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Antimicrobial Agents and Chemotherapy, February 2006, p. 439-444, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.439-444.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Rifalazil Pretreatment of Mammalian Cell Cultures Prevents Subsequent Chlamydia Infection

Robert J. Suchland,¹ Kara Brown,² David M. Rothstein,² and Walter E. Stamm^1*

Division of Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington 98195,¹ ActivBiotics, Inc., Lexington, Massachusetts 02421²

Received 12 August 2005/ Returned for modification 6 September 2005/ Accepted 28 November 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia species are widely disseminated obligate intracellular pathogens that primarily cause urogenital, ocular, and respiratory infections. In these studies, we show that exposing mammalian cells to antibacterial agents prior to Chlamydia inoculation protects the host cells against subsequent challenge by chlamydiae (the protective effect [PE]). Rifalazil exhibited a considerably stronger PE than did azithromycin, rifampin, doxycycline, and ofloxacin. Specifically, 0.002 µg/ml rifalazil incubated for 1 day with a monolayer of McCoy cells was sufficient to protect against a challenge 2 days later with Chlamydia trachomatis serovar D (UW-3). The PE was observed with five different mammalian cell lines and with a variety of C. trachomatis and Chlamydia pneumoniae isolates. The duration of the PE was 6 to 12 days for rifalazil (depending on the cell line), a maximum of 3 days for azithromycin, and less than a day for the other drugs tested. For rifalazil, the PE was shown to be mediated by inhibition of the chlamydial RNA polymerase since mutants with altered RNA polymerases had correspondingly altered PEs. These results suggest that rifalazil may be unique in its ability to prevent infection with obligate intracellular pathogens for a considerable time after treatment. This characteristic may be of particular public health value in reducing reinfection with chlamydiae.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Chlamydia species are widely disseminated obligate intracellular pathogens that primarily cause urogenital, ocular, and respiratory infections. Chlamydia trachomatis infection is the most prevalent bacterial sexually transmitted disease and can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility in women. It is also the leading cause of preventable blindness (trachoma) worldwide (12). Chlamydia pneumoniae is a frequent cause of respiratory infections (5).

Rifalazil [3'-hydroxy-5'-(4-isobutyl-1-piperazinyl)benzoxazinorifamycin] is a novel rifamycin derivative that inhibits the bacterial DNA-dependent RNA polymerase (3). It exhibits high potency against mycobacteria, gram-positive bacteria, Helicobacter pylori (4, 11), and both C. pneumoniae and C. trachomatis (11). MICs of 0.00025 to 0.0025 µg/ml have been reported against both Chlamydia species, depending on the methodology used (7, 10, 11, 15). Thus, rifalazil is more potent against chlamydiae in cell culture than any compound that has so far advanced to human clinical trials. For example, the MICs of rifalazil are 50 to 500 times lower than those of azithromycin, the standard of care for urogenital infections caused by C. trachomatis (1) and a frequently used regimen for treatment of respiratory infections caused by C. pneumoniae (6). Rifalazil also appears more efficient in eradicating chlamydiae from cell cultures (minimum bactericidal concentration) than azithromycin and other drugs (13, 15). Consistent with its potent in vitro activity, a recent clinical trial showed that rifalazil is effective in treating males infected with C. trachomatis (B. Batteiger, W. McCormack, and W. Stamm, unpublished data).

Rifalazil is also able to enter mammalian cells and has a long half-life in animals and humans (11), characteristics that suggest the possibility of a protracted effect of rifalazil against chlamydiae. In view of this, we investigated the ability of rifalazil to inhibit the growth of chlamydiae in cultured cells when exposure of the cells to the drug occurred prior to infection. We have termed this phenomenon the protective effect (PE). In these studies, we show that rifalazil has the unique ability to enter the host mammalian cell and exert an inhibitory effect on chlamydia infection that may last up to 12 days after exposure of a monolayer to the drug.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organisms. We used C. trachomatis strains D/UW-3 (a laboratory reference strain) J/UW-9640, F/UW-9353, and E/UW-9383 (clinical isolates).

