Characterization Of The Multidrug Resistance Gene Cfr In Methicillin-resistant Staphylococcus Aureus (MRSA) From Animals And Humans In China

Methicillin-resistant Staphylococcus aureus(MRSA)is an important pathogenic bacterium in public health,which called "Super bugs".The cfr gene encodes an A2503 23 S rRNA methyltransferase that results in resistance to five unrelated classes of antimicrobial agents: phenicols,lincosamides,oxazolidinones,pleuromutilins and streptogramin A antibiotics.In recent years,cfr-positive MRSA is usually isolated from animals,people and environment,arousing public concern.In this study,a total of 128 Methicillin-resistant Staphylococcus aureus(MRSA)were screened from animals and human samples in seven provinces of China to present the transmition mechanism and prevalence of the multiresistant gene cfr as well as compare the cfr-positive MRSA with cfr-negative MRSA in resistance.In this study,a total of 128 Methicillin-resistant Staphylococcus aureus(MRSA)were screened from animals and human samples in seven provinces of China,such as Guangdong,Henan,Hebei,Fujian,Shandong and Hunan and so on,we focus on the studies of cfr-positive MRSA.All MRSA isolates were screened for presence of the cfr gene and Other ARGs by PCR.The result show that we identified 20 cfr-positive MRSA strains,the most frequently identified ARGs were fexA,aadAI and tetL with detection rates ?92%.These were followed by ermC(85.16%),aph(4')-Ia(84.37%),ereB(76.56%)and tetM(72.65%).Compared with ermC,the ermA(33.59%)and ermB(11.71%)gene levels were low.In addition,the optrA gene was present in 43(33.59%)of the 128 MRSA isolates.Interestingly,the prevalence of other ARGs among cfr-positive MRSA isolates was higher than in cfr-negative isolates.This was especially the case for optrA,ereB,aac(3')-IIc and aph(3')-IV.Furthermore,all these carried fexA and ermC.The optrA and ermA genes were present in 18 and 11 isolates,respectively.Antibiotic minimum inhibitory concentrations(MIC)were determined by using the agar dilution method and microdilution broth method for 128 MRSA in 15 antibiotics.In general,most(>80%)showed resistance to erythromycin,clindamycin,tetracycline,azithromycin,tylosin,ciprofloxacin,ampicillin,cefotaxime,gentamicin and florfenicol.Relatively low resistance rates were observed for trimethoprim–sulfamethoxazole(43.75%),rifampicin(21.09%).All the isolates were susceptible to vancomycin,daptomycin and linezolid.The resistant rate >80% of 10 antibiotics for 20 cfr-positive MRSA with 15 resistance patterns.In addition,the resistance rates of the 20 cfr-positive MRSA were higher than for cfr-negative MRSA except for linezolid,vancomycin and daptomycin.None of the cfr-positive MRSA isolates was resistant to linezolid.However,compared with the linezolid MIC of the control strain S.aureus ATCC 29213,the proportion of isolates with increased linezolid resistance in cfr-positive MRSA were higher than cfr-negative MRSA(40% vs 6.5%,respectively).All MRSA strains were classified according to multilocus sequence typing(MLST)and typed through amplification of the polymorphic X-region of the Staphylococcus protein A(spa)gene.In the 128 MRSA isolates,we found 8 different STs and ST9 predominated(82.0%,105/128).Spa-typing analysis of these 128 identified 7 different types and t899(80.5%,103/128)was the most prevalent.Among cfr-positive MRSA isolates,three different STs and four different spa types were found.ST9(85%,17/20)and t899(75%,15/20)were the most prevalent ST and spa types,respectively.To further compare the genetic relatedness among cfr-positive and-negative MRSA isolates,PFGE was conducted.Twenty cfr-positive and 10 cfr-negative MRSA isolates were successfully typed by PFGE.We obtained 30 different PFGE profiles(> 100% similarity).These PFGE results suggested that most of these strains were clonally unrelated.S1-PFGE and Southern blot hybridizations revealed that the cfr genes were located on plasmids with sizes of ~50 kb(n=11)and ~7.1-kb(n=2)among the 13 transformants.The regions surrounding cfr genes were in two different genetic contexts among the 13 cfr-carrying transformants by using PCR mapping and inverse PCR based on cfr flanking regions.Type I was found in two transformants among which the complete nucleotide sequences of the 7,057-bp circular plasmids harboring cfr were obtained,the rep and ?pre/mob genes were upstream of the cfr gene while the pre/mob and ermC were present downstream.Type II was the most common structure observed in 11 of the 13 transformants among which the cfr gene was located on ~50 kb plasmids,The cfr-carrying region including an IS21-558 insertion and cfr was integrated into the Tn558 element,which come into being a Tn558 variant that cutoff the transposase genes tnpA and tnpB.Those two type genetic environments of cfr were similar to the plasmids that have been reported.In a short,the resistance of MRSA in our study is seriously,while the resistance of cfr-positive MRSA higher than the cfr-negative MRSA.Plasmid,insertion sequence and transposon were the important mobile element mediated the dissemination of cfr gene.Resistant genes like ermC and fexA co-transmission with cfr gene can promote the resistance of MRSA isolates.In this study,cfr gene was detected among MRSA isolated from different sources and regions,which brought a potential hazard for public and human health.