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Antimicrob Agents Chemother. 2005 May; 49(5): 1787–1793. doi: 10.1128/AAC.49.5.1787-1793.2005. | PMCID: PMC1087634 |
Bactericidal Activity of First-Choice Antibiotics against Gamma Interferon-Induced Persistent Infection of Human Epithelial Cells by Chlamydia trachomatis Nathalie Reveneau, Deborah D. Crane, Elizabeth Fischer, and Harlan D. Caldwell* Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840 Received July 30, 2004; Revised October 25, 2004; Accepted January 20, 2005. Chlamydia trachomatis is responsible for clinically important chronic inflammatory diseases of humans, including trachoma and pelvic inflammatory disease. Persistent infection of mucosal sites may contribute to the development of these chronic inflammatory diseases. Standard clinical therapy results in satisfactory cure rates of acute infections; however, chronic infection associated with persistence has been suggested to be less responsive to antibiotic therapy. We report the efficiency of two first-line chlamydial antibiotics, azithromycin and doxycycline, under conditions of eradication of C. trachomatis persistent infection using the in vitro model of gamma interferon (IFN-γ)-mediated persistence and reactivation from persistence. Doxycycline was superior in eradicating acute (minimal bactericidal concentration [MBC]100 = 2.5 to 5.0 μg/ml) compared to persistent (MBC100 = 10 to 50 μg/ml) infection. In contrast, azithromycin was significantly more effective in eradicating persistent infection (MBC100 = 2.5 to 5.0 μg/ml) than acute infection (MBC100 = 10 to 50 μg/ml). The superior bactericidal effect of azithromycin against persistent infection was found to correlate with the enhanced uptake of the drug by IFN-γ-treated infected epithelial cells. Based on these findings, we hypothesize that azithromycin should be a particularly efficacious anti-infective agent for the eradication of IFN-γ-induced chlamydial persistent infection in vivo. Chlamydia trachomatis is a human pathogen that exhibits a tropism for conjunctival and urogenital columnar epithelial cells ( 26). The organism is an obligate intracellular bacterium characterized by a unique biphasic developmental cycle in which infectious, metabolically inert elementary bodies (EB) differentiate into vegetative reticulate bodies (RB) within a vacuole, termed the inclusion ( 22). After a period of growth by binary fission, RB redifferentiate into infectious EB, and the release of infectious progeny occurs. Infection of the eye results in trachoma, a chronic inflammatory disease that is the leading cause of infectious blindness worldwide ( 27, 31). Infections of the genital tract are a major cause of sexually transmitted diseases, causing acute urethritis and cervicitis ( 26) that frequently progress into chronic inflammatory disease. The most significant of these is chronic salpingitis, an inflammatory disease of the fallopian tube(s) that can result in pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility ( 8). It is unclear whether reinfection alone or persistent infection consisting of altered forms of chlamydiae also contributes to the resulting pathological changes observed in chronic diseases. The current recommended antibiotic treatment for trachoma and urogenital infections is a single dose of azithromycin ( 13, 32). Alternative therapy, when azithromycin is not available or practical because of economic considerations, consists of the administration of topical tetracycline or a 7-day course of doxycycline for the management of active trachoma or genital infections, respectively ( 13, 32). These regimens have been shown to result in satisfactory cure rates of acute infections ( 14, 16, 19, 23); however, chronic diseases have been suggested to be less responsive to antibiotic therapy ( 24). Although productive chlamydial infection is the norm, chlamydiae are difficult to culture from patients with obstructive infertility or with progressive ocular scarring despite the detection of chlamydial antigens and nucleic acids indicating the presence of persisting organisms ( 11, 18, 30). In fact, persistent infection characterized by unculturable chlamydial forms has been proposed as being responsible for the induction of the sustained inflammatory response leading to debilitating pathological changes ( 3, 21). “Chlamydial persistence” has been described as a long-term association between chlamydiae