Temperature and contact surfaces are critical factors influencing biofilm formation of microbial communities derived from both lab and natural environments. However, there is limited understanding regarding how different temperatures impact transfer dynamics of individual microbial species between meat surfaces in dual-species biofilms. In this study, we evaluated the biofilm-forming capabilities of various meat-derived Escherichia coli and Pseudomonas strains on pork surfaces at 15 and 25 ℃. The results revealed that lower temperature had a significant negative effect on the growth fitness of E. coli compared to Pseudomonas. Two robust biofilm-forming strains, E. coli C-13 and P. fluorescens S_1-2 3, were selected under both temperature conditions to further explore their interactions in dual-species biofilms on meat surfaces. The results showed that E. coli exhibited a competitive growth advantage at 25 ℃, while Pseudomonas displayed enhanced growth at 15 ℃, indicating temperature-dependent competition patterns. Additionally, three established mathematical models were utilized to simulate the transfer dynamics of the two strains within mono- and dual-species biofilms. As anticipated, the numbers of transferred cells progressively declined with increased imprinting reactions between the meat surfaces. Interestingly, the transfer rates of both strains markedly improved in dual-species vs. mono-species biofilms at 15 ℃, highlighting the influential role of inter-species interactions on transfer dynamics of microbes that may cross-contaminate meats. Our findings advance current understanding of mono- and dual-species biofilm development on meat surfaces under different temperature conditions. They also offer scientific evidence to support strategies for controlling microbial cross-contamination during meat processing to ensure product safety.

This paper aims to investigate the biofilm formation and related genes expression of Escherichia coli isolated from pork. E. coli was isolated from pig slaughtering line, carcass surfaces and pork meat and identified using selective agar plates and specific polymerase chain reaction (PCR). After culture at 15 ℃ for 72 h, the biofilm forming abilities of the isolates were evaluated by means of microplate crystal violet staining. Furthermore, representative strains were selected to explore the changes of biofilm biomass, the composition of extracellular polymeric substances (EPS), and biofilm related-genes expression by means of reverse transcription real-time PCR in the process of biofilm formation. The results showed that a total of 31 strains of E. coli were isolated and identified in this study, among which 11 were obtained from pig slaughtering line, 4 from the surface of pig carcasses, and 16 from raw pork meat. The results of crystal violet staining showed that there were significant differences in the biofilm-forming ability among the 31 isolates, and 64.52% of them had weak biofilm-forming ability, one of which was then selected to study the biofilm formation process. The biofilm biomass of strain D_4-18 on the microplate increased continuously during 168 h incubation at 15 ℃. After incubation for 72 and 168 h, EPS were secreted by strain D_4-18, and the gene expression levels of papC、fimH、csgA genes were 0.095、0.933、0.435 copies/cm², respectively. With the increase of culture time, the contents of protein and polysaccharide in loose EPS (L-EPS) and the protein content in bound EPS (B-EPS) increased significantly (P < 0.01). After incubation for 168 h, the expression of the papC and fimH genes increased, while csgA gene expression did not change significantly, indicating that these genes play different regulatory roles in the biofilm formation of E. coli.

The interaction between Escherichia coli O157:H7 and the common food spoilage bacterium Pseudomonas spp. on their motility and biofilm-producing ability was investigated when cultured together. Furthermore, reverse transcription real-time quantitative polymerase chain reaction was used to analyze the changes of virulence genes (stx1, stx2, hly and eae) expression in representative strains of E. coli O157:H7 during mixed species biofilm formation. The results showed that the motility of the three tested strains of E. coli O157:H7 was lower than that of the four strains of Pseudomonas when they were cultured separately (P < 0.05). When co-cultured with E. coli O157:H7, the swimming motility of Pseudomonas was inhibited considerably. The selective culture results showed that the two species did not promote each other’s biofilm-producing ability when co-culturing for 72 h. Instead, biofilm formation by the four Pseudomonas strains was significantly inhibited by the three strains of E. coli O157:H7 (P < 0.05). Furthermore, it was found that the expression of four virulence genes of E. coli O157:H7 CICC21530 per unit volume in the mixed culture and per until area in the mixed biofilm were lower than that in the single culture and the single biofilm, respectively (P < 0.05), and the same was true for that per unit cell number, indicating that the expression of virulence genes in E. coli O157:H7 was inhibited by Pseudomonas during mixed culture and mixed biofilm formation. It was also found that the expression of the four virulence genes in the biofilm was higher than that in planktonic cells (P < 0.05), indicating that the virulence of the bacterial cells increased after biofilm formation. This study can provide a scientific basis for revealing the interaction between food-borne pathogens and spoilage bacteria during mixed biofilm formation and for risk assessment, prevention and control.

In this study, the survival of the main serotypes O157:H7 and O26:H11 of Shiga toxin-producing Escherichia coli (STEC) inoculated in fermented sausage during processing was investigated, and the changes of virulence gene expression in the bacteria and the product at different production stages were analyzed by reverse transcription real-time polymerase chain reaction (RT-real-time PCR). The results showed that there was no significant difference in the count of lactic acid bacteria, lactic acid content, pH, or water activity (a_w) value between STEC inoculated and uninoculated groups at most production stages. During sausage production, the growth of E. coli O157:H7 and O26:H11 was inhibited significantly (P < 0.05) and decreased by 1.51 and 1.39 (lg (CFU/g)), respectively, but the two strains showed different inhibition processes. After eliminating the difference in bacterial counts, it was found that the virulence gene expression in the bacterial cells was significantly up-regulated (P < 0.05) at the late stage of production. The expression of most virulence genes per unit mass of sausage increased significantly (P < 0.05) at the early stage of fermentation, and all virulence genes were highly expressed in the final product. Therefore, although STEC could be inhibited effectively during fermented sausage production, virulence gene expression was enhanced in the bacterial cells and was at a high level in the final product.

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 D_4-18 + Pseudomonas Y_2-2 10, and E. coli C_-13 + Pseudomonas Y_2-1 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.