| With the rapid development of antimicrobial resistance in bacteria,antibiotics as the main therapy against infection are facing great challenges,especially for infections caused by Staphylococcus aureus.Antibacterial peptides（AMPs）with broad antibacterial activity,have become one of the important research objects of new antibacterial drugs.Under certain specific conditions,bacteria can also selectively acquire resistance to antimicrobial peptides,but the mechanism of bacteria obtain antimicrobial peptide resistance remain unclear.In this study,three mutant genes（ndh,sagB,braS）that screened in a AMPs-resistant strain BA01611nz R were used to study their roles in the production of AMP resistance in S.aureus.In this study,multidrug-resistant S.aureus as a wild type（WT）was used to constuct the mutation strains（BA01611-△ndh,BA01611-△sagB,BA01611-△braS）and complementation strains（BA01611-△ndh+ndh,BA01611-△sagB+sagB,BA01611-△braS+braS）.Then the minimum inhibitory concentration（MIC）of different antibacterial peptides（NZ21114,Nisin,Bacitracin）were tested,and related phenotypic changes were study further to reveal the role of ndh,sagB,braS genes in development of AMP resistance in S.aureus.The main findings were shown as follows:（1）The MIC values of BA01611-△ndh on antibacterial peptides NZ2114,Bacitracin and Nisin were all increased.In order to further study the cause of MIC changes,the production of intracellular reactive oxygen species（ROS）was measured in WT and mutation strians.It was found that the intracellular ROS in WT increased significantly after exporsure to Bacitracin and Nisin,while BA01611-?ndh decreased significantly with or without AMPs.In addition,BA01611-?ndh+ndh were basically similar to WT.These results suggest that S.aureus may achieve AMP resistance through regulated the intracellular ROS metabolic pathways by ndh gene.（2）The MIC value of BA01611-△sagB on NZ2114 and Bacitracin were decreased,while on Nisin was increased.In addition,the growth curve of BA01611-?sagB showed obvious growth inhibition in the presence of AMPs（NZ21114,bacitracin）,and then showed a rapid growth trend.The ability to form biofilms of BA01611-△sagB was enhanced in the absence of AMPs.Whether in the presence or absence of AMPs,BA01611-ΔsagB was consistent with WT for Triton X-100 induced autolysis.The resistance of S.aureus to AMPs may be realized through the involvement of sagB in the biofilm formation process.（3）The MIC values of BA01611-△braS on NZ2114 and Bacitracin decreased,while Nisin were not changed.In addition,the growth curve of BA01611-?braS showed obvious growth inhibition in the presence of AMPs（NZ21114,Bacitracin）,and then showed a rapid growth trend.The ability to form biofilms of BA01611-△braS was weakened in the absence of AMPs.Whether in the presence or absence of AMP,BA01611-ΔbraS was consistent with WT for Triton X-100 induced autolysis.The resistance of S.aureus to AMPs may be achieved through the Bra RS two-component regulatory system.In summary,the related phenotypic changes during the development of antimicrobial resistance are verified by mutated ndh,sagB,and braS,thereby demonstrating the potential molecular mechanism of AMPs resistance in S.aureus.It may be the following three ways:1)the role of ndh in the electronic respiratory chain regulate indirectly the intracellular ROS metabolic pathways;2)sagB effect AMPs resistance by changed cell wall synthesis process;3)braS involved in the Bra RS two-component regulatory system.These findings are expected to provide data support for prevention and treatment of AMPS in cow mastitis,provide theoretical basis for reducing AMP resistance. |
