Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other abiotic surfaces (polystyrene and stainless steel). Therefore, this RTCA technology opens new opportunities in the biofilm research arena and its application could be further explored for other bacterial genera as well as for different bio-active molecules.

栖息于多样生态系统中的细菌，常以聚集细胞群落的形式生长，该群落结构有利于细菌的存活与定殖。不同的胞外聚合物（extracellular polymeric substances, EPS）可包裹该多物种群落，形成可附着于生物或非生物表面的生物被膜（biofilm）。然而，这种广泛存在且演化成功的细菌生存方式，会对人类活动产生负面影响：诸多致病菌与腐败菌形成的生物被膜极难被清除。因此，开发新型抗生物被膜生物活性分子已成为当前的研究热点，而简单、可靠且快速的筛选方法正是该领域亟需的工具。本研究成功验证了一种基于阻抗的检测方法——该方法最初用于贴壁真核细胞的研究——可实时监测三种模式细菌的单物种生物被膜形成过程。xCelligence实时细胞分析仪（RTCA）搭载涂覆有金微电极的专用微孔E板，能够通过检测贴附细胞的附着情况改变阻抗信号。本研究中，该技术可成功区分金黄色葡萄球菌与表皮葡萄球菌的生物被膜产生菌株与非产生菌株；同时仅在培养基中添加蔗糖时，才能检测到变形链球菌的生物被膜形成。此外，无论生物被膜基质聚合物的组成类型如何，不同的阻抗值均可实现受试产生物被膜菌株的区分。最后，本研究持续监测了噬菌体（bacteriophage）phi-IPLA7与噬菌体编码的内溶素（endolysin）LysH5对葡萄球菌生物被膜形成的抑制效果，以及该抗菌处理对预形成生物被膜的清除作用。基于阻抗法得到的实验结果，与结晶紫染色、细菌计数等标准方法的检测结果，以及在其他非生物表面（聚苯乙烯与不锈钢）上得到的结果均呈现高度相关性。综上，该RTCA技术为生物被膜研究领域提供了全新的研究思路，其应用场景可进一步拓展至其他细菌属以及各类生物活性分子的相关研究中。