ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1992,
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In Vitro Evaluation of Activities of Azithromycin, Erythromycin, and Tetracycline against Chlamydia trachomatis and Chlamydia pneumoniae LAURA E. WELSH,1 CHARLOTTE A. GAYDOS,1 AND THOMAS C. QUINN' 2* Division of Infectious Diseases, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205,1* and Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208922 Received 12 September 1991/Accepted 4 December 1991 The in vitro activities of azithromycin (CP-62,993; Pfizer), erythromycin, and tetracycline were evaluated by inhibiting Chlamydia trachomatis and Chlamydia pneumoniae, formerly TWAR, propagation in vitro in McCoy cells, HeLa cells, and HL cells. Eleven clinical isolates of C. trachomatis (serovars D, E, F, J, K, and L2) and four strains of C. pneumoniae were tested with an inoculum of 103 inclusion-forming units in a 96-well microtiter plate. The MIC ranges of these antimicrobial agents against C. trachomatis were as follows: azithromycin, 0.125 to 0.5 ,ug/ml; erythromycin, 0.25 to 0.1 ,ug/ml; and tetracycline, 0.0625 to 1.0 ,ug/ml. The MBC ranges, calculated from passage into antibiotic-free medium, were as follows: azithromycin, 0.125 to 4.0 ,ig/ml; erythromycin, 0.5 to 8.0 ,ug/ml; and tetracycline, 0.0625 to 4.0 ,ug/ml. The MIC ranges for C. pneumoniae in both HeLa and HL cells were as follows: azithromycin, 0.125 to 1.0 ,Ig/ml; erythromycin, 0.0625 to 1.0 ,ug/ml; and tetracycline, 0.125 to 1.0 ,ig/ml. The MBC ranges were as follows: azithromycin, 0.25 to 1.0 ,ug/ml; erythromycin, 0.25 to 1.0 ,ug/ml; and tetracycline, 0.125 to 4.0 ,Ig/ml. From the results of this in vitro study, azithromycin appears to be an effective antibiotic comparable to tetracycline and erythromycin for use in the treatment of both C. trachomatis and C. pneumoniae infections.
form, was obtained from Pfizer Central Research, Groton, Conn. Azithromycin was reconstituted in 1.0 ml of absolute methanol and then diluted in 9.0 ml of sterile distilled H20 for a concentration of 1,000 ,ug/ml. Bacterial strains. Two different clinical isolates of serovars D, E, F, J, and K and a clinical isolate of serovar L2 of C. trachomatis were obtained from the Baltimore City Sexually Transmitted Diseases Clinics. These were recent clinical isolates from uncomplicated genital infections. C. pneumoniae TW183, AR39, and AR388 were obtained from the Washington Research Foundation, Seattle, and C. pneumoniae VR1310 (CDC CWL029) was obtained from the American Type Culture Collection, Rockville, Md. The clinical isolates were passed three or fewer times to achieve titers for MIC testing and serovar typing. Susceptibility assays. The in vitro antimicrobial effects on C. trachomatis and C. pneumoniae were determined by tissue culture assay. Antibiotic-free McCoy cells were used for C. trachomatis. For C. pneumoniae, antibiotic-free HeLa and HL cells were used. Antibiotic-free cell monolayers in 96-well microtiter plates were pretreated with DEAEdextran (Sigma, St. Louis, Mo.) in phosphate-buffered saline for 30 min at 35°C with 5.0% CO2 in air (15). No dextran pretreatment was used for HL cells. The dextran was aspirated, and the cells were inoculated with 0.1 ml of 103 inclusion-forming units of each chlamydial strain or isolate. Cultures were centrifuged for 60 min at 600 x g at 35°C and then incubated at 35°C with 5.0% CO2 for 30 min (17). The inoculum was aspirated and replaced with 0.2 ml of overlay medium per well. This medium consisted of RPMI 1640 (Whittaker Bioproducts, Walkersville, Md.), 10% fetal bovine serum (Hyclone, Logan, Utah), 1% glucose, 1.0 ,ug of cycloheximide (Sigma) per ml, and serial twofold dilutions of each antimicrobial agent (17). Tetracycline, erythromycin, and azithromycin were diluted from 1.0 to 0.31 ,ug/ml. Six
Chlamydia trachomatis infections are recognized as the most prevalent sexually transmitted bacterial infections in the United States, with an estimated 4 million cases annually (2, 11). Chlamydia pneumoniae has been associated with 12% of cases of outpatient pneumonia, 5% of cases of bronchitis, and at least 1% of cases of pharyngitis among university students in Seattle, Wash. (6). Tetracycline or doxycycline has been the treatment of choice for chlamydial infections involving both C. trachomatis and C. pneumoniae. However, erythromycin has frequently been recommended as an alternative antibiotic regimen. Unfortunately, both tetracycline and erythromycin must be taken for 7 days for adequate efficacy, a fact that frequently results in noncompliance or inadequate treatment. Azithromycin is a 15-member ring macrolide antibiotic whose preliminary in vitro susceptibility studies have indicated superior activity compared with erythromycin when tested against various strains of C. trachomatis and C. pneumoniae (5, 17). Azithromycin can be administered as a single dose because of its long half-life. Since previous in vitro susceptibility studies have been limited to standard laboratory strains, we initiated a study to compare the in vitro activities of azithromycin, erythromycin, and tetracycline against 11 recent clinical isolates of C. trachomatis and four strains of C. pneumoniae. MATERIALS AND METHODS Antimicrobial agents. Tetracycline hydrochloride and erythromycin lactobionate were obtained in powder form from U.S.P.C., Inc., Rockville, Md. Tetracycline and erythromycin were reconstituted in sterile distilled H20 for a concentration of 1,000 ,ug/ml. Azithromycin, also in powder *
Corresponding author. 291
292
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WELSH ET AL.
