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Antimicrobial Agents and Chemotherapy, November 2006, p. 3908-3913, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00639-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vitro Antianaerobic Activity of DX-619, a New Des-Fluoro(6) Quinolone

Kaori Tanaka,* Hiroshige Mikamo, Ken'ichi Nakao, and Kunitomo Watanabe

Division of Anaerobe Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan

Received 24 May 2006/ Returned for modification 30 June 2006/ Accepted 28 July 2006


   ABSTRACT
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The in vitro activity of DX-619, a new des-F(6) quinolone, against anaerobic bacteria was evaluated. DX-619 showed potent activity against Bacteroides, Prevotella, Fusobacterium, Micromonas, Actinomyces, and Clostridium spp., with MIC50s/MIC90s of ≤0.03 to 0.25/≤0.03 to 1 µg/ml, respectively. DX-619 was also active against imipenem-resistant Bacteroides spp., with MIC50s/MIC90s of 0.25/1 µg/ml, respectively.


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Older fluoroquinolones, such as ciprofloxacin and ofloxacin, are inactive or only partially active against anaerobic bacteria (7, 11, 12, 15, 20, 23, 25, 26). Newer quinolones, such as sparfloxacin and levofloxacin (LVX), have improved in vitro activities against anaerobic bacteria but still have limited activities against certain gram-positive and gram-negative anaerobic bacilli (3, 7, 8, 11, 12, 15, 23, 25). Some of the newer fluoroquinolones are much more active to both gram-positive and gram-negative anaerobic bacteria (3, 8, 11, 12, 15, 20, 26). DX-619 is a new derivative of quinolone, defined as a des-F(6) quinolone that lacks the six-position fluorine characteristic of the previous generation of fluoroquinolones. It has potent antibacterial activity against multidrug-resistant, gram-positive aerobic bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (2, 5). Other compounds of this new type of quinolone showed potent activities against gram-positive and gram-negative aerobic and anaerobic bacteria (23, 25). There have been few reports on the antibacterial activity of DX-619 against anaerobic bacteria (16). We evaluated the in vitro activity of DX-619 against anaerobic gram-positive and gram-negative species in comparison with that of several reference agents.

For the investigation of the anaerobic antibacterial spectrum, a total of 71 gram-positive and gram-negative reference strains (64 species in 25 genera) of anaerobic bacteria and some fastidious microaerophilic anaerobes were examined. Those reference strains include strains obtained from the American Type Culture Collection (ATCC; Virginia), the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany), the Japan Collection of Microorganisms (JCM; Saitama, Japan), the National Collection of Type Cultures (NCTC; Wiltshire, United Kingdom), and VPI (Virginia Polytechnic Institute and State University, Virginia) and some characteristic clinical strains belong to GAI (the culture collection of our laboratory). A total of 268 clinical strains isolated from various sources (including intra-abdominal infection, head and neck space infection, pleuropulmonary infection, and soft-tissue infection) between 1994 and 2004 were also studied. Isolates were identified by standard criteria (9, 10, 22). In addition, the activities of DX-619 against 11 stock strains of the imipenem (IPM)-resistant Bacteroides fragilis group (10 Bacteroids fragilis and 1 B. thetaiotaomicron strain) were also studied.

The antibacterial agents used in this study were obtained as powders of known potency from their respective manufacturers. DX-619 was obtained from Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan. We used four antianaerobic agents, IPM (Banyu Pharmaceutical Co., Ltd., Tokyo, Japan), cefmetazole (CMZ), clindamycin (CLI), and metronidazole (MNZ) (Sigma-Aldrich Japan, Tokyo, Japan), as the reference agents for DX-619. One of the most popular fluoroquinolones, LVX (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan), and a popular cephalosporin for outpatient therapy, ceftriaxone (Sigma-Aldrich Japan, Tokyo, Japan), were also examined. The susceptibility of MNZ was examined only for reference strains.

The MICs were determined by an agar dilution method in accordance with CLSI (NCCLS) document M11-A5 (17). Brucella HK agar (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) supplemented with 5% laked sheep blood was used as the test medium. The test strains (105 CFU/spot) were inoculated and incubated at 35°C in an anaerobic chamber (82% N2, 10% CO2, 8% H2). B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 were used as quality control strains.

The results of the susceptibility test on the reference strains are listed in Tables 1 and 2. Overall, DX-619 showed potent activities against both gram-positive and -negative anaerobic reference strains. Most strains were inhibited at 0.5 µg/ml or less of this agent, including beta-lactam-resistant strains (B. fragilis GAI 0558, 7955, and 10150). DX-619 was more active than CMZ, ceftriaxone, LVX, and MNZ. Also, DX-619 was more active than CLI and almost as active as or more active than IPM against gram-positive cocci and Clostridium spp. In gram-negative organisms, DX-619 was slightly less active than IPM and CLI against Prevotella spp. It was, however, more active than IPM and CLI against the Bacteroides fragilis group.


