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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2004, p. 2081–2084 0066-4804/04/$08.00⫹0 DOI: 10.1128/AAC.48.6.2081–2084.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved. Vol. 48, No. 6 In Vitro Activities of Garenoxacin and Levofloxacin against Chlamydia pneumoniae Are Not Affected by Presence of Mycoplasma DNA Raymond P. Smith,1,2 Aldona L. Baltch,1,2* William J. Ritz,1 Andrea N. Carpenter,3 Tanya A. Halse,3 and Lawrence H. Bopp1 Stratton Veterans Affairs Medical Center,1 Albany Medical College,2 and Wadsworth Laboratories, New York State Department of Health,3 Albany, New York Received 4 November 2003/Returned for modification 12 January 2004/Accepted 10 February 2004 We studied 20 Chlamydia pneumoniae isolates obtained from respiratory sites and atheroma tissue of patients from various geographic areas to determine the susceptibilities of these isolates to a new desfluoroquinolone, garenoxacin, and to levofloxacin. In addition, we assessed the cultures with these isolates by PCR for the presence or absence of Mycoplasma sp. DNA. Both the MIC at which 90% of isolates are inhibited (MIC90) and the minimal bactericidal concentration at which 90% of isolates are killed (MBC90) for garenoxacin were 0.06 ␮g/ml, and both the MIC90 and the MBC90 for levofloxacin were 2.0 ␮g/ml. The activity of garenoxacin against C. pneumoniae was 32-fold greater than that of levofloxacin. Mycoplasma sp. DNA was detected by PCR in 17 of 20 cultures. Mycoplasma amplicons from five Mycoplasma DNA-positive C. pneumoniae cultures were sequenced and found to represent four Mycoplasma species. Our data demonstrate that C. pneumoniae cultures frequently contain Mycoplasma DNA and that its presence in C. pneumoniae cultures does not appear to affect the susceptibility results for the two fluoroquinolones that we tested. Chlamydia pneumoniae is a common cause of acute respiratory illnesses, including pharyngitis, sinusitis, bronchitis, and pneumonia, in children and adults (10). In addition, chronic C. pneumoniae infection is implicated in diseases such as atherosclerosis and its complications (16). C. pneumoniae respiratory infections are usually treated with a macrolide, an azalide, or tetracycline antimicrobials (11). However, fluoroquinolone antibacterial agents are known to be active against C. pneumoniae in vitro. A small number of fluoroquinolones have been moderately successful (70 to 80% bacterial elimination) for the treatment of culture-proven C. pneumoniae infections in humans (12). Garenoxacin (BMS-284756) is a new des-F(6)-quinolone with excellent activity against common respiratory pathogens (9). We studied the in vitro activities of garenoxacin against 20 C. pneumoniae isolates obtained from both respiratory tract and atheroma tissue sources. In addition, because the presence of Mycoplasma is common in reference C. pneumoniae cultures (13, 18) and is known to affect a variety of cell functions (5, 22), we were concerned about the potential effect of the presence of Mycoplasma in HEp-2 cells on antibiotic susceptibility testing with C. pneumoniae (5, 18). Therefore, we tested the original cultures, as well as the amplified preparations of C. pneumoniae used for MIC testing, for the presence of Mycoplasma DNA using PCR targeting the 16S rRNA gene (6, 26). We then identified, using DNA sequencing techniques and comparison of the sequences to those in sequence databases, the species of Mycoplasma present in selected cultures. * Corresponding author. Mailing address: Infectious Disease Research, Infectious Disease Section (III D), Stratton VA Medical Center, Albany, NY 12208. Phone: (518) 626-6416. Fax: (515) 626-6564. E-mail: [email protected]. 