and their host cells in which these microorganisms remain in a viable but culture-negative state ( 4). In vitro, persistent infections can be established by treatment with gamma interferon (IFN-γ) ( 2) or penicillin ( 12) or by deprivation of certain nutrients ( 15, 25). Murine models of infection, as well as studies in human patient populations, identify IFN-γ-secreting CD4 + and CD8 + T cells as primary mediators of protective immunity against chlamydial infections ( 17, 29). The inhibitory effect of IFN-γ on chlamydial growth in vitro is well described. IFN-γ affects human cells by inducing indoleamine 2,3-dioxygenase (IDO), which catalyzes the initial step in the degradation of l-tryptophan to N-formylkynurenine and kynurenine ( 9). The resulting depletion of intracellular pools of tryptophan by IDO starves chlamydiae of this essential amino acid, leading to the development of persistent forms ( 1, 10). Persistent chlamydiae exhibit morphologically abnormal RB unable to differentiate into infectious EB. These cryptic persistent forms rapidly retransform back to normal RB and infectious EB when host tryptophan pools return to normal levels ( 5). Although not definitively known, it is likely that this cytokine-induced persistent growth exists in vivo and is a primary mediator of chronic inflammation. Hence, treatment strategies that more effectively eradicate these persistent forms could be beneficial in the management of chlamydial diseases. Here, we investigate the efficacy of the two front-line antichlamydial antibiotics in eradicating acute and IFN-γ-mediated persistent infection in vitro. We show, using this model, marked differences between doxycycline and azithromycin in their ability to eradicate acute and persistent infection. We believe this in vitro model of persistence is relevant to in vivo infection; hence, our findings could have important implications in the management of human chlamydial infections. IFN-γ treatment and reactivation. HeLa 229 epithelial cells were grown in TC24 culture plates at a density of 4 × 105 cells per ml at 37°C in 5% CO2 in DME H-21 (UCSF Cell Culture Facility, San Francisco, Calif.) containing 4 mg of tryptophan/liter supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, Utah), 2 mM l-glutamine (Gibco Invitrogen Corp., Carlsbad, Calif.), 10 mM HEPES (Gibco), 1 mM MEM sodium pyruvate solution (Gibco), 55 μM β-mercaptoethanol (Gibco), and 10 μg of gentamicin/ml. Monolayers were formed in the presence or absence of 50 U of recombinant human IFN-γ (R&D Systems, Minneapolis, Minn.)/ml for 24 h. Monolayers were then infected with C. trachomatis serovar D at a multiplicity of infection of 0.2 in SPG (10 mM sodium phosphate [pH 7.2], 0.25 M sucrose, 5 mM l-glutamic acid). The plates were then centrifuged at 550 × g for 1 h, rocked at 37°C for 30 min, and incubated in the presence or absence of IFN-γ. After incubation with IFN-γ for 12, 24, 48, or 72 h, cultures were reactivated by removing IFN-γ and pulsing the monolayers with medium containing 10× tryptophan (40 mg/liter) (Fig. ). The cultures were then incubated for various intervals before they were harvested as described below. Recoverable IFU. After reactivation, monolayers were harvested, cells lysed, and chlamydial suspensions were used to infect fresh HeLa 229 monolayers. After 30 h, infected cells were methanol fixed and stained for enumeration of recoverable inclusion-forming units (IFU) by immunofluorescence assay using a chlamydial anti-lipopolysaccharide antibody, followed by a secondary fluorescein isothiocyanate-conjugated antibody. The results were expressed as the mean of four replicates ± the standard deviation. Transmission electron microscopy. C. trachomatis-infected HeLa cells were seeded on 13-mm Thermanox coverslips (Nunc, Naperville, Ill.) and fixed at various times postinfection (p.i.) with 4% (wt/vol) paraformaldehyde-2.5% (wt/vol) glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.4). The samples were postfixed with 0.5% (wt/vol) osmium tetroxide-0.8% (wt/vol) potassium ferricyanide, followed by 1% (vol/vol) tannic acid and stained overnight at 4°C en bloc in 1% (wt/vol) uranyl acetate. Samples were dehydrated with a graded ethanol series and embedded in Spurr's resin. Thin sections were cut with an RMC MT-7000 ultramicrotome (Ventana, Tucson, Ariz.) and stained with 1% uranyl acetate and Reynold's lead citrate before they were observed at 80 kV with a