TABLE 1. In vitro activity of tetracycline, azithromycin, and erythromycin against clinical isolates of C. trachomatis Isolate
Serovar
Tetracycline
Concn (Lg/ml) of: Azithromycin
Erythromycin
no .
396 443 448 677 021 256 176 701 173 457 422
D D E E F F J J K K L2
MIC
MBC
MIC
MBC
MIC MBC
0.125 0.125 0.125 0.125 0.0625 0.125 0.0625 0.0625 1.0 1.0 0.0625
1.0 1.0 1.0 4.0 2.0 2.0 0.0625 0.5 2.0 2.0 0.5
0.5 0.25 0.25 0.5 0.125 0.5 0.25 0.125 0.5 0.5 0.25
1.0 0.5 4.0 0.5
1.0 0.5 0.5 0.5
4.0 2.0 0.125 2.0
0.25
2.0 4.0 0.5
1.0 0.5 0.25 1.0 1.0 0.5
4.0 1.0 8.0 0.5 4.0 2.0 2.0 0.5 2.0 4.0 1.0
inoculated wells were overlaid with each dilution of antibiotic (1, 13). Positive and negative controls were overlaid with 0.2 ml of the above medium free of all antibiotics per well. Plates were then incubated for 48 h at 35°C with 5.0% CO2 in air. The same chlamydial strain or isolate served as the inoculum for a positive control in each assay. The inoculum for a negative control in each assay was RPMI 1640 medium without antibiotics. Following incubation, culture medium was aspirated and three wells per dilution were fixed and stained with a fluorescein-conjugated monoclonal antibody to C. trachomatis (Syva Corp., Palo Alto, Calif.) and C. pneumoniae (Kallestad Diagnostics, Chaska, Minn.). Chlamydial inclusion bodies were counted with a Zeiss fluorescent microscope (Zeiss, Oberkochen, Germany) and a 20x objective. An average of 14 inclusion-forming units per low-power field were seen. The MIC was determined to be the lowest concentration at which complete inhibition of inclusion formation was observed (17). The remaining wells were passed by scraping the cell layer and reinoculating it onto fresh cell monolayers without antibiotics (13). The cultures were incubated for another 48 h in the previously described antibiotic-free overlay medium. After staining with fluorescein-conjugated monoclonal antibody, the MBC was determined to be the lowest concentration of antimicrobial agent preventing inclusion formation after passage (17). When the MBC was greater than 1.0 p,g/ml, the culture assay was performed again using overlay medium with serial dilutions of antibiotic from 8.0 to 0.5 ,ug/ml. RESULTS
Tetracycline was the most effective antimicrobial agent for C. trachomatis, followed by azithromycin and then erythromycin (Table 1). C. trachomatis serovars F and K were most resistant to tetracycline, erythromycin, and azithromycin, with the MBCs for both isolates of each serovar ranging from 2.0 to 4.0 ,ug/ml. The MBC of erythromycin for one isolate of C. trachomatis serovar D was 4.0 ,ug/ml, and the MBC of tetracycline for one isolate of serovar E was 4.0 ,ug/ml. The MBC range of azithromycin and erythromycin for another isolate of C. trachomatis serovar E was 4.0 to 8.0 ,ug/ml. Serovar J was relatively sensitive to tetracycline, erythromycin, and azithromycin, with an MBC range of 0.0625 to 2.0 ,ug/ml. C. trachomatis serovar L2 was the most sensitive
to all three antibiotics, with an MBC range of 0.0625 to 1.0
p.gIml.
For C. pneumoniae isolates, azithromycin was more effective than tetracycline or erythromycin. Two C. pneumoniae isolates, Ar388 and VR1310, were more resistant to tetracycline than they were to erythromycin or azithromycin, with MBCs of 2.0 to 4.0 Rg/ml. The other C. pneumoniae isolates, TW183 and AR39, were more sensitive to tetracycline, erythromycin, and azithromycin, with MBC ranges of 0.125 to 1.0 p.g/ml. Little difference was observed between MICs and MBCs in HeLa cells and HL cells.