2081 Downloaded from https://journals.asm.org/journal/aac on 11 January 2022 by 3.239.7.17. MATERIALS AND METHODS Microorganisms. Twenty cultures of C. pneumoniae were tested. Eight isolates were obtained from the American Type Tissue Collection (ATCC; Manassas, Va.): ATCC 53592 (AR-39), ATCC VR-1310, ATCC VR-1360, ATCC VR-1356, ATCC VR-2282, ATCC VR-1435, ATCC VR-1355, and ATCC VR-1452. Eleven isolates were obtained from the University of Washington, Seattle: AR388, AR277, AR231, AR427, LR65, Ka50, Ka66, AC14, AC51, ER115, and AR458. Isolate A03SEG was obtained from the University of Louisville, Louisville, Ky. Aliquots of each isolate were prepared for susceptibility testing as described previously (4). Antimicrobial agents. Garenoxacin (Bristol-Myers Squibb Laboratories, Wallingford, Conn.) and levofloxacin (Ortho-McNeill Pharmaceuticals, Raritan, N.J.) were prepared in accordance with the suppliers’ instructions. Filter-sterilized antibiotic solutions were prepared fresh for each daily experiment and were used immediately. Determinations of MICs and MBCs. Susceptibility testing was performed at the Infectious Disease Research Laboratory, Stratton Veterans Affairs Medical Center, as described previously (11, 17). Briefly, HEp-2 cell monolayers in 96-well plates were inoculated with C. pneumoniae at a multiplicity of infection of 1 (103 inclusion-forming units/well), centrifuged at 1,700 ⫻ g, and incubated for 1 h at 37°C. Following this incubation, the medium (minimal essential medium [Gibco BRL, Grand Island, N.Y.] containing Earle’s salts and L-glutamine and supplemented with 2% NaHCO3 and 10% heat-inactivated fetal bovine serum [HyClone, Logan, Utah]) was removed by aspiration and replaced with fresh medium containing 1 ␮g of cycloheximide per ml and serial twofold dilutions of the test antibiotics. Following incubation for 72 h at 37°C, the inclusions were stained with fluorescein-labeled conjugated monoclonal antibody (Bio-Rad Laboratories, Redmond, Wash.) and counted with a fluorescence microscope. The MIC was defined as the lowest antibiotic concentration (in micrograms per milliliter) at which no inclusions were seen. For minimum bactericidal concentration (MBC) determination, the cultures were frozen at ⫺70°C and then thawed. Fresh HEp-2 cell monolayers were then inoculated with the resulting lysates, incubated for 72 h, fixed, and stained; and the inclusions were counted as described above. The MBC was defined as the lowest antibiotic concentration (in micrograms per milliliter) at which no inclusions were observed after passage of cell lysates to antibiotic-free, fresh HEp-2 cell monolayers. All conditions were studied in duplicate, and each experiment was run at least three times. Mycoplasma PCR testing. Mycoplasma PCR testing was performed at the Wadsworth Laboratories, New York State Department of Health, Albany. Briefly, 500-␮l aliquots of C. pneumoniae cell cultures were placed in microcentrifuge tubes for PCR analysis. These aliquots were concentrated by centrifugation at 13,000 ⫻ g for 10 min at room temperature, followed by the removal of 2082 SMITH ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Susceptibilities of 20 C. pneumoniae isolates to garenoxacin and levofloxacin and presence of Mycoplasma DNA in C. pneumoniae cultures from three sources Garenoxacinb Levofloxacinc Isolatea Source Site MIC (␮g/ml) MBC (␮g/ml) MIC (␮g/ml) MBC (␮g/ml) Mycoplasma DNA 53592 (AR-39) VR-1310 VR-1360 VR-1356 VR-2282 VR-1435 VR-1355 VR-1452 A03SEG AR388 AR277 AR231 AR427 LR65 Ka50 Ka66 AC14 AC51 ER115 AR458 ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC U.d Louisville U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington U. Washington Throat Throat Sputum Nasopharynx Conjunctiva Throat Nasopharynx Atheroma Atheroma Throat Throat Throat Throat Throat Throat Throat Throat Throat Throat Throat 0.015 0.03 0.03 0.03 0.015 0.015 0.03 0.03 0.015 0.03 0.03 0.03 0.06 0.06 0.06 0.015 0.03 0.015 0.015 0.03 0.015 0.03 0.03 0.125 0.03 0.015 0.03 0.03 0.015 0.03 0.03 0.03 0.06 0.06 0.06 0.015 0.03 0.015 0.015 0.03 0.5 0.5 0.5 1.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 2.0 2.0 1.0 0.5 2.0 1.0 0.5 2.0 0.5 0.5 0.5 2.0 0.5 0.5 0.5 0.5 0.5 0.5 1.0 1.0 4.0 4.0 1.0 0.5 2.0 1.0 1.0 2.0 ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ a All isolates except Ka50, Ka66, AC14, and AC51 were originally from the United States; isolates Ka50 and Ka66 were originally from Finland, and isolates AC14 and AC51 were originally from Japan. b The MIC50, MIC90, and MIC range for garenoxacin are 0.03, 0.06, and 0.015 to 0.06 ␮g/ml, respectively; the MBC50, MBC90, and MBC range for garenoxacin are 0.03, 0.06, and 0.015 to 0.125 ␮g/ml, respectively. c The MIC50, MIC90 and MIC range for levofloxacin are 0.5, 2.0, and 0.5 to 2.0 ␮g/ml, respectively; the