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Mycoplasma pneumoniae is the causative agent of upper respiratory tract infections and pneumonia in humans (6). Recently the efficacy of new quinolones has been demonstrated in vitro against various respiratory pathogens, including M. pneumoniae (2, 4, 11). In this report, we compared the in vitro activity of moxifloxacin, a new member of the fluoroquinolone group which has potent antimicrobial activities against gram-positive and gram-negative bacteria (3, 9), with those of other fluoroquinolone as references against M. pneumoniae.

A total of 105 strains of M. pneumoniae isolated from throat swabs from patients with pneumonia from 1986 (and earlier) to 2000 were used. These strains were subcultured fewer than five times in liquid medium and stored at 80°C until use. The antimicrobial agents used were moxifloxacin (Bay12-8039; Bayer Yakuhin Ltd.), sparfloxacin (SPFX; Dainippon Pharmaceutical Co., Ltd.) levofloxacin (LVFX; Daiichi Pharmaceutical Co., Ltd.), and ciprofloxacin (CPFX; Bayer Yakuhin Ltd.). The MICs and MBCs against each drug were determined by serial dilutions of the drugs in broth medium as described previously (5, 8). Briefly, each strain of M. pneumoniae cultured for 5 days was diluted to 10⁵ CFU/ml and 200 µl of each sample was cultured in a sealed microtiter plate (Nunc Co., Ltd., Roskilde, Denmark) at 37°C. After 2 days of cultivation, twofold dilutions of the antimicrobial agents were added to samples of each strain. After an additional 2 days of cultivation, two and three 10-fold dilutions of each sample were used for plating on agar plates to provide a range of 500 to 1,000 CFU/10 µl of the sample. The number of mycoplasmas in broth without drugs reached approximately 10⁷ CFU/ml. MIC is defined as the lowest concentration of an antimcrobial agent inhibiting more than 99% of the mycoplasmal colonies with the control. The definition of MBC most often used in clinical microbiology is the lowest drug concentration that kills 99.9% of the bacterial population in a liquid medium.

As shown in Fig. 1a, moxifloxacin (MIC for 50% of isolates [MIC₅₀], 0.15 µg/ml; MIC for 90% of isolates [MIC₉₀], 0.3 µg/ml) was more active than SPFX (MIC₅₀, 0.3 µg/ml; MIC₉₀, 1.25 µg/ml), LVFX (MIC₅₀, 1.25 µg/ml; MIC₉₀, 2.5 µg/ml), and CPFX (MIC₅₀, 5.0 µg/ml, MIC₉₀, 5.0 µg/ml). The MBCs and their distributions among the strains for these agents are shown in Fig. 1b; the MBCs of moxifloxacin (MBC₅₀, 0.15 µg/ml; MBC₉₀, 0.3 µg/ml) were markedly lower than those of SPFX (MBC₅₀, 0.6 µg/ml; MBC₉₀, 2.5 µg/ml), LVFX (MBC₅₀, 2.5 µg/ml; MBC₉₀, 5.0 µg/ml), and CPFX (MBC₅₀, 5.0 µg/ml; MBC₉₀, 5.0 µg/ml).

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Susceptibilies of 105 strains of M. pneumoniae to moxifloxacin and other fluoroquinolones. (a) MICs. (b) MBCs. Symbols: , moxifloxacin; , SPFX; , LVFX; , CPFX. The numbers of strains are indicated at the bottom.

Next, we compared the susceptibility of strains by the year of isolation. We collected the 105 strains into groups containing 39 strains isolated prior to 1986, 24 strains isolated between 1986 and 1990, 37 strains isolated between 1991 and 1995, and 5 strains isolated between 1996 and 2000. The MBC/MIC ratio for moxifloxacin was 4 in one strain isolated between 1986 and 1990 and in one strain isolated between 1991 and 1995, with MIC of 0.08 µg/ml and MBC of 0.3 µg/ml. The MBC/MIC ratio for SPFX was 4 or more in two strains isolated prior to 1986, one strain isolated between 1986 and 1990, five strains isolated between 1990 and 1995, and five strains isolated between 1996 and 2000, but the MBCs for these 13 strains were also comparatively low (between 0.6 and 2.5 µg/ml). Similarly, for two strains isolated between 1986 and 1990 and one strain isolated between 1991 and 1995, the MBC/MIC ratio was more than 4 for LVFX; the LVFX MBCs of one strain isolated prior to 1986, one isolated between 1986 and 1990, one isolated between 1991 and 1995, and one isolated between 1996 and 2000 were high (in the range between 5 and 20 µg/ml). The MBC/MIC ratio for CPFX was more than 4 for one strain isolated prior to 1986 and one strain isolated between 1996 and 2000. The MICs and MBCs for four strains isolated between 1991 and 1995 and three strains isolated between 1996 and 2000 were in the range between 5 and 20 µg/ml.

Moxifloxacin is a fluoroquinolone designed to have a broad spectrum of in vitro activities against gram-positive and gram-negative organisms. Addition of SS-2,8-diazabicyclo[4,3,0]non-8-yl at position 7 and a methoxyl substituent at position 8 of the quinolone nucleus differentiates moxifloxacin from other fluoroquinolone compounds (1). The purpose of the present study was to evaluate the in vitro activity of moxifloxacin against M. pneumoniae. The MIC₅₀s and MBC₅₀s of fluoroquinolones for the isolates were similar to those for isolates obtained during prior to 2000. We reported that the MBC₅₀/MIC₅₀ ratios for macrolides and tetracyclines were markedly higher, with a range of 37 to 2,000, as described previously (2, 5). In the present study, we showed that the MBC₅₀/MIC₅₀ ratio for moxifloxacin was lower in 105 strains of M. pneumoniae isolated from 1985 (and earlier) to 2000 than were those of other fluoroquinolones. These results suggest that development of fluoroquinolone resistance of M. pneumoniae has not been recognized, although the LVFX and CPFX MICs and MBCs for several strains isolated between 1991 and 2000 were relatively high. Niitu et al. reported that the development of erythromycin resistance in M. pneumoniae was accompanied by cross-resistance to other macrolide antibiotics in vitro and in vivo (7). Toumanen et al. reported that the extension of bactericidal activity to slowly growing bacteria like mycobacteria may be a major advance in efforts to improve the chemotherapy of infectious diseases (10).

Fluoroquinolones might show antimycoplasmal activities against macrolide- and tetracycline-resistant strains of M. pneumoniae, because of their different mechanisms of action from those of the other classes of drugs. Fluoroquinolone antimicrobial agents show excellent penetration into lung tissue, in particular bronchial secretions (2, 5). The proved susceptibility of fluoroquinolones, especially moxifloxacin, described above and the characteristics of fluoroquinolones in terms of organ distribution might be useful in treating respiratory tract infections caused by M. pneumoniae.

To clarify the correlation between the activities of fluoroquinolones against M. pneumoniae and the clinical effects of these antibiotics on mycoplasma infections, further work is needed.