In vitro activity of pazufloxacin, tosufloxacin and other quinolones against Legionella species

Futoshi Higa*, Morikazu Akamine, Shusaku Haranaga, Masato Tohyama, Takashi Shinzato, Masao Tateyama, Michio Koide, Atsushi Saito and Jiro Fujita

First Department of Internal Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa 903-0215, Japan


* Corresponding author. Tel: +81-98-895-1144; Fax: +81-98-895-1414; E-mail: fhiga{at}med.u-ryukyu.ac.jp

Received 10 May 2005; returned 25 July 2005; revised 21 September 2005; accepted 3 October 2005


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Objectives: The activities of pazufloxacin and tosufloxacin against Legionella spp. were evaluated in vitro and compared with those of other quinolones, macrolides and azithromycin.

Methods: The conventional MICs were determined by the microbroth dilution method. Intracellular activities of drugs were evaluated by a cfu count. The minimal extracellular concentration inhibiting intracellular growth of bacteria (MIEC) was determined by a colorimetric cytopathic assay.

Results: MICs of pazuloxacin and tosufloxacin at which 90% (MIC90) of isolates are inhibited in 76 different Legionella spp. strains (38 ATCC strains and 38 clinical isolates) were 0.032 and 0.016 mg/L, whereas the MIC90s of levofloxacin, ciprofloxacin, garenoxacin, erythromycin, clarithromycin and azithromycin were 0.032, 0.032, 0.032, 2.0, 0.125 and 2.0 mg/L, respectively. Pazufloxacin and tosufloxacin at 4x MIC inhibited intracellular growth of Legionella pneumophila SG1 (80-045 strain), as did other quinolones, clarithromycin and azithromycin, whereas erythromycin at 4x MIC did not. MIECs of pazufloxacin, tosufloxacin, levofloxacin, ciprofloxacin and garenoxacin for the strain were 0.063, 0.004, 0.016, 0.032 and 0.008 mg/L respectively, which were superior to those of macrolides and azithromycin. Pazufloxacin showed potent activity against three additional clinical isolates of L. pneumophila SG1, one clinical isolate each of L. pneumophila SG3 and SG5, as well as Legionella micdadei, Legionella dumoffii and Legionella longbeachae SG1.

Conclusions: Pazufloxacin and tosufloxacin, as well as other quinolones, were more potent than macrolides and an azalide. Present data warrant further study on the efficacy of these drugs in the treatment of Legionella infections.

Keywords: intracellular activity , MIEC , MIC , Legionnaires' diseases , fluoroquinolones


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Legionella species, which are Gram-negative facultative intracellular bacteria, are important pathogens that cause a potentially fatal type of community-acquired pneumonia1 and hospital-acquired pneumonia.2 The bacterium survives and multiplies within professional phagocytes3 and alveolar epithelial cells.4 ß-Lactams, which do not efficiently penetrate into the host cells, are not effective in Legionella infections. A combination of erythromycin and rifampicin used to be recommended for Legionella infections,5 based on retrospective analyses of the first identified outbreak in Philadelphia.6 Recently, fluoroquinolones and azalides have been reported to be more potent against Legionella spp. than erythromycin. To date, fluoroquinolones and an azalide are the choice for treatment of Legionella infections.7,8

Pazufloxacin mesilate is a fluoroquinolone synthesized by Toyama Chemical Co. Ltd (Tokyo, Japan). This drug has good in vitro and in vivo activity against a broad range of bacteria, especially Gram-negative bacteria.9,10 Clinical trials showed its intravenous injection formula was effective in treating respiratory infections11 and the drug has been approved and is available in Japan. Tosufloxacin has good antimicrobial activity against respiratory pathogens and shows good clinical efficacy in respiratory infections.12 These drugs might be useful for treatment of Legionella infection as well.

In this study, the in vitro activity of pazufloxacin and tosufloxacin against Legionella spp. was evaluated using two methods: extracellular activity (MICs by the microdilution method) and intracellular activity (cfu counting method and colorimetric cytopathic assay13), and the activity was compared with other quinolones, macrolides and an azalide.


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Bacteria

Bacterial strains used in the study included 38 ATCC reference strains of different Legionella spp. and serotypes (listed in Table 1) and 38 clinical isolates. The latter were the strains isolated from clinical specimens and collected in our laboratory, which included 29 strains of Legionella pneumophila SG1, four strains of L. pneumophila SG5, two strains of Legionella micdadei, and one strain of L. pneumophila SG3, Legionella dumoffii and Legionella longbeachae SG1. These strains were stocked in our laboratory and used for MIC determination. Clinical isolates of Legionella spp. were identified on the basis of their biochemical profile, direct fluorescent antibody test (m-Tech, Alpharetta, GA, USA) and a DNA hybridization technique using photobiotin-labelled bacterial DNA.14 The bacteria were cultured on buffered charcoal yeast extract supplemented with {alpha}-ketoglutarate (BCYE-{alpha}; Difco Laboratories, Detroit, MI, USA) plates at 35°C for 2–3 days for use in each experiment.


