Department of Immunology and Food Microbiology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Str., 10-748 Olsztyn, Poland; Department of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Plac Cieszyński 1, 10-726 Olsztyn, Poland.
Department of Immunology and Food Microbiology, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Tuwima 10 Str., 10-748 Olsztyn, Poland.
Int J Food Microbiol. 2021 Dec 16;360:109441. doi: 10.1016/j.ijfoodmicro.2021.109441. Epub 2021 Oct 17.
Biofilms pose a serious challenge to the food industry. Higher resistance of biofilms to any external stimuli is a major hindrance for their eradication. In this study, we compared the growth dynamics and benzalkonium chloride (BAC) resistance of dual species Listeria monocytogenes-Escherichia coli 48 h biofilms formed on stainless steel (SS) coupons surfaces under batch and fed-batch cultures. Differences between both operational culture conditions were evaluated in terms of total viable adhered cells (TVAC) in the coupons during 48 h of the mixed-culture and of reduction of viable adhered cells (RVAC) obtained after BAC-treatment of a 48 h biofilm of L. monocytogenes-E. coli formed under both culture conditions. Additionally, epifluorescence microscopy (EFM) and confocal scanning microscopy (CLSM) permitted to visualize the 2D and 3D biofilms structure, respectively. Observed results showed an increase in the TVAC of both strains during biofilm development, being the number of E. coli adhered cells higher than L. monocytogenes in both experimental systems (p < 0.05). Additionally, the number of both strains were higher approximately 2.0 log CFU/coupon in batch conditions compared to fed-batch system (p < 0.05). On the contrary, significantly higher resistance to BAC was observed in biofilms formed under fed-batch conditions. Furthermore, in batch system both strains had a similar reduction level of approximately 2.0 log CFU/coupon, while significantly higher resistance of E. coli compared to L. monocytogenes (reduction level of 0.69 and 1.72 log CFU/coupon, respectively) (p < 0.05) was observed in fed-batch system. Microscopic image visualization corroborated these results and showed higher complexity of 2D and 3D structures in dual species biofilms formed in batch cultures. Overall, we can conclude that the complexity of the biofilm structure does not always imply higher resistance to external stimuli, and highlights the need to mimic industrial operational conditions in the experimental systems in order to better assess the risk associated to the presence of pathogenic bacterial biofilms.
生物膜对食品工业构成严重挑战。生物膜对外界刺激的更高抵抗力是其消除的主要障碍。在这项研究中，我们比较了在不锈钢（SS）片表面上形成的李斯特菌-大肠杆菌双物种 48 小时生物膜在分批和补料分批培养下的生长动态和苯扎氯铵（BAC）抗性。在混合培养的 48 小时内，通过比较在补料分批培养下形成的李斯特菌-大肠杆菌 48 小时生物膜的 BAC 处理后存活附着细胞（RVAC）的减少，评估了两种操作培养条件之间的差异。此外，荧光显微镜（EFM）和共聚焦扫描显微镜（CLSM）分别允许可视化 2D 和 3D 生物膜结构。观察到的结果表明，两种菌株的 TVAC 在生物膜发育过程中均增加，附着细胞的数量在两种实验系统中大肠杆菌均高于李斯特菌（p<0.05）。此外，与补料分批系统相比，在分批条件下两种菌株的数量大约高出 2.0 log CFU/片（p<0.05）。相反，在补料分批条件下形成的生物膜对 BAC 的抗性明显更高。此外，在分批系统中，两种菌株的减少水平相似，约为 2.0 log CFU/片，而在补料分批系统中大肠杆菌的抗性明显高于李斯特菌（分别减少 0.69 和 1.72 log CFU/片）（p<0.05）。显微镜图像可视化证实了这些结果，并显示出在分批培养中形成的双物种生物膜的 2D 和 3D 结构的更高复杂性。总体而言，我们可以得出结论，生物膜结构的复杂性并不总是意味着对外界刺激的更高抵抗力，并强调需要在实验系统中模拟工业操作条件，以更好地评估与致病性细菌生物膜存在相关的风险。