In addition, C. trachomatis L2/432 (LGV parent strain) and rifamycin-resistant mutants L2/UW-73 (H471 224 N change in RpoB), L2/UW-60 (I517 224 M), and L2/UW-153 (H471 224 N and I517 224 M) were used as previously indicated (14). C. pneumoniae strains TW-183 (laboratory reference strain) and CWL-029 (clinical isolate) were used. HL, HeLa, McCoy, Hep-2, and BGMK mammalian host cell lines were used for inoculation.

Antibacterial compounds. Rifampin, azithromycin, doxycycline, and ofloxacin were obtained from Sigma, and rifalazil was obtained from the ActivBiotics chemical collection. All agents were dissolved in dimethyl sulfoxide at 10 mg/ml and then diluted appropriately.

Antimicrobial susceptibility testing. MICs were tested by inoculating Chlamydia strains onto McCoy cell monolayers in 96-well microtiter plates as described previously (15). Cells were maintained in antimicrobial-free growth medium consisting of minimal essential medium with 10% fetal bovine serum and 220 mg of L-glutamine/liter added. The inoculum size of infectious chlamydial organisms was 10,000 to 50,000 inclusion-forming units per well. Within 30 min of addition of chlamydiae, the monolayer was centrifuged with a Beckman model J-6 M centrifuge at 1,200 x g for 1 h at 37°C. Following the removal of supernatant, the same growth medium containing 1 µg/ml cycloheximide and the appropriate concentration of rifampin or rifalazil was applied in a volume of 100 µl. Cells were incubated at 37°C in 4% CO₂ for 48 h and fixed with methanol. Chlamydial inclusions were detected by fluorescence with genus-specific monoclonal antibody CF-2 (Washington Research Foundation, Seattle).

Drug protection determination. Monolayers of McCoy, HeLa, Hep-2, BGMK, and HL cells were grown at 37°C in 48-well microtiter plates in antimicrobial-free growth medium as described above. Cells were then exposed to twofold dilutions of rifalazil, ofloxacin, azithromycin, or rifampin for 5 min to 72 h. Unless otherwise noted, the 24-h incubation time was the standard we used. Cells were then centrifuged at 1,200 x g at 37°C for 1 h. Following the removal of supernatant, monolayers were rinsed three times with phosphate-buffered saline and the above-described growth medium containing 1 µg/ml cycloheximide but otherwise drug free (i.e., containing no antibacterial agents) was added. The mammalian cell monolayer was incubated for up to 12 days in the absence of antibacterial agents prior to infection as indicated. For prolonged incubations prior to infection, cells were transferred to fresh, antibacterial-free medium every 48 h. C. trachomatis or C. pneumoniae was then added at a multiplicity of infection of 0.5. After infection, cells were incubated at 37°C in 4% CO₂ for 48 h and fixed with methanol. Chlamydial inclusions were detected by fluorescence with genus-specific monoclonal antibody CF-2 (Washington Research Foundation, Seattle). The minimal protective concentration was defined as the lowest concentration of compound that produced no morphologically normal inclusions by one passage in shell vials in antimicrobial-free medium.

Top Abstract Introduction Materials and Methods Results Discussion References

 
PE as a function of exposure time. In order to determine if exposing mammalian cells to rifalazil prior to infection would result in inhibition of chlamydial growth, rifalazil was added to monolayers of McCoy cells for various times, and then after appropriate washing steps, fresh medium containing cycloheximide was added as described in Materials and Methods. After being transferred to antibacterial-free medium, cells were immediately infected with elementary bodies of C. trachomatis serovar D (UW-3) and the infected cells were incubated for 2 additional days. It was found that incubation with rifalazil for 5 min at 0.032 µg/ml just prior to infection was sufficient to prevent the formation of chlamydial replicating forms (Table 1). A concentration of 64 µg/ml azithromycin was required for 5 min of incubation to obtain a comparable effect. When incubations with either rifalazil or azithromycin were prolonged, there was a decrease in the concentration required to prevent subsequent chlamydial infection. After 24 h of incubation with rifalazil, only 0.002 µg/ml prevented infection, whereas 4 µg/ml azithromycin was required for a similar effect (Table 1). Because the major PE was observed after 24 h of exposure, this incubation time was chosen as the standard time for subsequent experiments.

