline and rifampin. The effects of three different combinations were compared by One-Way ANOVA analysis, and it illustrated no statistically important difference (When α=0.05, P=0.755). It was regretful that correlating the ∑FIC index with different serovars clearly remained extremely challenging understakings. Neverless, antagonism effects were observed in most of all test results when minocyline was combined with azithromycin, moxifloxacin and rifampin. These results were coincidence to reference strains.
Doxycycline and azithromycin are recommended as primary drugs in the treatment of genital Chlamydia infections. Azithromycin is one of the most widely prescribed drugs in the treatment of chlamydial infections. Doxycycline is cheaper than other antibiotics and has been used extensively for a long period. Rifampicin is a potent inhibitor of DNA-dependent RNA polymerase, and has been shown to be highly active against C. trachomatis in vitro studies [7, 8]. Long-term treatment of in vitro Chlamydial infection, however, results in the emergence of resistance [9, 10]. This strongly indicates that rifampin should not be used alone for the treatment of Chlamydial infections due to the potential favoring of the development of resistance. Fluoroquinolones are found to be among the least active antibiotics in vitro and this is due to the quinolones not being reliably effective against Chlamydial infection or not having been adequately evaluated . Some regimens have been shown to result in satisfactory cure rates in clinical trials [12, 13, 14]. While many C. trachomatis infections appear to be cleared by treatment with antibiotics, follow-up screening reveals a substantial fraction of previously treated subjects who are infected because of reinfection, treatmet failure, or the relative difficulty of eliminating latent infections .
Recently, clinical treatment failures (deï¬ned as Chlamydia persistence 1 month after treatment) following macrolide therapy have been reported and attributed to multidrug-resistant C. trachomatis strains , however, the potential for C. trachomatis to develop antimicrobial resistance has not been well studied. Additionally, we could not find drug resistance gene in clinical isolates from patients whose treatment are failure. Therefore, relapsing Chlamydial infections are a common problem, even though patients are often treated appropriately [17, 18]. There are also reports of recurrent infections after appropriate antibiotic treatment which appeared to be a result of the persistence of Chlamydia [19, 20]. And the clinical documents at Tianjin institute of sexually transmitted diseases in China from 1998 to 2005 indicate that C. trachomatis are also found following standard treatment: the treatment failure rate (deï¬ned as Chlamydia persistence proved by direct immunofluorescence staining 1 month after treatment) of the 836 patients who are treated with macrolides accoraccording to guideline is 36.44%; the treatment failure rate of the 3730 patients who are treated with quinolones according to guideline is 32.97%; the treatment failure rate of the 2050 patients who are treated with tetracyclines according to guideline is 34.54%.
The good activity of antibiotics alone in vitro as found in this study and our clinical documents suggests that they can play a significant role in the treatment of acute Chlamydia infections. However, their efficacy in the re-treatment of chronic genital Chlamydia infections and in the treatment of CP and PID is controversial. Patients with such pathology might represent a specific group for antibiotics combinations treatment. The effect of combined antibiotics on the growth of C. trachomatis in vitro has been tested infrequently [21, 22]. The combination of rifampin and azithromycin reveal more encouraging results. Development of resistance is prevented when cells are treated with azithromycin and rifampinp . Similar observations are made by Jones et al. , who report that subinhibitory concentrations of erythromycin and oxytetracycline inhibite the development of resistant strains under conditions in which such resistance would otherwise have emerged. In our study, slightly more than one half of all test results show synergy and additivity effects in combination of azithromycin with moxifloxacin, doxycycline and rifampin, and their MICs in combinations decline from one quarter to three quarters, in result to improved drug activity. When Chlamydia-infected patients may be exposed to intermittent or incomplete treatment, combination treantment will compensate for it. Besides, the MIC of isolates from persistence infection patients is higher in vitro, and this situation will lead to recurrence after standard therapy . Therefore, reasonable antibiotics combination may cure clinical insidious persistence infections, and reduce resistance and complication emergence. However, errant combination will induce serious harmful consequences, such as the combination between minocyline and azithromycin, moxifloxacin and rifampin. Although we cannot be sure that the combination treatment is indeed effective in killing Chlamydia, as discussed above for azithromycin, such a combination may prove to be more useful than azithromycin alone. Finally, we can assume that such a combination may possibly represent a new treatment strategy.
Funding: This study was supported by National Natural Science Foundation of China. (No. 30872285)