Antibiofilm properties of selected Lactobacillus species and their mechanism of action against Listeria monocytogenes

Abstract

The disastrous effects of the foodborne pathogen Listeria monocytogenes have been observed overtime by global listeriosis outbreaks claiming many lives. The spread of this pathogen is being reported at high levels with persistence spanning over several years in food-processing environments. The main source of contamination occurs in these very environments where L. monocytogenes present on surfaces comes into contact with food products and later infect consumers. The situation is exarcebated by the fact that L. monocytogenes forms biofilms, one very powerful virulence mechanism, that adhere to processing surfaces. The shortfall of commonly used antibiotics and sanitizers as treatment against L. monocytogenes biofilms due to antimicrobial resistance leads to a major safety crisis within the food industry. This limitation of antimicrobial agents sparked an interest in research of lactic acid bacteria (LAB) with certain probiotic properties as an alternative antibiofilm agent. Research has highlighted that the use of substances produced by LAB achieves inhibition and dispersal of L. monocytogenes biofilms. The antimicrobial substances of LAB have been revealed to showcase antilisterial activity, contributing to the combat of biofilm formation on surfaces. It is however known that the properties of probiotics cannot be generalized. Taking these into consideration the current study aimed to determine whether selected Lactobacilli had the potential as an alternative control measure for biofilms formed by L. monocytogenes isolated from the food or food environments. The initial step of the current study was to screen L. monocytogenes strains for biofilm formation abilities. Then subsequently, the capabilities of cell free supernatant (CFS) of selected LAB strains (Lactobacillus acidophilus La14 150B, Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus ATCC 4356) to inhibit as well as disperse the formation of biofilms of L. monocytogenes strains was investigated. L. monocytogenes ATCC 19115 and L. monocytogenes Cuc (originally isolated from cucumber) were classified as moderate biofilm formers, while L. monocytogenes 243 and L. monocytogenes Avo (isolated from avocado) were classified as strong biofilm formers. The strains isolated from the food processing environments were better biofilm formers that than the positive control L. monocytogenes ATCC 19115. After determining these categorizations, the L. monocytogenes strains were treated with CFS of LAB. All CFS managed to inhibit the formation of biofilms across all strains, significantly decreasing their biofilm former categories from either moderate or strong to weak (p < 0.05). L. acidophilus La14 150B performed better overall in inhibiting the biofilms. Cocktails of the CFS of LAB were prepared and additionally used as treatment. The cocktail ABC (L. acidophilus La14 150B + L. plantarum + L. rhamnosus ATCC 4356) had more efficient inhibition capabilities with both L. monocytogenes Avo and L. monocytogenes Cuc having the lowest recorded optical density values post treatment. The following step in this experiment explored the dispersal capabilities of the CFS of LAB. Though the preformed biofilms were not completely dispersed following treatment, the CFS were able to change the classification of all the L. monocytogenes strains into weaker biofilm former categories. L. acidophilus La14 150B was the most efficient of all LAB in removal of biofilms with significantly reduced optical density values (p < 0.05). Then, different cocktails of the CFS were prepared and assessed for their biofilm removal capabilities. It was deduced that all three CFS of LAB were able to significantly disperse the biofilms (p < 0.05). Collectively, the cocktail ABC (containing CFS of the three LAB test strains) had the greatest efficiency in dispersal abilities. From these results it was evident that cocktails were superior to individual CFS in the inhibition and dispersal of L. monocytogenes biofilms. The observed inhibition and dispersal abilities of CFS of LAB (L. acidophilus La14 150B, L. plantarum and L. rhamnosus ATCC 4356) raised an interest in seeking to investigate their potency in removing L. monocytogenes biofilms adhered to different surfaces similar to those in contact with food in food processing environments. Scanning electron microscopy (SEM) revealed that both L. monocytogenes ATCC 19115 and L. monocytogenes 243 have a different attachment style on the hydrophilic stainless steel in contrast to the hydrophobic polyvinyl chloride (PVC). A two-layered biofilm structure with a honey comb complex was observed for both these L. monocytogenes strains on stainless steel which was noticeably absent on PVC. Moreover, for the removal of L. monocytogenes biofilms on stainless steel coupons by CFS of LAB, all treatments managed to disperse the aggregated structures resulting in isolated cells. The antibiofilm properties of the different LAB was evidenced by a decline in the cell-clusters of L. monocytogenes ATCC 19115 and L. monocytogenes 243. On PVC, the three CFS exhibited the ability to interfere with and disrupt the aggregation of L. monocytogenes cells to each other as scattered cells were observed post treatment. Overall, L. acidophilus La14 150B dispersed the L. monocytogenes ATCC 19115 and L. monocytogenes 243 biofilms on both the stainless steel and PVC surfaces with the highest efficiency. In order to determine the mechanism by which CFS of LAB interfered with the L. monocytogenes biofilm formation capabilities, the study further investigated how expression of the L. monocytogenes prfA gene is affected by the presence of CFS of LAB. The results demonstrated that the presence of all CFS of LAB (L. acidophilus La14 150B, L. plantarum and L. rhamnosus ATCC 4356) caused a significant downregulation in the expression of prfA (p < 0.05). This downregulation affects the coding of the PrfA regulator protein and disrupts L. monocytogenes biofilm formation. In correlation with the results observed for SEM analysis, the CFS of L. acidophilus La14 150B exhibited the highest antagonistic behavior, with expression of prfA recorded at a low 23% after treatment. The results of the present study suggest that the CFS of LAB contain specific substances with antilisterial activity and antibiofilm properties. The presence of these substances contained within the CFS of LAB negatively affects the virulence gene prfA, decreasing the ability of L. monocytogenes to form biofilms. The study is of importance to the various food processing facilities and the food industry as it provides a potential safe alternative that can be used to limit any further outbreaks due to contamination of food products by L. monocytogenes. Cell free supernatants with demonstrated antibiofilm properties could be incorporated in industries and stipulated as the standard control measure for L. monocytogenes biofilms. Furthermore, for an enhanced effective treatment the cocktails of such CFS could be very beneficial. Thus cell free supernatants of lactic acid bacteria can be used to curb the formation of L. monocytogenes biofilms on food processing surfaces in the food industry and thereby contribute to improved food safety.