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  		TABLE 1. Antimicrobial activities of DX-619 and other reference compounds against gram-positive anaerobic bacteria and facultative anaerobic bacteria

 

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  		TABLE 2. Antimicrobial activities of DX-619 and other reference compounds against gram-negative anaerobic bacteria and facultative anaerobic bacteria

 
Table 3 shows the in vitro activities of DX-619 and reference agents against clinical strains frequently isolated in anaerobic infection. The results are expressed as MIC50s, MIC90s, and percentages of isolates susceptible at the indicated drug concentrations. Against three species of the B. fragilis group and Fusobacterium spp., the most active agent was DX-619, with MIC90s of 0.25 to 1 µg/ml. For the B. fragilis group, IPM was the second-most-active agent, with MIC90s of 1 to 4 µg/ml. Although CLI still showed strong activity against 68% of B. fragilis isolates, with a MIC50 of 1 µg/ml, the MIC50 of DX-619 (0.125 µg/ml) was threefold lower than that of CLI. In Fusobacterium spp., overall, the strains examined were sensitive to all six test agents and quite sensitive to DX-619. Against Prevotella, IPM and CLI showed superior activities. DX-619 was the third-most-active agent in this group but also showed potent activity, inhibiting all strains with a concentration of ≤1 µg/ml.


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  		TABLE 3. In vitro activities of DX-619 and other reference compounds against clinical isolates of anaerobic bacteria and facultative anaerobic bacteria

 
DX-619 was quite active for gram-positive organisms. Similarly to Fusobacterium strains, strains of Micromonas micros and Propionibacterium acnes were sensitive to six test agents, including DX-619. DX-619 inhibited those strains at ≤0.03 µg/ml (M. micros) and 0.125 µg/ml (P. acnes). The Actinomyces spp. included species variously susceptible to beta-lactams, but all strains were sensitive to DX-619 and CLI, with MIC90s of 1 µg/ml. As for clostridia, Clostridium perfringens and other Clostridium spp. had similar susceptibilities and most of the strains tested were susceptible to all six agents. Against these two groups, DX-619, IPM, and CMZ had potent activities (from highest to lowest, in that order), with MIC90s of ≤0.25 µg/ml. On the other hand, most of the agents, including IPM and CMZ, were less active against Clostridium difficile. The MIC90s for IPM and CMZ were 16 and 64 µg/ml, respectively, but DX-619 was very active against C. difficile and inhibited all C. difficile isolates at a concentration of 2 µg/ml (Fig. 1).


Figure 1
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  		FIG. 1. Antimicrobial activities of DX-619 and other compounds against 27 strains of C. difficile.

 
The IPM-resistant B. fragilis group strains (11 strains) and LVX-resistant anaerobic gram-positive cocci (14 strains) were also examined (Table 4). All but one strain (90.9%) of the IPM-resistant B. fragilis group were inhibited by ≤1 µg/ml of DX-619. The MIC90s for the other agents were 16 µg/ml for LVX and equal to or more than 128 µg/ml for four other agents. DX-619 was also active against LVX-resistant, gram-positive cocci. The MIC90 for DX-619 in this group was 2 µg/ml, sixfold lower than that for LVX.


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  		TABLE 4. In vitro activities of DX-619 and other reference compounds against resistant anaerobic strains

 
In the present study, DX-619 showed excellent activity against clinically important anaerobic species. Many antibacterial agents have poor or moderate activity against the B. fragilis group. Against the three major species of the B. fragilis group (B. fragilis, B. thetaiotaomicron, and Bacteroides distasonis)examined here, DX-619 was the most active agent (MIC90s of 0.5, 0.5, and 1 µg/ml, respectively), followed by IPM. As for the anti-Bacteroides activities of the other test agents, CMZ was less active against B. thetaiotaomicron and B. distasonis but active against B. fragilis. This is a known tendency of cephamycins (4). CLI still showed strong activity against 68% of B. fragilis isolates, with a MIC50 of 1 µg/ml. The resistance rate of CLI based on the CLSI breakpoint (≥8 µg/ml) was higher in B. thetaiotaomicron (52%) and B. distasonis (44%) than in B. fragilis (32%). Strains of these species examined in this report were isolated from 2000 to 2002. In 1991, the resistance rates of CLI were 29.0% in B. fragilis and 31.7% in B. thetaiotaomicron, though a different breakpoint (≥6.25 µg/ml) and different test media (Gifu anaerobic media) were used in that report (24). This suggests that resistance to CLI is increasing, especially in Bacteroides species other than B. fragilis. In Japan, the most popular antianaerobic agent, metronidazole, is not available as an antibacterial agent. Under such a situation, DX-619, which is also active against IPM-resistant B. fragilis and B. thetaiotaomicron, would be one of the reliable agents.