Philips CM-10 electron microscope (FEI, Hillsboro, Oreg.). Images were acquired with an AMT digital camera system (Advanced Microscopy Techniques, Chazy, N.Y.) and processed by using Adobe Photoshop, version 7.0 (Adobe Systems, Inc., San José, Calif.). Antibiotic treatment. HeLa 229 cells were grown and infected in the presence or absence of IFN-γ as described above. Various concentrations of azithromycin (Pfizer Labs, New York, N.Y.) or doxycycline hyclate (Sigma-Aldrich Co., St. Louis, Mo.) were added 12 h p.i. in IFN-γ-treated and untreated infected cells. At that time point, persistence of chlamydiae is induced in IFN-γ-treated cells ( 6). A control without antibiotic in the presence or absence of IFN-γ was included. Infected cells were cultivated with antibiotics for 36 h, washed three times (10 min each) in Hanks balanced salt solution (Gibco) to remove IFN-γ and antibiotic, and incubated in fresh medium containing 40 mg of tryptophan/liter for an additional 48 h (reactivation period). Recoverable IFU were determined for each experimental condition as described above. For each concentration of antibiotic used, the log 10 recoverable IFU values were normalized against the no-IFN-γ control cultures. The results were expressed as the mean of four replicates ± the standard deviation. Uptake of [3H]azithromycin by IFN-γ-treated chlamydia-infected HeLa 229 cells. HeLa 229 cells were grown and infected at a multiplicity of infection of 3 in the presence or absence of IFN-γ as described above. [3H]azithromycin was kindly provided by Pfizer Global Research and Development, Groton, Conn., and [3H]doxycycline was purchased from ARC, St. Louis, Mo. Cells were incubated with a solution containing 1.65 μCi of [3H]azithromycin (9.9 Ci/mmol) or 1.46 μCi of [3H]doxycycline (5 Ci/mmol) plus nonradioactive azithromycin or doxycycline at a final concentration of 2.5 μg/ml (3.35 or 5.85 nmol/ml, respectively). At each time point, the monolayers were washed five times with Hank's balanced salt solution to remove extracellular antibiotic. The cells were then lysed with 1% Triton X-100 (Sigma-Aldrich Co.), and the amount of radioactivity in the lysate was determined by liquid scintillation counting (LS 6500; Beckman Coulter, Fullerton, Calif.). To determine the amount of radioactive background (nonspecific binding to the plastic), [3H]antibiotic was added to an empty well and removed by washing, and the well was treated with 1 ml of 1% Triton X-100. The radioactivity in the preparation was subtracted from this background. At each time point after addition of antibiotic, for each condition, two extra wells were used to determine the cell viability. Monolayers were washed five times with Hank's balanced salt solution and treated with 0.5 ml of 0.5% trypsin-5.3 mM EDTA (pH 7.4; Gibco) for 5 min at 37°C. The trypsin action was stopped with 1 ml of fetal bovine serum containing growth medium. Trypan blue stain 0.4% (Gibco) was added to cell suspension to give a dilution factor of 2, and viable cells were counted by using a hemocytometer. The amount of radioactivity for each condition was normalized to 105 viable cells. The results were expressed as the mean ± the standard deviation of triplicate samples. Statistical analysis. Statistical analyses were performed with the Microsoft Excel software. The two-tailed unpaired Student t test was used to determine the significance of the differences between groups. Differences were considered significant at a P value of <0.05. In vitro model of chlamydial persistent infection We first performed experiments to determine the temporal kinetics of persistence and reactivation from persistence. These experiments were designed to determine the longest time period of exposure to IFN-γ that resulted in a persistent growth phenotype that could be maximally reactivated to normal infectious progeny following cytokine removal, thus allowing us to examine the bactericidal effect of antibiotics on persistent growth for the greatest time period. Figure describes how these experiments were performed. Chlamydia-infected HeLa cells were incubated with or without IFN-γ for various periods of time (0 to 72 h), the IFN-γ was removed, and the cells were pulsed with medium containing excess tryptophan to promote reactivation from persistent growth. The cells were lysed and processed for recoverable IFU at different time points (6 to 72 h) after reactivation. There was no effect on