Effect Of Plectasin-Derived Antimicrobial Peptides On S. Aureus Mastitis And The Study On Mechanism Of Antimicrobial Peptides Resistance Development

For the dairy industry,mastitis is the most costly disease and affects animal health and welfare.Staphylococcus aureus（S.aureus）is one of the most important pathogens causing bovine mastitis.Now,intramammary administration of antibiotics is the most common method to treat bovine mastitis.However,antibiotic therapy has many disadvantages,such as low cure rates and residue in milk.There is an urgent need to develop alternatives to antibiotics for the treatment of S.aureus bovine mastitis.Antimicrobial peptides（AMPs）are considered to be one of the most powerful candidates for alternatives to antibiotics in the future due to their broad bactericidal spectrum,no residues,and antibacteria by multi-target sites.The development of AMPs as antibacterial agents has become a research hotspot.However,the development of any new antibacterial agent must fight with the evolution of bacterial resistance.Some studies have found that bacteria can use a variety of strategies to fight AMPs.But there is a relatively limited understanding on how bacteria would acquire heritable resistance to AMPs.The problem of AMPs-resistance has become an important factor hindering the large-scale application of AMPs.Therefore,it’s an urgent need for basic research on mechanism of AMPs-resistance.This study first evaluated the efficacy of plectasin-derived AMPs in the treatment of S.aureus mastitis in vitro and in vivo,then we studied the mechanism of S.aureus resistance to AMPs.The main results were as follows:1.Effective antimicrobial activity of plectasin-derived AMPs against S.aureus isolated from milk in vitro.We used S.aureus isolated from milk as test strains to study the antimicrobial activity of AMPs（NZ2114 and MP1102）in culture medium,milk and bovine mammary epithelial cells.The results showed that NZ2114 and MP1102 had lower minimum inhibitory concentrations（MICs）against eight strains of S.aureus while most strains（6/8）had tetracycline resistance.NZ2114 and MP1102 at the concentration of 100μg/mL reduced the number of S.aureus by almost 100%within 4 h in processed bovine milk and reduced the number of internalized S.aureus in cultured mammary epithelial cells.These results demonstrated that NZ2114 and MP1102 had the potential to treat a range of diseases caused by intracellular S.aureus infections,including bovine mastitis.2.Evaluated plectasin-derived AMPs in a mouse model of the S.aureus-induced mastitis.In this study,we evaluated the bactericidal and anti-inflammatory effects of AMPs in a mouse model of mastitis induced by S.aureus strain isolated from milk.The lactating mice were randomly divided into five groups:snipped only group,S.aureus+PBS group,S.aureus+NZ2114 group,S.aureus+MP1102 group,and S.aureus+tetracycline group.The results showed both AMPs significantly reduced the S.aureus load and concentrations of TNF-a and IL-6 in mammary glands,compared to PBS treatment group.The S.aureus-induced inflammatory cell infiltration was significantly reduced by both AMPs.Our results suggest that NZ2114 and MP1102 may be used to treat S.aureus-induced mastitis.3.Mechanism of AMPs-resistance development in S.aureus in vitro.In order to study the mechanism of AMPs resistance in S.aureus development,two strains were treated with AMPs in vitro and the AMP resistant isolates were whole-genome sequenced.The two strains after 35 serial passages with AMP treatment exhibited a heritable AMP-resistance.Through the whole genome sequencing and functional analysis on the resistant strains,we found mutations in genes associated with cell wall metabolism and genes of transcriptional regulator and some other mutations.What’s more,three of the four resistant strains had the same gene-ndh mutation.The membrane-bound respiratory enzyme,which encoded by ndh,is involved in the transmission of the electronic chain and is involved in the reactive oxygen species（ROS）production in the bacterium.By the gene complementation of all five mutant genes in one of the resistant strains BA01611^nzR,we found the mutations caused by AMPs were responsible for the change of the AMPs-resistance.These results demonstrate that the acquisition of AMPs-resistance in S.aureus is the result of a combination of multiple mutant genes,and ROS may play an important role in this process.4.Role of ROS in the evolution of AMPs-resistance in S.aureus.The intracellular ROS production and the rifampicin mutation rate after AMPs treatment were measured.The results showed that the intracellular ROS of S.aureus increased significantly after treatment with AMP,which caused an increase in the mutation rate of rifampin.There was a significant linear relationship between the two（R²=0.7702）.However,the AMPs did not increase the rifampicin mutation rate in the absence of oxygen and iron ions,and the rifampicin mutation rate of iron homeostasis unbalanced strain BA01611Δfur was significantly higher than that of AMP-treated wild strains.These results indicate that ROS plays an important role in the evolution of AMPs-resistance in S.aureus and it’s a way to reduce the probability of bacterial AMPs-resistance by reducing bacterial ROS levels.In summary,this study first shows that AMPs have potential for the treatment of bovine mastitis.Then,in exploring the mechanism of AMPs-resistance development in S.aureus,a number of new gene mutations related to resistance to antimicrobial peptides were discovered and identified;it seems that ROS plays an important role in the evolution of S.aureus AMPs-resistance.In addition,our results also suggest that AMPs can be used in combination with iron chelators in the future to reduce bacterial mutation rates and hinder AMPs-resistance development.Finally,this study provides a scientific basis for the rational use of AMPs in the animal husbandry and the reduction of bacterial AMPs-resistance.