1 Chemotherapy Division, Mitsubishi Kagaku Bio-Clinical Laboratories, 3-30-1 Shimura, Itabashi-ku, Tokyo 174-8555; 2 Pharmaceuticals Research Division, Mitsubishi Pharma Corporation, Kanagawa, Japan
Received 29 October 2001; returned 15 January 2002; revised 10 April 2002; accepted 2 May 2002
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Abstract |
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Introduction |
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Materials and methods |
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One hundred and fifty-five strains of H. pylori, including 30 CAM-resistant5 strains (MIC 1.56 mg/L) and 25 MNDZ-resistant6 strains (MIC
6.25 mg/L), were isolated from patients with peptic ulcers. The specimens were obtained between 1997 and 1998 at several hospitals in Japan and were cultured using selective and non-selective agar media, as described previously.7 Twenty-nine reference and clinically isolated strains of different species were also used: Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228, Micrococcus luteus ATCC9341, Streptococcus pyogenes S-23, Streptococcus pneumoniae ATCC33400, Enterococcus faecalis ATCC19433, Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC10031, Citrobacter freundii ATCC8090, Enterobacter cloacae ATCC13047, Proteus vulgaris ATCC13315, Proteus mirabilis ATCC21100, Pseudomonas aeruginosa ATCC27853, Acinetobacter calcoaceticus NCTC7844, Haemophilus influenzae ATCC9334, Bacillus subtilis ATCC6633, Neisseria gonorrhoeae WHO-A, Peptostreptococcus anaerobius ATCC27337, Peptostreptococcus magnus ATCC29328, Propionibacterium acnes ATCC11829, Bacteroides fragilis ATCC25285, Bacteroides vulgatus ATCC29327, Fusobacterium nucleatum ATCC25586, a Prevotella intermedia clinical isolate, Clostridium difficile ATCC9689, Clostridium perfringens NCTC4969, Veillonella parvula ATCC10790, a Campylobacter jejuni clinical isolate and a Campylobacter fetus clinical isolate. All strains were stored at 80°C until required.
Antimicrobials
Stock solutions of BAS-118 (Mitsubishi Pharma Co., Osaka, Japan), CAM (Taisho Pharmaceutical Co., Ltd, Tokyo, Japan), MNDZ (Sigma Chemical Co., St Louis, MO, USA) and amoxicillin (Sigma Chemical Co.) were prepared from powders of known potency in accordance with the manufacturers instructions.
Susceptibility testing
The MICs of BAS-118 for H. pylori and other species were determined by an agar dilution method according to the guidelines established by the Japanese Society of Chemotherapy.8 For the H. pylori strains, BAS-118 and the other drugs were tested at concentrations of 500.003 mg/L and 1000.025 mg/L, respectively. For the other species, the drugs were tested at concentrations of 40.004 mg/L. The test strains (5 x 103 cfu/spot or 5 x 105 cfu/spot) were inoculated on to agar plates containing two-fold serial dilutions of the test agents with a multi-point inoculator (Sakuma Seisakusho, Tokyo, Japan). The agar plates inoculated with the test strains, including H. pylori, were cultured under the appropriate conditions for optimal growth: H. pylori and Campylobacter were inoculated on to Blood agar base No. 2 (Oxoid, Unipath Ltd, Basingstoke, UK) with 5% horse blood and incubated at 35°C for 72 h in a microaerophilic atmosphere; S. pneumoniae and H. influenzae were inoculated on to MuellerHinton agar (Difco Laboratories, Detroit, MI, USA) with 5% heat-lysed horse blood and incubated at 35°C for 18 h in 10% CO2; N. gonorrhoeae was inoculated on to GC agar (Becton Dickinson, Cockeysville, MD, USA) and incubated at 35°C for 24 h in 10% CO2; anaerobes were inoculated on to Brucella HK agar (Kyokuto Pharmaceutical Industrial Co., Ltd, Tokyo, Japan) supplemented with 5% defibrinated horse blood and incubated anaerobically at 35°C for 48 h; and all other aerobes were inoculated on to MuellerHinton agar and incubated in air at 35°C for 18 h. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibited visible bacterial growth.
Serial passage experiment
Five strains of H. pylori were subjected to a serial passage experiment for CAM and BAS-118 as described by Haas et al.9 The strains were each transferred with a swab on to the agars containing 0.5 x, 1 x and 2 x MIC of each drug. These agar plates were then incubated for 72 h under the above-mentioned conditions. This process was repeated serially until either no growth occurred or the tenth passage. The MICs of CAM and BAS-118 were determined at every passage.
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Results |
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Discussion |
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In conclusion, the potent and selective activity against H. pylori shown by BAS-118 may overcome the drawbacks of the present triple combination eradication regimen, including side effects and resistant strains. Moreover, the mechanism of its anti-H. pylori activity is expected to be novel because there have been no reports for structurally related compounds and because BAS-118 showed a very narrow antibacterial spectrum.
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Footnotes |
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References |
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