Data_Sheet_1_3D spatial organization and improved antibiotic treatment of a Pseudomonas aeruginosa–Staphylococcus aureus wound biofilm by nanoparticle enzyme delivery.PDF

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (α-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes α-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.

由铜绿假单胞菌（Pseudomonas aeruginosa）和金黄色葡萄球菌（Staphylococcus aureus）感染的慢性伤口是全球范围内的重要健康问题，因为这些病原体嵌入在由多糖、蛋白质、脂质与细胞外DNA构成的网络（即生物膜（biofilm））中，该结构会阻碍抗生素的递送并提升细菌的抗菌耐受性。亟需研究能够提升抗生素穿透性与疗效的治疗方案。在此背景下，本研究的核心目标为探究铜绿假单胞菌与金黄色葡萄球菌之间的相互关系，以及该关系如何影响抗菌治疗，并研究功能化银纳米颗粒能否改善抗生素疗法。我们采用了优化后的体外伤口模型，该模型可模拟体内伤口环境，用于共培养铜绿假单胞菌与金黄色葡萄球菌生物膜。我们使用由抗生素（庆大霉素（gentamycin）与环丙沙星（ciprofloxacin））以及游离的或经酶类（α-淀粉酶（α-amylase）、纤维素酶（cellulase）、脱氧核糖核酸酶I（DNase I）或蛋白酶K（proteinase K））功能化修饰的生物膜分散剂构成的抗菌联合疗法，对该体外伤口生物膜进行处理，以评估其抗生物膜效能。通过共聚焦显微镜与电子显微镜，我们对伤口样生物膜内的铜绿假单胞菌与金黄色葡萄球菌的相互作用与共定位情况进行了检测与详细表征。研究结果表明，抗生素单药治疗效果不佳：其对混合生物膜中的两种细菌存在差异化影响——使用环丙沙星时，铜绿假单胞菌会战胜金黄色葡萄球菌；而使用庆大霉素时则结果相反。与之相反，双重抗生素联合疗法可有效降低两种细菌的数量，同时维持种群平衡。此外，脱氧核糖核酸酶I纳米颗粒治疗展现出强效的抗生物膜效果：在联合抗生素使用时，可将铜绿假单胞菌与金黄色葡萄球菌的存活率分别降至0.017%与7.7%。结果显示，使用经脱氧核糖核酸酶I功能化修饰的纳米颗粒可增强抗菌治疗效果，其细菌存活率降幅优于单独使用抗生素。α-淀粉酶与纤维素酶展现出一定的抗生物膜效果，但相较于脱氧核糖核酸酶I治疗效果较弱。蛋白酶K则未展现出显著的抗生物膜效果。最后，我们提出了一个三维共定位模型：金黄色葡萄球菌聚集体嵌入生物膜结构中，这可能与抗生物膜疗法对该类细菌的低疗效相关。因此，设计结合抗生物膜酶类与抗生素的临床治疗方案，或许对根除慢性伤口感染至关重要。