The ability of Listeria monocytogenes to form biofilm on surfaces relevant to the mushroom production environment.

Due to its ubiquitous nature, Listeria monocytogenes is a threat to all fresh fruits and vegetables, including mushrooms, which are Ireland's largest horticultural crop. Although fresh cultivated mushrooms (Agaricus bisporus) have not been previously linked with listeriosis outbreaks, the pathogen still poses a threat to the industry, particularly due to its ability to form biofilms. This threat is highlighted by the multiple recalls of mushroom products caused by L. monocytogenes contamination and by previous studies demonstrating that L. monocytogenes is present in the mushroom production environment. In this study, the biofilm formation potential of L. monocytogenes strains isolated from the mushroom production environment was investigated on materials and at temperatures relevant to mushroom production. A preliminary assessment of biofilm formation of 73 mushroom industry isolates was undertaken using a crystal violet assay on polystyrene microtitre plates. The biofilm formation of a subset (n = 7) of these strains was then assessed on twelve different materials, including materials that are representative of the materials commonly found in the mushroom production environments, using the CDC biofilm reactor. Vertical scanning interferometry was used to determine the surface roughness of the chosen materials. All the strains tested using the CDC biofilm reactor were able to form biofilms on the different surfaces tested but material type was found to be a key determining factor on the levels of biofilm formed. Stainless steel, aluminium, rubber, polypropylene and polycarbonate were all able to support biofilm levels in the range of 4-4.9 log10 CFU/cm2, for seven different L. monocytogenes strains. Mushroom industry-specific materials, including growing nets and tarpaulins, were found to support biofilms levels between 4.7 and 6.7 log10 CFU/cm2. Concrete was found to be of concern as it supported 7.7 log10 CFU/cm2 of biofilm for the same strains; however, sealing the concrete resulted in an approximately 2-log reduction in biofilm levels. The surface roughness of the materials varied greatly between the materials (0.7-3.5 log10 Ra) and was found to have a positive correlation with biofilm formation (rs = 0.573) although marginally significant (P = 0.051). The results of this study indicate that L. monocytogenes can readily form biofilms on mushroom industry relevant surfaces, and additionally identifies surfaces of specific concern, where rigorous cleaning and disinfection is required.

Lactococcus lactis subsp. lactis as a natural anti-listerial agent in the mushroom industry.

Mushroom growth substrates from different commercial producers of mushrooms (Agaricus bisporus) were screened for the presence of bacteria with potential for use as biocontrol agents for controlling Listeria monocytogenes in the mushroom production environment. Eight anti-listerial strains were isolated from different sources and all were identified using 16s rRNA gene sequencing as Lactococcus lactis subsp. lactis. Whole-genome sequencing of the Lc. lactis isolates indicated that strains from different sites and substrate types were highly similar. Colony MALDI-TOF mass spectrometry found that these strains were Nisin Z producers but inhibitory activity was highly influenced by the incubation conditions and was strain dependant. The biofilm forming ability of these strains was tested using a crystal violet assay and all were found to be strong biofilm formers. Growth of Lc. lactis subsp. lactis using mixed-biofilm conditions with L. monocytogenes on stainless steel resulted in a 4-log reduction of L. monocytogenes cell numbers. Additional sampling of mushroom producers showed that these anti-listerial Lc. lactis strains are commonly present in the mushroom production environment. Lc. lactis has a generally regarded as safe (GRAS) status and therefore has potential for use as an environmentally benign solution to control L. monocytogenes in order to prevent product contamination and to enhance consumer confidence in the mushroom industry.