| In order to study the drug resistance characteristics of swine fecal Escherichia coli carrying the tigecycline resistance gene tet（X4）,the environment of the tet（X4）tigecycline resistance gene and the function of the tet（X4）gene,195 swine fecal samples and 56 swine fecal soil samples were collected from large-scale swine farms in Sichuan Province.A total of 200 strains of E.coli were isolated,including 149strains from swine feces and 51 strains from soil,The resistance phenotypes of E.coli isolates to 12 antibiotics including tigecycline were determined.The distribution of drug resistance gene,virulence gene and tet（X4）gene carried by 13 strains of tigecycline resistant E.coli were studied by second generation whole genome sequencing.The minimal inhibitory concentration and multiple drug resistance of tet（X4）positive E.coli to tigecycline were analyzed.Two strains of tet（X4）positive E.coli from different sources were selected for three generations of whole genome sequencing,and the genetic environment of the plasmid gene carrying tet（X4）was analyzed.Finally,the in vitro degradation activity of tetracycline by Tet（X）was preliminarily verified by constructing a genetically engineered strain.The main results and findings of this study are as follows:（1）195 swine fecal samples and 56 swine fecal soil samples were collected from large-scale swine farms in Sichuan Province.A total of 200 strains of E.coli were isolated,and the isolation rate was 79.68%.The degree of resistance of E.coli isolates to 12 kinds of antibiotics is different.200 strains of E.coli are highly resistant to tetracycline,amoxicillin,sulfadiazine and erythromycin,all of which are more than75%.All of the E.coli from swine fecal and soil are resistant to tetracycline.The highest resistance rate of E.coli from swine fecal to amoxicillin and sulfadiazine is84.56%and 95.30%,respectively,and the highest resistance rate of E.coli from soil to erythromycin is 90.20%.The multi-drug resistance of E.coli isolates is serious.The main drug resistance types of E.coli from swine fecal and soil are resistant to four and five.The highest multi drug resistance rate of E.coli from swine fecal is 97.32%.The main resistance types of tigecycline resistant E.coli from different sources were resistant to five（30.77%）and six（30.77%）.13 strains of tigecycline resistant E.coli produced 10 drug resistance profiles.（2）The second generation whole genome sequencing analysis found that among13 tigecycline resistant E.coli strains,the detection rate of tet（X4）,a tetracycline resistance gene,was the highest,accounting for 76.92%.The tet（X4）resistance gene encodes Tet（X）protein and has the function of inactivating tetracycline.The detection rate of bla TEM-1B inβ-lactam resistance genes was the highest（76.92%）.The highest detection rate of qnr S1 among quinolone resistant genes was 69.23%.sul3 has the highest detection rate among sulfonamide resistant genes,at 61.54%.The detection rate of flo R in chloramphenicol resistant genes is the highest,at 76.92%.Among 13 strains of tigecycline resistant E.coli,the detection rate of csg A and fim H was the highest,which were 100%.The detection rate of ast A in toxin virulence genes is the highest,at 23.08%.The detection rate of hly E in hemolysin type virulence genes is the highest,at 100%.The highest detection rate of tra T was 53.85%among the virulence genes of anti-serum survival factors.The detection rates of irp2,fyu A,iro N,and sit A in iron uptake toxicity genes were the highest,at 23.08%.In 10 strains of tet（X4）positive E.coli,the insertion sequence ISVsa3,IS470,flo R,rdm C and other genes exist upstream and downstream of the tet（X4）resistance gene,which is highly similar to other tet（X4）carrying sequences.The tet（X4）resistance gene may be located in the plasmid.The phenotypic analysis of drug resistance of tet（X4）positive E.coli showed that swine fecal E-5 and soil E-236 had the highest level of drug resistance to tigecycline,and there were many kinds of multi drug resistance.（3）By sequencing the whole genome of three generations of tet（X4）positive E.coli E-5 and E-236,and analyzing their genome sequences,it was found that two strains of E.coli carried four different types of plasmids,respectively named p LKYW51,p LKYW52,p LKYW261,p LKYW262,with plasmid sizes of 94399 bp,57933 bp,191683 bp,78911 bp,and plasmid types of p0111,Inc X1,Inc FIA（HI1）/Inc HI1A/Inc HI1B（R27）,Inc FIB,Plasmids carry a large number of antibiotic resistance genes and insertion sequences.The plasmids p LKYW52 and p LKYW261carry a large number of antibiotic resistance genes,including tet（X4）.The genetic environment of tet（X4）gene was analyzed,and it was found that there were insertion sequences such as ISCR2（ISVsa3）and IS26 in the upstream and downstream of tet（X4）gene.It is speculated that the plasmids p LKYW52 and p LKYW261 may have the ability to mediate multiple drug resistance,including tigecycline resistance,and may have horizontal transmission potential.（4）The gene engineering strain WH320-p HIS1525-tet（X4）was successfully constructed by using the tet（X4）positive E.coli E-5 as the gene source strain,the Bacillus megaterium expression line WH320-p HIS1525 system,TA cloning and other genetic engineering technologies.The WH320-p HIS1525-tet（X4）genetically engineered strain was induced by xylose,and Tet（X）crude enzyme solution was collected.The improved microbial method was used to demonstrate that the expressed Tet（X）crude enzyme solution has the ability to degrade tetracycline.The study showed that in swine fecal E.coli drug resistance rate of tetracyclines,β-lactam and other antibiotics is high,and the phenomenon of multiple drug resistance is serious.There are a large number of antibiotic resistance genes and virulence genes in tigecycline resistant E.coli,with a high positive rate.Most of tigecycline resistant E.coli carried tet（X4）resistance genes.A large number of antibiotic resistance genes and insertion sequences such as tet（X4）were found on plasmids in two strains of tet（X4）positive E.coli from different sources,and insertion sequences such as ISCR2（ISVsa3）and IS26 were found upstream and downstream of tet（X4）gene,indicating that insertion sequences such as ISCR2（ISVsa3）may play a key role in the horizontal transmission of tet（X4）.Genetic engineering technology was used to construct a WH320-p HIS1525-tet（X4）genetically engineered strain and xylose induction was performed,which preliminarily demonstrated that the expressed Tet（X）crude enzyme solution can reduce tetracycline activity,providing resources for further in-depth research in the future. |
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