De Bleeckere Amber, Van den Bossche Sara, De Sutter Pieter-Jan, Beirens Tine, Crabbé Aurélie, Coenye Tom
Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium.
Laboratory of Medical Biochemistry and Clinical Analysis, Ghent University, Ghent, Belgium.
Biofilm. 2023 Feb 6;5:100106. doi: 10.1016/j.bioflm.2023.100106. eCollection 2023 Dec.
The presence of biofilms in cystic fibrosis (CF) patients suffering from chronic lung infections contributes to the failure of antimicrobial therapy. Conventionally, the minimal inhibitory concentration (MIC) is determined to assess the antimicrobial susceptibility of a pathogen, however this parameter fails to predict success in treating biofilm-associated infections. In the present study we developed a high throughput method to determine the antimicrobial concentration required to prevent biofilm formation, using a synthetic cystic fibrosis sputum medium (SCFM2). Biofilms were grown in SCFM2 for 24 h in the presence of antibiotics (tobramycin, ciprofloxacin or colistin), whereafter biofilms were disrupted and a resazurin staining was used to quantify the number of surviving metabolically active cells. In parallel, the content of all wells was plated to determine the number of colony forming units (CFU). Biofilm preventing concentrations (BPCs) were compared to MICs and minimal bactericidal concentrations (MBCs) determined according to EUCAST guidelines. Correlations between the resazurin-derived fluorescence and CFU counts were assessed with Kendall's Tau Rank tests. A significant correlation between fluorescence and CFU counts was observed for 9 out of 10 strains investigated, suggesting the fluorometric assay is a reliable alternative to plating for most isolates to determine biofilm susceptibility in relevant conditions. For all isolates a clear difference between MICs and BPCs of all three antibiotics was observed, with the BPCs being consistently higher than the MICs. Additionally, the extent of this difference appeared to be antibiotic-dependent. Our findings suggest that this high throughput assay could be a valuable addition to evaluate the antimicrobial susceptibility in biofilms in the context of CF.
患有慢性肺部感染的囊性纤维化（CF）患者体内生物膜的存在导致抗菌治疗失败。传统上，通过测定最低抑菌浓度（MIC）来评估病原体的抗菌敏感性，然而该参数无法预测生物膜相关感染的治疗成功率。在本研究中，我们开发了一种高通量方法，使用合成囊性纤维化痰液培养基（SCFM2）来确定防止生物膜形成所需的抗菌浓度。生物膜在含有抗生素（妥布霉素、环丙沙星或黏菌素）的SCFM2中培养24小时，之后破坏生物膜，并使用刃天青染色来量化存活的代谢活跃细胞数量。同时，将所有孔的内容物进行平板接种以确定菌落形成单位（CFU）的数量。将生物膜预防浓度（BPC）与根据欧洲抗菌药物敏感性试验委员会（EUCAST）指南测定的MIC和最低杀菌浓度（MBC）进行比较。使用肯德尔等级相关检验评估刃天青衍生荧光与CFU计数之间的相关性。在所研究的10株菌株中，有9株观察到荧光与CFU计数之间存在显著相关性，这表明对于大多数分离株而言，荧光测定法是在相关条件下确定生物膜敏感性的一种可靠替代平板接种的方法。对于所有分离株，观察到所有三种抗生素的MIC和BPC之间存在明显差异，BPC始终高于MIC。此外，这种差异的程度似乎取决于抗生素。我们的研究结果表明，这种高通量测定法可能是评估CF背景下生物膜抗菌敏感性的一项有价值的补充方法。
