Novartis Vaccines and Diagnostics, Siena, Italy.
J Bacteriol. 2013 Feb;195(3):545-55. doi: 10.1128/JB.01980-12. Epub 2012 Nov 21.
Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.
生物膜内的细菌受到多种压力的保护，包括免疫反应和抗菌剂。细菌病原体的生物膜形成能力与抗生素耐药性和慢性复发性感染的增加有关。尽管已经对几种肠道病原体的生物膜形成进行了充分研究，但对于厌氧肠道物种的生物膜形成知之甚少。专性厌氧菌艰难梭菌引起艰难梭菌感染（CDI），这是一个主要的医疗保健相关问题，主要是由于复发性感染的发生率很高。当正常肠道微生物群被抗生素破坏时，艰难梭菌会在肠道中定殖;然而，在感染期间涉及肠道定殖的因素或过程尚不清楚。我们证明临床艰难梭菌菌株，即菌株 630 和高毒力菌株 R20291，在体外形成结构良好的生物膜，其中 R20291 积累了大量的生物膜。显微镜和生化分析显示，多层细菌被包裹在含有蛋白质、DNA 和多糖的生物膜基质中。通过使用同源突变体，我们表明与毒力相关的蛋白 Cwp84、鞭毛和一个假定的群体感应调节因子 LuxS 对于艰难梭菌的最大生物膜形成都是必需的。有趣的是，一个转录因子 Spo0A 的突变体，该转录因子控制孢子形成，在生物膜形成中存在缺陷，这表明孢子形成和生物膜形成之间可能存在联系。此外，我们证明了艰难梭菌生物膜中的细菌对高浓度万古霉素更具耐药性，万古霉素是一种常用于治疗 CDI 的药物。我们的数据表明，艰难梭菌的生物膜形成是一个复杂的多因素过程，可能是艰难梭菌在宿主中持续存在的关键机制。