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TABLE 1. PE when C. trachomatis serovar D (UW-3) is inoculated immediately after exposure and washing, as a function of time of exposure

Magnitude of the PE with McCoy cells as the host. The PE was studied in McCoy cell monolayers after 24 h of incubation with doxycycline, azithromycin, rifampin, ofloxacin, or rifalazil, while the incubation time in compound-free medium was varied. As expected, the concentration required to produce the PE was elevated compared with the MIC. When McCoy cell monolayers were challenged with bacteria immediately after removal of the antibacterial agents, the protective doses were 128, 64, and 8 times the MICs of rifampin, azithromycin, and rifalazil, respectively. Rifalazil, the most potent compound (i.e., the one with the lowest MIC), also had the lowest n-fold elevation in concentration required to achieve the PE. Thus, rifalazil's protective dose was considerably lower than those of the other compounds (0.5 µg/ml for rifampin, 8 µg/ml for azithromycin, but 0.002 µg/ml for rifalazil) (Table 2). Furthermore, the PE of rifalazil was more durable. When the incubation in antibacterial-free medium was increased to 2 days, the protective dose of rifalazil was still 0.002 µg/ml. The PE was detectable for at least 12 days (within 256 times the MIC) for rifalazil with either McCoy cells (Table 2) or HeLa cells (Table 2) as hosts. In contrast, the protective dose rose to more than 256 times the MIC after 3 to 4 days for azithromycin and after 2 days for doxycycline in McCoy cells (Table 2) and HeLa cells (Table 2). Because cycloheximide, which inhibits mammalian cell protein synthesis, is included in the PE procedure as described in Materials and Methods, we decided to determine if the absence of cycloheximide would have an effect on the PE. When cycloheximide was not added and McCoy cells were exposed to rifalazil, the PE was not changed when measured from 1 to 7 days (data not shown), showing that the PE is independent of cycloheximide treatment.

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TABLE 2. PE resulting from 24 h of exposure to the indicated antibacterial, as a function of incubation time prior to inoculation with C. trachomatis serovar D (UW-3)a

Host range of the PE. It was possible that the PE would only be observed with McCoy cells. Table 2 shows that the PE is a more general phenomenon. The PE was the most pronounced in HL and Hep-2 cells against C. trachomatis serovar D (UW-3) for rifalazil, rifampin, and azithromycin (Table 2). McCoy and HeLa cell monolayers were also capable hosts for the PE. However, BGMK cells were less able to support the PE. For example, the amount of rifalazil required to inhibit growth was more than 256 times the MIC beyond 4 days of incubation in rifalazil-free medium (Table 2). McCoy cells were thus chosen for use in subsequent experiments.

The MICs of most compounds against C. trachomatis serovar D were constant in different cell types (0.00025, 0.004, 0.064, and 0.5 µg/ml, respectively, for rifalazil, rifampin, doxycycline, and ofloxacin). However, as reported previously, the MICs of azithromycin did change markedly, depending on the cell type (15). The MIC was lowest when chlamydiae were inoculated into HL and HeLa cells (0.008 and 0.016 µg/ml, respectively) and increased when Hep-2 and McCoy cells were hosts (0.064 and 0.125 µg/ml, respectively). In BGMK cells, the azithromycin MIC was higher at 1.0 µg/ml. As might be expected, the protective dose for azithromycin was considerably reduced in BGMK cells (to 4 µg/ml) when the PE was immediately assessed and was not observed at all beyond 1 day.

