Epidemiological Characteristics Of Bacitracin Resistance Cluster BcrRABD In Enterococcus From Animal

Bacitracin is a peptide antibiotic used for the treatment of local infection caused by gram-positive bacterium.While in China,bacitracin also widely used for prevented food animal diseases.As an animal feed-additive,bacitracin could promote animal growth,improve feed conversion rate,and inhibit the reproduction of harmful pathogenic bacteria in food animal intestine.However,with the large-scale use of bacitracin in the veterinary,the problem caused by bacterial resistance is more and more seriously.Now,in Enterococcus the primary resistance mechanism has been reported to be the presence of the bcrABDR cluster,and the ATP-binding cassette(ABC)transporter BcrAB were proposed to mediate major active to efflux bacitracin.But now in China,there are few studies to investigate the bacitracin resistance rate,or the transmission and distribution of cluster bcrRABD.This will bring many potential risks to the treatment in veterinary clinics.Therefore,in this study we intend to investigate the bacitracin resistance in enterococci from the food animals,and detected the prevalence and the transmission mechanism of bacitracin resistance cluster bcrRABD.To provides a scientific reference data for the assessment of bacitracin veterinary clinical application and avoid the generation and dissemination of bacitracin resistance enterococcus under antibiotic stress.In this study,we isolated 109 Enterococcus from chicken fecal samples in six provinces(Guangdong,Guangxi,Hunan,Hainan,Jiangxi,and Zhejiang)in 2014.229 Enterococcus from four pig farms in Guangdong,in 2015.The bacitracin resistance cluster bcrRABD were detected by PCR.The results showed that 37 strains of chicken-source enterococcus carrying the bcrRABD cluster,the detection rate was 33.9%.In Hunan,Guangxi and Zhejiang province the detection rate of the cluster was about 83.3%,57.6% and 50% higher than in Guangdong,Haian and Jiangxi province(22.9%,36.3% and 0%).In four pig farms,67 enterococcus were detected as bcrRABD-positive strains,the detection rate was 29.2%.Among 67 strains,60 strains were from pig,4 strains from human and 3 strains from the wastewater around the farm.At the same time,two gene type of bcrRABD cluster were detected.The amino acid similarity of the four genes bcrR,bcrA,bcrB,and bcrD were about 90% respectively,but these two different sequences did not show significant differences in bacitracin resistance phenotypes.Pulsed-field gel electrophoresis(PFGE),multilocus sequence typing(MLST),S1-PFGE,Southern hybridization,conjugal transfer/electrotransfer,and inverse PCR were used to study the transmission mechanism of bcrRABD in positive strains from chicken and pig farm.The results of PFGE showed that the 37 bcrRABD-positive strains from chicken farms were belong to 26 groups.In group A,the 8 strains were from three different province.The MLST result showed that most of bcrRABD-positive strains were belong to ST16(14/37).S1-PFGE with Southern blot resulted showed that all of cluster bcrRABD were located on the plasmids.The conjugation result showed that seven strains carried the bcrRABD-positive plasmid were successfully transferred to recipient strain.Inverse PCR result showed that in 17 of the 37 strains the bcrRABD cluster was flanked by two copies of the insertion sequence ISEnfa1 located in the same orientation,and a cyclic intermediate contain one ISEnfa1 and one bcrRABD cluster could be formed via the homologous recombination of the insertion sequence ISEnfa1.In the pig farm the PFGE result showed that the 64 E.faecalis were belong to 45 groups and the three Enterococcus hirae were belong two groups.In group 1,7,17,18,24,29,30,34,and group 42 contains strains from pigs in same farm,in group 43 contains pig source strain and environment source strain from different farms.The conjugation result showed that one strain from worker and 14 strains from pigs carried the bcrRABD-positive conjugative plasmid.Cyclic intermediates were detected in 31 of the 67 strains.In the sequencing analysis of bcrRABD-positive conjugative plasmid,we discovered a novel conjugative plasmid pEF123 which can mediate the horizontal transfer of bcrRABD cluster in E.faecalis.The whole sequence of plasmid pEF123 was blast in Genbank sequence database the result showed that the plasmid with the highest coverage was also only 38%.Therefor we annotationed the framework region and drug resistance region of the plasmid,and analyzed the formation of drug resistance regions.The analysis of plasmid pEF123 showed that plasmid was a novel sex bacteriocin sensing plasmid.In the framework structure of the plasmid,the replication region and conjugation region was conservative,but there was a new structure in the stability region.At the same time,plasmid pEF123 containing two multi-resistant regions.Insertion sequence IS1216 E plays a very important role in the formation of plasmid resistance regions.The plasmid resistance region was formed through the interaction between IS1216 E,Tn6248,Tn551 and the own homologous recombination of the IS1216 E.Blasting in the Genebank database,it was found that there were 30 species of strains carrying the bcrRABD cluster.The clusters carried in these strains were mainly divided into three evolutionary branches,and five kinds of mobile elements showed closer connection to the transfer of the cluster bcrRABD.In vitro time-kill curve test of the bcrRABD cluster cloned strains to bacitracin showed that after the insertion of the segement ISEnfa1-bcrRABD-ISEnfa1 into the recipient bacteria,bacitracin will lose the ability to inhibit the recipient bacteria,at the same time the growth rate of the recipient bacteria will not be affected.This study systematically investigated the transmission of bacitracin resistant genes in animal souce enterococcus.The results showed that the bcrRABD gene cluster can be spread by positive clone strains,horizontal transmation of plasmids,and the ISEnfa1-mediated mobile elements,in different provinces and pig farm.To provide a reference for assessing the impact of bacitracin use in livestock on the spread of resistance to animal-origin pathogens.At the same time provide scientific guidance to slow down the development of multidrug-resistant enterococci,exploring new resistance control strategies.