QCM-D-Based Metadata on Adhesion and Dispersal Patterns of Three Bacterial Species Recovered from Dairy Pasteurizer Surfaces Following Cleaning and Disinfection

In a research project "KILLFILM," conducted at the host institute (Flanders Research Institute for Agriculture Fisheries and Food: ILVO) in Belgium and funded by Flanders' Food, a significant number and diversity of bacterial species were recovered from food contact surfaces in the milk processing industries following cleaning and disinfection (C&D) procedures. This study involved identified dominant bacteria on the surface of dairy pasteurizers following C&D and included Stenotrophomonas rhizophila (B68), Bacillus licheniformis (B65), and Microbacterium lacticum (B30). Bacterial biofilm forming ability in monoculture and mixed culture biofilms was confirmed on polystyrene and stainless steel surfaces. We also used Quartz crystal microbaance with dissipiation (QCM-D) to study adherence and dispersal signals for the three above mentioned bacteria using the QCM-D E4 instrument (Q-sense Gothenburg, Sweden). 5 MHz AT-cut quartz crystals with silica (SiO2) and stainless steel coatings were purchased from Biolin Scientific (Gothenburg, Sweden). The sensors were cleaned according to the method described earlier Derick et al., 2023. In each experiment, we initially stabilized the frequency shift and energy dissipation signals for at least 20 minutes in a cell-free medium, ensuring a stable baseline. After this, we introduced a suspension of cells to the sensor, maintaining a flow rate of 100 µl/min for 60 minutes. Following the cell addition, the cells were allowed to interact with the sensor surface in a static state for 18-48 h. During this period, we continuously monitored the frequency shift and energy dissipation at a controlled room temperature of approximately 19 ± 0.5 °C. For comparison, we conducted similar measurements using cell-free buffer solutions. Unless specified otherwise, our data analysis primarily focused on the 7th overtone. This particular overtone is more sensitive to the presence of the cell body compared to lower overtones, which might detect bacteria-free medium or appendages. Higher overtones, in contrast, are more sensitive to changes at the cell-body/chip interface, including any trapped medium and cell appendages. The protocol was adapted from Derick et al., 2023. In the metadata each strain has been represented by its strains name: B68, B30 and B65. S1, S3 and S4 represent three different compartments in which the samples were run at the same time. BHI broth was for all experiments. Reference: Derick et al. https://onlinelibrary.wiley.com/doi/abs/10.1002/pssa.202300310.本研究项目名为“KILLFILM”，由比利时佛兰德斯农业渔业与食品研究所（Flanders Research Institute for Agriculture Fisheries and Food，缩写ILVO）主导开展，佛兰德斯食品（Flanders' Food）提供资助。研究团队从乳制品加工厂经清洁消毒（Cleaning and Disinfection, C&D）流程后的食品接触表面，分离得到大量且物种多样性丰富的细菌菌株。本研究鉴定了巴氏杀菌机表面经C&D处理后留存的优势细菌，包括嗜麦芽窄食单胞菌（Stenotrophomonas rhizophila, B68）、地衣芽孢杆菌（Bacillus licheniformis, B65）与乳酸微杆菌（Microbacterium lacticum, B30）。团队验证了单菌种及混合菌种生物膜在聚苯乙烯与不锈钢表面的形成能力。 此外，本研究使用耗散型石英晶体微天平（Quartz Crystal Microbalance with Dissipation, QCM-D），搭配瑞典哥德堡Q-sense公司的QCM-D E4仪器，针对上述三种细菌的黏附与扩散信号开展研究。实验所用的5 MHz AT切割石英晶体，带有二氧化硅（SiO₂）与不锈钢涂层，购自瑞典哥德堡的Biolin Scientific公司。传感器的清洗流程参照Derick等人2023年发表的方法进行。 每次实验初始阶段，先在无细胞培养基中稳定频移与能量耗散信号至少20分钟，以获得稳定的基线信号。随后向传感器体系中注入细胞悬液，维持流速100 µl/min，持续60分钟。完成细胞添加后，使细胞在静态条件下与传感器表面相互作用18至48小时。在此期间，在约19±0.5℃的可控室温下，持续监测频移与能量耗散数据。为进行对照，团队同时使用无细胞缓冲液开展了相同的测量流程。除非另有说明，本研究的数据分析主要聚焦于第7次泛音：相较于低阶泛音（可能检测到无细菌的培养基或细胞附属结构），该泛音对细胞体的存在更为敏感；而高阶泛音则对细胞-芯片界面的变化（包括截留的培养基与细胞附属结构）更为敏感。本实验方案改编自Derick等人2023年的研究。 元数据中，各菌株以其菌株编号标注：B68、B30与B65。S1、S3与S4代表三个并行运行样品的独立舱室。所有实验均采用BHI肉汤（脑心浸液肉汤）作为培养基。 参考文献： Derick et al. https://onlinelibrary.wiley.com/doi/abs/10.1002/pssa.202300310.