| Vibrio harveyi is a conditioned pathogen of aquatic animals. Excess use of antibiotics to control vibriosis, however, lead to the drug resistance of vibriosis as well as a increasing number of drug-resistant bacteria, which caused huge economic losses to aquaculture. Thus,disease control has become more difficult. Studies on the resistant mechanism of pathogenic bacteria found that antibiotics can cause oxidative stress in the pathogenic bacteria cells, so as to damage or kill cells. So in addition to classic resistant mechanism,the removal of ROS by GSH antioxidant system may be a non-classical mechanism for pathogenic bacteria to develop drug resistance.This study using Genome DNA of vibrio harveyi ZJ0603 as templates, cloned and analyzed the gene of gs, gcs and gr with PCR. Results showed that gs gene contained a 951 bp ORF, encoding 316 amino acids with a predicted relative molecular weight of 35.19 kDa. gcs gene contained 1335 bp ORF, encoding 444 amino acids with a predicted relative molecular weight of 50.37 kDa. gr gene contained 1356 bp ORF, encoding 451 amino acids with a predicted relative molecular weight of 48.90 kDa. Blast analysis revealed that gs,gcs and gr gene shared homology of 99%, 91% and 98% respectively with V.parahaemolyticus, and 98 %, 90% and 98% respectively with V.alginolyticus.Three prokaryotic expression plasmid pET- gs, pET- gcs and pET- gr were constructed respectively. After the optimization analysis was carried out on the conditions of protein expression, Drug resistance of three recombinant bacteria to doxycycline and norfloxacin was determinated under the condition of best expression. The results showed that the resistance of recombinant bacteria to doxycycline increased.Antibiotics resistant bacteria to two kinds of antibiotics were respectively screened using the drug sensitive experiment and testing tube double dilution method. Using RT-PCR technique, analyzed the relative expression of related genes in GSH antioxidant system in drug sensitive bacteria and drug resistance bacteria. Results showed that expressive level of genes in the drug resistant bacteria was significantly higher than that in the sensitive bacteria.The result of the activity of related enzymes in GSH antioxidant system and the GSH content of the sensitive and drug resistance bacteria was measured byspectrophotometry.The results showed that the enzyme activity and GSH content of the same strain were distinctly different before and after screening, i.e., the enzyme activity and GSH content of drug-resistant bacteria were significantly higher than those of sensitive bacteria. Under the stimulus of antibiotics, OH · content and enzyme activity changed significantly in both sensitive bacteria and drug resistance bacteria. And the increased multiples of OH·content and enzyme activity of sensitive bacteria were higher than that of resistant bacteria. It was shown that antibiotics caused oxidative stress in cells, and drug-resistant bacteria were not susceptible to oxidative damage.Enzyme activity in sensitive bacteria and drug-resistance bacteria as well as MIC to norfloxacin were determined after adding GSH or BSO respectively to further validate their function.The results showed that the drug resistance of sensitive and resistant bacteria to norfloxacin was increased, and as well as the activity of enzyme related to GSH after adding GSH;and the activity of GCS and drug resistance to norfloxacin was decreased in sensitive and resistant bacteria after adding BSO.In a word, the norfloxacin and doxycycline can induced oxidative stress in vibrio harveyi, there is a certain correlation between the formation of drug resistance to this two kinds of antibiotic and the GSH antioxidant system. |
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