DISCUSSION
Tetracycline and erythromycin are considered to be the drugs of choice for treatment of C. trachomatis and C. pneumoniae infections. Several clinical trials have established the efficacy and essential therapeutic equivalence of tetracycline, doxycycline, and minocycline. There is now little difference in the costs of tetracycline and doxycycline; therefore, the latter is preferred because of better compliance (1, 3). Erythromycin is recommended for treatment of chlamydial infections when the patient is pregnant or nursing a child or is an adult unable to tolerate tetracycline (3, 4). Standard regimens of tetracycline, doxycycline, or erythromycin appear to be effective against chlamydial infections in most situations, producing a decrease in symptoms and preventing further transmission. However, in some cases it appears that these antimicrobial agents suppress the organism without eradicating it, causing a chronic persistent infection (9). The eradication rate is 94% with tetracycline and 91% with erythromycin (14). The mean peak concentration of erythromycin in serum is 1.73 ,ug/ml, and that of tetracycline is 2.6 ,ug/ml (17). Of patients being treated with erythromycin, 8 to 10% have treatment-related side effects, compared with 7% of patients being treated with tetracycline. All of these side effects are gastrointestinal (14). In one study, several C. trachomatis isolates were resistant to tetracycline, doxycycline, erythromycin, sulfamethoxazole, and clindamycin but were sensitive to rifampin, ciprofloxacin, and ofloxacin (10). This resistance is not surprising, given the current practice of treating most documented or suspected C. trachomatis and C. pneumoniae infections with tetracycline or erythromycin, and continued surveillance for antibiotic resistance is warranted to determine the extent of this problem. Azithromycin is a 15-member azalide which has a long half-life in serum and high tissue affinity. After one 500-mg dose of azithromycin, concentrations in tissue are 400 times those achieved in serum (17). The mean peak concentration of azithromycin in serum is 0.37 ,uglml, and the half-life in serum is 68 h (12). This long half-life in serum and high tissue affinity make it possible to administer azithromycin in a single dose. In one of the clinical trials, azithromycin was given as a single 1-g dose and as a 500-mg dose on day 1 followed by single 250-mg doses on days 2 and 3. Doxycycline was given as a 100-mg dose twice per day for 7 days. There was 100% eradication of C. trachomatis after 1 week with both dosage regimens of azithromycin and 95% eradication with the doxycycline regimen. Eight percent of azithromycin-treated patients and 6% of the doxycyclinetreated patients reported treatment-related side effects, all of which were mild and related to the gastrointestinal tract (8). Since doxycycline and erythromycin must be administered several times a day for 7 days, there should be better patient
ACTIVITY OF AZITHROMYCIN AGAINST CHLAMYDIA SPP.
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TABLE 2. In vitro activity of tetracycline, azithromycin, and erythromycin against C. pneumoniae strains in HeLa and HL cells Concn (,ug/ml) of:
TW183 TW183 (duplicate test) AR39 AR388 VR1310
MIC
Erythromycin
Azithromycin
Tetracycline Strain
MIC
MBC
HeLa
HL
HeLa
HL
0.25 0.125 1.0 1.0 0.25
0.25 0.25 0.5 0.5 0.25
0.25 0.125 1.0 2.0 2.0
1.0 1.0 1.0 4.0 1.0
MIC
MBC
MBC
HeLa
HL
HeLa
HL
HeLa
HL
HeLa
HL
0.125 0.125 0.125 0.25 0.125
0.125 0.125 0.125 0.25 0.125
0.25 0.25 1.0 0.5 0.5
0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.25 0.125 0.125
0.125 0.125 0.125 0.0625 0.125
0.25 0.25 1.0 1.0 0.5
0.5 0.5 1.0 1.0 0.25
compliance and therefore more effective treatment of chlamydial infections with one dose of azithromycin. In this study, the MIC of azithromycin for C. pneumoniae varied from 0.125 to 0.25 ,ug/ml and the MBC varied from 0.25 to 1.0 jig/ml. All differences between HeLa cells and HL cells for the MICs were within one dilution (Table 2), except for AR39, for which there was a twofold difference, with the MIC being 0.125 jig/ml in HL cells and 1.0 ,ug/ml in HeLa cells. Similarly, all MBC results were within a onetube variation, except for AR39, for which the MBC was 0.25 ,ug/ml in HL cells and 1.0 jig/ml in HeLa cells. The MIC for TW183 was 0.25 ,ug/ml in both HL and HeLa cells, which is in contrast to that observed by Chirgwin et al. of 0.06 ,ug/ml in HeLa cells (5). Reasons for this remain unknown, although slight differences in methods may have influenced these results. Since less inoculum (0.1 ml) is used for microtiter wells than shell vials (0.2 ml), this may account for differences seen between laboratories. In addition, these are clinical isolates from a different area of the United States and may represent population differences in our strains. Our results remained reproducible when done in triplicate on two different occasions. No dextran pretreatment was used for HL cells, and 30 jig/ml for 30 min was used for HeLa cells. Chirgwin et al. (5) pretreated HeLa cells for 10 min, although this would not seem to explain a two-dilution