[Co-existence of resistance genes and their association with the genetic marker of integrons among multi-resistant Escherichia coli isolates].

OBJECTIVE: To investigate co-existence of resistance genes (beta-lactamases, BLs, and aminoglycoside-modifying enzymes, AMEs) and their association with the genetic marker genes of Class I, II, III integrons carried by multiresistant Escherichia coli isolates. METHODS: We used VITEK-GNS to determine the susceptibility of 136 isolates to 14 antibiotics, disc agar diffusion test to confirm ESBL-producing isolates, PCR to analyze BLs, AMEs and integrons genes, conjugation and plasmids extraction to locate the methylase genes. RESULTS: We found that 70.59% of the isolates produced ESBLs. They showed stronger resistance against 9 antibiotics than isolates without ESBLs in 14 antibiotics. PCR amplification showed that the positive rate of BLs, AMEs and qacEdelta1-sul1 was 96.32% , 100% and 94.12%, respectively, but Class II, III integrons genes were negative. Only one strain was oprD2 gene negative. 90.44% of the isolates were both positive for BLs and qacEdelta1-sul1 genes, and 94.12% for AMEs and qacEdelta1-sul1 genes, but there was no statistical significance. 90.44% of the isolates were all positive for the 3 genes. 12 strains carried 16S rRNA methylase genes including armA (2.21%), rmtB (7.35%) while rmtA, rmtC, rmtD were negative. The conjugation assay and plasmids mapping results showed that the methylase genes were located on the 23 kb plasmid, and the efficiency of transformation was 83.3%. CONCLUSIONS: The results suggested that there was a tight correlation between the 3 genes (BLs, AMEs and qacEdelta1-sul1) and the incidences of multi-resistance of Escherichia coli, but there was no correlation of the incidence of multi-resistance with Class II, III integrons. 16S rRNA methylase genes harboured plasmids of -23 kb which transformed other isolates within the same strains efficiently.

[Expression, purification and application of bla(TEM-116) extended-spectrum beta-lactamase].

To produce TEM-116 extended-spectrum beta-lactamase (ESBL) from recombinant bacteria in a cost-effective way, we purified and renatured the recombinant TEM-116 ESBL from the inclusion bodies by Ni(2+)-NTA affinity and gel filtration chromatography through subcloning the bla(TEM-116) into expression vector pET28a(+), transforming into Escherichia coli BL21(DE3) and inducing with IPTG. We characterized the purified protein that had the molecular weight of 30 kDa and specific activity of 476 IU/mg. The recombinant TEM-116 ESBL showed higher efficiency in eliminating penicillin and cephalosporin in vitro and in vivo. Specifically, the recombinant TEM-116 ESBL could eliminate 7000 mg penicillin G (PG) when used at 10.0 IU in 1 L fermentation medium. When used at 320.0 IU, it could also degrade a mix of PG, ampicillin and cefazolin each at 200 mg in 1 L of urine. In milk, 1.0-2.5 IU of the recombinant enzyme could remove 80 U/L of PG. The recombinant enzyme was fully active at the temperature ranged from 4 degrees C to 37 degrees C. Furthermore, the recombinant enzyme used at 2.0x10(4)-2.3x10(4) IU/(kg bw) (body weight) eliminated 8.0x10(4)-9.1x10(4) microg/(kg bw) PG in mouse models in vivo. The recombinant TEM-116 ESBL has the potential as a tool enzyme in food and environmental protection to eliminate harmful residues of antibiotics.

[Conserved region analysis of aac(3)-II gene from E. coli aminoglycoside resistance strain].

According to standard K B method, bacteriostatic tests were performed to screen out aminoglycoside resistance bacteria from 47 strains of isolated E.coli. To analyze correlations between the degree of E.coli aminoglycoside resistance and aac(3)-II gene conserved region, PCR amplified aac(3)-II gene conserved regions and were analyzed by DNA sequencing. The results showed that there were two species of aac(3)-II gene type including 65G and 84T or 65A and 84C in the samples. Strains with high activity of modifying enzyme to gentamicin all were 65G and 84T aac(3)-II gene type.