Ito Akinobu, Taniuchi Asami, May Thithiwat, Kawata Koji, Okabe Satoshi
Department of Urban and Environmental Engineering, Hokkaido University, Sapporo, Japan.
Appl Environ Microbiol. 2009 Jun;75(12):4093-100. doi: 10.1128/AEM.02949-08. Epub 2009 Apr 17.
Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 microg/ml), kanamycin (25 microg/ml), and ofloxacin (10 microg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.
生物膜被认为对抗菌剂具有高度抗性。已经提出了几种机制来解释生物膜的这种高抗性，包括抗菌剂进入生物膜的渗透受限、由于营养限制导致的生长缓慢、参与一般应激反应的基因表达以及生物膜特异性表型的出现。然而，由于这些因素的组合涉及大多数生物膜研究，因此仍然难以完全理解生物膜对抗生素的抗性机制。在本研究中，排除了抗菌剂进入生物膜的渗透受限和由于营养限制导致的生长缓慢的影响，研究了生物膜中大肠杆菌细胞的抗生素敏感性。将三种不同的抗生素，氨苄青霉素（100微克/毫升）、卡那霉素（25微克/毫升）和氧氟沙星（10微克/毫升），直接应用于在流动池中形成的成熟生物膜深层的细胞，这些生物膜在去除生物膜表层后形成。抗生素处理分析结果表明，氧氟沙星和卡那霉素对生物膜细胞有效，而氨苄青霉素不能杀死细胞，导致氨苄青霉素处理停止后生物膜重新生长。活/死染色显示，在成熟生物膜的深层出现了一小部分抗性细胞，并且这些细胞即使在氨苄青霉素处理24小时后仍然存活。此外，为了确定生物膜细胞中哪些基因被诱导，从而增加对氨苄青霉素的抗性，在生物膜形成的不同阶段，即附着、菌落形成和成熟阶段，分析了全局基因表达。结果表明，在生物膜形成过程中基因表达发生了显著变化，部分是由rpoS表达诱导的。基于实验数据，观察到的生物膜抗性可能归因于成熟生物膜深层而非年轻菌落生物膜中氨苄青霉素抗性亚群的形成，并且这些亚群的产生和抗性得益于生物膜特异性表型，如生长缓慢和rpoS介导的应激反应的诱导。