difference. Inoculum sizes for each study also varied slightly. Perhaps small variations in this inoculum size are significant. The results of the present in vitro study indicate that azithromycin may be effective for C. trachomatis and C. pneumoniae infections. Other studies have shown that azithromycin is more effective than erythromycin against penicillinase-producing strains of Neisseria gonorrhoeae (16). This drug could be used as treatment for both C. trachomatis and N. gonorrhoeae because these infections often occur concurrently. However, treatment trials have indicated that a 2-g dose of azithromycin is more effective in treating N. gonorrhoeae infections (7). Unfortunately, the 2-g dose of azithromycin was associated with a high frequency of gastrointestinal side effects. Consequently, the optimum dose and efficacy for the treatment of gonorrhea is not yet fully established. Azithromycin has also been shown to be highly effective in vitro against Mycoplasma pneumoniae (16). This is important, because the clinical presentation of M. pneumoniae and C. pneumoniae infections may be similar, but the organisms are difficult to isolate (5), and most of these infections are diagnosed by serological methods. Since azithromycin is effective against both M. pneumoniae and C. pneumoniae, empiric therapy should be active against both. The in vitro efficacy of azithromycin against C. trachomatis and C. pneumoniae indicates that this is an effective
antimicrobial agent for these infections. The long half-life in serum, high levels in tissue, and single-dose treatment regimen will certainly be an advantage for azithromycin because they may result in greater patient compliance. There will probably be more effective treatment of chlamydial infections because a single 1-g dose of azithromycin can effectively eradicate the organism. The additional in vitro efficacy of azithromycin against N. gonorrhoeae and M. pneumoniae, both found concurrently with chlamydial infections, suggests that azithromycin could be used as a one-drug regimen for the treatment of these pathogens. With these advantages, azithromycin appears to be an effective antimicrobial agent comparable to tetracycline and erythromycin for use in treatment C. trachomatis and C. pneumoniae infections. REFERENCES 1. Blackman, H. J., C. Yoneda, C. R. Dawson, and J. Schachter. 1977. Antibiotic susceptibility of Chlamydia trachomatis. Antimicrob. Agents Chemother. 12:673-677. 2. Centers for Disease Control. 1985. Chlamydia trachomatis infections, policy guidelines for prevention and control. Morbid. Mortal. Weekly Rep. 34(Suppl):53-73. 3. Centers for Disease Control. 1989. Sexually transmitted diseases treatment guidelines. Mortal. Weekly Rep. 34(Suppl.):1-43. 4. Centers for Disease Control. 1989. Treatment of uncomplicated urethral endocervical or rectal Chlamydia trachomatis infections. Morbid. Mortal. Weekly Rep. 38(Suppl.):28-29. 5. Chirgwin, K., P. M. Roblin, and M. R. Hammerschlag. 1989. In vitro susceptibilities of Chiamydia pneumoniae (Chlamydia sp. strain TWAR). Antimicrob. Agents Chemother. 33:1634-1635. 6. Grayston, J. T., C. C. Kuo, S. P. Wang, and J. Altamn. 1986. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N. Engl. J. Med. 315:161. 7. Handsfield, H. H. 1991. New macrolides in the treatment of sexually transmitted diseases, p. 45, abstr. C-20-272. Int. Soc. STD Res. 9th Int. Meet. International Society for STD Research, Banff, Alberta, Canada. 8. Johnson, R. B. 1990. Azithromycin treatment of genital chlamydial infections, p. 534-536. In W. R. Bowie, H. D. Caldwell, R. P. Jones, P. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.), Chlamydial infections. Cambridge University Press, New York. 9. Jones, R. B. 1990. Treatment of Chlamydial trachomatis infections of the urogenital tract, p. 509-518. In W. R. Bowie, H. D. Caldwell, R. P. Jones, P. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.), Chlamydial infections. Cambridge University Press, New York. 10. Jones, R. B., B. Van Der Pol, D. H. Martin, and M. K. Shepard. 1990. Partial characterization of Chiamydia trachomatis isolates resistant to multiple antibiotics. J. Infect. Dis. 162:1309-1315. 11. Judson, F. N. 1985. Assessing the number of genital chlamydial infections in the United States. J. Reprod. Med. 30:269-272. 12. Martin, D. H. 1991. A multicenter, randomized trial of single-
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dose azithromycin vs. multidose doxycycline for Chlamydia trachomatis genital tract infection, p. 47, abstr. C-20-082. Int. Soc. STD Res. 9th Int. Meet. International Society for STD Research, Banff, Alberta, Canada. 13. Mourad, A., R. L. Sweet, N. Sugg, and J. Schachter. 1980. Relative resistance to erythromycin in Chlamydia trachomatis. Antimicrob. Agents Chemother. 18:696-698. 14. Scheibel, J. H., J. K. Kristensen, and B. Hentzer. 1982. Treatment of chlamydial urethritis in men and Chiamydia trachomatis-positive female partners: comparison of erythromycin and tetracycline in treatment course of one week. Sex. Transm. Dis.
9:128-131.
ANTIMICROB. AGENTS CHEMOTHER. 15. Stamm, W. E., M. Tam, M. Koester, and L. Cles. 1983.
