Study To Plasmid-Mediated Genotypes In Multiple Drug- Resistance Escherichia Coli

Objective To investigate ESBLs-producing genotypes and drug-resistance of CTX-M-22,CTX-M-24, CTX-M,CTX-M-3,SHV,TEM in plasmid-mediated multiple -drug resistance Escherichia coli(E.coIi),which were isolated from the patients in Affiliated Hospital of LuZhou Medical College,and to provide theoretical basis. Methods 1. The Kerby-Bauer disk diffusion method from the National Committee for clinical Laboratory Standards, NCCLS) of America identified what kinds of drug-resistance in 71 E.coil. Antibacterials included cephalosporins, aminoglycosides and fluoquinolones, control group from ATCC 25922 of standard E.coil strains.2. PCR technique:Firstly, the plasmid DNA were refined according to specification. CTX-M-22, CTX-24, CTX-M, CTX-M-3, SHV, TEM genes were amplified by PCR technique. Genes size was detected by agarose gel electrophoresis and sequencing. The volume of PCR reaction is 35μl (15μl of 2×PCR MasterMix,7μl of refined DNA template,5ul of each primer,8ul of ddH2O). Amplification was carried out as follows:initial denaturation at 94℃ for 3min and denaturation at 94℃ for 30s, annealing at 55℃ for 30s and extension at 72℃ for 30s, amount to 30 cycles, and a final step of 72℃ extension again for 5min. PCR products were visualized in the ultraviolet light after 2% agarose gels electrophoresis made in 0.5×TBE ethidium bromide, and photographed with gelatin camera.3. Sequencing:The PCR products were sequenced, and then compared with known sequences. Results 1. A total of 71 strains of E.coil were isolated, the fully susceptible was 6 strains(8.57%), the one drug-resistant was 16 strains (17.14%), the double drug-resistant was 19 strains (27.14%) and the multiple-drug resistant was 30 rains (42.86%) respectively.2. The plasmids with genotypes CTX-M-22, CTX-M-24, TEM, SHV, CTX-M, CTX-M-3 among multiple-drug resistance E.coil had 9 (12.86%),24 (80.0%),11 (36.67%),1 (3.33%),23 (76.67%),21 (70.0%), respectively.3. Polygenic of CTX-M-22+CTX-M-24, CTX-M+CTX-M-3, CTX-M-24+CTX-M had 6 (20%),12 (30.0%),12 (30.0%), respectively. A strain(3.33%) included five kinds of drug-resistance genotypes. No genes was 3 strains (10.0%). Chi-square test showed that the difference between CTX-M family genes (CTX-M,CTX-M-3, CTX-M-22,CTX-M-24) and other group of SHV,TEM merged together was satistically significant (P<0.001). The difference between strains with CTX-M-24, CTX-M-3, TEM, CTX-M, CTX-M-24+CTX-M genes to susceptibility of AMK and TM was satistically significant (P<0.05). The difference between strains with TEM, CTX-M, CTX-M-3 genes to susceptibility of CFX and CPZ showed satistically significant (P<0.05). Conclusions Plasmid-mediated TEM, SHV, CTX-M, CTX-M-3, CTX-M-22, CTX-M-24 genotypes occurrs in E.coil with multiple-drug resistance in LuZhou. The main in single ESBLs-producing genes have CTX-M, CTX-M-3, CTX-M-24. The major polygenes in multi ESBLs-producing have CTX-M+CTX-M-3, CTX-M-24+CTX-M and special case that is 3 kinds of different genotypes at same plasmid in E.coil with multiple-drug resistance.