Study On The Antibacterial Mechanism Of Lactobionic Acid Against Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus(S.aureus),a common foodborne pathogen,food poisoning caused by it ranks second in global food poisoning incidents.S.aureus is sensitive to antibiotics and can easily cause resistance.Methicillin-resistant S.aureus(MRSA)is one of the multi-drug resistant bacteria that seriously threatens human health.It has a high detection rate and a higher mortality rate and has become a public health issue of global concern.At present,commonly used anti-MRSA drugs have also developed drug-resistant bacteria,so it is necessary to find new antibacterial agents to prevent and control the spread and pollution of MRSA.Lactobionic acid(LBA)is a high value-added organic acid that naturally occurs in fermented dairy products,and its antibacterial activities have gradually attracted attention.Our previous study found that LBA not only had inhibitory effects on common food-borne spoilage and pathogenic bacteria,but also has a good antibacterial activity against multi-drug resistant bacteria MRSA.However,the mechanism of the action of LBA against MRSA were not clear.In this paper,MRSA was used as the target strain,from the cell level,protein level and metabolic level,to deeply analyze the inhibitory effect,the site of action and the mode of action of LBA on MRSA,then layer by layer to explore the mechanism of LBA against MRSA.On this basis,the preservative application of LBA on fresh whole milk was evaluated.The main findings are as follows:(1)The antibacterial activity of LBA against MRSA were measured by minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC),growth curve and time-kill curve,etc.,and the results indicated that LBA exhibit an antibacterial and bactericidal properties against MRSA,and the MIC and MBC were 18.75 mg/m L and 50mg/m L,respectively.The alkaline phosphatase activity assay,flow cytometry,the leakage of protein and K~+,and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that LBA could disrupt the integrity of the cell wall and membrane,increase the membrane permeability,cause the leakage of cell contents,and influence the content and activity of bacterial proteins.Scanning electron microscopy and transmission electron microscopy results showed obvious morphological and ultrastructural changes in the bacterial cells,further confirming the cell wall and membrane damages caused by LBA.DNA gel retardation and competitive binding experiments indicated that LBA could interact with bacterial DNA via intercalation,to disturb the normal cellular functions.In addition,LBA also exhibited antibiofilm formation activity.(2)Sequential window acquisition of all theoretical mass spectra(SWATH-MS)was used for quantitative proteomics to analyze 100 differentially expressed proteins(28up-regulated and 72 down-regulated)after LBA treatment.Furthermore,multiple experiments were conducted to validate the results of the proteomic analysis including reactive oxygen species(ROS),virulence-associated gene expression,and the relative quantification of target proteins and genes by parallel reaction monitoring and quantitative real-time PCR.The results speculate that LBA destroy the integrity of cell wall by cleaving teichoic acid and increasing the net negative charge of the cell wall.LBA inhibit the activity of endogenous antioxidants,leading to the accumulation of ROS in MRSA cells that cause a series of injuries including oxidative stress,DNA damage,cell wall rupture,and increased cell membrane permeability.LBA causes MRSA genome instability by blocking DNA repair pathway.LBA interfere with the de novo synthesis of purine nucleotides,which means that LBA affect purine nucleotide synthesis.LBA inhibit protein synthesis by inhibiting RNA synthesis and reducing m RNA decoding speed and accuracy.LBA affects the energy and nutrient supply of MRSA by inhibiting Co A biosynthesis,hindering carbohydrate metabolism and phosphorylated sugar production,arginine synthesis and glutamate transport.LBA hinders carbohydrate metabolism and phosphorylated sugar production,arginine synthesis and glutamate transport by inhibiting the biosynthesis of Co A,and ultimately affects the energy and nutritional supply of MRSA.LBA can reduce the virulence,the biofilm production and the adhesion to the host of MRSA,which means that LBA reduce the pathogenicity of MRSA to the host.LBA interferes with the conformational change of the ribosomal 50S subunit,indicating that it can reduce the resistance of MRSA to lactobionic acid.(3)Liquid chromatography mass spectrometry-chromatography(LC-MS)was used for non-target metabolomics to analyze 98 differentially expressed metabolites(74 up-regulated and 24 down-regulated)after LBA treatment.Furthermore,the activities of phosphofructokinase and aconitase and the ATP content were measured to validate the results of the metabolomic analysis.The results speculate that LBA can break down teichoic acid by inducing the expression of wall phosphoric acid,accelerate cell wall lysis,thus destroying the integrity of the cell wall.LBA may change the composition of fatty acids in cell membranes by down-regulating the ratio of unsaturated fatty acids/saturated fatty acids(UFAs/SFAs);or reduce endogenous antioxidant activity and induce ROS accumulation;or open the ion channel of the cell membrane that cause an increase of the membrane permeability,thus destroying the integrity of the cell membrane,causing the cell contents leakage,and eventually leading to the cell damage or death.LBA block deoxynucleotide synthesis,inhibit DNA synthesis and repair,cause instability of the MRSA genome,and lead to cell damage or death.LBA promote the catabolism of purine nucleotides and block the de novo synthesis of purine nucleotides,interfere with the synthesis of purine nucleotides,and then affect the synthesis of nucleic acids and proteins.LBA inhibit glycolysis and tricarboxylic acid cycle pathways,block ATP synthesis,cause insufficient energy supply,and lead to cell death.LBA inhibits riboflavin metabolism,affecting the physiological functions and cell viability of cells.LBA interferes with electron transfer,suggesting that lactobionic acid can reduce ATP synthesis.(4)The pasteurized fresh whole milk was used as a model system to examine the antibacterial activity of LBA against MRSA in a real food matrix;the sensory properties of fresh whole milk stored for 12 days were evaluated to assess the effect of LBA treatment on whole milk quality.The results showed that LBA significantly inhibited the total number of MRSA colonies during storage of fresh whole milk at 4?,delaying the deterioration of sensory qualities.