Soares Anaïs, Roussel Valérie, Pestel-Caron Martine, Barreau Magalie, Caron François, Bouffartigues Emeline, Chevalier Sylvie, Etienne Manuel
GRAM 2.0, EA 2656, Normandie University, UNIROUEN, Rouen, France.
Microbiology Department, Rouen University Hospital, Rouen, France.
Front Microbiol. 2019 Nov 13;10:2603. doi: 10.3389/fmicb.2019.02603. eCollection 2019.
Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a biofilm escapes ciprofloxacin treatment. PA14 mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.
生物膜通常对抗生素具有顽固性，其耐受和持续存在等机制尚未完全阐明。我们旨在研究生物膜如何逃避环丙沙星治疗。用超最低抑菌浓度（supra-MIC）的环丙沙星处理PA14成熟生物膜。通过荧光素二乙酸酯测定法定量细胞活力。通过对存活的可培养细胞计数来确定群体动态。使用共聚焦激光扫描显微镜（CLSM）分析生物膜，并通过RT-qPCR对未处理和处理后的生物膜中参与严谨反应、毒素-抗毒素HigB/HigA和III型分泌系统（T3SS）的基因表达进行定量。环丙沙星处理后，细菌计数呈双相时间杀灭曲线，最初有所减少。处理24小时后，与未处理的生物膜相比，总体细胞活性和可培养细胞密度显著降低。在<1%的存活细胞中未分离出耐药突变体。仅在抗生素暴露1小时后，就出现了对持续存在的表型适应，这是通过参与严谨反应和毒素-抗毒素系统的基因过表达实现的，而编码T3SS的基因表达保持不变。环丙沙星暴露4小时后，严谨反应基因恢复到其基础表达水平。长时间暴露于环丙沙星后，观察到基质结构发生深度改变，变得更薄且失去了蘑菇状聚集体，这与胞外多糖和细胞外DNA的生物体积减少有关。这些结果表明，环丙沙星可能首先诱导大多数细菌细胞的死亡，但同时激活严谨反应机制，促使亚群向持续存在表型转变。一旦持续存在表型表达，尽管生物膜基质出现意外改变，环丙沙星仍无法根除生物膜。
