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DIAGN MICROBIOL INFECT DIS 1992;15:371-373

In vitro Activity of -Lactam Drugs and Sulbactam Against Chlamydia

trachomatis John Segreti, Kathi S. Kapell, and Gordon M. Trenholme

We tested the in vitro activity of ampicillin, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, and sulbactam against 18 recent clinical isolates of Chlamydia trachomatis and two ATCC strains. Ampicillin (MICso, 256 tag/ml) and sulbactam (MICso, 128 p~g/ml) demonstrated some activity against C. trachomatis, but cefoperazone had little to no ac-

tivity. At 2-3 dilutions below the MIC, C. trachomatis treated with ampicillin or sulbactam, but not cefoperazone, formed small inclusions that remained small on passage onto antibiotic-free McCoy cells. It appears that ampicillin and sulbactam suppress rather than kill C. trachomatis.

Chlamydia trachomatis is probably the most common

therefore, tested the in vitro activity of ampicillin, ampicillin-sulbactam, cefoperazone, and cefoperazone-sulbactam against 20 recent clinical isolates of

sexually transmitted bacterial pathogen. It is the major cause of urethritis and epididymitis in men, and cervicitis and salpingitis in women. It is well known that the commonly used doses of penicillins or other ~-lactams used to treat gonococcal infections usually fail to eradicate a concomitant chlamydial infection (Stamm et al., 1984). However, preliminary studies suggest that some ~-lactams might be effective in treating C. trachomatis. A few human trials have shown favorable results in the treatment of C. trachomatis infections with amoxicillin (Bowie et al., 1981; Crombleholme et al., 1990; Martin et al., 1986). Sulbactam is a ~-lactamase inhibitor that extends the spectrum of activity of ampicillin to include penicillinase-producing Neisseria gonorrhoeae, Bacteroides spp., and many Enterobacteriaceae. Ampicillin-sulbactam in combination has, therefore, become increasingly popular in the treatment of pelvic inflammatory disease. However, the activity of sulbactam alone and in combination with ampicillin or cefoperazone against C. trachomatis has not been previously reported. We, From Rush Medical College, Chicago, Illinois, USA. Address reprint requests to Dr. J. Segreti, Section of Infectious Disease, Rush-Presbyterian-St. Luke's Medical Center, 600 South Paulina, Suite 143 Academic Facility, Chicago, IL 60612, USA (present address). Received 1 May 1991; revised and accepted 14 August 1991. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/92/$5.00

C. trachomatis.

Ampicillin, sulbactam, and cefoperazone were obtained from Pfizer Research Laboratories. These drugs were prepared as stock dilutions in concentrations of 1280 ~g/ml according to their stated potency. Stock solutions were stored in -70°C for a maximum of 2 weeks. Drugs were diluted to the appropriate concentration with medium containing Hank's balanced salt solution, amino acids, vitamins, 1% glutamine, 10% inactivated fetal calf serum, 5.4 g glucose/L, and 1 ~g cycloheximide/ml. Dilutions were made on the day of use. A total of 20 strains of C. trachomatis were tested including one D strain (ATCC VR-885), one LGV-2 strain (ATCC VR-902B), and 18 clinical isolates. All clinical isolates were of genital origin that had been passed fewer than 10 times in the laboratory. All isolates were passed an additional two times in antibiotic-free medium before susceptibility testing and were stored at -70°C until the time of use.

The antimicrobial susceptibility of C. trachomatis was determined by using 96-well dilution plates as previously described (Segreti et al., 1990). McCoy cell monolayers, 24 hr old and grown in antibioticfree medium, were inoculated with a dilution of C. trachomatis test strain known to yield 500-1000 in-

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TABLE I

J. Segreti et al.

In vitro Susceptibility of 20 Chlamydia trachomatis Strains MIC

MBC

Drug

Range

50

90

Range

50

90

Ampicillin Ampicillin-sulbactam Sulbactam Cefoperazone Cefoperazone-sulbactam

128->256 128->256 128->256 256->256 128->256

256 256 128 >256 256

>256 >256 >256 >256 >256

64->256 64->256 64->256 256->256 128->256

256 256 128 >256 256

>256 >256 >256 >256 >256

MIC, minimum inhibitory concentration; and MBC, minimum bactericidal concentration.

