Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms
Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms
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DOI：
10.1128/aac.48.7.2659-2664.2004
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发表时间：
2004-07-01
影响因子：
4.9
通讯作者：
Stewart, PS
中科院分区：
文献类型：
--
作者：
Borriello, G;Werner, E;Stewart, PS
研究了氧限制在保护铜绿假单胞菌生物膜中免受抗生素杀伤的作用。当暴露于妥布霉素、环丙沙星、羧苄青霉素、头孢他啶、氯霉素或四环素12 h时，成熟（48 h）菌落生物膜中的细菌被杀死的效果很差。氧微电极显示，这些生物膜包含大的缺氧区域。氧气渗透约50 μ m的生物膜，其平均210 μ m厚。活性蛋白质合成的区域通过使用可诱导的绿色荧光蛋白可视化。该区域也限于与生物膜的空气界面相邻的约30 μ m宽的窄带。成熟生物膜中的细菌表现出仅0.02 h（-1）的比生长速率。这些结果表明，48小时的菌落生物膜是生理异质性和生物膜中的大多数细胞占据氧限制，静止相状态。相比之下，4小时的菌落生物膜中的细菌仍然在生长，活跃，并且当它们在空气中受到挑战时对抗生素敏感。当4小时-老的菌落生物膜在厌氧条件下的挑战，抗生素的杀伤水平降低相比，有氧生长的控制。氧气限制可以解释70%或更多的保护提供给48小时的菌落生物膜的所有抗生素测试。硝酸盐改良剂刺激了在厌氧条件下生长的未处理对照铜绿假单胞菌分离株的生长，但降低了生物体对抗生素的敏感性。局部氧限制和硝酸盐的存在可能有助于降低铜绿假单胞菌生物膜的亲和性，从而引起体内感染。
The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 mum into the biofilms, which averaged 210 mum thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band, approximately 30 mum wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h(-1). These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.