MBC50, MBC90, and MBC range for levofloxacin are 0.5, 2.0, and 0.5 to 4.0 ␮g/ml, respectively. d U., University of. a 1% agarose gel alongside 2 ␮l of a Low DNA Mass Ladder (catalog no. 10068-013; Invitrogen Life Technologies). The PCR products were sequenced at the Wadsworth Center Molecular Genetics Core Facility. The sequences were compared to those in the GenBank database (Wisconsin package [Genetics Computer Group], version 10.0-UNIX) and the database of the National Center for Biotechnology Information (NCBI). RESULTS Table 1 lists the sources of the C. pneumoniae isolates, the anatomic sites from which they were originally isolated, the susceptibilities to garenoxacin and levofloxacin (MICs and MBCs), and the presence or absence of Mycoplasma sp. DNA. Two isolates were from Finland, and two isolates were from Japan; all other isolates were from the United States. All but two isolates were from the respiratory tract. Cultures of 17 of 20 isolates (85%) contained Mycoplasma DNA. C. pneumoniae isolates were approximately 32-fold more susceptible to garenoxacin than to levofloxacin. There was no evidence that the presence of Mycoplasma sp. DNA affected the results of antimicrobial agent susceptibility testing with C. pneumoniae. Table 2 shows the identities (determined by comparison of the sequences of the 16S rRNA gene PCR amplicons with the sequences in the GenBank and NCBI databases) of the Mycoplasma spp. found in cultures of five of the C. pneumoniae isolates used in this study. Cultures of one atheroma tissue isolate and four respiratory tract isolates were positive for Mycoplasma DNA. The Mycoplasma amplicons from these cultures were sequenced. Of these, two, Ka50 (isolated in Finland) and AR51 (isolated in Japan), tested positive for Myco- Downloaded from https://journals.asm.org/journal/aac on 11 January 2022 by 3.239.7.17. 480 ␮l of the medium and the addition of 30 ␮l of sterile water to the remaining 20 ␮l of medium and the pellet. The samples were then vortexed and heated to 95°C for 15 min. Ten-microliter aliquots of the lysates were then used directly for PCR amplification. Mycoplasma genus-specific PCR was performed with a total reaction mixture volume of 100 ␮l. The reaction mixture contained each primer at a final concentration of 0.5 ␮M (primer Myco-F [5⬘-GGGAGCAAACAGG ATTAGATACCCT-3⬘] and primer Myco-R [5⬘-TGCACCATCTGTCACTCTG TTAACCTC-3⬘]) (26); each deoxynucleoside triphosphate at a final concentration of 200 ␮M; and 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 2.5 U Amplitaq Gold (Applied Biosystems, Foster City, Calif.) (final concentrations). Thermocycler (9600; Applied Biosystems) conditions consisted of an initial incubation of 95°C for 9 min, followed by 40 cycles of 94°C for 45 s, 55°C for 60 s, and 72°C for 2 min (6). An additional incubation at 72°C for 10 min was added to complete the extension. To prevent amplicon contamination, aerosolbarrier tips were used. The reagent mixtures were prepared in a limited-access PCR clean room and were moved to a sample preparation room for addition of cell lysates. The samples were then taken to a PCR instrumentation room for amplification. A negative control with no template was included, as was a control known to be positive. The amplified products were analyzed on a 2% GTG agarose gel containing ethidium bromide and were visualized at maximal levels of intensity with a Gel Doc 1000 gel analysis system (Bio-Rad Laboratories, Hercules, Calif.). The oligonucleotides were synthesized in the Wadsworth Center Molecular Genetics Core Facility, New York State Department of Health. All 20 C. pneumoniae preparations generated by growth in HEp-2 cells for use for susceptibility testing were evaluated by PCR for the presence of Mycoplasma DNA. In addition, 7 of these 20 preparations (5 that tested positive and 2 that tested negative for Mycoplasma DNA) were diluted 1:10 and retested. Finally, the original cultures (those obtained from outside laboratories) of these seven isolates were tested. Sequencing of PCR products. Sequencing of the PCR products was performed at the Wadsworth Laboratories, New York State Department of Health. The PCR products of Mycoplasma spp. from five C. pneumoniae cultures were purified by using ExoSAP-IT, a commercial reagent (U.S. Biochemicals, Cleveland, Ohio), by using two hydrolytic enzymes to remove unwanted deoxynucleoside triphosphates and primers in a PCR product mixture. The clean PCR products were analyzed for purity and concentration by running 2 ␮l of each product on GARENOXACIN AND CHLAMYDIA PNEUMONIAE TABLE 2. Identities of Mycoplasma species determined with DNA recovered by PCR from five C. pneumoniae cultures Isolate AR427 AR427 A03SEG A03SEG AC51 Ka50 AR231 Mycoplasma species M. M. M. M. M. M. M. timonea indiensea argininib gateaeb hominis hominis cloacale % Identity 99.2 99.6 98.7 99.6 98.5 a The Mycoplasma DNA from C. pneumoniae isolate AR427 had 99.2% identity with both M. timone and M. indiense. b The Mycoplasma DNA from C. pneumoniae isolate AO3SEG had 99.6% identity with both M. arginini and M. gateae. plasma hominis DNA, although the sequences of the two varied slightly. The other three Mycoplasma-positive C. pneumoniae cultures were originally isolated in the United States and contained the DNA of Mycoplasma species not considered to be clinically significant. For two of these isolates (AR427 and A03SEG), the sequence data generated equal percentages of identity with two different Mycoplasma species. DISCUSSION The primary purpose of this study was to determine the garenoxacin and levofloxacin susceptibilities of C. pneumoniae isolates obtained from a variety of respiratory and nonrespiratory anatomic sites and originating from geographically diverse locations. The susceptibilities of 20 isolates originating in Finland, Japan, and the United States were determined. The susceptibility testing method used in this study was that of Malay and colleagues (17), originally described by Hammerschlag (11). All but three isolates tested in our study were different from those tested previously (17). In the two studies the MIC at which 90% of isolates are inhibited (MIC90) and the MBC at which 90% of isolates are killed (MBC90) were the same, i.e., 0.06 ␮g/ml for garenoxacin and 2.0 ␮g/ml for levofloxacin. Three isolates tested in both studies had the same susceptibilities in both studies. Three other studies reported on the garenoxacin MICs for small numbers of C. pneumoniae isolates (8, 9, 27). The methods used in those studies either differed from those used in our study or were not reported, and therefore, a comparison of the results must be made with caution because of possible methodological differences (11, 24). However, all garenoxacin MICs reported have ranged from ⬍0.008 to 0.015 ␮g/ml, and MBCs were equal to the MIC or were 1 twofold dilution higher, while the levofloxacin and moxifloxacin MICs were higher, usually 2 to 5 twofold dilutions for MIC testing (8, 9, 27). The second purpose of this study was to evaluate cultures of C. pneumoniae isolates from various sources (Table 1) by PCR for the presence of Mycoplasma DNA. Aliquots from cultures of 17 of 20 (85%) C. pneumoniae isolates were positive for Mycoplasma DNA. Mycoplasma DNA amplicons from selected positive cultures were sequenced, and the sequences were compared to those in the GenBank and NCBI databases. The cultures of one isolate from Finland (strain Ka50) and one isolate from Japan (strain AR51) contained DNA whose sequences most closely matched that of M. hominis (99.6% and 2083 98.7% identities, respectively), while the cultures of three isolates from the United States contained DNA whose sequences most closely matched those of Mycoplasma species not considered to be of clinical significance. Contamination of chlamydial stock cultures with Mycoplasma spp. has been recognized in research laboratories for many years (5, 13, 18). Methods to detect Mycoplasma spp. in chlamydia cultures, as well as tissue culture cell lines and media, include immunofluorescence and direct DNA detection methods, primarily PCR (19, 25). It is unclear whether C. pneumoniae isolates and Mycoplasma spp. coexist in clinical specimens or whether Mycoplasma spp. are acquired in the laboratory. The results of assays for biological activity, such as assays for the production of inflammatory mediators (14, 21), nitroblue tetrazolium reduction assays (7), and assays for the uptake of specific small molecules such as nucleosides, have been shown to be affected (20) by the presence of Mycoplasma