Functional Research of Cassette Chromosome Recombinase C Mediated Staphylococcal Cassette Chromosome mec Istand Excision / 中国医科大学学报

ABSTRACT

Objective To construct 2 recombinant plasmids carrying cassette chromosome recombinase C(ccrC)and ccrAB respectively and introduce the plasmids into methicillin-resistant Staphlococcus aureus(MRSA)strain 81/0432,and to observe the precise excision of Staphylococcal cassette chromosome mec(SCCmec)island from bacterial chromosome.Methods ccrC and ccrAB genes were amplified with chromosomal DNAs isolated from MRSA strains 81/0342 and N31S as PCR templates.We constructed recombinant plasmids pSR5C and pSR2AB by cloning ccrC and ccrAB genes into temperature-sensitive plasmid pYI3,after introducing them into MRSA strains 81/0432 and N315 by electroporation.PCR was performed to identify SCCmec excision from the bacterial chromosome.The transformants were serial passaged for 10 days,and then the drug resistance of these rransformants was detected by replica experiment.Results The fragment length of ccrC gene was 1.9 kb,smaller than the fragment length of ccrAB from N315.The recombinant plasmids of pSR5C and pSR2AB were successfully constructed.After these 2 recombinant plasmids were introduced into MRSA strain 81/0342,type-V SCCmec island was excised from the chromosome and formed a closed circular structure in the bacteria.However,type-Ⅱ SCCmec island could be excised only in N31S strain after the expression of pSR2AB.Replica experiment verified that transformed strains of 0342(pSR2AB),0342(pSR5C),and N315 (pSR2AB)were mostly susceptible to ceftizoxim or tobramycin after the excision of SCCmec island.Conclusion cciC could serve as a recombinase as ccrAB,which could mediate precise excision of SCCmec island from the chromosome of type-V MRSA strain.This study shows that type-V SCCmec island is widely disseminated between Staphylococcus aweus strains in community setting.