The Study On Dissemination Of Methicillin Resistance In Mastitis-Inducing Staphylococci

Staphylococci are one of the major causes for bacterial infections both in humans and animals.Staphylococci could cause acute or chronic infections which are a serious threat to the health of livestock and cause great economic loss to the animal industry.It has been reported that overuse of antibiotics in animal production promotes emergence of methicillin-resistant staphylococci(MRS).Due to exogenous acquisition of a mobile element,staphylococcal cassette chromosome mec(SCCmec),MRS are resistant to virtually all?-lactam with the exception of the latest generation of cephalosporin.PBP2 a protein,encoded by gene mecA or mecC which is located in SCCmec,is involved in bacterial cell wall synthesis.PBP2 a confers MRS with ?-lactam resistance due to its low affinity to ?-lactam antibiotics.The common method to detect MRS in clinical is based on the resistant phenotype.The fact that SCCmec could transfer among different staphylococcal species promotes widely spread of MRS.Horizontal gene transfer is the most common way of antibiotic resistance dissemination.Excision from a donor genome is the initial and most crucial step for transfer of SCCmec to a recipient.Excision of SCCmec is mediated by a cassette chromosome recombinase(Ccr)which is encoded by gene ccr.However,how antibiotics regulate SCCmec excision and transfer remains unclear.Recently,more and more MRS strains containing the mecA gene but being susceptible to oxacillin are being reported.These strains are called oxacillin-susceptible mecA-positive staphylococci(OS-MRS)and they can escape detection from routine diagnostic laboratory tests because of their susceptible phenotypes and will be treated as methicillin susceptible staphylococci(MSS).Some of OS-MRS will convert into highly resistant staphylococci to oxacillin after repetitive exposure in a short time(oxacillin MIC ? 128 ?g/mL).This will cause therapeutic failure and make OS-MRS infection life-threatening.Thus,it is worthy toidentify critical regulatory factors involved in the regulation of phenotype conversion in OS-MRS.In the first experiment,we tested the presence of residual antibiotics in the 244 fresh bovine milk samples by standard methods.The results showed that more than 50% of the 244 milk samples contained antibiotics.Thirty nice randomly selected samples containing antibiotic residues were further determined for residual amounts of five selected antibiotics(ciprofloxacin,chloramphenicol,sulfadiazine,streptomycin and tetracycline).All 39 samples contained ciprofloxacin at the amount more than the Maximum Residue Limits(MRLs,European Union),while 11,9,13 and 2 out of 39 samples contained two,three,four and five antibiotics at the amount exceed the MRLs respectively.In the second experiment,we tried to identify how commonly used antibiotics,including?-lactam,glycopeptide,tetracycline,sulfonamide and quinolones,promoted the expression of ccr and excision of SCCmec.Among these antibiotics,DNA targeting drugs could promote ccr expression even at low concentrations.Assming that antibiotics could stimulate the expression of ccr by inducing SOS responses,we have shown that antibiotics at certain dasages could induce DNA damage in staphylococci and then activate SOS signal pathway.Activated SOS alleviated the repression of LexA on the ccr promoter and increased expression of ccr.Up-regulated expression of Ccr promoted the excision of SCCmec.In the third experiment,we aimed to explore the regulatory mechanism of a methicillin resistant phenotype in MRS.Staphylococcal isolates from bovine milk were used.Our results showed that the expression levels of regulator blaR1 and blaI were involved in the resistant phenotype and phenotypic conversion from being susceptible to being resistant.In OS-MRS isolates,expression of blaI and blaR1 was high.In the absent of oxacillin,high levels of BlaI could reduce the oxacillin resistance through repressing the expression of mecA.In the present of oxacillin,high levels of BlaR1 promoted the high expression of PBP2 a in a short time by alleviating the repression of BlaI and conferd OS-MRS with a high level of resistant phenotype.In conclusion,the results from the first experiment suggest that the regular monitoring of antibiotic residue in bovine milk is essential for avoiding the abuse of antibiotics in animal production.Our results in the second experiment clearly explained the mechanism by which many antibacterial agents promote methicillin resistance dissemination in staphylococci.Furthermore,our study provides a new direction for the development of new antibacterial agents which will inhibit the spread of antibiotic resistant genes.In the third section,ourresults suggest that in the process of screening and identifying MRS including OS-MRS isolates,the effect of bla gene complex should be taken into consideration.Furthermore,development of BlaR1-targeting inhibitor is one way to help cure the infection caused by MRS or OS-MRS.