| In recent years, the overuse of antibiotics in livestock farming and clinical medicine has caused the frequent occurrence of multi-drug resistant bacteria. However, the mechanism is not clear. Some researches show that Plasmids often carry some movable elements(e.g. transposons) to capture different resistance genes and produce resistance, which is one of the main mechanisms for the spread of antibiotic resistance genes. In this study, we aim to investigate the role of the plasmid in horizontal gene transfer. The main results are as following:Firstly, the identification of multidrug resistant bacteria isolation, and testing for drug resistance in the chicken farm manure: We use culture medium(LB medium or SS medium) with one of eight kinds of antibiotics(gentamicin, chloramphenicol, amoxicillin, cephalexin, ciprofloxacin, tetracycline, erythromycin and kanamycin) to isolate 32 multiple drug resistant bacteria from the chicken manure. These multiple drug resistant bacteria belong to 13 different genera, the most of which are gram-negative bacteria. In LB culture medium, G- bacteria and G+ bacteria accounted for 63% and 37% of these bacteria, respectively; in the SS medium, G- bacteria and G+ bacteria accounted for 87% and 13% of these bacteria, respectively. In these bacteria, the most bacteria occupied erythromycin resistance, but only a few bacteria occupied amoxicillin resistance. These multi-resistant bacteria belong to Proteobacteria, Actinobacteria and Firmicutes. Antibiotic resistance genes often co-exist with heavy metal resistance genes in the same mobile genetic elements. Our results have showed that 2 strains of 8 multiple antibiotic resistant bacteria displayed stronger resistance to Pb, Cu, Mn and Zn than the E.coil without antibiotics resistance.Secondly, the study focuses on the testing and genetic characterization of multiple drug resistant plasmids. To study the horizontal transfer of the drug-resistant genes, the plasmids from muti-resistant bacteria were extracted and transformed into strain DH5 a. The results have showed that the plasmid with a size of about 15 kb was found in DH5 a. The DH5 a carried the above size plasmid can grow in LB solid medium supplemented with tetracycline, kanamycin and cephalexin, suggesting that the plasmid may carry the resistance genes against these three antibiotics. Sequencing analysis has indicated that the plasmid has a size of 14591 bp and possess the similarity of 99 % with a plant binary shuttle expression vector pZGA22. By bioinformatic analysis, we have found that the plasmid carries resistance gene encoded with tetracycline(tetR_C, tetR_N), phosphotransferase enzymes(NTPII, NTPIII), and one superfamily domain Glo_EDI_BRP which can simultaneously control multiple antibiotic resistance and heavy metal resistance. All of these results has suggested that this plasmid probably mediated horizontal transfer of antibiotic resistance genes in the environment.And in the last, we have investigated the presence of this plasmid in different multi-resistant bacteria in chicken manure. We first performed an enzyme digestion for the 15 Kb plasmids in different multi-resistant bacteria. Electrophoresis pattern has suggested that all these 15 Kb plasmids might be the same plasmid. Next, based on the complete sequence of the plasmid, we have designed 12 pairs of primers to performed a PCR amplification. The results from sequencing and splicing have showed that the same plasmid does exist in Corynebacterium xerosis, Vagococcus fluvialis, Escherichia coli, Proteus sp. Oceanobacillus oncorhynchi, Citrobacter sp. All the data has suggested that the plasmid plays an important role in horizontal transfer of antibiotic resistance genes between different species of bacteria in the environment. |
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