Environment Quinolone Resistance Gene Detection Method And Its Application

Antibiotics have been widely used in clinical, agricultural production and animal husbandry, etc. since their discovery. Within the environmental problems that antibiotics have induced, resistant bacteria has become an important topic.In this work, water samples of Beijing Wenyu River Basin were sampled to investigate the resistance of E.coli strains to quinolones. The main tasks were as follows: detecting the resistance genes to tetracycline, sulfanilamide andβ-lactamases of total DNA in gram-negative bacteria on the membranes, investigating the rates of quinolone-resistant E.coli strains, testing the minimum inhibitory concentrations (MICs) of all 161 E.coli strains to quinolones, analysing the mutation points and virulence factors (VFs) in E.coli.Along the Wenyu River Basin, 12 sampling points were designed. By PCR combining with agarose gel electrophoresis method, the resistance genes of total DNA in the gram-negative bacteria which had been overnight cultured on the membranes were detected. Genes resistance to three antibiotics were detected, which were sulfonamides (sulⅠ, sulⅡ, sulⅢ), tetracycline (tetA, tetB, tetM) andβ-lactamases (TEM) resistance genes.The results showed that, resistance genes to sulfonamides, tetracyclines andβ-lactamases had a wide distribution along Wenyu River Basin. The Kunming Lake sampling point, which located on the upper reaches of other samples and was very far away from the living area,. contained all the other resistance genes except for tetB, sulⅢgenes. Some resistance genes were not all contained in 1, 6, 8 sampling points as well. Other sampling points, such as 5, 12, 18, 19, 20, 25 and 26, however, had positive results of all the resistance genes in addition to tetM. TetM was not detected in all points. From the detection of resistance genes in all the sampling points, a conclusion could be made that these types of antibiotic resistance genes were universally contained in the Wenyu River water. The distribution of the resistance genes did not manifold or reduce along the water flow, instead it can be influenced by many factors.At the same time, quinolone resistance-determining region (QRDR) of gyrA gene in E.coli were amplified in the PCR reaction using total DNA as templates, and the PCR productions were sequenced and were compared by Bioedit. But the amino acid sequences deduced had no resistance mutation points corresponding with the research already reported.According to American Society of Clinical Laboratory Standards Institute (CLSI, formerly known as NCCLS), Escherichia coli whose MIC to levofloxacin (LEV) or gatifloxacin (GAT) reaches 8 ug/mL are defined as resistant. So, the membranes containing E.coli were cultured on non-antibiotic containing plates and 8 ug/mL-quinolone containing plates separately. Numbers of E.coli strains growed on the different plates were counted to get the rates of LEV-resistance E.coli strains and GAT-resistance E.coli strains among the river. The incidence of LEV-resistant E.coli was between 2.3% ~ 31.3%, while the incidence of GAT-resistance E.coli strains was between 0.8%~ 57.8%. In order to further affirm the occurrence of quinolone resistance in the environment, we selected and cultured 161 E.coli strains from membranes of different sampling points cultured on different gradients of quinolone-containing plates.Resistance analysis of E.coli strains to quinolones included two aspects, resistance ability and resistance mechanism.First of all, the resistance ability of E.coli strains to quinolones were detected, that is, the minimum inhibitory concentration (MIC) to quinolones. The MICs of each E.coli strains to two types quinolone agents were determined by agar plate dilution method. The results showed that of the same bacteria strains, the MICs to the third-generation quinolone LEV, was significantly higher than to the fourth-generation quinolone GAT. There were two E.coli strains whose MICs of LEV were even as high as 128 ug/mL. The E.coli strains also had a certain ability of resistance to GAT, the highest MICs of which was 32 ug/mL.Secondly, the mutation points in the QRDRs of E.coli with different MIC levels were analysed. QRDRs of gyrA gene and parC gene were focused to search for the effect of mutation points on the resistance ability of E.coli to quinolone. Mutation analysis showed that, S83L mutation point presented universally, and it took place in both low MIC strains and high MIC strains, while the D87N mutation point appeared in resistance strians, and was a key mutation point for resistance; D87Y mutation point occurred mainly in the E.coli strains with MICs of 128 ug/mL and 64 ug/mL to LEV, so we suggested D87Y mutation point to be a key mutation point which enable E.coli strains to have high capacity of resistance.Resistance genes in E.coli and the resistant bacteria strains themselves are a kind of environmental pollution, and the pathogenic E.coli strains exacerbated the threat of this pollution. The E.coli strains are a normal intestinal flora in human and other animals, but some serotypes of E.coli can also lead to diseases, such as the urinary tract infection, gastro-intestinal infections, diarrhea, septicemia and so on. In this paper, multiple-PCR method were used to analysis 6 virulence genes in E.coli. The results showed that, in 161 E.coli strains, a rate of 31.7% strains contained at least one virulence factor, and virulence factors were relevant to antibiotic resistance: within all E.coli strains containing virulence factors, the rate of resistant to levofloxacin strains reached 80.4%, and the rate of gatifloxacin-resistant strains reached 52.9%. There was a high proportion of virulence E.coli strains that were resistant to quinolone agents.