Biofilms on equipment surfaces can be vectors for cross-contaminations in food processing facilities. A particular problem is that biofilms can protect pathogenic bacteria from daily cleaning and disinfection operations. In the present study, microflora were recovered from fresh-cut processing facilities, identified, and tested for biofilm forming potential. Subsequently, dual-species biofilms of selected isolates and Escherichia coli O157:H7 were investigated. Approximately 30% of the isolates were potent biofilm formers, producing large amounts of biomass. A hundred and seventeen tested isolates were identified into 23 genera, including plant related bacteria and coliforms with some opportunistic pathogens. Dual-species biofilms formed by Burkholderia caryophylli or

Ralstonia insidiosa and E. coli O157:H7 manifested increased biomass in comparison to their monocultures. Additionally, about a one-log unit increase of E. coli O157:H7 cell counts were observed for both dual-species biofilms. To test the effects of environmental factors on growth of R. insidiosa and E. coli O157:H7 in dual-species biofilms, factors tested included low temperature (10 oC), media with different composition of nutrient sources (10% TSB, M9, 1.25% Cantaloupe Juice) and a continuous culture system with limited nutrients. E. coli O157:H7 cell counts increased for all tested conditions. To examine bacterial localization within biofilms, confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) were used. Images showed distinct spatial distributions with E. coli O157:H7 commonly located at the bottom and also interspersed among R. insidiosa cells. To test the interactions with other pathogens, R. insidiosa was co-cultured with Listeria monocytogenes, Salmonella spp., and Shiga toxin-producing E. coli. Cells counts for 7 out of 9 tested pathogenic bacteria strains were increased (0.36-1.60 CFU log units). It is notable that the biomass formed by R. insidiosa and L. monocytogenes was much greater than those produced by other tested combinations. These results indicate that R. insidiosa could be a food safety risk in fresh-cut processing environments by providing protective habitats for pathogenic bacteria.