Resistance-related Genes In Staphylococcus Aureus From Bovine Mastitis And Phenotypic Heterogeneity In The Resistant Isolate

Bovine mastitis is the most common disease in dairy cows and has a major limitation on the healthy development of the dairy industry,causing dairy farms great economic damage.Staphylococcus aureus,which is known to be pathogenic in both human and livestock,is one of the main pathogens involved in bovine mastitis.In recent years,the overuse of antibiotics leads to the emergence and prevalence of the resistant S.aureus,which result in difficulties and increased costs in treatment of bovine mastitis.Moreover,the contaminated milk and environmental pollution caused by antibiotics and resistant bacteria seriously endanger the public health safety.Therefore,investigating the antibiotic resistance mechanism and the environmental adaptation mechanism of S.aureus are important for guiding the rational use of antibiotics in bovine mastitis and preventing the rapid spread of resistant bacteria.In this study,by using the gene-editing technology,cell culture technology,TEM,q RT-PCR and whole genomic sequencing,the resistant genes in bovine mastitis S.aureus were identified and analyzed,moreover,the effects of the phenotypic heterogeneity on bacterial antibiotic resistance and environmental adaptability were investigated,and the mechanism of this phenotypic heterogeneity was explored.The detailed results are described as follows:(1)Identification of the antibiotic resistant genes in the Methicillin-resistant Staphylococcus aureus(MRSA)from bovine mastitis.According to the existing RNA-seq data of the bovine mastitis MRSA BA01611 that was treated with oxacillin,15 genes were selected which functions are unclear in S.aureus and their RNA transcription levels were regulated by oxacillin,and the knockout vectors of these genes were constructed.Finally,9mutants were constructed successfully.The antibiotic resistance of these 9 mutants and other unexplored 7 mutants in our lab were analyzed.Results showed that there were 13 genes involved in antibiotic resistance in bovine mastitis MRSA,including one gene that was positively correlated with antibiotic resistance and 12 genes that were negatively correlated with antibiotic resistance.And three of these genes played a very important role in MRSA resistance,including the phosphotransferase gene lcpA belonging to Lyt R-Cps A-Psr family,the glutamate synthase git C belonging to the Lys R-type transcriptional regulator and an Mar R-type gene SAV0686.(2)The strain-specific effects of the phosphotransferase gene lcpA on phenotypes,such as antibiotic resistance and cell adhesion,in clinical isolates of S.aureus,and the isolation of a novel phenotypic variant Mu50△lcpA-LC.The lcpA null mutant strains of MRSA Mu50 and MSSA Newman were constructed,and a new phenotypic variant Mu50△lcpA-LC with high antibiotic resistance and large colony morphelogy was isolated from the Mu50△lcpA population.The antibiotic resistance assay revealed the strain-specific effect of lcpA on antibiotic resistance in these S.aureus strains: for BA01611 and Newman,deleting lcpA significantly decreased their resistance to most cell wall-targeting antibiotics and ribosome-targeting antibiotics;for Mu50,deleting lcpA did not affect resistance to most cell wall-targeting antibiotics,but decreased it resistance to ribosome-targeting antibiotics;for Mu50△lcpA-LC,deleting lcpA improved resistance to most cell wall-targeting antibiotics,while did not affect resistance to ribosome-targeting antibiotics.Moreover,phenotypic assays identified that lcpA also had strain-specific effects on other phenotypes in these strains: BA01611 lcpA null mutant had no changes in pigment level,but had increased biofilm formation and high ability to induce inflammatory cytokines,and had decreased adhesion ability and reduced resistance to Triton-X100;Mu50 lcpA null mutant had decreased pigment level,increased ability to stimulate inflammatory cytokines production,and had no changes in spreading,biofilm formation,cell adhesion and resistance to Triton-X100.In conclusion,these results showed that the lcpA had strain-specific effects on various phenotypes of S.aureus,including antibiotic resistance,etc.Therefore,these results suggested that the pathogenic mechanism of lcpA was complicated,and the Mu50△lcpA could improve its antibiotic resistance by producing phenotypic variant subpopulation Mu50△lcpA-LC.(3)The characterizations and mechanism of phenotypic heterogeneity of S.aureus Mu50△lcpA-LC.The phenotypes of Mu50△lcpA-LC were detected by TEM etc.techniques.The results showed that the Mu50△lcpA-LC had reduced pigment level,enlarged cell size,increased spreading ability and decreased biofilm formation,and could form a reversible non-pigment large colony on agar plates.And the Mu50△lcpA-LC had increased abilities to adhere to host cells and stimulate inflammatory cytokines production.Using the q RT-PCR and whole genome sequencing etc.techniques,we analyzed the acetate metabolism level,the transcription level of carbon metabolism genes,and performed the genomic sequence alignment.Results showed that Mu50△lcpA-LC accelerated its acetate cycle by upregulating related genes,especially the acs A,resulting in a downregulation of cid,which lead to less cell wall hydrolysis and cell lysis.The TCA cycle was elevated in Mu50△lcpA-LC,and the bacterial protein expression and secretion patterns were changed significantly,moreover,its expression level of spa encoding protein A was significantly increased,which might improve its ability to evade host immunity.Phenotypic heterogeneity of Mu50△lcpA-LC occurred might be due to the regulation of carbon metabolism.These results showed that Mu50△lcpA could improve environmental adaptability by producing a phase variant subpopulation Mu50△lcpA-LC with advantageous phenotypes including high bacterial expansion and antibiotic resistance etc.,and its phenotypic heterogeneity occurred might be due to the regulation of carbon metabolism,independently of gene mutations and environmental changes.In summary,this study identified 13 antibiotic resistance genes in MRSA from bovine mastitis,and demonstrated the strain-specific effects of the phosphotransferase gene lcpA on phenotypes in S.aureus clinical isolates.Moreover,a new resistant subpopulation Mu50△lcpA-LC with phenotype switching was identified from the Mu50△lcpA strain.The phenotypic switching of Mu50△lcpA-LC involved in bacterial expansion,antibiotic resistance and adhesion,etc.were characterized,and the mechanism of this phenotype switching was analyzed,which revealed the importance of the phenotypic heterogeneity that occurred within the bacteria group on the environmental adaptability of S.aureus.This work improves our understanding of the adaptability of S.aureus to environmental stress,provides material resources and basis for the research of antibiotic resistance of MRSA,and provides a theoretical basis for the treatment of persistent bovine mastitis induced by S.aureus.