| Vibrio harveyi is a serious bacterial pathogen to aquatic aninamals with high mortality.Clinical symptoms commonly exhibited is include the exophthalmos, surface bleeding,muscle ulceration in the reared fish. As the antibiotic have been widely used in marine aquaculture animals, the antibiotic resistance of V. harveyi is increased more seriously,especially the occurrence of multiple antibiotic resistance and superbacteria have already attracted extensive attention among researchers around the world. Resistant genes carried by bacterial plasmid frequently and can be transferred among homogeneous or heterogeneous genus, which can changed the antibiotic resistance and futher caused wide spraed of drug resistance gene.The present study investigated the tolerance of 41 strains with 40 common antibiotics,isolated from Mariculture area in South China. The results showed that 41 strains were resistant to amikacin,the resistance rate of 40 antibiotics to the strain was 4.9% ～100%,and the multidrug resistance ratio of all strains to 40 antibiotics ranged from 27.5% to 95.0% , 15 strains were resistant to at least 30 antibiotics. There were 41 antibiogram types totally. The clustering analysis showed that when the Squared Euclidean was 2, all trains were clustered into 7 species (i～vii). When the Squared Euclidean was 8, all trains were clustered into 2 species (Ⅰ、Ⅱ). The resistant species of group_Ⅱ_was more than that of in group I,and performed more antibiogram type. Therefore V. harveyi in seawater aquaculture waters in southern China had serious multidrug resistance, and the resistant strains of V. harveyi were resistant and varied, and the resistance patterns in different strains were various.In all strains we studied, there are 36 strains of bacteria carryied plasmid, with 87.80% detection rate, each strain carry 1～5 plasmid. Twenty-three different size plasmids were detected from 36 strains,ranging from 1.72～36.23 kb,and formed 20 plasmids map.After digestion with EcoR I, all strains still formed 20 kinds of plasmid digestion map. but their digestion map was distinct different, indicating that the size of plasmids were coincident but the sequence in these strains were different.In this paper, the plasmids were eliminated by high temperature and SDS. When the strain were cultivated to 30 generations with alternately treated at 37 ℃ (adding SDS) and 43 ℃, the plasmids of 21 strains were all eliminated, and the other 15 strains were not eliminated completely. According to the results of ERIC classification, the plasmid carried by GDH11385 (main genotype of Vibrio harveyi pathogens in South China) could be completely eliminated, while the other nine types of plasmids were partially eliminated.Indicating that the plasmid of GDH11385 strain was easy to be eliminated, The plasmid of GDH11385 was successfully sequenced, and the three plasmids carried were easily transferred between the strains, which made the bacteria competitive in the environment. It also proved that the strain had strong environmental competitive advantage, Which is manifested as the main Vibrio pathogens in the area.After elimination of plasmid, The resistance of aminoglycosides, β-lactam,sulfonamides, fluoroquinolones in 31 strains was significantly reduced. According to the drug resistance genes (ant (3")-I, aac (6’)-I, blaTEM, blaCTX-M, FloR, Sul I, qnrVC, qnrA,2/drugs) of the above four categories of drugs, we designed PCR primer. The results showed that the detection rates of positive strains were 78%, 63.4%, 14.6%, 46.3%, 68.3%,70.7% ,0 and 48.8% respectively. According to the results of drug resistance and PCR, the resistance gene of Vibrio harveyi in South China was polymorphic, and the above-mentioned drug resistance gene was not suitable for the drug resistance test in this area.The correlation analysis of strain resistance and plasmid map results showed that the drug-resistance pattern of HNH1105 and HNH1111 were different obviously, but there plasmid map were same. which indicated that the resistance of these two strains was encoded by the chromosome gene. The drug-resistance pattern and plasmid map of WC13DH51 and GDH11388 were different, which indicated that the resistance of these two strains was encoded by the plasmid gene. The drug-resistance pattern of strains were different obviously and there plasmid map were same or not, which indicated that no obvious correlation between antibiogram and plasmid map. In conclusion, the resistance of V. harveyi can be encoded by plasmid genes or chromosomal genes.In conclusion, the study showed the resistance characteristics and plasmid profile of 41 strains of V harveyi in southern China. And through the correlation analysis, we determined carrying of the antibiotic resistance gene in the bacteria It not only provides guidance for the prevention and control of major epidemics caused by Vibrio harveyi in southern China, but also has an important theoretical basis for clarifying the molecular mechanism of V. harveyi drug resistance. |
