CHEMOTHERAPY OCT. 1993

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Transcription:

CHEMOTHERAPY OCT. 1993

VOL. 41 NO. 10

CHEMOTHERAPY Table 1. Antibiotic permeability through biofilm of clinical isolates of Pseudomonas aeruginosa CAZ: ceftazidime, GM: gentamicin. Fig. 1. The effect of incubation periods on permeability of gentamicin through Pseudomonas aeruginosa 201-3 biofilm at a concentration of 50,ug/ml. CAZ: ceftazidime, GM: gentamicin. Fig. 2. Permeability of ceftazidime and gentamicin through Pseudomonas aeruginosa 201-3 biofilm.

VOL. 41 NO, 10 CAZ: ceftazidime, GM: gentamicin, Fig. 3. The effect of Pseudomonas aeruginosa slime on the standard curves of antibiotics. Fig. 4. The effect of slime on bactericidal activities of ceftazidime and gentamicin against Pseudomonas aeruginosa 201-3.

OCT. CHEMOTHERAPY 1068 (a) (b) (c) (d) (e) (f) (g) (h) Fig. 5. filter. Scanning electron microscopy of Pseudomonas aeruginosa 201-3 grown 1993 on the membrane

2) Anwar H, Dasgupta M K, Costerton J W: Testing the susceptibility of bacteria in biofilms to antibacterial agents. Antimicrob Agent Chemother 34: 2043 `2046, 1990 6) Costerton J W, Cheng K-J, Geesey G G, Ladd T I, Nickel J C, Dasgupta M, Marrie T J: Bacterial biofilms in nature and disease. Annu Rev Mi- crobiol 41: 435-464, 1987 7) Prosser BLaT, Taylor D, Dix B A, Cleeland R: Method of evaluating effects of antibiotics on bacterial biofilm. Antimicrob Agent Chemother 31: 1502-1506, 1987 10) Nickel J C, Ruseska I, Wright J B, Costerton J W: Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofllm on urinary catheter material. Antimicrob Agent Chemother 27: 619-624, 1985 11) Anwar H, van Biesen T, Dasgupta M, Lam K, Costerton J W: Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system. Antimicrob Agent Chemother 33: 1824 ` 1826, 1989 12) Anwar H, Costerton J W: Enhanced activity of combination of tobramycin and piperacillin for eradication of sessile biofilm cells of Pseudomonas aeruginosa. Antimicrob Agent Chemother 34: 1666 4671, 1990 13) Anwar H, Strap J L, Costerton j W: Establishment of aging biofilms: possible mechanism of bacterial resistance to antimicrobial therapy. Antimicrob Agent Chemother 36:1347 `1351, `

CHEMOTHERAPY 15) Obana Y: Pathogenic significance of Acinetobacter calcoaceticus: analysis of experimental infection in mice. Microbial lmmunol 30: 645 `157, 1986 16) Slack M P E, Nichols W W: Antibiotic penetration through bacterial capsules and exopolysaccharides. J Antimicrob Chemother 10: 368 `372, 1982 17) Gilbert P, Collier P J, Brown M R W: Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob Agent Chemother 34: 1865 `1868, 1990 18) Eng R H K, Padberg F T, Smith S M, Tan E N, Cherubin C E: Bactericidal effects of antibiotics on slowly growing and nongrowing bacteria. Antimicrob Agent Chemother 35: 1824 `1828, 1991 19) Murakawa T: Slime production by Pseudomonas aeruginosa IV. Chemical analysis of two varieties of slime produced by Pseudomonas aeruginosa, Japan J Microbial 17: 513 `520, 1973 20) Slack M P E, Nichols W W: The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudomonas aeruginosa. Lancet II: 502 `503, 1981 21) Tannenbaum C S, Hastie A T, Higgins M L, Kueppers F, Weinbaum G: Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics. Antimicrob Agent Chemother 25: 673 `675, 1984 A new method of measuring antibacterial agents through biofilms Machiko Naito, Tadahiro Matsushita and Totaro Yamaguchi Pharmacological Research Laboratory, Tanabe Seiyaku Co, Ltd, 2-2-50, Kawagishi, Toda, Saitama 335, Japan Takeshi Yokota Juntendo Medical College of Nursing The formation of biofilm is a cause of chronic infection because biofilm blocks the diffusion of antibacterial agents, preventing them from reaching bacteria. To assess the permeability of antibacterial agents through Pseudomonas aeruginosa biofilms, we devised a simple method using an Intercell(R) and a multi-well plate. P. aeruginosa cells were inoculated in an Intercell(R) placed on a brain heart infusion agar plate. Biofilm was formed on the membrane filter of the Intercell(R) during incubation at 37 Ž for 3 days. Scanning electron micrographs showed that the biofilm formed was about 100 pm in thickness and consisted of bacterial and spongy glycocalyx layers. The biofilmformed Intercell I was placed in a multi-well plate, and the surfaces of the biofilm were coated with agar to prevent peeling. A solution of antibacterial agent was added to the Intercell(R) and phosphate buffer was poured into the outside of the Intercell(R) until the levels of the solution on both sides were equal. After incubation at room temperature for 24 hours, the concentration of the antibacterial agents outside the Intercell(R) was measured by bioassay. The diffusion rate of gentamicin (200ƒÊg/ ml) in the presence of biofilm was 42% of that in the absence of biofilm. In addition, the slime prepared from P. aeruginosa lowered the antibacterial potency of gentamicin, and its bactericidal activity decreased with an increase in the concentration of the slime. These findings suggest that biofilm forms a barrier to diffusion of gentamicin, attributable to binding of the drug to alginate which is a primary constituent of P. aeruginosa biofilm. On the other hand, in the case of ceftazidime, no change was observed in the diffusion rate regardless of the presence or absence of biofilm. Furthermore, addition of P. aeruginosa slime did not affect the potency of ceftazidime and its bactericidal activity, suggesting that ceftazidime diffuses freely through biofilm.

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