Detection of Chlamydia trachomatis inclusions in McCoy cell cultures with fluorescein-conjugated monoclonal antibodies. J. Clin. Microbiol. 17:666-668. 16. Waites, K. B., G. H. Cassell, K. C. Canupp, and P. B. Fernandes. 1988. In vitro susceptibilities of mycoplasmas and ureaplasmas to new macrolides and aryl-fluoroquinolones. Antimicrob. Agents Chemother. 32:1500-1502. 17. Walsh, M., E. W. Kappas, and T. C. Quinn. 1987. In vitro evaluation of CP-62,993, erythromycin, clindamycin, and tetracycline against Chlamydia trachomatis. Antimicrob. Agents Chemother. 31:811-812.
p.
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In Vitro Evaluation of Activities of Azithromycin, Erythromycin, and Tetracycline against Chlamydia trachomatis and Chlamydia pneumoniae LAURA E. WELSH,1 CHARLOTTE A. GAYDOS,1 AND THOMAS C. QUINN' 2* Division of Infectious Diseases, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205,1* and Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 208922 Received 12 September 1991/Accepted 4 December 1991 The in vitro activities of azithromycin (CP-62,993; Pfizer), erythromycin, and tetracycline were evaluated by inhibiting Chlamydia trachomatis and Chlamydia pneumoniae, formerly TWAR, propagation in vitro in McCoy cells, HeLa cells, and HL cells. Eleven clinical isolates of C. trachomatis (serovars D, E, F, J, K, and L2) and four strains of C. pneumoniae were tested with an inoculum of 103 inclusion-forming units in a 96-well microtiter plate. The MIC ranges of these antimicrobial agents against C. trachomatis were as follows: azithromycin, 0.125 to 0.5 ,ug/ml; erythromycin, 0.25 to 0.1 ,ug/ml; and tetracycline, 0.0625 to 1.0 ,ug/ml. The MBC ranges, calculated from passage into antibiotic-free medium, were as follows: azithromycin, 0.125 to 4.0 ,ig/ml; erythromycin, 0.5 to 8.0 ,ug/ml; and tetracycline, 0.0625 to 4.0 ,ug/ml. The MIC ranges for C. pneumoniae in both HeLa and HL cells were as follows: azithromycin, 0.125 to 1.0 ,Ig/ml; erythromycin, 0.0625 to 1.0 ,ug/ml; and tetracycline, 0.125 to 1.0 ,ig/ml. The MBC ranges were as follows: azithromycin, 0.25 to 1.0 ,ug/ml; erythromycin, 0.25 to 1.0 ,ug/ml; and tetracycline, 0.125 to 4.0 ,Ig/ml. From the results of this in vitro study, azithromycin appears to be an effective antibiotic comparable to tetracycline and erythromycin for use in the treatment of both C. trachomatis and C. pneumoniae infections.
form, was obtained from Pfizer Central Research, Groton, Conn. Azithromycin was reconstituted in 1.0 ml of absolute methanol and then diluted in 9.0 ml of sterile distilled H20 for a concentration of 1,000 ,ug/ml. Bacterial strains. Two different clinical isolates of serovars D, E, F, J, and K and a clinical isolate of serovar L2 of C. trachomatis were obtained from the Baltimore City Sexually Transmitted Diseases Clinics. These were recent clinical isolates from uncomplicated genital infections. C. pneumoniae TW183, AR39, and AR388 were obtained from the Washington Research Foundation, Seattle, and C. pneumoniae VR1310 (CDC CWL029) was obtained from the American Type Culture Collection, Rockville, Md. The clinical isolates were passed three or fewer times to achieve titers for MIC testing and serovar typing. Susceptibility assays. The in vitro antimicrobial effects on C. trachomatis and C. pneumoniae were determined by tissue culture assay. Antibiotic-free McCoy cells were used for C. trachomatis. For C. pneumoniae, antibiotic-free HeLa and HL cells were used. Antibiotic-free cell monolayers in 96-well microtiter plates were pretreated with DEAEdextran (Sigma, St. Louis, Mo.) in phosphate-buffered saline for 30 min at 35°C with 5.0% CO2 in air (15). No dextran pretreatment was used for HL cells. The dextran was aspirated, and the cells were inoculated with 0.1 ml of 103 inclusion-forming units of each chlamydial strain or isolate. Cultures were centrifuged for 60 min at 600 x g at 35°C and then incubated at 35°C with 5.0% CO2 for 30 min (17). The inoculum was aspirated and replaced with 0.2 ml of overlay medium per well. This medium consisted of RPMI 1640 (Whittaker Bioproducts, Walkersville, Md.), 10% fetal bovine serum (Hyclone, Logan, Utah), 1% glucose, 1.0 ,ug of cycloheximide (Sigma) per ml, and serial twofold dilutions of each antimicrobial agent (17). Tetracycline, erythromycin, and azithromycin were diluted from 1.0 to 0.31 ,ug/ml. Six
