红色毛癣菌、犬小孢子菌生物被膜模型构建及其对特比萘芬敏感性观察

Construction of biofilm models of Trichophyton rubrum and Microsporum canis and observation of their sensibility to terbinafine

目的构建红色毛癣菌(T.rubrum)、犬小孢子菌(M.canis)体外生物被膜模型,并观察其对特比萘芬(TERB)的敏感性。方法取红色毛癣菌、犬小孢子菌标准菌,分别接种到96孔板中,35℃静置分别孵育6、3 h,制作红色毛癣菌、犬小孢子菌体外生物被膜模型,分别于培养24、48、72、96 h时倒置显微镜观察红色毛癣菌、犬小孢子菌生物被膜形态,采用XTT法观察红色毛癣菌、犬小孢子菌生物被膜生长情况,番红精法检测培养96 h时红色毛癣菌、72 h时犬小孢子菌成熟生物被膜中的细胞外基质(ECM,以OD492值表示),扫描电镜观察红色毛癣菌成熟生物被膜的形态。测定特比萘芬(TERB)对红色毛癣菌和犬小孢子菌的最低抑菌浓度(MIC)以及抑制50%和80%生物被膜的浓度(SMIC50、SMIC80)。结果培养48 h,两种菌株酵母相细胞与其延伸所形成的菌丝开始密集交织,培养72 h时两种菌株开始大量形成ECM并沿着菌丝形成团块聚集,可见多层膜状结构。红色毛癣菌和犬小孢子菌形成成熟生物被膜的时间分别为96、72 h。红色毛癣菌和犬小孢子菌成熟生物被膜OD492值分别为1.925±0.111、1.173±0.157。红色毛癣菌成熟生物被膜菌丝相互缠绕连接,形成精密的被膜网,菌丝外围清晰可见ECM。TERB对红色毛癣菌和犬小孢子菌浮游菌MIC分别是0.125、1μg/m L,而TERB对红色毛癣菌和犬小孢子菌成熟生物被膜的SMIC50、SMIC80均大于256μg/m L。结论成功构建体外红色毛癣菌和犬小孢子菌生物被膜模型。成熟的红色毛癣菌、犬小孢子菌生物被膜对TERB的敏感性低。

Objective To construct biofilm models of Trichophyton rubrum（T. rubrum） and Microsporum canis（M.canis） and to observe their sensibility to terbinafine（TERB）. Methods Standard isolates of T. rubrum and M. canis were inoculated in 96-well plates,at 35 ℃ for 6 h or 3 h,respectively,and then we constructed the biofilm models of T.rubrum and M. canis in vitro. The T. rubrum and M. canis biofim morphology at the incubation period of 24,48,72,96 h was observed by inverted microscope. The growing states of T. rubrum and M. canis biofilms were determined by using the XTT assay. Extracellular matrix（ECM） in the mature biofilms of T. rubrum and M. canis were calculated by safranin staining（expressed by OD492）. The characteristic of T. rubrum mature biofilm was analyzed by the scanning electron microscope（SEM）. Measuring and comparing the minimal inhibitory concentration（MIC） as well as the SMIC50,SMIC80（50%and 80% inhibitory concentration for biofilm） of T. rubrum and M. canis to TERB. Results Yeast cells and its extended product-hypha started to interweave intensively at the time of 48 h. Moreover,two isolates formed lots of ECM which gathered with the hypha at 72 h,and multilayer membranous structure was observed at this time. The mature time of T. rubrum and M. canis biofilms was 96 h and 72 h,respectively,and the OD492 of ECM for two isolates were 1. 925 ± 0. 111 and1. 173 ± 0. 157,respectively. The hypha of T. rubrum mature biofilm connected with each other,forming the intensive biofilm net. ECM appeared clearly at the periphery of hypha. The MIC of TERB against the planktonic cells of T. rubrum and M. canis were 0. 125 and 1 μg/m L,respectively,whereas the SMIC50 and SMIC80 of TERB against two strains biofilm were 256 μg/m L. Conclusion The biofilm models of T. rubrum and M. canis in vitro are constructed successfully. The mature biofilms of T. rubrum and M. canis have low sensitivity to TERB.