Romanova Nadya A, Gawande Purushottam V, Brovko Lubov Y, Griffiths Mansel W
Department of Food Science and Canadian Research Institute for Food Safety, University of Guelph, 43 McGilvray St., Guelph, Ontario, Canada N1G 2W1.
J Microbiol Methods. 2007 Dec;71(3):231-7. doi: 10.1016/j.mimet.2007.09.002. Epub 2007 Sep 14.
Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P=0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.
采用不同方法研究生物膜生长情况，包括结晶紫染色、ATP生物发光法和总活菌计数法。筛选了7株单核细胞增生李斯特菌及其8株衍生菌株形成生物膜的能力。适应苯扎氯铵（BC）和质粒消除均未显著影响生物膜形成能力。与血清型4相比，血清型1的单核细胞增生李斯特菌菌株形成生物膜的能力明显更强（P=0.0003）。为评估BC消除生物膜的效果，使用了生物膜形成能力最强和最弱的菌株（C719和LJH 381）。观察到，生物膜中的单核细胞增生李斯特菌菌株C719对BC的抗性至少比浮游形式高1000倍。生物膜中的细胞在BC处理后能够恢复并生长，从而成为再污染的来源。结果表明，ATP生物发光法与生物膜中单核细胞增生李斯特菌的细菌计数具有良好的相关性。相反，在高浓度BC存在的情况下，结晶紫染色与生物膜中的细菌生长不相关，但能提供附着在表面的活细菌和死细菌细胞浓度的信息。发现ATP生物发光法是快速评估消毒剂对生物膜消毒效果的可靠方法。另一方面，结晶紫染色被证明是监测生物膜去除情况的合适方法。我们的研究表明，对于李斯特菌生物膜，应使用浓度高于10 mg/ml的BC处理至少30分钟，以杀死生物膜中几乎所有的活细胞。然而，该浓度仍不足以从塑料表面去除生物膜。
