Inhibitory effect of lactocin AL705 on listeria monocytogenes biofilm under continuous flow nutrients conditions

Abstract

Listeria monocytogenes, which causes serious foodborne infections and public health problems worldwide, is one of the most important foodborne pathogens. Some strains of L. monocytogenes are able to form biofilm facilitating their persistence in the food-processing environments as a chronic source of contamination. Since abundant evidence indicates that the biofilm mode of life leads to increased resistance to antimicrobials/sanitizers, new and effective strategies to control pathogen biofilms as eco-friendly approaches involving lactic acid bacteria (LAB) and/or their bacteriocins have emerged. Therefore, the objective of this work was to evaluate the inhibitory effect of lactocin AL705 produced by Latilactobacillus curvatus CRL1579 on L. monocytogenes FBUNT biofilms under continuous nutrient flow using microscopic techniques. Continuous-flow biofilms were grown in a flow cell (76 mm x 18 mm, with 3 channels of 40 mm x 4 mm) at 10°C. Overnight-grown cultures (18 h at 30°C) were diluted in TSB (1%), and the flow chambers were inoculated. After 2-h bacterial adhesion, lactocin AL705 at a subinhibitory concentration (20 AU/ml) was added and TSB medium was pumped through the flow cell with a flow of 3 ml/h. The biofilms were washed to remove planktonic bacteria, specifically stained live/dead by flushing with a 1:1000 dilution of BacLight staining (SYTO9 /propidium iodide) and examined by fluorescence and Confocal Laser Scanning Microscopy (CLSM) at 3 and 6 days of incubation. Fluorescence micrographs of the untreated biofilms on glass surface displayed greater complex multilayered cells and strong adhering ability at 6 days of incubation than at 3. Developing biofilm-treated lactocin AL705 exhibited a structure composed of sparsed cells and a greater reduction of live cells at 3 days of incubation. By employing ImageJ software, the thresholding analysis revealed that there was a 43% reduction in cell adhesion at 3 days while 23% at 6 days in the presence of lactocin AL705. The CLSM images analyzed using the programme comstat2 (allows quantification of three-dimensional biofilm structure) showed the clumping and complex morphology of L. monocytogenes FBUNT biofilm in untreated control surfaces. Lactocin AL705 produced a visible reduction in the biofilm formation, specifically in the parameters biomass, average and maximum thickness of the biofilms. Furthermore, the bacteriocin caused the dispersing and disintegrating clumps along with collapsed microcolonies. In conclusion, anti-listeria bacteriocin from L. curvatus CRL1579 may be considered as novel anti-biofilm strategy for the control of persistent L. monocytogenes biofilms in the food industry. Unlike bactericidal strategies, the implementation of this approach would not impose any selective pressure for pathogen resistance development.