Besides that, DX-619 showed potent activity against Prevotella spp., Fusobacterium spp., and anaerobic gram-positive cocci. Species in those groups are frequently isolated from major sites of anaerobic infection, such as the head and neck space and pleuropulmonary site. As well as other infections involving anaerobes, those anaerobic infections are usually polymicrobial infections involving both anaerobic and aerobic bacteria. DX-619 has been reported to have excellent activity against aerobic gram-positive organisms and also strong activity against aerobic gram-negative rods, such as Escherichia coli and Klebsiella pneumoniae (5). These data indicate that DX-619 has the potential to be used as a single agent in the treatment of polymicrobial infections involving those anaerobic and aerobic species.

Gas gangrene is one of the severe anaerobic infections. The gas gangrene-related species C. perfringens (25 strains), Clostridium novyi A (4 strains), Clostridium septicum (3 strains), and Clostridium sordellii (1 strain) were overall sensitive to all agents tested and quite sensitive to DX-619, which inhibited those strains at MICs of ≤0.03 to 0.25 µg/ml. DX-619 might be useful for this anaerobic infection.

C. difficile is an important nosocomial pathogen that is acquired exogenously, and a variety of clinical outcomes ensue following infection, ranging from asymptomatic colonization to diarrhea to more-severe disease syndromes. It causes disease almost exclusively in the presence of exposure to antimicrobial agents. The most commonly implicated agents were CLI and cephalosporins, but recently, implication of fluoroquinolones was suggested (6, 13, 18). After 2002, outbreaks of a highly lethal type of C. difficile were observed in Canada and the United States (13, 14, 19). These strains were reported to be resistant to clinically used newer fluoroquinolones, such as moxifloxacin, gatifloxacin, and LVX (13, 14). Half of the C. difficile strains examined in this study were resistant to LVX and CLI, but all strains were susceptible to DX-619 (MIC, ≤2 µg/ml). The test strains included four moxifloxacin-resistant strains (the MICs were 8 µg/ml for one strain and 16 µg/ml for the others), and they were also susceptible to DX-619 (MICs of 0.5 to ≤2 µg/ml) (data not shown). These observations suggest that DX-619 may be a low-risk agent as an inducer of C. difficile-associated diarrhea.

A comparison of our results to the published data on other quinolones shows that, overall, DX-619 covers a wide range of the antianaerobic spectrum, as broad as that for sitafloxacin. Its potent antianaerobic activity seems to be close to that of sitafloxacin and comparable to that of trovafloxacin (1, 3, 8, 11, 15, 20, 21, 25, 26). Fujikawa et al. comprehensively studied the antibacterial activities of DX-619 and reference agents, including IPM and LVX (5). They reported antibacterial activity against Peptostreptococcus spp., C. difficile, and B. fragilis in that paper. Our results were largely consistent with their results. Their results indicated that the antibacterial activities of DX-619 against those anaerobes were more potent than those of garenoxacin, another desfluoroquinolone. During the process of the review of this paper, another study of the antianaerobic activity of DX-619 was published in Antimicrobial Agents and Chemotherapy (16). Molitoris et al. compared the activity of DX-619 and those of other antianaerobic agents (amoxicillin-clavulanate, linezolid, meropenem, and moxifloxacin). Though the comparator agents were different from those in this study, we could compare the results of DX-619 treatment for some groups of species tested in both studies (B. distasonis, B. fragilis, B. thetaiotaomicron, Fusobacterium spp., Prevotella spp., and Clostridium spp.). The ranges of MICs, MIC50s, and MIC90s for DX-619 were almost the same as those for these anaerobes.

In summary, this study showed a potent in vitro activity for DX-619 against clinically important gram-positive and gram-negative anaerobic bacteria. It was also active against LVX- and IPM-resistant strains. Our results indicate that DX-619 would be an effective agent against anaerobic bacteria resistant to other antianaerobic agents and has the potential to be a useful antianaerobic agent for treatment of severe anaerobic infection. To be useful for treatment of community-acquired infections, the novel des-F(6) quinolone DX-619 would have to exhibit higher levels of activity against both gram-positive and gram-negative anaerobic pathogens with various resistance profiles. Clinical studies, including pharmacokinetic/pharmacodynamic studies, will be required to clarify the role of DX-619 in the empirical treatment of community-acquired infections.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Anaerobe Research, Life Science Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan. Phone: 81-58-230-6553. Fax: 81-58-230-6551. E-mail: kktb{at}gifu-u.ac.jp. Back


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Antimicrobial Agents and Chemotherapy, November 2006, p. 3908-3913, Vol. 50, No. 11
0066-4804/06/$08.00+0     doi:10.1128/AAC.00639-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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