Transmission Mechanism Of Multidrug Resistance Gene Cfr In Swine Farm

The multiresistance gene cfr encodes a 23S rRNA methyltransferase that modifies the site A2503.Cfr confers resistance to phenicols,lincosamides,oxazolidinones,pleuromutilins,and streptogramin A antibiotics（the PhLOPS_A phenotype）,which are used extensively in the treatment of infections in both humans and animals;oxazolidinones,in particular,are regarded as the last defense against methicillin-resistant Staphylococcus aureus（MRSA）or vancomycin-resistant Enterococcus（VRE）in humans.The multiresistance gene cfr is widely distributed among various gram-positive and gram-negative species in livestock in China.In this study,we intended to determine the prevalence of cfr gene in staphylococci and E.coli in swine farm in China,and to study the transmission mechanism of cfr gene and characterize the cfr-carrying isolates.To better understand the epidemiology of cfr among Staphylococcus,254 isolates of Staphylococcus were subjected to prevalence and genetic analysis.Forty（15.7%）Staphylococcus isolates,including 38 Staphylococcus sciuri strains,one Staphylococcus chromogenes strain,and one Staphylococcus lentus strain.Most of the S.sciuri strains were clonally unrelated;however,clonal dissemination of cfr-positive S.sciuri was detected at the same farm.In eight randomly selected cfr-positive staphylococci,a cfr-harboring module（IS21-558-cfr-ΔtnpB）was detected in six S.sciuri isolates;cfr was bracketed by two copies of ISEnfa4 or IS256 in the remaining two S.sciuri isolates.Six（0.77%）cfr positive isolates were detected from 777 ESBLs E.coli strains saved in our laboratory and two（0.5%）cfr-carrying isolates from 398 E.coli strains collected from six swine farms in China in 2013.All the strains were resistant to florfenicol,ampicillin,streptomycin,tetracycline,and trimethoprim/sulfamethoxazole.The cfr-positive strains were displayed different PFGE patterns and genetically divergent,suggesting that the spread of the cfr gene in E.coli was not due to clonal dissemination but horizontal transfer.All the cfr genes were located on plasmids in which the majority were approximately 80-150kb in size by S1-PFGE-Southern.PCR-mapping was used to identify the genetic environment of cfr gene and showed 2 different types of genetic environment which were flanked by two IS26elements in the same or opposite orientation.Comparison with the plasmid pEC-01 from E.coli LYP-C-BCTb11 showed the part of segment between IS26 shared high homology and sμggested IS26 may have played an important role in the horizontal transfer of the cfr gene.Three conjuncations and three transformations plasmids were belonged to IncP（n=1）,IncA/C（n=1）,and F43:A-:B-（n=2）by PCR-based replicon typing and others were failed to be typed.Complete sequence analysis of the novel F43:A-:B-plasmid pHNEP28 revealed that it contains two multi-resistance regions:cfr together with floR,qnrS1 interspersed with IS26,ΔISCR2 and ISKpn19,and bla_TEM-1 together with tet（M）interspersed with IS26,ISApl1,ΔTn2,andΔIS1B.The coexistence of cfr with other resistance genes on a conjugative plasmid may contribute to the dissemination of these genes by co-selection.Complete sequence analysis of IncP plasmid pHNFP671 revealed that only one cfr gene accompanied with IS26 inserted into plasmid stability region.Continuous monitoring the prevalence of cfr gene in farm GDEP in 2014 and 2015,138cfr positive isolates were identified from 448（30.8%）faecal samples and 205 from 452（45.4%）nasal samples.Especially,the prevalence rate from fattening pigs was over 60%and from weaned pigs over 45%which were significantly higher than from piglets and sows.Of the 213 cfr-positive strains separated from nasal and faecal swabs,they are 166 gram positive isolates,44 gram negative isolates and 3 fungus isolates.Twenty-four clonal patterns of 36E.coli were typed by PFGE,and 8 from 14 randomly selected Proteus mirabilis isolated were successfully typed belonging to 3 types.cfr gene was widely spread in gram-positive and gram-negative bacteria in this farm.Thus,rational drug use and continued surveillance of cfr in swine farms are warranted.