PEs measured with different chlamydial isolates. A variety of strains were tested to determine whether the PE was possibly linked to a particular serovar or other strain characteristic or was more broadly applicable. The isolates studied included the commonly used laboratory strain C. pneumoniae TW 183, as well as a C. pneumoniae clinical isolate and three clinical isolates representing different serovars of C. trachomatis (Table 3). When rifalazil was tested for a PE against these strains, the results were similar to those obtained with C. trachomatis D (UW-3) (Table 2). However, there was in general a twofold elevation in the protective dose for all time points tested (compare Tables 3 and 2). The protective dose of azithromycin was considerably elevated compared with that of rifalazil (Table 3), again indicating that rifalazil showed a more robust PE.

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TABLE 3. PE against Chlamydia isolates in McCoy mammalian cell monolayers after 1 day of exposure to rifalazil or azithromycin

Rifalazil-treated cells were able to support growth of chlamydiae. It was important to determine if the PE observed with rifalazil could be attributed to inhibition of its target, bacterial RNA polymerase. An alternative explanation could have been that rifalazil affected an independent mammalian cell function that was required to support chlamydial growth and development. Therefore, we utilized a collection of mutants that we had previously isolated and characterized (14) which produce an altered RNA polymerase. The MICs of rifalazil against these mutants are elevated, so it is possible to test whether mutants could grow in mammalian monolayers treated to protect against wild-type chlamydiae. Table 4 shows that the MIC of rifalazil for the wild-type C. trachomatis L2 parent was 0.00025 µg/ml. The protective dose was 0.004 µg/ml after 2 days of incubation in rifalazil-free medium. If the rifalazil PE was mediated by an effect other than RNA polymerase inhibition, then similar pretreatment of monolayers with rifalazil would have resulted in a uniform protective dose of 0.004 µg/ml for mutant chlamydial strains or the wild-type strain. Instead, the protective doses were elevated in direct proportion to the starting MIC for each mutant. These results indicate that the PE is mediated through inhibition of RNA polymerase by rifalazil.

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TABLE 4. PE for McCoy mammalian cell monolayers exposed to rifalazil for 24 h and inoculated 2 days subsequently with C. trachomatis mutants expressing an RNA polymerase partially resistant to rifalazil

The PE summarized for different antibacterial agents. As indicated above, mammalian cells exposed to rifalazil were capable of retaining sufficient concentrations to inhibit the RNA polymerase activity of chlamydiae that infected cells several days after the mammalian cells were transferred to growth medium free of rifalazil. The magnitude of the PE of rifalazil was considerably greater than that of other compounds both in duration and in potency, as summarized in Fig. 1.

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FIG. 1. PE in HeLa cells against C. trachomatis infection after exposure to the indicated antibacterial, followed by growth in drug-free medium for the number of days indicated. The MICs are shown below the names of the antibacterials, and the protective concentrations at specified times are shown in the bars.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rifalazil has exceptional potency against both C. pneumoniae and C. trachomatis in standard cell culture MIC studies (7, 8, 11, 15). In fact, compared with other drugs, rifalazil uniquely has the capacity to eradicate C. trachomatis (13) and C. pneumoniae in cell culture (R. J. Suchland and W. E. Stamm, unpublished data). Rifalazil also has the benefit that it is less prone to resistance development than rifampin and retains substantial activity against strains that are highly resistant to rifampin (14). In this study, we investigated the possibility that rifalazil might also protect mammalian cells against subsequent infection by chlamydiae. Indeed, it appears that rifalazil does protect mammalian cells against infection for up to 12 days in cell culture. The PE was more pronounced for rifalazil than for the other antibiotics tested. The PE of azithromycin, for example, was limited to 2 or 3 days, and the protective dose after 2 days was 32 µg/ml, an increase of 256-fold compared with its MIC. Rifalazil, in contrast, had a protective dose of 0.002 to 0.008 µg/ml (depending on the chlamydial isolate), which represented only an 8- to 32-fold increase compared with the MIC. The PE was essentially independent of host cell and strain variation, in that the protective doses observed were mostly within a twofold range for rifalazil against a variety of C. pneumoniae and C. trachomatis isolates. Finally, we showed that rifalazil exerts its PE through inhibition of its specific target, bacterial RNA polymerase. To demonstrate this, we tested mutants containing an altered RNA polymerase which is less susceptible to rifalazil and observed an equivalent increase in the protective dose. Thus, the mammalian cells treated with rifalazil are competent to support infection if the infecting strain contains an RNA polymerase that is partially refractory to rifalazil inhibition.

