High throughput determination of the biofilm prevention concentration for Pseudomonas aeruginosa biofilms using a synthetic cystic fibrosis sputum medium.
High throughput determination of the biofilm prevention concentration for Pseudomonas aeruginosa biofilms using a synthetic cystic fibrosis sputum medium.
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作者：
患有慢性肺部感染的囊性纤维化（CF）患者中铜绿假单胞菌生物膜的存在导致抗微生物治疗的失败。常规地，确定最小抑菌浓度（MIC）以评估病原体的抗微生物剂敏感性，然而该参数不能预测治疗生物膜相关感染的成功。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（SCFM 2）来确定防止铜绿假单胞菌生物膜形成所需的抗菌剂浓度。在抗生素（妥布霉素、环丙沙星或粘菌素）存在下，生物膜在SCFM 2中生长24小时，之后生物膜被破坏，刃天青染色用于定量存活的代谢活性细胞的数量。平行地，将所有威尔斯孔的内容物铺板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EUCAST指南确定的MIC和最小杀菌浓度（MBC）进行比较。刃天青衍生的荧光和CFU计数之间的相关性用Kendall的Tau秩检验进行评估。在所研究的10种菌株中，观察到9种菌株的荧光和CFU计数之间存在显著相关性，表明荧光测定法是大多数铜绿假单胞菌分离株在相关条件下确定生物膜敏感性的平板接种的可靠替代方法。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是药物依赖性的。我们的研究结果表明，这种高通量测定可能是一个有价值的补充，以评估在CF的情况下，铜绿假单胞菌生物膜的抗菌药物敏感性。
The presence of Pseudomonas aeruginosa 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 P. aeruginosa 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 P. aeruginosa 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 P. aeruginosa biofilms in the context of CF.
DOI：
10.3390/antibiotics9120880
发表时间：
2020-12-09
期刊：
Antibiotics (Basel, Switzerland)
影响因子：
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作者：
Lozano C;López M;Rojo-Bezares B;Sáenz Y
通讯作者：
Sáenz Y