clusions per well. Plates w e r e centrifuged at 1000 g at 24°C for 60 rain, a n d t h e n overlaid with 0.1 ml of each d r u g solution to yield appropriate twofold dilutions. Ampicillin-sulbactam a n d c e f o p e r a z o n e sulbactam were tested at a 2:1 ratio. Each solution was tested in triplicate. Antibiotic-free controls were included on each plate. Cultures were incubated for 48 hr at 37°C in 5% CO2, fixed with absolute ethanol, and stained with fluorescein-conjugated m o u s e monoclonal antibody to C. trachomatis (Ortho Diagnostics, Raritan, NJ) according to the directions of the manufacturer. The m i n i m u m inhibitory concentration (MIC) was defined as the lowest concentration of antibiotic w i t h o u t inclusions. The m i n i m u m bactericidal concentration (MBC) was defined as the lowest concentration of antibiotic yielding no inclusion after passage onto 24-hr-old M c C o y cell monolayers g r o w n on antibiotic-free m e d i u m . The results are s h o w n in Table 1. For all the antimicrobials tested, the m e d i a n MIC for all 20 isolates exceeded 128 p,g/ml. Inclusion formation was unaffected by cefoperazone. H o w e v e r , both the ampicillin- and sulbactam-treated Chlamydia formed small inclusions 2-3 dilutions below the MIC. Interestingly, these inclusions r e m a i n e d small on passage onto antibiotic-free M c C o y cells. For all isolates except one, the results of the MICs and MBCs were identical. The MBC of one isolate was one dilution

below the MIC for ampicillin, sulbactam, and the combination. Of interest, sulbactam alone inhibited inclusion formation. In fact, sulbactam was more active than either ampicillin or c e f o p e r a z o n e against C. trachomatis. These results s u p p o r t previous studies s h o w i n g that some f3-1actams have a definite but incomplete inhibitory effect on C. trachomatis in vitro (Bowie, 1982; H a m m e r s c h l a g and Glyezer, 1983; H o w et al., 1985; Kuo et al., 1977). This s t u d y suggests that the addition of sulbactam to either ampicillin or cefoperazone does not e n h a n c e activity against C. trachomatis. Because b o t h sulbactam a n d ampicillin affected inclusion formation, h o w e v e r , ampicillin and sulbactam might suppress some C. trachomatis infections. The persistence of inclusion-forming units w h e n transferred to antibiotic-free McCoy cells suggests that these 13-1actams s u p p r e s s rather than kill C. trachomatis. This incomplete r e s p o n s e w o u l d preclude use of these f3-1actam agents if the desire is to eradicate C. trachomatis. Rather, standard antimicrobial agents, such as tetracyclines or macrolides, should be used for the t r e a t m e n t of C. trachomatis infections. In certain situations, for example, d u r i n g p r e g n a n c y , penicillins and ~3-1actamase inhibitors might be used to suppress Chlamydia until such time as m o r e effective t h e r a p y can be utilized. There does not appear to be any role for c e f o p e r a z o n e in the t r e a t m e n t of Chlamydia infections.

REFERENCES Bowie WR (1982) Lack of in vitro activity of cefoxitin, cefamandole, cefuroxime, and piperacillin against Chlamydia trachomatis. Antimicrob Agents Chemother 21:339340. Bowie WR, Alexander ER, Holmes KK (1981) Eradication of Chlamydia trachomatis from the urethras of men with nongonococcal urethritis by treatment with amoxicillin. Sex Transm Dis 8:79-81. Crombleholme WR, Schachter J, Grossrnan H, Landers DV, Sweet RL (1990) Amoxicillin therapy for Chlamydia trachomatis in pregnancy. Obstet Gynecol 75:752-758. Hammerschlag MR, Gleyzer A (1983) In vitro activity of a group of broad-spectrum cephalosporins and other

beta-lactam antibiotics against Chlamydia trachomatis. Antimicrob Agents Chemother 23:493-494. How SJ, Hobson D, Hart CA, Quayle E (1985) A comparison of the in vitro activity of antimicrobials against Chlamydia trachomatis examined by Giemsa and a fluorescent antibody stain. J Antimicrob Chemother 15:399404. Kuo CC, Wang SP, Grayston JT (1977) Antimicrobial activity of several antibiotics and a sulfonamide against Chlamydia trachomatis organisms in cell culture. Antimicrob Agents Chemother 12:80-83. Martin DH, Pastorek JG, Faro S (1986) In vitro and in vivo activity of parenterally administered beta-lactam anti-

Note

biotics against Chlamydia trachomatis. Sex Transm Dis 13:81-87. Segreti J, Hirsch DJ, Harris AA, Kapell KS, Orbach H, Kessler HA (1990) In vitro activity of tosufloxacin (A61827; T3262) against selected genital pathogens. An-

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timicrob Agents Chemother 34:971-973. Stamm WE, Guinan ME, Johnson C, Starcher T, Holmes KK, McCormack WM (1984) Effect of treatment regimens for Neisseriagonorrhoeae on simultaneous infection with Chlamydia trachomatis. N Engl J Med 310:545-549.

In vitro activity of beta-lactam drugs and sulbactam against Chlamydia trachomatis.

We tested the in vitro activity of ampicillin, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, and sulbactam against 18 recent clinical is...
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