PCR Detection Of Resistance Genes And Construction Of A Crl Gene Knock-out Mutant Of Forest Musk Deer Lung Pathogenic Escherichia Coli

Pneumonia and purulent disease is one of the major diseases of forest musk deer that affects the survival of musk deer breeding farms in the past decades. The morbidity and mortality rates are high. In the previous study, LPEC strains were found to be one of the principal bacterial pathogens. In this study, O serotype of 29 forest musk deer LPEC isolated and identified by our laboratory was carried out using glass plate agglutination method. The results showed that O serotype of 16 strains were identificated among 29 forest musk deer LPEC isolates, which belonged to 9 different serotypes.O78 was the dominant serotype with a positive rate of 43.75%?7/16?.20 resistance genes including sulfonamides, quinolones, tetracyclines, aminoglycosides and ?-lactamases were detected using PCR amplification method. The results showed that all of 29 LPEC isolates were multidrug resistant strains. Mainly detected resistance genes were sulfanilamides sul1, sul2 and sul3, quinolones aac ?6'?-1b, qnrB and lactamases blaTEM, blaCTX-M, aminoglycosides aac?3?-lia?ant?3?-la and tetracyclines tet?B?. The average resistant rates were 100%?96.55%?96.55%?100%? 86.21%?100%?65.52%?100%?96.55% and 58.62%, respectively. UPGMA contribution analysis showed that there was no significant correlation between the o serotype and drug resistance genes.In the research, the knock-out mutant strain LPEC O78-crl^- was constructed by using ?-Red homologous recombination method. And the comparative analysis of physiological characteristics, differences in virulence characteristics and hemagglutination activity between wild strain LPEC O78 and mutant strain LPEC O78-crl^-were carried out. The results of biochemical experiments and growth curve measure confirmed that the basic metabolism of LPEC O78-crl^- was not changed and the growth rate had no significant change. The median lethal dose (LD₅₀) results showed that the LD₅₀ of LPEC O78-crl^- was about 7 times lower than that of LPEC O78. The hemagglutination activity of LPEC O78 and LPEC O78-crl^- respectively was 2^-5 and 2^-4 which indicated that the knock-out of crl gene had a negative influence on red cell agglutination ability. The results have an important guiding significance for clinical scientific, rational drug using and laying a foundation for the further research of forest musk deer LPEC pathogenic mechanism.