Study On The Mechanism Of Tolerance To Ciprofloxacin In Staphylococcus Aureus

Healthy breeding is a new breeding model that improves the level of dairy cow breeding in China,improves milk quality,and enhances market competitiveness.Dairy cow mastitis is one of the common and multiple diseases that restrict this model and the development of dairy cattle breeding.About 80% of dairy cow mastitis in our farms is caused by pathogenic microorganisms.So far,more than 140 pathogenic bacteria causing mastitis have been found and Staphylococcus aureus is the major Gram-positive pathogen..It can colonize the breast tissue of dairy cows and cause infection,which is extremely difficult to eradicate.Antibiotics therapy are currently the most important metric to prevent and treat such infections,but the long-term and large-scale use of antibiotics promotes the resistance evolution of Staphylococcus aureus,which lead to the weakness or even loss of the therapeutic effect of antibiotics.Antibiotic tolerance is one of these strategies,and responsible for relapsing chronic infections.This experiment used Staphylococcus aureus Newman as the parental strain for tolerance induction,and isolate the ciprofloxacin tolerant S.aureus;determined the molecular mechanism of ciprofloxacin tolerance formation through genomics and proteomics.The main results are as follows:(1)Induction ciprofloxacin-tolerance Staphylococcus aureus strain Cip-T8-seq under laboratory conditions: Intermittent use of 20 times the MIC value of the Ciprofloxacin on wildtype bacteria.After 17 cycles induction,the tolerance phenotype was confirmed by the measurement of minimor inhibitition concentration and maximine duration of killing.(2)The mutation of savRS was responable for the formation of CIP tolerance: the tolerant strain was perfomed towhole genome sequencing,and the mutations were identified through genomecomparison with wildtype bacteria.the relationship between the mutant gene and the tolerance phenotype was confirmed through the knockout and he complementation of savRS and phenotype determination.The results showed that savRS Mutations might lead to tolerance formation through the changes in the surface activity or affinity of the toxin-antitoxin,which affects the expression of other genes and leads to the emergence of phenotypic tolerance.(3)Identify the downstream genes srr AB relating to tolerance phenotype through proteomics determination of tolerant strain: the proteomics of savRS mutant strain and wildtype strain were determined.The proteins which have significant changes in tolerant strain were indentified by function annotation using Uniprot protein database and Perseus database.and the relationships between gene matationsand phenotypic tolerance were verified by gene overexpression.Finally,the functional gene srrAB was confirmed to have a effect on the tolerance formation.(4)Cross-tolerance detection of tolerant strain(Cip-T8-seq): 11 antibiotics,including 2penicillin antibiotics(oxacillin and flucloxacillin),2 glycopeptide antibiotics(vancomycin and teicoplanin),2 quinolone antibiotics(ciprofloxacin and levofloxacin),1 lipopeptide antibiotic(daptomycin),2 carbapenem antibiotics(imipenem and meropenem),2cephalosporin antibiotics(ceftazolin and Cefpirome) were using to detect the multidrug tolerance phenotypes of The results showed that Cip-T8-seq was not tolerant to penicillins,glycopeptides,and cycloaliphatic peptide antibiotics,but tolerant to carbapenems and cephalosporinsand quinolone antibiotics.Therenfore,this study successfully isolated ciprofloxacin-tolerance S.aureus strain Cip-T8-seq under laboratory conditions,and identified the genetic mutation savRS in tolerance strains through genomics sequencing.Through the phenotype determination of Newman-?sav RS and Cip-T8-seq::savR strains,we confirmed that the the mutation of savRS might lead to the changesof the surface activity and affinity of the SavRS protein,which affects downstream genes expression and then lead to tolerance formation;the gene srrAB was confirmed to be related to tolerance and the regulatory relationship between savRS and srrAB was explored through q RT-PCR and EMSA.In addition,this study also tested the multiple tolerance phenotypes of Cip-T8-seq.