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Study on the Mechanism of the β-lactamase、integron and Novel Gene in Clinical Mult-resistant Acinetobacter Spp. Strains

Author: ZhangXiaoNi
Tutor: LiJiaBin
School: Anhui Medical University,
Course: Internal Medicine
Keywords: Plasmid OXA type β - lactamase Acinetobacter Resistance Isoelectric focusing (IEF) Southern Blotting Enzyme kinetics
CLC: R440
Type: Master's thesis
Year: 2010
Downloads: 76
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Abstract

Objective: To understand the Anhui Province multidrug-resistant Acinetobacter-β-lactamase resistance gene, and to reveal its related resistance mechanisms, clinical therapy. Learn Anhui Province multidrug-resistant Acinetobacter integrated sub-genes and their associated resistance genes. Understand the molecular characterization of novel plasmid-mediated OXA-146 type enzyme. MATERIALS AND METHODS: Anhui bacterial resistance monitoring center collected in September 2007 in 35 hospitals, inpatient and outpatient clinical the submission specimens, no repeated separation of Acinetobacter 176. Using MH agar double dilution method for 14 kinds of wild mushroom antimicrobial drug sensitivity test. Boiling method to extract bacterial DNA carbapenemase and extended-spectrum enzyme specific fragments I, II and Ⅲ type integrase gene amplification and type I integrase gene positive strains of multi-resistant strains with type I integration The sub-structure of gene amplification, to reveal the existence of a variety of mechanisms of resistance. PCR-positive strains metastasis bonding test, parallel coding primer PCR amplification. Production of new enzyme strains of several sequenced and identified after GenBank than the following experiment: PCR amplification of the wild-type strain and / or transfer bonding OXA-146 in the sub-carbapenemase-encoding gene fragment was cloned into pUC- 118 vector, expressed in the Escherichia coli JM109 competent cells, the nucleotide sequence was determined again results to GenBank ratio, and DNA sequence analysis. New enzyme coding gene was cloned into the expression vector pET28a was transformed into competent cells E. coli JM109 transformants were screened with antibiotics agar. Wild strains using agar dilution method, the zygote simultaneously transformed strains MIC determination. The sonicated bacteria transformed strains producing new enzymes extracted crude enzyme isoelectric focusing (IEF) determination of its pI. Crude enzyme purification, SDS-PAGE and Coomassie blue staining, the size of the determination of the molecular weight of the new enzyme kinetics of enzymatic reaction to a preliminary understanding of the dynamics of the new enzyme. Using Southern Blotting testing again to verify that the new enzyme is mediated by the plasmid encoding gene resides plasmid size and position. Results: 176 Acinetobacter variety of antimicrobial agents including carbapenem antibiotics, including 34 pairs, including Acinetobacter Acinetobacter 16, Bowman / hemolytic Acinetobacter 15, calcium acetate 2 Acinetobacter, the Loepfe Acinetobacter one. 34 pairs of the carbapenem antibiotic resistant strains also piperacillin, piperacillin / tazobactam, cefotaxime, ceftriaxone, ceftazidime, cefuroxime, aztreonam, gentamicin, ciprofloxacin resistance. 34 multi-drug resistant Acinetobacter in 19 OXA-23-positive, TEM-1 positive of 24, 10 CTX-M-1-positive, 10 CTX-M-9-positive, 23 class Ⅰ integrase-positive. At the same time containing OXA-23-type enzymes and I int2 17. 23 Ⅰ integron-positive strains 21 class I integron gene structure, measured not detected OXA-24-type enzyme, IMP, VIM, the SIM-type metal enzyme group. 19 blaOXA-23-positive products were cloned DNA sequencing BLASTn than 17 with the gene pool of the same name exactly the same genetic sequence, two additional mutations. Mutant resistant Acinetobacter AB1622 and calcium acetate Acinetobacter OXA-23-type enzyme AC518 gene bank at the same time between 832 and 833 points nucleotide insertion of three bases (GGC) in 221 nucleotide sites caused by an alanine insertion mutations, the sequences in GenBank (respectively FJ194460; FJ194494) was Jacoby, George A named OXA-146 enzymes. The study found that the wild strains containing the TEM-1 enzyme AC518 CTX-M-type enzyme Ⅰ integron wild strains AB1622 containing TEM-1-type enzymes and Ⅰ integron. AC518 and AB1622 Ⅰ integron structural gene contained arr3 aacA4 not contain OXA-146 enzyme, TEM-1-type enzyme, CTX-M-1-type enzyme. Other class I integron structure of genes containing common constituent aacA4, eatB8, aadA1, art3 integron resistance gene cassette of genes, explained the research Acinetobacter of aminoglycoside antibacterials, rifampicin, chloramphenicol prime resistant reason. Producing OXA-146 the enzyme strains of transfer bonding test and cloning expression test success, the wild strains zygote and transformed strains MIC results show: the wild-type strain and the zygote has almost the same resistance spectrum, and the conversion of strains on the sensitivity of some of the drugs significantly decreased compared with the previous two. Production OXA-146 type enzyme bacterial transformant isoelectric focusing and SDS-PAGE results showed that its isoelectric point is about 7.7. The Southern Blotting trial once again confirmed that the new enzyme from the plasmid-mediated and locate its coding gene located 52Kb plasmid. Conclusion: This study showed Anhui multi-drug resistant Acinetobacter β-lactam antibiotics, carbapenem antibiotics, aminoglycoside antibiotic resistance and producing OXA-23-type carbapenem enzyme TEM-1 type, CTX-M-1-type and CTX-M-9 extended-spectrum β-lactam enzymes and class I integrons containing aminoglycoside resistance gene. 19 blaOXA-23-positive product coding gene alignment analysis identified two mutations are the first in Anhui Province was found. Production the new enzyme strains exist a variety of mechanisms of resistance, and can be passed between the different strains by plasmid, it is necessary to strengthen the region's production of plasmid-mediated OXA-23-type enzyme strains of monitoring and to drug data exchange with other regions to the formation of a large drug monitoring network, they work to prevent the spread of resistance genes epidemiological significance.

Study on the Mechanism of the β-lactamase、integron and Novel Gene in Clinical Mult-resistant Acinetobacter Spp. Strains

Abstract

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