Detection Of Integron In ESBLs Producing Strains And Their Resistance Phenotype Correlaiton Analysis

In recent years with extensive use of Antimicrobial drugs on veterinary clinic,many pathogens, especially ESBLs positive strains, produced a wide range ofmulti-drug resistance, which made enormous difficulties to the veterinary clinical.Therefore, doing more research on resistant mechanism of ESBLs positive strains issignificance. In this study, bacterial drug sensitivity test and PCR detection were usedto research the role of extended spectrum β-lactamases (ESBLs) and the integron inthe ESBLs positive strains multiple drug resistance, in order to further clarify themechanism of resistance to ESBLs-positive isolates basis.First, the screening and identifying of20strains of ESBLs-positive isolates:69clinical isolates of pathogenic E. coli and Pseudomonas strains were identified byESBLs screening and confirmatory detection methods recommended by CLSI. Theresults showed that20strains in69strains of bacteria were detected ESBLs positivestrains. The detection rate is28.99%, of which16strains are pathogenic E. coli,3strains are Pseudomonas aeruginosa and1strain is Pseudomonas putida.Secondly, from the molecular level to detect the existence of integron in ESBLspositive strains. Genomic DNA was extracted by boiling bacterial as template, usingsimultaneous amplification ofⅠ,Ⅱ,Ⅲtype integrase gene of degenerate primers forPCR detection of ESBLs-positive isolates, combined with endonuclease restrictionfragment length polymorphism analysis to determine the integron type. The resultsshowed that20ESBLs positive isolated strains carry typeⅠintegron. In addition,YD1, YLN and LN11073strains carried bothⅠandⅡtype integron, which showedthat typeⅠintegron positive rate is100%while typeⅡis15%.TypeⅠintegron insertion gene cassettes sequence was amplified by PCR,sequence analysised of the drug-resistance gene cassettes.20ESBL integron-positivestrains were PCR amplified using classⅠintegron-resistance gene cassettes primersdesigned by Du XD. The PCR product was cloned into pMD-18T vector multiplecloning site then recombinant plasmid was cloned and sequenced. Log in GenBankand the sequencing results were BLAST to confirm the type of resistance genecassettes. The results showed that8strains in20strains tested bacteria amplified2different gene cassette insertion sequence, in JD4strains carryingⅠintegron genecassettes inserted sequence of about2315bp, the remaining seven bacterial carryingclassⅠgene cassette inserted sequence of about2065bp. The two gene cassettes ofdifferent sizes containing two resistance genes are mainly amino sugars dihydrofolatereductase and adenosine transferase, then the bacterias present antibiotics resistanceon the trimethoprim sulfadiazine and the aminoglycoside.Finally, analyzing the correlation among ESBLs, integrons and antibioticresistance phenotype. Using K-B disc agar diffusion method to detect DrugResistance of clinical-isolated69strains. Using the comparative analysis of statisticalmethods to detect the differences of the resistance rate between integron-positivestrains of ESBLs and negative, also between ESBLs integron-positive isolatescarrying resistance gene cassettes and do not carry resistance gene cassette. The results showed that Drug Resistance of ESBLs integron-positive strains wassignificantly higher than non-ESBLs integron-negative strains, two showed significantdifferences (P <0.001), but the Drug Resistance between ESBLs integron-positiveisolates carrying resistance gene cassettes and do not carry resistance gene cassettewas no significant difference.In this study20ESBLs-positive strains were selected from69clinical isolates.TypeⅠintegron positive rate is100%while typeⅡis15%. The results showed thatDrug Resistance of ESBLs integron-positive strains was significantly higher thannon-ESBLs integron-negative strains, but the Drug Resistance between ESBLsintegron-positive isolates carrying resistance gene cassettes and do not carryresistance gene cassette was no significant difference.