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

peer-reviewedMushroom 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

Examining the efficacy of mushroom industry biocides on Listeria monocytogenes biofilm

Aims: The aim of this study was to test the efficacy of new and currently used biocides in the mushroom industry for inactivating L. monocytogenes biofilm. Methods and results: A lab‐scale study was initially carried out to test the efficacy of eleven biocidal products against a cocktail of five L. monocytogenes strains that were grown to three‐day biofilms on stainless steel coupons. Biocidal efficacy was then tested under clean and dirty conditions based on the EN 13697:2015 method. The results for the biocides tested ranged between 1.7‐log to 6‐log reduction of biofilm, with only the efficacy of the sodium hypochlorite‐based biocide being significantly reduced in dirty conditions. A pilot‐scale trial was then carried out on a subset of biocides against L. monocytogenes on concrete floors in a mushroom growing room and it was found that biocide efficacy in lab‐scale did not translate well in pilot‐scale. Conclusions: Biocides that are used in the mushroom industry and potential alternative biocides were determined to be effective against L. monocytogenes biofilm in both lab‐scale and pilot‐scale experiments. Significance and impact of the study: This study has direct impact for the industry as it provides information on the efficacy of currently used biocides and other biocidal products against L. monocytogenes, an added benefit to their primary use

Listeria monocytogenes in the mushroom production environment: biofilm formation and control measures

Mushrooms are Ireland’s most valuable horticultural crop and as such, proactive steps must be taken to prevent association with foodborne pathogens which can cause reputational and economic loss to the industry. Listeria monocytogenes is one of the major foodborne pathogens and while it has a relatively low incidence rate, it is one of the deadliest, especially for the immunocompromised individuals. This pathogen has been found to be present within the mushroom production environment which is of concern to the industry. The persistence of L. monocytogenes in different environments has been attributed to its ability to form biofilms. However, prior to this work, the biofilm forming ability of L. monocytogenes on mushroom industry relevant surfaces was undetermined. Furthermore, the mushroom industry generally employs the use of biocides that are specifically marketed for disease control within the mushroom production environment, but their efficacy against L. monocytogenes biofilms was unknown. In this work, it was established that mushroom industry isolates of L. monocytogenes can form biofilms on most of the surfaces that can be encountered within the mushroom production environment, especially on the concrete floors. In addition, the biocidal products that are approved for use within the mushroom production environment were found to be effective against L. monocytogenes biofilm. It was also found that biocidal products with more environmentally friendly active ingredients such as peracetic acid or plant-derived compounds could be effective alternative biocides for the mushroom industry. Interestingly, a reduction in efficacy was observed from the biocidal products when tested on concrete floors within the mushroom production environment. Lab-scale experiments revealed that nisin-producing Lactococcus lactis strains derived from the mushroom production environment had the potential to be utilised as a biocontrol agent in the form of a competitive exclusion organism against L. monocytogenes. However, in a similar pattern to the biocidal products, this efficacy did not translate well in pilot-scale trials as it was found to be ineffective when applied during a standard mushroom cropping process. Despite the ineffectiveness of the biocontrol agents tested in pilot-scale conditions, the L. monocytogenes numbers decreased naturally over the duration of the cropping cycle. Due to the rich microbial diversity present, this decrease in L. monocytogenes numbers may have been caused by other species producing anti-listerial agents. In order to better understand the microbial community dynamics during cropping and to identify other potential biocontrol agents, a shotgun metagenomic sequencing study was carried out which revealed other potential bacteriocin producing bacteria and clearly illustrated the changes in species dominance which occur over time. All in all, this body of work has provided beneficial information for the mushroom industry in relation to L. monocytogenes, as it identified surfaces of specific concern and determined the efficacy of biocides against the biofilm form of L. monocytogenes. Moreover, alternative biocontrol options were tested and while shown to be ineffective in this case, their use is promising as the application method tested did not have a negative effect on the crop yield. The sequencing dataset provides valuable information on the indigenous microflora present during production and how they may influence product quality and safety