Formation And Resistance Of Biofilms Of Vibrio Parahaemolyticus And Listeria Monocytogenes Formed At Different Temperatures

Listeria monocytogenes and Vibrio parahaernolyticus are important foodborne pathogens.The bacteria in the form of biofilm have been proved to enter into viabe but non culturable(VBNC)state.The biofilm formation is closely related to bacterial resistance.At present,most of the researches on biofilms are concentrated on biofilms formed at room temperature,and the survival mechanisms of pathogenic bacteria in biofilm at low temperature are still undefined.In this study,the biofilm formations of L.monocytogenes and V.parahaernolyticus at 32°C and 10°C were investigated and the biofilms were treated with different concentrations of antibacterial agents.The crystal violet staining,XTT assays,PMA-q PCR,fluorescence microscopy and laser confocal microscopy were used to evaluate the removal effects.The formation of VBNC cells in mature biofilm of L.monocytogenes under different conditions was also investigated.The effects of low temperature storage on the resistance of V.parahaemolyticus and L.monocytogenes biofilms were explored.This study has great significance for controlling food spoilage,eliminating the residues of food-borne pathogens and ensuring food safety.The results showed as followed:The biofilm formations of L.monocytogenes and V.parahaemolyticus at different temperatures were investigated.The results showed that L.monocytogenes and V.parahaernolyticus could form stable biofilms at 32?and 10?,and the biomass of biofilm formed at 32?was significantly higher than that at 10°C.V.parahaernolyticus and L.monocytogenes could formed considerable biomass and metabolic activity at 32?,16 h and 10?,6 d,and the numbers of live V.parahaernolyticus were 5.56±0.62CFU/cm~2and 5.48±0.1 lg CFU/cm~2.The live cell numbers of L.monocytogenes were8.81±0.42 lg CFU/cm~2and 9.09±0.05 lg CFU/cm~2 at 32°C,12h and 10°C,19d respectively.PMA-q PCR method were established for quantitative detection of viable cells.The primers of PMA-q PCR were designed based on the tlh gene of V.parahaernolyticus and hly gene of L.monocytogenes.The results showed that the suitable sample treatment condition was that the cells were treated with PMA at the final concentration of 40?mol/L for5 min and light exposure for 5 min.The dection range for V.parahaernolyticus and L.monocytogenes were 2.6×10~3?2.6×10~9and 1.9×10~3?1.9×10~9CFU/m L,respectively.This method can significantly distinguish viable from dead cells and quantitatively detect viable cells of V.parahaernolyticus and L.monocytogenes.Studying on the effects of six antibacterial agents at four concentrations(0.5?4 MIC)against L.monocytogenes biofilms formed at different temperatures.The results showed that lavender essential oil and 84 disinfectant presented significant removal effects on biofilm.After three hours of treatment with four concentrations of lavender essential oil,the removal rates of biofilms formed at 32°C and 10°C were 62-82%and 25-56%,respectively.The elimination rates of four concentrations of the 84 disinfectant against warm biofilms and cold biofilms were 47%-78%and 36-56%,respectively.The biofilm were observed by LIVE/DEAD cell kit and laser confocal microscope.From the graph we knew that lavender essential oil displayed good effect on L.monocytogenes biofilm.The amount of extracellular polysaccharide was significantly reduced and the dead cell counts were increased notably,the biofilm thickness was decreased.Investigating the effects of six antibacterial agents at four concentrations(0.5?4 MIC)on V.parahaemolyticus biofilms formed at different temperatures.The results showed that after three hours of treatment with four concentrations of lavender essential oil,the removal rates of biofilms formed at 32°C and 10°C were 58?80%and 36?72%,respectively.The elimination rates of four concentrations of the 84 disinfectant against warm biofilms and cold biofilms were 20?72%and 32?57%,respectively.Compared with the 84 disinfectant,lavender essential oil had stronger removal effect on V.parahaemolyticus.The resistance of cold biofilm was stronger than that of warm biofilm.The biofilms were observed by LIVE/DEAD cell kit and laser confocal microscope.The results indicated that the amount of extracellular polysaccharide was significantly reduced and the dead cell counts were increased remarkably,the biofilm thickness was decreased.The effects of heat treatment on V.parahaemolyticus and L.monocytogenes biofilms formed at different temperatures were studied.The biofilms formed at different temperatures were treated with wet heat at 60°C,100°C,121°C and dry heat at 121°C.The results showed that after treatments,the residual viable cell counts of L.monocytogenes biofilms formed at 32°C and 10°C were 3.7-5.8 lg CFU/cm~2 and 5.7-6.3 lg CFU/cm~2.The residual viable cell counts of V.parahaemolyticus biofilms formed at 32°C and 10°C were 1.9-2.7 lg CFU/cm~2 and 3.3-4.3 lg CFU/cm~2.Heat treatments could not completely remove the biofilm of the two bacteria.V.parahaemolyticus was more sensitive to high temperatures than L.monocytogenes.The formations of VBNC cells in mature biofilm of L.monocytogenes under different conditions were investigated.4MIC of lavender essential oil induced the biofilm cells entered into the VBNC state at 14 days,and the cell concentration was 4.77 lg CFU/cm~2.MIC and 2MIC of 84 disinfectant induced biofilm cells entered into the VBNC state within40 days,the viable cell concentration was above 5.45 lg CFU/cm~2.