| Objective:Daptomycin is a cyclic lipopeptide antibiotic,which is a cell membrane-active compound that has a strong bactericidal effect.Daptomycin is mainly used for the treatment of complex infections caused by methicillin-resistant Staphylococcus aureus(MRSA).The mechanism of daptomycin resistance in Staphylococcus aureus(S.aureus)is currently unknown.Studying the mechanisms of resistance to daptomycin in S.aureus can both provide ideas for the development of new drugs and study plans targeting resistance relative targets to slower resistance development.In this study,we induced S.aureus resistance to daptomycin in vitro,and then investigated the phenotypic characteristics and resistance mechanism of the resistant strains.Methods:1.MRSA from different parts of China were collected,and the MRSA daptomycin MIC was determined by broth microdilution methods according to the Clinical and Laboratory Standards Institute(2020)recomendation.2.Using ALE(Adaptive Laboratory Evolution)strategy,continuously culture S.aureus USA300,Newman and SA75 in sub-inhibitory concentration of daptomycin,in vitro induced them resistant to daptomycin.3.Using drug susceptibility test to determine daptomycin-induced resistant strains susceptibility change to other antimicrobials.4.Determine the growth and virulence phenotype of daptomycin-induced resistant strains relative to wild strains: The growth change of S.aureus after daptomycin resistance was observed by measuring the growth curve and placing equal amounts of resistant strains and their corresponding wild type strains in the same environment to competitively growth.In vitro and in vivo virulence change were examined by measuring the ability of wild and daptomycin-induced resistant strains to lysis rabbit erythrocytes,and the pathogenic ability in wax worms and mouse skin infection model.5.Determine daptomycin resistance-related phenotypes of daptomycin-induced resistant strains relative to wild strains: Mmeasuring S.aureus cell wall thickness by transimission electron microscope;measuring bacterial surface charge by observing the ability of S.aureus to bind to cytochrome C;measuring S.aureus autolysis ability by using Triton X-100 to induce;measuring biofilm formation ability by using crystal violet stain method;measuring membrane potential by using membrane probe Di OC2(3).6.Newman and it’s daptomycin-induced resistant strain Newman-DNS were send to transcriptome sequencing.Using RT-q PCR to identifiy the expression changes of genes associated with daptomycin resistance.7.Find mutation sites that may be associated with daptomycin resistance by analyzing the SNP results of whole genome sequencing.8.Gene editing was performed after daptomycin associated site mutations were identified.Strains with single site mutations were constructed in wild type strain by homologous recombination method and then reverted the mutation.Result:1.The 407 MRSA isolates were all sensitive to daptomycin,with daptomycin MIC range of 0.06 μg/m L-1 μg/m L,Geometric MIC of 0.25μg/m L,MIC50 of0.25μg/m L,and MIC90 of 0.5μg/m L.The propotion of MRSA isolates with MIC value of 0.06μg/m L,0.125μg/m L,0.25μg/m L,0.5μg/m L and 1μg/m L were 3.19%,24.57%,50.37%,15.97% and 5.90%,respectively.2.After induced in sub-inhibitory concentration of daptoycin,the daptomycin MIC of USA300 increased from 0.25μg/m L to 16 μg/m L,Newman increased from 0.25μg/m L to 32 μg/m L and SA75 increased from 0.25 μg/m L to 32 μg/m L.When consecutivey culture 15 times without daptomycin,it remained resistant to daptomycin and showed some stable daptomycin resistance.3.Daptomycin-induced resistant strains showed reduced sensitivity to glycopeptides and gentamicin,and increased sensitivity to β-lactams antimicrobials.4.Compared with wild type strain,daptomycin-induced resistant strains were grow slower,the lysis of rabbit erythrocytes ability was weakened,the lethality of wax worms was reduced,and the severity of skin abscess in mice was weakened.Daptomycin-induced resistant S.aureus exhibited slower growth and weaken virulence both in vitro and in vivo.5.Daptomycin-induced resistant strains had daptomycin relative phenotypic changes,such as thicken cell wall,increased positive charge on the cell surface,enhanced autolytic activity,enhanced biofilm formation capacity,and reduced initial cell membrane potential.6.The expression of membrane phospholipid metabolism related genes mpr F,cls2 and autolysis related gene lyt M were upregulate in three daptomycin-induced resistant strains compared to their wild type strains.The transcriptomic result of Newman and its corresponding inducible resistant strains showed significant changes in gene expression associated with the sae RS two-component regulatory system,and PCR sequencing found partial deletion of the sae R,indicating that the significant reduction in virulence of the induced resistant strains corresponding to Newman was likely related to mutation in the sae R.7.mpr F S295 L daptomycin relative site mutation was found in USA300 daptomycininduced resistant strain;mpr F L341 F and cls2 F60 S daptomycin relative site mutations were found in Newman daptomycin-induced resistant strain;and mpr F L826 F and cls2 F60 L site mutations were found in SA75 daptomycin-induced resistant strain.8.Previous studies have demonstrated the role of mpr F S295 L and mpr F L826 F in bacterial resistance to daptomycin,but the role of mpr F L341 F,cls2 F60 S and cls2F60 L did not identified.Mutation of the mpr F L341 F site can increase the daptomycin MIC of Newman from 0.25 μg/m L to 4 μg/m L,restored the mutation and the strain daptomycin MIC declined to 0.25 μg/m L.This indicated that mpr F L341 F site mutation can directly alter the sensitivity of Newman to daptomycin.Deletion of sae R genes did not alter bacterial sensitivity to daptomycin but can enhance the competitive ability in the daptomycin environment.Conclusion:1.Daptomycin has a good in vitro antibacterial effect for MRSA isolates in China.2.Daptomycin-induced resistant strains grow slower and have waken virulence.S.aureus resistance to daptomycin maybe associated with thicken cell wall,increased cell surface positive cell charge,increased biofilm formation capacity,and decreased initial membrane potential.3.L341 F site mutation of the mpr F gene can directly reduce the susceptibility of S.aureus Newman to daptomycin. |
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