| Escherichia coli(E.coli)is the most common gram-negative bacterial pathogen and is highly susceptible to changes in environmental stress.The serotypes of some E.coli are zoonotic serotypes which need to prevent from public health.There have been many outbreaks of food-borne or water-borne diseases all over the world caused by strains with multi-drug resistance.Antibiotics have been used in the control and treatment of E.coli infections for years,and also been used as feed additives to promote animal growth and development.However,excessive use and abuse of antibiotics forced strains to develop their resistance.During the spread of bacterial drug resistance,the integration and recombination mechanism of the integrin-gene cassette system can make the resistance gene spread quickly,so it has attracted widespread attention from researchers.Type I integron have three different genetic regions: two conserved segments(5'-CS and 3'-CS)and a variable region that are capable of capturing and expressing multiple genes from the environment.5'-CS carries an intase gene Int I,promoters Pc and P2,and recombination sites att I1 and att C(secondary recombination sites are also called 59 bases or 59-be).3'-CS contains qac E?1-sul I gene(encoding resistance to sulfonamide and quaternary ammonium salt)and orf5 gene whose function is unknown.The gene cassette can exist in a free circular form,cannot be replicated but can be integrated in the variable region between 5'-CS and 3'-CS and expressed using an integrin promoter,thereby conferring strains with aminoglycosides,?-lactams,trimethoprim and chloramphenicol resistance,which also considered as the adaptive evolution of bacteria.In this study,large-scale pig farms in 4 provinces were selected,and Escherichia coli from pigs was used as the research object.A total of 330 swine rectal swabs and stool samples were collected for the identification and serotype determination of Escherichia coli.18 antibiotic susceptibility tests were performed on the isolated strains using the paper diffusion method(K-B)recommended by CLSI.The PCR method was used to detect type I integrants and their gene cassettes.SPSS was then used to analyze the correlation between the drugresistant phenotype and genotype of the strains.The main findings are as follows:(1)A total of 330 samples were taken from four provinces of Hebei,Shaanxi,Shanxi and Anhui,and 264 strains of Escherichia coli were isolated,with an isolation rate of 80%.O antigen single factor serum was used to determine the serotypes of 189 strains,including O8,O9,O20,O45,O64,O101,O138,O141,O147,O149,and O157.The top three serotypes were O8(18.5%),O138(18.5%),and O101(12.2%),and the lowest serotype was O45(2.7%).(2)The K-B method was used to test the sensitivity of strains of 18 antibacterial drugs.Results show that there were three types of antibacterial drugs with higher resistance,including tetracycline antibiotic tetracycline(63.6%),doxycycline(62.9%),and sulfafurazole(59.5%).The four antibiotics with lower resistance are erythromycin(22.3%),neomycin(22.3%),ciprofloxacin(13.3%)and norfloxacin(11.4%).A total of 184(69.7%)E.coli isolates showed MDR,11 MDR patterns covered 3 to 13 antibiotics,and the most common type of MDR was 6 to 7 antibiotics.(3)PCR detection of type I integrase and gene cassette carrying status of isolated strains,SPSS analysis of the correlation between type I integrase and strain resistance phenotype,and gene analysis of gene cassette positive strains.197 strains of 264 E.coli were detected to carry the type I integrase(74.6 %),The most common resistance to integrase-positive strains is doxycycline(70.1%),tetracycline(69.0%),sulfamethoxazole(67.0%),amoxicillin(66.5%),sulfafurazole(64.5%),florfenicol(54.3%)and ampicillin(53.3%).Comparing the differences in the resistance phenotypes of integrase-positive and negative strains,it was found that in addition to kanamycin,amikacin,and sulfafurazole,integrase-positive strains showed higher resistance beyond the negative strains(P <0.01).70 strains(30.5%)carried the gene cassette,containing 12 different gene cassettes,and endowed with aminoglycoside antibiotics(aad A1,aad A2,aad A5,aad A16,aad A1-1,aad A1-2),trimethoprim(dfr A1,dfr A7,dfr A12,dfr A17)and resistance to chloramphenicol(cml A1).An unknown type of resistance gene cassette(orf F)was also discovered.A total of 17 types of gene cassette combinations were found,including 11 simple gene cassette combinations and 5 gene cassette variants with different insertion sequences of dfr A12-orf F.Among all strains carrying gene cassettes,the most common combination of gene cassettes is dfr A17-aad A5(19 strains in total,accounting for 27.14% of all gene cassette-positive strains).Analyzed the relationship between the gene cassettes combination carried by the gene cassettes positive strain and its drug resistance phenotype,result shows that strains resistant to sulfonamides / trimethoprim,aminoglycosides and florfenicol(82.9%,58/70)were found to have corresponding gene cassettes(dfr,aad and cml gene cassettes).The results of this study will provide a reference for large-scale farms to carry out epidemic prevention against different serotype strains,provide guidance for the selection of sensitive drugs for the prevention and treatment of E.coli.Provide basic data for further research on the role of the integron-gene cassette system in the multi-drug resistance spread of strains. |
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