BACKGROUND The majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time. RESULTS The collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide- 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2-1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation. CONCLUSIONS The collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

中文翻译：

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体外胶原蛋白灌注伤口生物膜模型；及其在抗微生物研究和微生物代谢组学中的应用。

背景技术医学上相关的生物膜的大多数体外研究涉及在无生命的固体表面上生物膜的发展。然而，体内感染由生物膜在组织的半固体基质上生长或悬浮在组织的半固体基质中组成，因此当前的模型不能有效地模拟体内环境的性质。本文介绍了一种在系统中培养伤口相关微生物的体外方法的开发，该系统将半固体胶原蛋白凝胶基质与模拟伤口液的连续流动相结合。这使得能够在更紧密地模拟动态伤口环境的条件下培养伤口相关的可再生稳态生物膜。为了证明使用该模型，头孢他啶对成熟和发育中的铜绿假单胞菌生物膜均具有抗菌动力学，被评估。此外，我们通过选择离子流管质谱（SIFT-MS）实时监测铜绿假单胞菌生物膜产生的氨和氰化氢，显示了该模型系统在研究微生物代谢组学中的潜在应用。结果胶原伤口生物膜模型促进伤口状态下稳态可复制的铜绿假单胞菌生物膜的生长。连续培养30 h可达到1010 cfu玻片-1的最大生物膜密度，并在其余实验过程中保持不变。与未治疗的对照组相比，以临床相关剂量的头孢他啶治疗可导致72 h的生物膜密度降低1.2-1.6 log。处理导致丧失复杂的生物膜结构和细菌细胞的形态变化，使用共聚焦显微镜可视化。当使用SIFT-MS监测生物膜时，氨和氰化氢水平在12小时分别达到2273 ppb（±826.4）和138 ppb（±49.1）的峰值，并且在整个实验中均可检测到。结论胶原蛋白伤口生物膜模型已被开发出来，以促进在类似伤口的条件下可再生生物膜的生长。我们已经成功地将这种方法用于：（1）评估抗菌功效和动力学，清楚地证明了生物膜培养物中抗菌素耐受性的发展；（2）表征铜绿假单胞菌生物膜产生的挥发性代谢物，证明该方法在代谢组学研究中的潜在用途。4）和138 ppb（±49.1），并且在整个实验过程中均可检测到。结论胶原蛋白伤口生物膜模型已被开发出来，以促进在类似伤口的条件下可再生生物膜的生长。我们已经成功地将这种方法用于：（1）评估抗菌功效和动力学，清楚地证明了生物膜培养物中抗菌素耐受性的发展；（2）表征铜绿假单胞菌生物膜产生的挥发性代谢物，证明该方法在代谢组学研究中的潜在用途。4）和138 ppb（±49.1），并且在整个实验过程中均可检测到。结论胶原蛋白伤口生物膜模型已被开发出来，以促进在类似伤口的条件下可再生生物膜的生长。我们已经成功地将这种方法用于：（1）评估抗菌功效和动力学，清楚地证明了生物膜培养物中抗菌素耐受性的发展；（2）表征铜绿假单胞菌生物膜产生的挥发性代谢物，证明该方法在代谢组学研究中的潜在用途。清楚地证明了生物膜培养物中抗微生物耐受性的发展；（2）表征铜绿假单胞菌生物膜产生的挥发性代谢物，证明该方法在代谢组学研究中的潜在用途。清楚地证明了生物膜培养物中抗微生物耐受性的发展；（2）表征铜绿假单胞菌生物膜产生的挥发性代谢物，证明该方法在代谢组学研究中的潜在用途。