Chlamydia trachomatis infections are recognized as the most prevalent sexually transmitted bacterial infections in the United States, with an estimated 4 million cases annually (2, 11). Chlamydia pneumoniae has been associated with 12% of cases of outpatient pneumonia, 5% of cases of bronchitis, and at least 1% of cases of pharyngitis among university students in Seattle, Wash. (6). Tetracycline or doxycycline has been the treatment of choice for chlamydial infections involving both C. trachomatis and C. pneumoniae. However, erythromycin has frequently been recommended as an alternative antibiotic regimen. Unfortunately, both tetracycline and erythromycin must be taken for 7 days for adequate efficacy, a fact that frequently results in noncompliance or inadequate treatment. Azithromycin is a 15-member ring macrolide antibiotic whose preliminary in vitro susceptibility studies have indicated superior activity compared with erythromycin when tested against various strains of C. trachomatis and C. pneumoniae (5, 17). Azithromycin can be administered as a single dose because of its long half-life. Since previous in vitro susceptibility studies have been limited to standard laboratory strains, we initiated a study to compare the in vitro activities of azithromycin, erythromycin, and tetracycline against 11 recent clinical isolates of C. trachomatis and four strains of C. pneumoniae. MATERIALS AND METHODS Antimicrobial agents. Tetracycline hydrochloride and erythromycin lactobionate were obtained in powder form from U.S.P.C., Inc., Rockville, Md. Tetracycline and erythromycin were reconstituted in sterile distilled H20 for a concentration of 1,000 ,ug/ml. Azithromycin, also in powder *
Corresponding author. 291
292
ANTIMICROB. AGENTS CHEMOTHER.
WELSH ET AL.
TABLE 1. In vitro activity of tetracycline, azithromycin, and erythromycin against clinical isolates of C. trachomatis Isolate
Serovar
Tetracycline
Concn (Lg/ml) of: Azithromycin
Erythromycin
no .
396 443 448 677 021 256 176 701 173 457 422
D D E E F F J J K K L2
MIC
MBC
MIC
MBC
MIC MBC
0.125 0.125 0.125 0.125 0.0625 0.125 0.0625 0.0625 1.0 1.0 0.0625
1.0 1.0 1.0 4.0 2.0 2.0 0.0625 0.5 2.0 2.0 0.5
0.5 0.25 0.25 0.5 0.125 0.5 0.25 0.125 0.5 0.5 0.25
1.0 0.5 4.0 0.5
1.0 0.5 0.5 0.5
4.0 2.0 0.125 2.0
0.25
2.0 4.0 0.5
1.0 0.5 0.25 1.0 1.0 0.5
4.0 1.0 8.0 0.5 4.0 2.0 2.0 0.5 2.0 4.0 1.0
inoculated wells were overlaid with each dilution of antibiotic (1, 13). Positive and negative controls were overlaid with 0.2 ml of the above medium free of all antibiotics per well. Plates were then incubated for 48 h at 35°C with 5.0% CO2 in air. The same chlamydial strain or isolate served as the inoculum for a positive control in each assay. The inoculum for a negative control in each assay was RPMI 1640 medium without antibiotics. Following incubation, culture medium was aspirated and three wells per dilution were fixed and stained with a fluorescein-conjugated monoclonal antibody to C. trachomatis (Syva Corp., Palo Alto, Calif.) and C. pneumoniae (Kallestad Diagnostics, Chaska, Minn.). Chlamydial inclusion bodies were counted with a Zeiss fluorescent microscope (Zeiss, Oberkochen, Germany) and a 20x objective. An average of 14 inclusion-forming units per low-power field were seen. The MIC was determined to be the lowest concentration at which complete inhibition of inclusion formation was observed (17). The remaining wells were passed by scraping the cell layer and reinoculating it onto fresh cell monolayers without antibiotics (13). The cultures were incubated for another 48 h in the previously described antibiotic-free overlay medium. After staining with fluorescein-conjugated monoclonal antibody, the MBC was determined to be the lowest concentration of antimicrobial agent preventing inclusion formation after passage (17). When the MBC was greater than 1.0 p,g/ml, the culture assay was performed again using overlay medium with serial dilutions of antibiotic from 8.0 to 0.5 ,ug/ml. RESULTS
Tetracycline was the most effective antimicrobial agent for C. trachomatis, followed by azithromycin and then erythromycin (Table 1). C. trachomatis serovars F and K were most resistant to tetracycline, erythromycin, and azithromycin, with the MBCs for both isolates of each serovar ranging from 2.0 to 4.0 ,ug/ml. The MBC of erythromycin for one isolate of C. trachomatis serovar D was 4.0 ,ug/ml, and the MBC of tetracycline for one isolate of serovar E was 4.0 ,ug/ml. The MBC range of azithromycin and erythromycin for another isolate of C. trachomatis serovar E was 4.0 to 8.0 ,ug/ml. Serovar J was relatively sensitive to tetracycline, erythromycin, and azithromycin, with an MBC range of 0.0625 to 2.0 ,ug/ml. C. trachomatis serovar L2 was the most sensitive
to all three antibiotics, with an MBC range of 0.0625 to 1.0
p.gIml.
