Study On The Effect Of Caffeic Acid On Reducing Drug Resistance Of Pathogenic Bacteria In Raw Milk And Its Mechanism

Cow mastitis is one of the important diseases threatening the dairy industry,which can decrease yield and quality of milk,and seriously hinder the development of global dairy industry.Klebsiella pneumoniae,Staphylococcus aureus and Listeria monocytogenes infection are the main factors leading to bovine mastitis.So far,the treatment of mastitis caused by pathogenic bacteria mainly depends on antibiotics.However,the irrational use and abuse of antibiotics accelerate the development of drug resistance,and lead to the emergence of multi drug resistant bacteria.Therefore,effective measures are urgently needed to restore the sensitivity of pathogenic bacteria to antibiotics.At present,the global strategies to deal with bacterial resistance mainly include:strengthening drug resistance monitoring,rational use of antibiotics,developing new antibiotics and restoring or enhancing the antibacterial activity of existing antibiotics.Natural phenolic compounds have many advantages,such as wide source,less side effects,not easy to produce drug resistance,and synergistic antibacterial effect when combined with antibiotics.Therefore,they have broad application prospects in dealing with the problem of bacterial drug resistance.As one of the important phenolic compounds,caffeic acid(CA)is widely present in fruits,vegetables and grains.It has broad-spectrum antibacterial activity against Escherichia coli,S.aureus,Pseudomonas aeruginosa and other common pathogenic bacteria.Combined with antibiotics,it can reduce bacterial resistance and enhance the sensitivity of multidrug-resistant bacteria to commonly used antibiotics.Therefore,in order to explore whether CA can improve the susceptibility of major drug-resistant pathogens in raw milk to antibiotics and its mechanism,the drug resistance spectrum of three pathogenic bacteria in raw milk was determined by drug sensitive disk method,and the antibacterial activities of antibiotics and natural products(CA,anthocyanins and cinnamaldehyde)were determined by broth dilution method,and then screen out the natural products that have synergistic antibacterial effects with antibiotics and their optimal concentration combination through a checkerboard test.Secondly,the change of the number of living bacteria after the optimal concentration combination and single antibacterial agent were compared by the analysis of sterilization time,and the effect of natural products combined with antibiotics on reducing the drug resistance of pathogenic bacteria in raw milk was analyzed.Finally,the activity of β-lactamase was determined by spectrophotometry,fluorescence staining method was used to determine the integrity of the cell membrane,the activity of efflux pumps were determined by fluorescence spectrophotometry,the structure changes of cell membrane and biofilm were observed by microscope,and fluorescence quantitative PCR(RT-qPCR)was used to quantify the expression of drug resistance related genes(antibiotic hydrolase gene,efflux pump gene and porin gene)to study the mechanism of reducing drug resistance of CA and SMZ alone or in combination on three pathogenic bacteria in raw milk,so as to provide a theoretical basis for solving the problem of bacterial drug resistance and further exploring the mechanism of reducing bacterial drug resistance,It provides a new strategy for the development and application of CA and other phenolic compounds as safe and effective new synergistic antibacterial agents.The main results are as follows:1.K.pneumoniae(K.p 305),S.aureus(S.a 302)and L.monocytogenes(L.m6)were resistant to 22,15 and 16 kinds of antibiotics,respectively,and the minimum inhibitory concentrations(MICs)value of antibiotics were>512 μg/mL.The drug resistance spectrum of K.p 305 was the widest.CA and anthocyanins had good antibacterial effect on the three strains,while cinnamaldehyde had almost no antibacterial effect on K.p305.2.CA could significantly enhance the antibacterial activity of antibiotics against three classes of drug-resistant bacteria in raw milk by reducing the MICs of antibiotics by ≥4096 times,while anthocyanins could only reduce the MICs of four kinds of antibiotics by 4 times,thus enhancing the sensitivity of three classes of drug-resistant bacteria to antibiotics.After CA combined with SMZ or chloramphenicol(CHL)for 24 h,compared with single antibiotic or CA,the number of viable cells of K.p 305 was decreased by at least 2.62 log and 2.61 log,respectively,S.a 302 decreased by 2.18 log and 2.03 log,respectively,and that of L.m6 decreased by 2.71 log and 2.38 log,respectively,which indicated that CA combined with SMZ/CHL had synergistic bactericidal effect on K.p 305,S.a 302 and L.m6.3.SMZ(1/256 MIC)combined with CA(1/2 MIC)significantly inhibited the βlactamase activity of K.p 305 and S.a 302,and the specific activity of the enzyme decreased to 108.2±42.2 and 205.1±49.5 nmol/min/mg,respectively,and had almost no effect on L.m6.The concentration dependent inhibition of CA on the efflux pump activity of three drug-resistant bacteria was observed by enhancing the accumulation of SMZ in the bacteria.Among them,1/2 MIC CA could increase the accumulation of SMZ in K.p 305,S.a 302 and L.m6 by 75%,69%and 69%,respectively.4.The changes of SYTO 9 and PI fluorescence intensity were observed by scanning electron microscope and confocal laser scanning microscope(CLSM)to explore the effect of CA combined with SMZ on the membrane structure of three strains of drugresistant bacteria in raw milk.The results showed that the combination of CA(1/2 MIC)and SMZ(1/256 MIC)could synergistically destroy the integrity of cell membrane,enhance the permeability of cell membrane and increase the intake of SMZ.The combination of CA and SMZ could kill bacteria cooperatively,which caused three classes of drug-resistant bacteria lost water and wrinkled and released a lot of cell contents,and the cells were disintegrated.5.The inhibitory rates of CA(1/2 MIC)combined with SMZ(1/256 MIC)on L.m6,K.p 305 and S.a 302 biofilms were 84.6%,81.1%and 75.3%,respectively.The inhibition rate of their combination on EPS and extracellular protein was positively correlated with the inhibition rate of biofilm.The inhibitory rates on EPS of L.m6,K.p 305 and S.a 302 were 51.8%,51.1%and 50.8%respectively,and the inhibitory rates of extracellular protein were 52.6%,51.3%and 50.8%respectively;In addition,their combination could significantly destroy the structure of the biofilm,significantly disperse the aggregated microbial community,accelerate the SMZ to reach the target,enhance the bactericidal activity of antibiotics,and finally kill the bacteria in the biofilm.6.The combination of CA(1/2 MIC)and SMZ(1/256 MIC)can down-regulate the antibiotic hydrolase genes of K.p 305,S.a 302 and L.m6 respectively by 2-17 times,310 times and 4-9 times,respectively,thus enhancing the antibiotics sensitivity of the three strains.The efflux pump gene was down regulated by 4-8 times,3-6 times and 6-8 times respectively,which greatly inhibited the efflux pump system from actively pumping SMZ.In addition,their combination could down regulate the sulfamethoxazole resistance gene(sul2)of the three strains by 12.75 times,10.05 times and 9.42 times respectively,which indicated that CA and SMZ had synergistic antibacterial effect.The effective bactericidal effect of sulfonamide antibiotics on three classes of drug-resistant bacteria was significantly improved.In conclusion,this study shows that CA can reduce the drug resistance of three kinds of pathogens in raw milk by reducing the MICs of antibiotics.CA mainly reduced the drug resistance of three kinds of pathogens by inhibiting the activities of βlactamase,activities of efflux pump and the formation of biofilm,destroying the integrity of cell membrane structure and down regulating the expression of drug resistance-related genes.