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Table 1. List of ATCC reference strains of Legionella spp. used in this study

 
Antimicrobial agents

The following antimicrobial agents were obtained as powders of known potency from their respective manufacturers: pazufloxacin, tosufloxacin and garenoxacin (Toyama Chemicals Ltd, Tokyo, Japan); ciprofloxacin (Bayer Yakuhin Ltd, Osaka, Japan); levofloxacin (Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan); erythromycin base (Wako Pure Chemicals, Osaka, Japan); clarithromycin (Taishotoyama Pharmaceutical Co. Ltd, Tokyo, Japan); and azithromycin (Pfizer Pharmaceutical Ltd, Tokyo, Japan).

MIC determination

The MIC of each antimicrobial agent for Legionella was determined by the microdilution method15 using ACES-buffered yeast extract supplemented with {alpha}-ketoglutarate (BYE-{alpha}) broth. Bacterial suspension (50 µL) was inoculated into BYE-{alpha} broth (50 µL) containing serial dilutions of the antibiotics in a 96-well microplate (inoculum size: 5 x 104 cfu/well). The MIC was defined as the minimum concentration of the drug that inhibited visible bacterial growth after culture at 35°C for 2 days. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as control strains.

Intracellular antimicrobial activity testing

A murine macrophage J774.1 cell line (purchased from Riken Cell Bank, Tsukuba, Japan) was permissive for intracellular growth of Legionella and was used as the in vitro infection model for evaluating the intracellular activity of the antimicrobials.16,17 The macrophage cells were maintained in RPMI 1640 (Nipro, Osaka, Japan) supplemented with 10% fetal bovine serum in humidified air containing 5% CO2 at 37°C. For experiments, the cells were subcultured in RPMI 1640 supplemented with 5% fetal calf serum in a 96-well flat-bottom tissue culture plate (1 x 105 cells/well). The macrophages were then infected with Legionella (1 x 106 cfu/well). After culture for 12 h, the extracellular fluids and bacteria were aspirated, and the remaining cells were washed once. Antimicrobials were added to each well at the indicated concentrations, and the cells then incubated for 3 days. The number of viable Legionella in each well was determined by cfu counting as previously described.13

The activity of drugs against intracellular L. pneumophila was also evaluated using a previously described quantitative colorimetric assay system13,17 with minor modifications. The viability of macrophages was quantified using a rapid colorimetric assay employing the tetrazolium dye procedure. Treatment of Legionella-infected J774.1 cells with 2-fold dilutions of the drug allowed measurement of the dose–response curve of each drug against the cytopathic effect of the bacteria.13,17

Statistical analysis

Distributions of MICs between different groups were analysed by ANOVA and Scheffe's test using software (Statview, SAS Institute Inc., NC, USA). A P-value <0.05 is considered as significant.


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MICs

The MICs of various antimicrobials were evaluated for standard and clinical strains of Legionella. MIC50 and MIC90 of pazufloxacin for Legionella spp. bacteria were both 0.032 mg/L, which was identical to those of levofloxacin and ciprofloxacin, but lower than those of erythromycin, clarithromycin and azithromycin (Table 2). MICs of tosufloxacin were lower than those of other quinolones (Table 2). MICs of azithromycin for non-L. pneumophila were significantly higher than those for L. pneumophila SG1 and L. pneumophila SG2-14 (Table 3), while no difference was detected in MIC distributions of other drugs between L. pneumophila SG1, L. pneumophila SG2-14 and non-L. pneumophila.


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Table 2. MICs for Legionella spp. (76 isolates) of pazufloxacin, tosufloxacin and comparators using the microbroth dilution method

 

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Table 3. MIC50/MIC90 of various antimicrobials for Legionella spp.a

 
Intracellular activity of drugs

The activity of drugs was evaluated using the J774.1 macrophage in vitro infection model (Figure 1). MICs for L. pneumophila SG1 (strain 80-045) of pazufloxacin, tosufloxacin, levofloxacin, ciprofloxacin, garenoxacin, erythromycin, clarithromycin and azithromycin were 0.032 (mg/L), 0.016, 0.032, 0.032, 0.016, 0.125, 0.063 and 0.125, respectively. This strain grew well within J774.1 macrophages and the mean log increase in bacteria during culture for 3 days was 2.61. Pazufloxacin, as well as other quinolones and clarithromycin, inhibited the intracellular growth of L. pneumophila at 4x MIC, while erythromycin at 4x MIC did not.