chlamydial infectivity when cells were incubated in the presence of IFN-γ for 12 h (Fig. ). In contrast, dramatic reductions in IFU (2 to 5 log10) were observed in cultures treated for 24 (Fig. ), 48 (Fig. ), and 72 h (Fig. ). There was rapid recovery of infectious progeny after the removal of cytokine and pulsing with tryptophan in the 24- and 48-h treated cultures (Fig. ). In contrast, only minimal recovery in infectious progeny was observed after 72 h of exposure (Fig. ). The morphological characteristics of chlamydiae were evaluated for each time point by transmission electron microscopy. No morphological changes were detected in chlamydial structure between untreated and IFN-γ-treated cells at 12 h p.i. (data not shown), a finding consistent with recoverable IFU shown in Fig. . In contrast, however, important differences in chlamydial morphology and development were observed between IFN-γ-treated and control cultures at 24 and 48 h p.i. (Fig. ). In untreated cells at 24 h p.i. (Fig. ), inclusions contained typical large RB that developed into normal-appearing larger inclusions filled with a mixture of RB, intermediate bodies, and highly condensed EB at 48 h p.i. (Fig. ). In contrast, inclusions in IFN-γ-treated cells at 24 h p.i. were much smaller and occupied by enlarged highly pleomorphic RB (Fig. ). Inclusions retained these highly atypical developmental forms and properties at 48 h p.i. (Fig. ) and 72 h p.i. (data not shown). Of interest to the goals of the present study was our observation that the effect of IFN-γ treatment at 48 h p.i. was reversible. After removal of the cytokine, inclusions became much larger and were packed with typical chlamydial RB and EB developmental forms (Fig. ) with a morphological appearance similar to that of untreated cultures (Fig. ). Based on these collective results, the 48-h IFN-γ treatment period was chosen to study the effect of antibiotics on in vitro chlamydial persistent growth. Bactericidal activity of antibiotics on IFN-γ-induced C. trachomatis persistent infection. To investigate whether persistent infection was more resistant to antibiotics than active infection, we assayed chlamydial growth in cells treated with or without IFN-γ in the absence or presence of various concentrations of azithromycin or doxycycline. The antibiotic concentrations used in these experiments were chosen based on the published literature to include a range of concentrations that, in theory, would reflect both bacteriostatic and bactericidal activities, the latter being considered particularly relevant because of our focus to relate our findings to the successful management of chlamydial persistent infection in vivo. A schematic of the experimental protocol used in these studies is shown in Fig. . Figure show the recoverable IFU obtained from IFN-γ-treated and untreated chlamydia-infected HeLa cells at multiple drug concentrations. Azithromycin treatment resulted in a dose-dependent reduction in recoverable IFU in both normal and persistently infected cells. Notably, the reduction in IFU was markedly greater (1.0 to 1.5 log10) at each concentration of azithromycin in persistently infected cultures compared to normal cultures (P < 0.005). Of particular importance to the present study was the striking difference in minimal bactericidal concentration (MBC) values between treated persistent and normal cultures. The MBC for persistently infected cultures was between 2.5 and 5.0 μg/ml versus 10 to 50 μg/ml for nonpersistent infection. Thus, persistent chlamydial organisms were significantly more sensitive to eradication by azithromycin than were actively growing chlamydiae. Completely contrasting results were observed in comparative experiments with doxycycline (Fig. ). Although we observed a significant dose-dependent reduction in recoverable IFU in doxycycline-treated persistently infected and normally infected cultures (1.5 to 2.0 log10, P < 0.005), the trend in the sensitivity of persistent and normal chlamydial growth phenotypes to the drug was completely opposite from that of azithromycin. For example, the doxycycline MBC for persistently infected cultures was between 10 and 50 μg/ml, whereas the MBC for normal growth cultures was between 2.5 and 5.0 μg/ml. This result demonstrated that persistent chlamydial organisms were more resistant to doxycycline than were actively growing normal organisms. Table 1 shows the MBC 90 and MBC 100 for azithromycin and