when cell cultures containing Mycoplasma spp. are used in research studies. There is also evidence that a relationship exists between the number of Mycoplasma organisms infecting cells in culture and the ability of the cell culture to support the growth of C. pneumoniae (15). Furthermore, immunodiagnostic testing for infection with C. pneumoniae could be affected by the presence of Mycoplasma sp. antigens in immunofluorescence test systems thought to be composed of chlamydial antigens only (13). In our study, in which HEp-2 cells were carefully monitored for the presence of Mycoplasma spp. at set intervals, there was no evidence for the presence of Mycoplasma other than in the cultures of C. pneumoniae isolates obtained from outside laboratories or in subcultures produced from them. Among the five Mycoplasma DNA-positive C. pneumoniae cultures selected for DNA sequencing, the data indicated that a clinically significant Mycoplasma, M. hominis, was present in two isolates (one isolate each from Finland and Japan). However, the sequences of the M. hominis DNA from those two C. pneumoniae isolates differed slightly. The DNA of three Mycoplasma species not considered to be of clinical significance was present in the three C. pneumoniae isolates from cultures obtained from the United States. For the five C. pneumoniae isolates which were positive for Mycoplasma DNA and for which the Mycoplasma PCR amplicons were sequenced, the contents of both the original vials containing C. pneumoniae received from outside laboratories and the aliquots of C. pneumoniae grown in our laboratory from the organisms in the original vials were positive by PCR for the presence of Mycoplasma DNA. In addition, we tested the contents of the original vials for two isolates that were PCR negative for Mycoplasma DNA following amplification in our laboratory. The contents of these original vials were PCR negative for Mycoplasma DNA. Finally, of the five Mycoplasma amplicons that were sequenced, each had a different sequence. These data all indicate that the Mycoplasma DNA detected in our C. pneumoniae cultures was present in the isolates when they arrived in our laboratory and support the idea that Mycoplasma contamination of C. pneumoniae cultures and/or the cell lines used to grow them is very common. We did not find evidence that the presence of Mycoplasma DNA affected the susceptibilities of C. pneumoniae to garenoxacin or levofloxacin. In this study we found that garenoxacin was more active than Downloaded from https://journals.asm.org/journal/aac on 11 January 2022 by 3.239.7.17. VOL. 48, 2004 SMITH ET AL. ANTIMICROB. AGENTS CHEMOTHER. levofloxacin against C. pneumoniae. There are several possible explanations for the difference in activity. First, the intracellular concentration of garenoxacin may be significantly higher than that of levofloxacin. The intracellular concentration of levofloxacin in human monocytes in vitro has been studied previously (23). To our knowledge, garenoxacin has not been studied in a similar system. However, results from two studies with these drugs in which comparable methods were used suggest that garenoxacin penetrates human phagocytic cells to a slightly greater extent than levofloxacin (2, 3). Second, garenoxacin also has greater activity than levofloxacin in an in vitro assay of inhibition of C. pneumoniae DNA gyrase activity (1). Finally, there may be differential penetration of the two drugs into HEp-2 cells, which were used in the susceptibility assays. No data regarding the latter possibility are available. The presence of Mycoplasma DNA in C. pneumoniae cultures did not appear to influence the antimicrobial susceptibility testing results for C. pneumoniae. ACKNOWLEDGMENTS This research was supported by Bristol-Myers Squibb and in part by the Medical Research Service of the U.S. Department of Veterans Affairs. 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Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl. Environ. Microbiol. 58:2606–2615. 27. Weller, T. M. A., J. M. Andrews, G. Jevons, and R. Wise. 2002. The in vitro activity of BMS-284756, a new des-fluorinated quinolone. J. Antimicrob. Chemother. 49:177–184. Downloaded from https://journals.asm.org/journal/aac on 11 January 2022 by 3.239.7.17. 2084