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Figure 1. Inhibition of intracellular multiplication of L. pneumophila SG1 80-045 by various antimicrobials. Infected J774.1 macrophages were washed, and then antimicrobials were added to the wells (concentrations shown in parentheses as multiplications of MIC). Bacterial growth after 3 days of culture was determined. Each column represents the mean ± SD for 3–7 wells and the data are shown as the ratio of cfu at 72 h and cfu at time 0. Mean cfu on day 0 was 2.2 x 104/well.

 
Pazufloxacin inhibited the cytotoxicity of intracellularly multiplying L. pneumophila at a concentration of 0.063 mg/L (Figure 2). In a previous study,12 the minimum extracellular concentration inhibiting intracellular growth of Legionella (MIEC) was defined as the minimum concentration inhibiting >50% of the cytopathic effect of L. pneumophila. According to this definition, the MIECs of pazufloxacin, tosufloxacin, levofloxacin, ciprofloxacin, garenoxacin, erythromycin, clarithromycin and azithromycin for L. pneumophila SG1 strain 80-045 were 0.063, 0.004, 0.016, 0.032, 0.008, 0.5, 0.125 and 0.25 mg/L, respectively. MICs and MIECs of pazufloxacin for an additional three clinical isolates of L. pneumophila SG1, one clinical isolate each of L. pneumophila SG3 and SG5, L. micdadei, L. dumoffii and L. longbeachae SG1 were determined, which showed that the drug was potent against all these strains (Table 4).



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Figure 2. Inhibition of cytopathic effects of intracellular L. pneumophila SG1 80-045 by various antimicrobials. Infected J774.1 macrophages were washed, and then antimicrobials were added to the wells. After 3 days of culture, the viable J774.1 macrophages were quantified using a colorimetric assay. Dose–response curves of each drug against the cytopathic effect of bacteria are shown. Each point represents the mean ± SD for quadruplicate wells. The figure shows the representative result of three different experiments.

 

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Table 4. MIC and minimal concentration inhibiting intracellular growth of bacteria (MIEC) of pazufloxacin for eight clinical isolates of Legionella spp.

 

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Pazufloxacin9,10 and tosufloxacin12 have broad and potent antibacterial activity against respiratory pathogens including Gram-positive bacteria such as staphylococci and streptococci, and Gram-negative bacteria such as members of the family Enterobacteriaceae and Haemophilus spp. In addition, the present study showed that pazufloxacin and tosufloxacin had potent antimicrobial activity against both extracellular and intracellular Legionella species.

In this study, the antimicrobial activity of quinolones tested against Legionella was superior to that of erythromycin. This finding supports previous reports on levofloxacin,18 ciprofloxacin19 and garenoxacin.20,21 In addition, the present study first showed that the potent activities of pazufloxacin and tosufloxacin against intracellular Legionella were superior to that of erythromycin. This study also revealed a little heterogeneity among in vitro activities of quinolones tested against Legionella. The extracellular activity (MIC) of pazufloxacin was almost the same as those of levofloxacin and ciprofloxacin, but the ratio of MIEC and MIC for pazufloxacin (2) was slightly higher than those for levofloxacin (0.5), ciprofloxacin (1) and garenoxacin (1). The relative higher MIEC of pazufloxacin may be due to less penetration of the drug into macrophages than other quinolones. One study showed that pazufloxacin distributed into rat muscle cells, but was less concentrated within the cells than ciprofloxacin and ofloxacin.22 The ratio of MIEC and MIC for tosufloxacin (0.25) was lower than those of other quinolones, suggesting this drug has excellent intracellular potency.

Present in vitro results suggested possible clinical efficacy of quinolones. In fact, clinical efficacy of levofloxacin has been confirmed,8 and that of ciprofloxacin has been reported.23,24 A case report suggested that intravenous injection of pazufloxacin mesilate was effective in Legionella infections,25 but further studies are required to verify the efficacy of the drug. Tosufloxacin and garenoxacin may be useful in Legionella infection and warrant further clinical evaluations.

In summary, our study showed that pazufloxacin and tosufloxacin were potent in vitro against Legionella spp., as were other quinolones, and more potent than macrolides and azithromycin. Present data suggested possible efficacy of these drugs in treatment of Legionella infections.


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None to declare.


    Acknowledgements
 
We thank Ms Marie Suzuki for her skilled technical support.


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