doxycycline in treating persistently infected and normal chlamydial cultures. The MBC 90 of azithromycin was identical for persistently infected and normal cultures (MBC 90 = 0.25 to 1.0 μg/ml). On the other hand, the MBC 90 of doxycycline was 20-fold higher for persistently infected cultures (MBC 90 = 1 μg/ml) than for normal cultures (MBC 90 = 0.05 μg/ml); however, an interesting finding was that azithromycin was more effective in eradicating persistent infection (MBC 100 2.5 to 5.0 μg/ml) than actively growing chlamydiae (MBC 100 10 to 50 μg/ml). This was in marked contrast to doxycycline, which was more efficacious in eradicating growing bacteria (MBC 100 = 2.5 to 5.0 μg/ml) versus persistent organisms (MBC 100 = 10 to 50 μg/ml). | TABLE 1.MBCs of azithromycin and doxycycline on C. trachomatis in vitro |
Uptake of [3H]azithromycin by IFN-γ-treated infected HeLa cells. To investigate why azithromycin had a greater bactericidal activity against persistent forms than on actively growing bacteria, we analyzed the uptake of [3H]azithromycin by infected HeLa 229 cells treated with IFN-γ. We also measured the uptake of [3H]doxycycline as a control. Untreated and IFN-γ-treated cells were incubated with a mixture of radioactive and unlabeled antibiotic. HeLa cells were assayed for intracellular antibiotic at different time points during the incubation period. Irrespective of treatment, incubation of HeLa 229 cells with azithromycin was associated with a rapid increase in the uptake of drug by 4 h (Fig. ). Of interest, the concentration of azithromycin in IFN-γ-treated cells increased dramatically by 36 h, whereas in untreated cells there was no significant change in intracellular drug concentration. The difference obtained at 36 h in the intracellular concentration of azithromycin between untreated and IFN-γ-treated cells was highly significant (P = 0.0004). In contrast, there was no similar accumulation of doxycycline in IFN-γ-treated cells over time (Fig. ). Thus, these findings are consistent with those reported in Fig. , where we showed that chlamydia-infected IFN-γ-treated cultures were more sensitive to the growth-inhibitory action of azithromycin than doxycycline. We investigated here the ability of two first-line antichlamydial antibiotics, azithromycin and doxycycline, to eradicate C. trachomatis serovar D persistent infection using an in vitro model of IFN-γ-mediated persistence and reactivation from persistence. Our results demonstrate that azithromycin is significantly more effective in eradicating persistent chlamydiae than actively growing organisms. In contrast, doxycycline was found to be more efficacious in eradicating normally replicating chlamydiae than it was against abnormal persistent forms of the organism. We have focused on defining concentrations of antibiotics that result in eradication of infection in both acute and persistent infection models. We have used the IFN-γ model of in vitro persistence ( 2) because it has been well characterized both biologically ( 1, 5) and transcriptionally ( 6) and is patently relevant to in vivo infections, in which the cytokine has been well documented as an important mediator of chlamydial immunity ( 17, 20). Collectively, we believe these are very important aspects of our work in correlating the findings to the clinical setting. For example, if IFN-γ is the major factor in the induction of persistent infection and eradication of persistent infection is paramount to curing chlamydial chronic disease, our in vitro model clearly has important parallels in the antibiotic management of diseases such as chronic trachoma and salpingitis. Considering this to be accurate, we clearly show that the MBC 100 for azithromycin and doxycycline are markedly different for the treatment of persistent and acute in vitro chlamydial infection, respectively (Fig. and Table 1). Our findings differ from those recently reported by Wyrick and Knight ( 33), who showed that persistent chlamydial organisms generated in human endometrial epithelial cells by exposure to penicillin were more refractory to azithromycin. These differences in results might be explained by the mechanisms used to study persistence. Although penicillin-induced persistent chlamydiae share some of the morphological and biologic characteristics of IFN-γ-induced persistent forms, there are some very important differences in gene expression profiles between these two mechanisms that