In terms of a more general role in testing antibacterial activity, we believe that the PE is uniquely suited for studying the durability of the antibacterial activity of a drug against intracellular pathogens. The PE cannot be assessed for bacteria which cannot reside inside host cells because there is no opportunity for preincubation of the antibacterial in the absence of its target under normal circumstances. Whereas it may be possible to expose extracellular bacteria directly to an antibacterial and only later induce production of the antimicrobial target, it is only under such specialized circumstances that the PE could be demonstrated for extracellular bacteria. For the study of intracellular pathogens, however, the PE could prove to be a very useful assay that may predict the clinical effectiveness of an antimicrobial against intracellular pathogens.

The ability of rifalazil to exert a PE is assumed to be due to its propensity to concentrate inside mammalian cells and be retained for days after exposure. Rifalazil is concentrated inside mammalian cells, including in monolayers (unpublished data). In addition, we have shown that the chlamydial RNA polymerase remains the target of rifalazil in the PE as described above, and in fact, the PE of partially rifalazil-resistant mutants is elevated in proportion to the MIC for mutant chlamydiae (Table 4). Therefore, the logical conclusion is that preexposure of mammalian cells to rifalazil results in retention of sufficient rifalazil to inhibit the RNA polymerase target days later, upon subsequent infection with chlamydiae.

If our cell culture studies are predictive of an in vivo phenomenon, the PE would imply that protection against infection could persist for days after dosing with an antibiotic. However, it is important to point out that our cell culture experiments undoubtedly differ greatly from the in vivo environment. Thus, we counsel caution in predicting a linear relationship between the PE observed in cell culture and cure rates that might be observed in vivo. For example, cell culture experiments are conducted in the presence of cycloheximide, which prevents growth of mammalian cells. Although the PE of rifalazil is unchanged in mammalian cells not treated with cycloheximide (data not shown), the half-life of rifalazil in such cell cultures might be longer than the half-life in replicating cells in an animal. Still, these results suggest that subjects treated with rifalazil might be protected from reinfection for a longer time relative to other antibacterials such as azithromycin. It would be interesting to test this hypothesis in an animal model to determine whether the PE also occurs in vivo. Of interest, in a recent clinical trial of men with chlamydial nongonococcal urethritis who were tested 5 weeks after single-dose treatment with either azithromycin or rifalazil for persistent or recurrent chlamydial infection, the rifalazil group had a higher proportion of chlamydia-free men than the azithromycin-treated group (Batteiger et al., unpublished).

Finally, the PE may be an important and unique attribute of a drug from the public health perspective in that treated patients may be protected against reinfection for days after treatment. Recurrent infections play an important role in the epidemiology and sequelae of C. trachomatis infection (2, 9, 16), with both trachoma and sexually transmitted infections. A prospective multicenter cohort study of more than 1,100 women showed that 13.4% had a persistent infection or became reinfected after a median of 4.3 months (16). In another study, it was estimated that 6% of the men and 10% of the women were infected at 1 month posttreatment (9). The effect of the long PE provided by rifalazil upon the subsequent risk of reinfection could be directly tested and would be of interest to determine.

 
We thank Joli Carswell for excellent technical support, members of the University of Washington Chlamydia Laboratory for technical assistance, and Christo Shalish for computer expertise in constructing Fig. 1.

This study was supported in part by a grant from ActivBiotics, Inc.

 
* Corresponding author. Mailing address: 1959 NE Pacific Street, University of Washington, Box 356523, Seattle, WA 98195. Phone: (206) 616-4170. Fax: (206) 616-4898. E-mail: wes{at}u.washington.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, February 2006, p. 439-444, Vol. 50, No. 2
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.2.439-444.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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