Behavior of Foodborne Pathogens Listeria monocytogenes and Staphylococcus aureus in Mixed-Species Biofilms Exposed to Biocides

Abstract

In nature and man-made environments, microorganisms reside in mixedspecies biofilms, in which the growth and metabolism of an organism are different from these behaviors in 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 one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus. The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed fivespecies biofilms on stainless steel, the two pathogens remained at a constant level of ϳ10 5 CFU/cm 2 . The mixed five-species biofilms as well as the pathogens in monospecies 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 by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the fivespecies biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated 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 to identify 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 are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

Characteristics of Listeria Monocytogenes Strains Persisting in a Meat Processing Facility over a 4-Year Period

“…Different strains of L. monocytogenes also exhibited high MBCs for PAA in the range of up to 0.5% [28,29,30,31]. In contrast, others concluded that PAA was able to significantly reduce L. monocytogenes from multispecies biofilms at 0.15% [32] and 0.3% [30], respectively.…”

“…Different strains of L. monocytogenes also exhibited high MBCs for PAA in the range of up to 0.5% [28,29,30,31]. In contrast, others concluded that PAA was able to significantly reduce L. monocytogenes from multispecies biofilms at 0.15% [32] and 0.3% [30], respectively. The MIC of PAA for other organisms seemed to be much lower, in the range of 0.01–0.03% for Gram-positive and Gram-negative flora isolated from citrus fruit [33], 0.0003% for Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa [34], and 0.0001–0.001% for P. aeruginosa , S. aureus , E. coli , and S. epidermidis [35].…”

The Role of Bacterial Biofilm in Antibiotic Resistance and Food Contamination

“…Biofilm is an association of microorganisms that are firmly attached to the biotic or abiotic surface, encased within an extracellular polymeric substance (EPS) matrix, and that can show new character with respect to gene expression, protein synthesis, growth rate, and metabolic activities [44,45]. Biofilm production can be influenced by a number of factors such as surface conditions, chemical and physical growth factors, cellular structures, and any other challenges.…”

Listeria monocytogenes Virulence, Antimicrobial Resistance and Environmental Persistence: A Review

“…Biofilms are reportedly behind the pathogen's increased tolerance to quaternary ammonium compounds due to changes in membrane fluidity [75]. Biofilms can also protect L. monocytogenes from the action of biocides, but the underlying mechanism of biocide protection is yet to be clarified [76].…”