In nature and man-made environments, microorganisms reside in mixed-species biofilm where behavior is modified compared to the single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms leading to health risk for humans. Here, we developed two mixed-five-species biofilms that included the foodborne pathogens Listeria monocytogenes or Staphylococcus aureus, respectively. The five species, including the pathogen, were isolated from a single food-processing environmental sample thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105 CFU/cm2 The mixed-five-species biofilms as well as the pathogens in mono-species biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased 4.6 log cycles in mono-species biofilm, but the pathogen was protected in the five-species biofilm and decreased only 1.1 log cycles. Sessile cells of L. monocytogenes were affected equally as a mono-biofilm or as a member in the mixed-species biofilm; decreasing by three log cycles when exposed to 0.0375 % peracetic acid. When the pathogen was exchanged in each associate microbial community, S. aureus was eradicated while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection, and the species playing the protective role in microbial communities of biofilms. Importance: This study demonstrates that foodborne pathogens can be established in mixed species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed species biofilm. Biocide treatment and resistance is a challenge for many industries and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.
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