For C. pneumoniae isolates, azithromycin was more effective than tetracycline or erythromycin. Two C. pneumoniae isolates, Ar388 and VR1310, were more resistant to tetracycline than they were to erythromycin or azithromycin, with MBCs of 2.0 to 4.0 Rg/ml. The other C. pneumoniae isolates, TW183 and AR39, were more sensitive to tetracycline, erythromycin, and azithromycin, with MBC ranges of 0.125 to 1.0 p.g/ml. Little difference was observed between MICs and MBCs in HeLa cells and HL cells.
DISCUSSION
Tetracycline and erythromycin are considered to be the drugs of choice for treatment of C. trachomatis and C. pneumoniae infections. Several clinical trials have established the efficacy and essential therapeutic equivalence of tetracycline, doxycycline, and minocycline. There is now little difference in the costs of tetracycline and doxycycline; therefore, the latter is preferred because of better compliance (1, 3). Erythromycin is recommended for treatment of chlamydial infections when the patient is pregnant or nursing a child or is an adult unable to tolerate tetracycline (3, 4). Standard regimens of tetracycline, doxycycline, or erythromycin appear to be effective against chlamydial infections in most situations, producing a decrease in symptoms and preventing further transmission. However, in some cases it appears that these antimicrobial agents suppress the organism without eradicating it, causing a chronic persistent infection (9). The eradication rate is 94% with tetracycline and 91% with erythromycin (14). The mean peak concentration of erythromycin in serum is 1.73 ,ug/ml, and that of tetracycline is 2.6 ,ug/ml (17). Of patients being treated with erythromycin, 8 to 10% have treatment-related side effects, compared with 7% of patients being treated with tetracycline. All of these side effects are gastrointestinal (14). In one study, several C. trachomatis isolates were resistant to tetracycline, doxycycline, erythromycin, sulfamethoxazole, and clindamycin but were sensitive to rifampin, ciprofloxacin, and ofloxacin (10). This resistance is not surprising, given the current practice of treating most documented or suspected C. trachomatis and C. pneumoniae infections with tetracycline or erythromycin, and continued surveillance for antibiotic resistance is warranted to determine the extent of this problem. Azithromycin is a 15-member azalide which has a long half-life in serum and high tissue affinity. After one 500-mg dose of azithromycin, concentrations in tissue are 400 times those achieved in serum (17). The mean peak concentration of azithromycin in serum is 0.37 ,uglml, and the half-life in serum is 68 h (12). This long half-life in serum and high tissue affinity make it possible to administer azithromycin in a single dose. In one of the clinical trials, azithromycin was given as a single 1-g dose and as a 500-mg dose on day 1 followed by single 250-mg doses on days 2 and 3. Doxycycline was given as a 100-mg dose twice per day for 7 days. There was 100% eradication of C. trachomatis after 1 week with both dosage regimens of azithromycin and 95% eradication with the doxycycline regimen. Eight percent of azithromycin-treated patients and 6% of the doxycyclinetreated patients reported treatment-related side effects, all of which were mild and related to the gastrointestinal tract (8). Since doxycycline and erythromycin must be administered several times a day for 7 days, there should be better patient
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TABLE 2. In vitro activity of tetracycline, azithromycin, and erythromycin against C. pneumoniae strains in HeLa and HL cells Concn (,ug/ml) of:
TW183 TW183 (duplicate test) AR39 AR388 VR1310
MIC
Erythromycin
Azithromycin
Tetracycline Strain
MIC
MBC
HeLa
HL
HeLa
HL
0.25 0.125 1.0 1.0 0.25
0.25 0.25 0.5 0.5 0.25
0.25 0.125 1.0 2.0 2.0
1.0 1.0 1.0 4.0 1.0
MIC
MBC
MBC
HeLa
HL
HeLa
HL
HeLa
HL
HeLa
HL
0.125 0.125 0.125 0.25 0.125
0.125 0.125 0.125 0.25 0.125
0.25 0.25 1.0 0.5 0.5
0.25 0.25 0.25 0.25 0.25
0.25 0.25 0.25 0.125 0.125
0.125 0.125 0.125 0.0625 0.125
0.25 0.25 1.0 1.0 0.5
0.5 0.5 1.0 1.0 0.25
compliance and therefore more effective treatment of chlamydial infections with one dose of azithromycin. In this study, the MIC of azithromycin for C. pneumoniae varied from 0.125 to 0.25 ,ug/ml and the MBC varied from 0.25 to 1.0 jig/ml. All differences between HeLa cells and HL cells for the MICs were within one dilution (Table 2), except for AR39, for which there was a twofold difference, with the MIC being 0.125 jig/ml in HL cells and 1.0 ,ug/ml in HeLa cells. Similarly, all MBC results were within a onetube variation, except for AR39, for which the MBC was 0.25 ,ug/ml in HL cells and 1.0 jig/ml in HeLa cells. The MIC for TW183 was 0.25 ,ug/ml in both HL and HeLa cells, which is in contrast to that observed by Chirgwin et al. of 0.06 ,ug/ml in HeLa cells (5). Reasons for this remain unknown, although slight differences in methods may have influenced these results. Since less inoculum (0.1 ml) is used for microtiter wells than shell vials (0.2 ml), this may account for differences seen between laboratories. In addition, these are clinical isolates from a different area of the United States and may represent population differences in our strains. Our results remained reproducible when done in triplicate on two different occasions. No dextran pretreatment was used for HL cells, and 30 jig/ml for 30 min was used for HeLa cells. Chirgwin et al. (5) pretreated HeLa cells for 10 min, although this would not seem to explain a two-dilution difference. Inoculum sizes for each study also varied slightly. Perhaps small variations in this inoculum size are significant. The results of the present in vitro study indicate that azithromycin may be effective for C. trachomatis and C. pneumoniae infections. Other studies have shown that azithromycin is more effective than erythromycin against penicillinase-producing strains of Neisseria gonorrhoeae (16). This drug could be used as treatment for both C. trachomatis and N. gonorrhoeae because these infections often occur concurrently. However, treatment trials have indicated that a 2-g dose of azithromycin is more effective in treating N. gonorrhoeae infections (7). Unfortunately, the 2-g dose of azithromycin was associated with a high frequency of gastrointestinal side effects. Consequently, the optimum dose and efficacy for the treatment of gonorrhea is not yet fully established. Azithromycin has also been shown to be highly effective in vitro against Mycoplasma pneumoniae (16). This is important, because the clinical presentation of M. pneumoniae and C. pneumoniae infections may be similar, but the organisms are difficult to isolate (5), and most of these infections are diagnosed by serological methods. Since azithromycin is effective against both M. pneumoniae and C. pneumoniae, empiric therapy should be active against both. The in vitro efficacy of azithromycin against C. trachomatis and C. pneumoniae indicates that this is an effective
antimicrobial agent for these infections. The long half-life in serum, high levels in tissue, and single-dose treatment regimen will certainly be an advantage for azithromycin because they may result in greater patient compliance. There will probably be more effective treatment of chlamydial infections because a single 1-g dose of azithromycin can effectively eradicate the organism. The additional in vitro efficacy of azithromycin against N. gonorrhoeae and M. pneumoniae, both found concurrently with chlamydial infections, suggests that azithromycin could be used as a one-drug regimen for the treatment of these pathogens. With these advantages, azithromycin appears to be an effective antimicrobial agent comparable to tetracycline and erythromycin for use in treatment C. trachomatis and C. pneumoniae infections. REFERENCES 1. Blackman, H. J., C. Yoneda, C. R. Dawson, and J. Schachter. 1977. Antibiotic susceptibility of Chlamydia trachomatis. Antimicrob. Agents Chemother. 12:673-677. 2. Centers for Disease Control. 1985. Chlamydia trachomatis infections, policy guidelines for prevention and control. Morbid. Mortal. Weekly Rep. 34(Suppl):53-73. 3. Centers for Disease Control. 1989. Sexually transmitted diseases treatment guidelines. Mortal. Weekly Rep. 34(Suppl.):1-43. 4. Centers for Disease Control. 1989. Treatment of uncomplicated urethral endocervical or rectal Chlamydia trachomatis infections. Morbid. Mortal. Weekly Rep. 38(Suppl.):28-29. 5. Chirgwin, K., P. M. Roblin, and M. R. Hammerschlag. 1989. In vitro susceptibilities of Chiamydia pneumoniae (Chlamydia sp. strain TWAR). Antimicrob. Agents Chemother. 33:1634-1635. 6. Grayston, J. T., C. C. Kuo, S. P. Wang, and J. Altamn. 1986. A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infections. N. Engl. J. Med. 315:161. 7. Handsfield, H. H. 1991. New macrolides in the treatment of sexually transmitted diseases, p. 45, abstr. C-20-272. Int. Soc. STD Res. 9th Int. Meet. International Society for STD Research, Banff, Alberta, Canada. 8. Johnson, R. B. 1990. Azithromycin treatment of genital chlamydial infections, p. 534-536. In W. R. Bowie, H. D. Caldwell, R. P. Jones, P. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.), Chlamydial infections. Cambridge University Press, New York. 9. Jones, R. B. 1990. Treatment of Chlamydial trachomatis infections of the urogenital tract, p. 509-518. In W. R. Bowie, H. D. Caldwell, R. P. Jones, P. Mardh, G. L. Ridgway, J. Schachter, W. E. Stamm, and M. E. Ward (ed.), Chlamydial infections. Cambridge University Press, New York. 10. Jones, R. B., B. Van Der Pol, D. H. Martin, and M. K. Shepard. 1990. Partial characterization of Chiamydia trachomatis isolates resistant to multiple antibiotics. J. Infect. Dis. 162:1309-1315. 11. Judson, F. N. 1985. Assessing the number of genital chlamydial infections in the United States. J. Reprod. Med. 30:269-272. 12. Martin, D. H. 1991. A multicenter, randomized trial of single-
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