Preliminary Synergistic Inhibition Mechanism Of Pediocin PA-1 And L-lactic Acid Against Aeromonas Hydrophila

As bacteriostatic metabolites of lactic acid bacteria,bacteriocin and lactic acid have the potential to synergistically inhibit Gram-negative bacteria.The present study explored the inhibitory effects of Pediococin PA-1,the typical representative of class IIa bacteriocins,combined with L-lactic acid at sub-inhibitory concentration,against the aquatic Gram-negative pathogen-Aeromonas hydrophila.We revealed the mechanism of combined inhibition under the cellular and molecular level.The results of the study are as follows:（1）The synergistic treatment of PA-1 and L-lactic acid has a growth inhibitory and lethal effect on Aeromonas hydrophila.The minimum inhibitory concentrations of PA-1 and L-lactic acid against Aeromonas hydrophila strains ATCC 35654 and CICC 10500 were 3.5 mmol/L L-lactic acid+50μmol/L PA-1 and 3.5 mmol/L L-lactic acid+30μmol/L PA-1,respectively.The synergistic treatment of the two substance could significantly prolong the growth lag period of the two strains,reduce the OD₆₀₀max value and the maximum specific growth rate during the plateau period.When treated Aeromonas hydrophila with PA-1 and L-lactic at the minimum inhibitory concentration for 8 h,the mortality of the two strains was more than 60%.（2）The synergistic treatment of PA-1 and L-lactic acid could damage the inner and outer membranes of Aeromonas hydrophila.The strain treated with L-lactic acid at sub-inhibitory concentration could cause the releases of lipopolysaccharide from the outer membrane,and the release amount increases with the increase of L-lactic acid concentration;laser confocal imaging shows that PA-1 can contact the bacteria after lactic acid treatment.The synergistic treatment of PA-1 and L-lactic acid could increase the permeability of cell membranes, leakaged of intracellular nucleic acid,protein and ATP,and caused the dissipation of transmembrane electromotive force and proton motive force;SEM and TEM images proved that PA-1 and L-lactic acid are synergistic The treatment caused damage to the inner and outer membranes of bacterial cells,leakaged of intracellular material,abnormal DNA structure,and cell deformation.（3）The synergistic treatment of PA-1 and L-lactic acid affects the gene transcription level of Aeromonas hydrophila.PA-1 and L-lactic acid co-treatment at the logarithmic growth phase Aeromonas hydrophila ATCC 35654 bacterial cell for 20 minutes caused the bacterial cell:glycine betaine transport system and multiple drug efflux pump gene transcription levels to be down-regulated,suggesting internal homeostasis imbalance and decreased drug resistance;energy metabolism,cell wall synthesis,flagella synthesis,and the up-regulation of Ton B system gene transcription levels are the positive responses of the bacteria to the stress.The results of this study provide the theoretical basis for the synergistic inhibition of bacteriocins and lactic acid to G^-bacteria,and provided a basis for the application of antimicrobial peptides and bacteria producing bacteria as substitutes for antibiotics in aquaculture.