deserve comment. First, IFN-γ has an effect on both host cell and chlamydial gene expression, whereas penicillin affects only chlamydiae. IFN-γ regulates the expression of a multitude of genes, many of whose products function in mediating host immune responses ( 28). In human epithelial cells, IFN-γ induces IDO, which has been shown to be an important response mediator in the inhibition of chlamydial growth by the deprivation of host tryptophan pools. Of interest, global transcriptional analysis of chlamydial gene expression during IFN-γ-mediated persistence showed a dramatic increase in the expression of the trpRBA operon ( 6). This coordinated transcriptional response by chlamydiae to sense the effects of the cytokine on host cells strongly implicates an important and physiologically relevant interaction between host and pathogen. Similar transcriptome analysis has not been reported for penicillin-induced persistent infection, but it is unlikely that the drug has an analogous effect on either the host or chlamydiae. Furthermore, IFN-γ induces the expression of HLA class II molecules in HeLa 229 cells (data not shown), suggesting that other host functions are likely modified or changed after treatment. Such a cytokine-induced alternation in host physiology might be mirrored by differences in endocytic or pinocytic processes that could account for the increased uptake of radiolabeled azithromycin into IFN-γ chlamydia-affected cells (Fig. ). Yet again, such host responses are not likely induced by exposure to penicillin. Of note, however, similar to the Wyrick and Knight penicillin studies ( 33), we found that persistent chlamydial forms were more resistant to doxycycline (Fig. ). Thus, it is possible that the persistent growth forms demonstrate differences in susceptibility or resistance to antibiotics depending on how the drug is taken up by cells and/or its disposition following incorporation into cells. Because azithromycin is efficiently and stably accumulated into IFN-γ-treated infected cells (Fig. ), it could be more available to interact with the inclusion-harboring persistent chlamydia growth forms. A similar effect of IFN-γ on enhanced azithromycin uptake has been previously reported with macrophages infected with Mycobacterium tuberculosis ( 7). C. trachomatis causes a spectrum of clinically distinct manifestations ranging from acute self-limiting infections to chronic inflammatory diseases. Relapsing infection is associated with the notion of a persistent state of chlamydial infection. Indeed, persistence has been suggested as a mechanism that leads to the inflammatory response, contributing to the resulting pathological changes observed in patients with chronic diseases. Moreover, a persistent infection may serve as an important reservoir for new infections. This may require alternative therapeutic approaches for the management of chlamydial infections. We think that the IFN-γ-mediated persistence model is relevant considering the role of IFN-γ in vivo. IFN-γ is a key cytokine in the development of antichlamydial protective immunity ( 17, 20). Consequently, chlamydiae likely subsist in an environment rich in IFN-γ in vivo. If the in vitro observation of IFN-γ-mediated persistence holds for human infection, then chlamydiae may establish persistent infections forming morphologically aberrant nonculturable forms at the site of chronic diseases. Moreover, changes in the level of IFN-γ are likely to occur in vivo, which could lead to the development of a mixed population of bacteria. Our findings could have important clinical applications, because they indicate that azithromycin would be particularly efficacious against persistent chlamydial infection. In contrast, doxycycline may not be as effective in treating persistent infection. This raises the possibility that azithromycin would be effective therapy for the management of both acute and persistent infections, whereas doxycycline might be more effective for the former. With the assumption that chronic chlamydial infections such as pelvic inflammatory disease or trachoma involve persistent infection and uncomplicated infections are a mixture of acute and persistent infections we hypothesize that overall azithromycin would be more effective for the treatment of chlamydial infections than doxycycline. We thank Pfizer, Inc., for providing [3H]azithromycin. We thank T. 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