P. Aeruginosa Biofilms and Burn Wound Infections

由多重耐药细菌引起的医院感染尤其难以治愈，对健康构成重大风险，并给经济带来巨大负担。这种医院感染的一个主要原因是形成生物膜的铜绿假单胞菌，它主要感染免疫功能受损的人和严重烧伤创面的人。尽管铜绿假单胞菌是院内感染的主要原因之一，但对其体内生物被膜表型以及阻止伤口愈合和促进感染部位铜绿假单胞菌持续存在的细菌因素知之甚少。我们最近发现，与浮游生物相比，铜绿假单胞菌生物膜表现出截然不同的表型，有800多种蛋白质和2000多个基因在生物膜中差异表达。定义体外生物被膜表型的毒力特征包括EXOS-T、ExoA、LasB和OprF-I，它们最近与伤口感染相关。使用猪烧伤创面模型，我们还证明了细菌形成了基质包裹的、抗菌素耐药的微菌落。创面中铜绿假单胞菌生物膜的形成与铜绿假单胞菌感染创面与非感染创面相比蛋白质表达的变化有关。有趣的是，蛋白质表达分析还显示，在体内铜绿假单胞菌生物膜中，被发现在体外被抑制的蛋白质的表达增加。在这个R21提案中，我们打算通过使用新兴技术和大胆而积极的方法来挑战现有的生物膜模型，以研究铜绿假单胞菌生物膜在烧伤创面感染中的作用，并确定体内铜绿假单胞菌生物膜的表型。据我们所知，这是解决如此复杂任务的第一次尝试。我们的发现表明存在着新奇和更多的 伤口中致病的体内生物膜表型。我们假设生物膜的形成与感染的临床症状相关，在体内铜绿假单胞菌生物膜比体外生物膜显示出一种新的、更具毒性的表型。为了验证我们的假设，我们建议使用体内感染-生物膜模型来表征基质包裹的、具有抗菌素耐药性的铜绿假单胞菌生物膜的时间形成，并将我们的发现与伤口感染的迹象相关联。我们还建议表征体内铜绿假单胞菌生物被膜的表型，这种表型仅在 微生物区系正常，免疫细胞和炎性细胞存在。我们预计，我们的发现将通过提高我们对铜绿假单胞菌发病机制的理解和确定可能作为治疗干预目标的毒力因素，来影响当前根除铜绿假单胞菌生物被膜感染的治疗策略。

Nosocomial infections by multiple antibiotic resistant bacteria are especially difficult to cure, pose a significant health risk and place an enormous burden on the economy. A leading cause of such nosocomial infections is the biofilm-forming Pseudomonas aeruginosa that primarily infects immune compromised individuals and those with severe burn wounds. In spite of P. aeruginosa being among the leading cause of nosocomial infections, little is known about the in vivo biofilm phenotype and the bacterial factors that prevent wound healing and promote persistence of P. aeruginosa at the infection site. We recently discovered that P. aeruginosa biofilms display a profoundly different phenotype compared to their planktonic counterparts with more than 800 proteins and 2000 genes being differentially expressed in biofilms. Among the virulence traits that defined the in vitro biofilm phenotype were ExoS-T, ExoA, LasB, and OprF-I that have recently been correlated with wound infections. Using a porcine burn wound model, we also demonstrated that bacteria formed matrix-encased, antimicrobial resistant microcolonies. The formation of P. aeruginosa biofilms in wounds correlated with a change in protein expression in P. aeruginosa-infected wounds as compared to non-infected wounds. Interestingly, the protein expression analysis also revealed an increased expression of proteins in in vivo P. aeruginosa biofilms that were found to be repressed in biofilms in vitro. In this R21 proposal, we intend to challenge current biofilm models by using emerging technologies and bold and aggressive approaches to investigate the role of P. aeruginosa biofilms in burn wound infections and to determine the P. aeruginosa in vivo biofilm phenotype. To our knowledge, this is the first attempt to tackle such a complex task. Our findings indicate the presence of novel and more virulent in vivo biofilm phenotypes in wounds. We hypothesize that biofilm formation correlates with clinical signs of infections and that in vivo P. aeruginosa biofilms display a new and more virulent phenotype than in vitro biofilms. To test our hypotheses, we propose to characterize the temporal formation of matrix-encased, antimicrobial resistant P. aeruginosa biofilms using an in vivo infection-biofilm model and to correlate our findings with signs of wound infections. We also propose to characterize the in vivo P. aeruginosa biofilm phenotype that is only displayed when normal microflora, and immunological and inflammatory cells are present. We anticipate that our findings will impact current treatment strategies to eradicate Pseudomonas biofilm infections by improving our understanding of P. aeruginosa pathogenesis and by identifying virulence factors that may be targeted for therapeutic intervention.