Abstract: This study aimed to explore mixed-species biofilm formation (BF) between meat-derived Escherichia coli and Pseudomonas. The biofilm-forming capacity of isolates was evaluated by means of microplate crystal violet staining and the process of mixed-species biofilm formation between representative strains was investigated. The dynamic changes of biofilm amount, microstructure and the composition of extracellular polymeric substances (EPS) were examined. Absolute quantitative real-time PCR was used to analyze the difference in the expression of BF-related genes. The results showed that there were significant inter-strain differences in the biofilm-forming capacity of E. coli and Pseudomonas (P < 0.05), in which the strains with strong biofilm-forming capacity accounted for 25.81% and 23.08%, respectively. The results of plate counting showed that the BF processes of E. coli D4-18 + Pseudomonas Y2-2 10, and E. coli C-13 + Pseudomonas Y2-1 1 were different on stainless steel sheet, and the interaction between the two species changed with BF time. Similar changes in the microstructure were observed by confocal laser scanning microscope (CLSM). The secretion of protein and polysaccharide in EPS was inhibited during mixed-species BF; the amount of protein decreased by 31% and 48%, respectively, and the amount of polysaccharide decreased by 38% and 56%, respectively, at 72 and 168 h of culture compared with the total EPS of the two single-species biofilms. The results of gene expression per unit cell number showed that the expression of BF-related genes was significantly up-regulated in the biofilm compared with the planktonic cells (P < 0.05). Compared with the single-species biofilm, the papC and csgA genes of E. coli in the mixed biofilm were significantly up-regulated at 72 and 168 h (P < 0.01), while the phoR and cbrA genes of Pseudomonas were significantly down-regulated at 72 h (P < 0.01). The absolute amount of genes expression per unit area in mixed-species BF changed compared with that in single-species BF. This study can provide a scientific basis for understanding the mixed-species BF of meat-derived spoilage microorganisms and for the risk assessment and control of microbial contamination in meat production and processing.

Key words: meat-derived Escherichia coli; meat-derived Pseudomonas; mixed-species biofilm; interaction; gene expression