Study On AmpC Gene And Fluoroquinolones Gene Mutation In Escherichia Coli

Objective To learn the case of Escherichia coli(E.coli) carrying amp C gene and Fluoroquinolones genes mutation in Luzhou. And investigate to the correlation between the amp C and Fluoroquinolones genes. And analysis of amp C gene positive strains and Fluoroquinolones genes mutation strains resistant to commonly used fluoroquinolone antimicrobial drugs. Methods 1.Polymerase chain reaction(PCR) technique: PCR used bacterial genomic DNA extraction kit according to instructions extracted bacterial genomic DNA was used as PCR template. Five pairs of primers were designed and synthesized for amplification. Amplified products were subjected to agarose gel electrophoresis, and the photographic image processor to 254 nm wavelength UV light. 2. Gene sequencing: PCR products were recovered by measuring the concentration of DNA nucleic acid quantification instrument qualified(greater than 300 ng / μl), and sequenced by the Shanghai Sangon biotechnology companies. 3. Kerby-Bauer disk diffusion method: 3.1 Identification of high-producing-Amp C strains: If the diamerter of inhibition zone of cefoxitin was smaller than 18 mm as suspicious Amp C-producing strains. Cefoxitin three-dimensional test methods to detec E.coli β- lactamase can hydrolysis cefoxitin(eclipse ring phenomenon) to validate the enzyme production strains.3.2 Kerby-Bauer disk diffusion method : Determinating the sensitivity of the clinical isolates of Escherichia coli to OFX、LOM、CIP、NA、NOR、FOX、CTX、FEP、ATM、IPM、S、GEN total 12 kinds of antimicrobial agents. Results 1. The amp C gene positve case: 71 E. coli amp C gene positive detection had 61(85.92%). 2. Genetic variation :There were 49 stains appearing S83 L amino acids substitutions and 36 appearing D87 N amino acids substitution to Gyr A subuni. Gyr B subunit had gene mutations, but there was no amino acid substitution. Par C subunits appeared 34 S80 I and 8 A56 P replacement. Par E subunits appeared 6 E84 G, 4 S458 A and 3 A426 V replacement. There were 51strains(92.72%) carrying amp C gene and Fluoroquinolones gene mutation. 3. Amp C beta-laetamase-producing strains: 10 strains were suspected Amp C-producing, accounting for 16.39%. There are 8 strains producing Amp C beta-lactamase, accounting for 13.11%. 4. Antimicrobial susceptibility test results of 71 E.coli strains:(1) All the 71 strains were sensitive to imipennem. Lots of them were sensitive to cefoxtin, aztreonam, cefepime and fluoroquinolone drugs. They had a lower susceptibility to the other antibiotics.(2)The resistant rate of amp C gene-positive strains for NA, NOR, OFX was higher than that of amp C gene negative strains(P <0.05). The resistant rate of high-Amp C-producing strains resistance to ciprofloxacin was higher than that of non-Amp C-producing strains(P <0.05).(3) Multidrug-resistantEscherichia coli had 21 strains(38.18%), the amp C gene-positive strains with the quinolone gene mutations account for 71.43%. Amp C-producing strains accounted for 38.10%. In addition, the phenomenon of cross-resistance strains to quinolones was also more common, the strains resistanted to more than three quinolones had 22(40%).(4)The mutation sites of multi-drug resistant strains compared with the double resistance did not differ much, but the difference was the emergence of amp C positive and gyr A + par C + par E gene mutations. There was a difference compared to all sensitive and single resistant strains, which was only individual locus mutation. Conclusions 1. The amp C gene positive strains accounting for 85.92% in clinical isolates of E. coli strains, 90.48% in multi-drug resistant strains, the high-Amp C-prducing strains accounting for 11.27%, but the main distribution in multi-drug resistant strains. 2. Quinolone gene mutation is common in clinical isolates, multi-site mutation is mainly concerned with the mutation of gyr A and par C. In multi-drug resistant strains, the mainly mutation of gyr A, par C and amp C-positive. 3. The drug resistance rate of amp C-gene-positive strains are higher than that of amp C gene-negative strains to NA, NOR and OFX. The drug resistance rate of the high-Amp C-prducing strains to ciprofloxacin are higher than that of non-Amp C-producing strains.