| Drug resistance of animal-derived bacteria is a major threat to public health and food safety,and has been widely concerned.The intestinal tract of food-animals,which suffers the long-term selective pressure of antibiotics,is considered as the “generator” and “amplifier” of the spread of antibiotic resistance genes(ARGs).The intestinal flora is composed of abundant and diverse bacterial species,which provides a good environment for the horizontal exchange of antibiotic resistance genes.However,little is known about the spreading mechanism of ARGs in the food-animal intestine.In this study,floR-resistant strains from one chicken intestinal flora were collected,and the distribution pattern and transmission mechanism of floR gene in intestinal tract were investigated in strain and species-level,which provided scientific data for understanding the generation,transmission and distribution of ARGs in food-animal intestinal tract,and theoretical basis for further controlling the spread of ARGs.In this study,intestinal flora was taken as the research object,and the fecal samples at different time points(D1,D8,D15,D22,D29,D35,D41 and D46)in the whole life of one chicken were tracked and collected.Using antibiotic-containing chromogenic Agar,florfenicol-resistant Gram-negative strains were isolated and collected to establish the resistance-strain library.On this basis,the floR gene was screened by PCR,and the floR-carrying strains were identified by MALDI-TOF MS technology.PFGE was used to identify the genetic relationship of the isolated strains.Representative strains of different PFGE types were subjected to conjugation experiment and minimum inhibitory concentrations(MICs)tests.Whole genome sequencing(WGS)and bioinformatics analysis were used to obtain the full-length of the floR-carrying vectors for the transconjugants and to draw the genetic structures of floR transmission in the chicken intestinal flora.The results showed that:(1)A total of 500 strains were collected by chromogenic Agar supplemented with florfenicol(15 mg/L),PCR screening,MALDI-TOF MS identification showed that all strains carried floR gene,including 427 strains of Escherichia coli(85.4%),48 strains of Klebsiella pneumoniae(9.6%)and 25 strains of Enterobacter cloacae(5.0%).39 E.coli strains,2 K.pneumoniae strains and 18 E.cloacae strains were isolated on D1 day and 53 E.coli strains 11 K.pneumoniae strains,3 E.cloacae strains were isolated on D8.and 45 E.coli strains 7 K.pneumoniae strains,1 E.cloacae strains were collected on D15.and 64 E.coli strains,2 K.pneumoniae strains,2 strains of E.cloacae strains were isolated on D22.56 E.coli strains and 8 K.pneumoniae strains were isolated on D29,30 E.coli strains and3 K.pneumoniae strains were isolated on D35,73 E.coli strains and 15 K.pneumoniae strains were isolated on D41,and only 67 E.coli strains were isolated on D46.(2)The genetic relationship of floR-positive strains was analyzed by pulsed-field gel electrophoresis(PFGE).The results showed that 500 strains were divided into 53 different PFGE patterns,of which 40 different E.coli PFGE types,12 different K.pneumoniae PFGE patterns and 1 E.cloacae PFGE pattern were identified.In addition,the phylogenetic tree analysis showed that some strains with highly similar PFGE patterns(>80.0%)could be isolated at all sampling points,indicating that these floR strains could stably plant and clonally spread in the intestinal flora.The MIC results showed that the resistance rate of 53 representative strains to florfenicol and chloramphenicol was 100.0%.High resistance rates were observed for cefpodoxime(90.6%),tetracycline(98.1%),streptomycin(96.2%)and ciprofloxacin(96.2%).These strains showed 64.2% and 84.9% resistance rates to gentamicin and co-trimoxazole respectively.Resistance rates of amikacin,polymyxin and tigecycline were observed for 32.1%,28.3%,22.6%,respectively.No drug-resistant strains were detected.(3)In order to investigate the transferability of floR gene and its vectors,conjugation experiments of the53 strains with different PFGE types were performed.Transconjugants were successfully obtained for 24 strains of E.coli with transfer frequency ranging between 3.67×10-5-9.04×10-3.The success rate of conjugation was 45.3%(24/53).The MIC test result showed that the MIC values of all the transconjugants to florfenicol and chloramphenicol increased by 16 times,and some transconjugants showed resistance to tetracycline,streptomycin,cefpodoxime,gentamicin,co-trimoxazole and ciprofloxacin indicated that cotransfer of other resistance genes occurred in the conjugation process.(4)In order to illustrate the transmission vectors and gene environments of floR,24 transconjugants and2 strains failed to transfer were subjected to WGS and bioinformatics analysis.The results showed that the floR gene could be located both on different plasmids and genome.(1)The floR gene of the 2 strains failed to transfer was located on a new genetic element of 77 kb,which was inserted into the t RNA(Phe)(GAA)gene of E.coli,which formed direct repeats(DRs)at both ends.The resistance gene clusters carried by the element included floR,tet A,tet R,aph6-Ic and aph3-I genes.Sequence alignment showed that this element also existed in the genome of E.coli H9 strains(Gen Bank accession number: CP029180),suggesting that it had potential transferability.(2)In most of the strains,the vectors of floR are plasmids with the sizes ranging from 99 kb to 180 kb,which can be divided into five types.Each plasmid contained plasmid transfer modules.Five plasmid types contained many drug resistance genes,such as floR,sul3,bla TEM-1,bla CTX-M,tet A,tet R,fos A3 and aph-3-Ib.Some plasmids contained the gene cluster iro NEDCB that promotes iron absorption,the manganese transporter sitabcd and the potassium tellurite resistance gene encoded by the ter gene cluster.In addition,bioinformatic analysis showed that some of the five different types of plasmids had novel and unique structures,which different from previously-reported floR plasmids.The five types of plasmids in this study contained a large number of tnp genes and IS sequences,which may play an important role in the formation of the hybrid plasmids mediated by fragment exchange and recombination between different plasmids.In summary,this study preliminarily explored the distribution characteristics of floR gene in chicken intestinal flora.The floR gene has rich host diversity at species-level and strain-level,suggesting that the distribution and diffusion of floR gene in chicken intestinal flora could be mediated by horizontal transfer and clonal transmission.A variety of floR gene vectors co-exist in the same intestine also shows the complexity of ARG transmission in the intestinal tract.In addition,Co-existence of floR gene and other ARGs in the same transfer element increases the risk of co-transfer of multiple drug resistance.Systematic research on distribution characteristics and transmission patterns of important ARGs and their carriers in intestine should be further strengthened to provide scientific basis for drug resistance prevention and control. |
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