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.