Study Of L-lactate Dehydrogenase Multidrug Resistance Mechanism On Staphylococcus Xylosus

Staphylococcus xylosus(S.xylosus)is the main isolated strain of cow mastitis caused by coagulase-negative staphylococcus,which has attracted much attention because of its increasing separation rate and serious multidrug resistance phenomenon.T he problem of bacterial drug resistance is a global focus.The resistant mechanism is regulated by the bacterial metabolic pathway.L-lactate dehydrogenase(ldh)is a metabolically related protein and our previous study has shown that it is related to tylosin-resistant S.xylosus.However,whether it is related to the multidrug resistance of S.xylosus and its potential mechanism are still unclear.Our study used green fluorescent protein(gfp)as a screening marker for deleting or supplementing ldh gene based on the tylosin-resistant S.xylosus.After that,we tested the drug resistance phenotypes of macrolides,amides,lincamides,tetracyclines and aminoglycoside antimicrobial agents commonly used in veterinary medicine to clarify whether L-lactate dehydrogenase could affect the multidrug resistance of S.xylosus.In the end,the variation of the key genes,enzyme activity and metabolite content in the tricarboxylic acid cycle(TCA cycle)and thioredoxin system were determined to clarify whether L-lactate dehydrogenase influenced the multidrug resistance through these pathways.The specific results are as follows:(1)Using gfp gene as the screening marker,ldh gene-deleted strain and ldh complement strain were successfully constructed.(2)After deleting the ldh gene,the bacterial resistance to five kinds of antimicrobial agents was increased,in which the MIC of bacteria was increased 10 times to tylosin,32 times to erythromycin,512 times to lincomycin,160 times to florfenicol,8 times to chlortetracycline and oxytetracycline,1024 times to streptomycin and 2 times to gentamicin.However,compared to the ldh gene deletion strain,the MIC of tylosin,erythromycin,florfenicol,chlortetracycline and oxytetracycline were decreased two-fold,and the MIC of lincomycin,streptomycin and gentamicin were decreased four-fold on the ldh complement strain.(3)After deleting the ldh gene,the content of lactic acid was increased significantly,the content of pyruvate and NADH were decreased significantly.However,the above indexes were restored on the ldh complement strain.(4)After deleting the ldh gene,the activity of thioredoxin oxidoreductase was significantly increased,the m RNA level of thioredoxin in bacteria was significantly increased and the content of reactive oxygen species was decreased significantly.However,the activity of thioredoxin oxidoreductase and the m RNA levels of thioredoxin in bacteria were restored on the ldh complement strain.Taken together,we hypothesize that L-lactate dehydrogenase is related to the multidrug resistance of S.xylosus.After deleting the ldh gene,S.xylosus might promote multidrug resistance by inhibiting the TCA cycle and regulating the thioredoxin system in bacteria.