| In recent years, the blind pursuit of high- yield, high-density culture, technology disorder, drug abuse, disease epidemics, poor seed quality, environmental pollution and other reasons, caused a great impact on shrimp farming. In order to explore the prevalence, drug resistance and pathogenicity of Vibrio in shrimp, which were preserved by our laboratory and isolated from pathogenetic shrimps, fishes and crabs in Shanghai, Jiangsu, Zhejiang, Fujian, Hainan and other places, polymerase chain reaction(PCR) was used to detect the integrons and drug resistance genes in these isolates; minimal inhibitory concentration(MIC) method was used to detect the drug-resistance of these vibrio. Right judgments for clinical guidance should be made according to the drug-resistant genotype and phenotype of the bacteria in the drug-resistant farmed areas, antibiotics should be used rationally to prevent bacterial disease. The study would provide a theoretical basis for clinical guidelines. Samples were collected in five coastal provinces, including hemolymph, hepatopancreas, muscle of Peaneus vannamei and Scylla and samples from body surface、ulcers、intestinal tract, abdominal dropsy and blood isolated from the Perch, tongue sole, turbot, Blowfish..1. Drug resistance analysisThe MIC values of 194 strains of 8 antibiotic and drug resistance gene were detected to study the resistance of the isolated strains, compare the correlation of resistant phenotypes and genotypes for resistance and summarize the resistance of Vibrio in various regions with simple. The MIC values of 194 strains showed that the erythromycin and SMZ-TMP had highest resistance rate which were 60.8% and 58.2%, respectively and for streptomycin, 156 strains were distrubuted in intermediary scope and florfenicol had the lowest drug resistance rate which was 7.7%. Vibrio isolated from different animals of 8 kinds of antibacterial drug resistance rates varied and strains isolated from fish got higher resistant rate, crab’s lowest and of florfenicol test and oxytetracycline resistant rate were 0. The integrated gene of 1、 2、3 and 4 type of integrated sub of 194 strains were also detected.The results showed that the two and three kinds of integrated enzymes were not detected, and the detection rate of a class of integrated enzymes was 95.4%. In 194 strains of bacteria, 11 strains contains a integron conserved regions, the detection rate was 5.67%, but V-69, V-76, gate three strains of bacteria not only has complete integron, but also has a variable region gene cassettes. After T-A cloning and sequencing of variable region PCR positive 1308 bp, 1308 bp and 1315 bp were obtained, three strains of Vibrio containing the same gene cassettes and rifampicin ADP ribosyl transfer enzyme(rifampin ADP-ribosylating transferase and arr-3) and dihydrofolate reductase(Dihydrofolate reductase small) two resistance gene cassettes, respectively mediated by rifampin and trimethoprim resistance. 2. Virulence gene detection of Vibrio vibrio from prawn sourceVirulence genes of lab saved 74 Vibrio parahaemolyticus strains of shrimp source, recently reported vice hemolytic Vibrio pir A and gene pir B and Vibrio and type III secretion system T3SS1 and t3ss2 were detected in this chapter. The infection of shrimp with serval Vibrio parahaemolyticus strains was also tested. The pathogenicity of Vibrio was judged according to the semi lethal concentration of 24 h.. Detection results showed that TDH and TRH gene was no detected, and TLH 61 strains, the detection rate was 82.4%, T3SS1 detection rate is higher, the highest virulence genes VP1670 detection rate highest was 62.2%; and t3ss2 detection rate is lower, the lowest virulence genes VPA1362 detection rate was 0%. The detection of virulence factors of pir A and pir B in Vibrio parahaemolyticus showed the detection rate was 23% and 20.3%, respectively. 3. Multiple drug resistance and pathogenicity of Vibrio V-76A pathogenic Vibrio alginolyticus strain, named as V-76, was isolated from ceca contents of a diseased Litopenaeus vannamei collected from Fuqing, Fujian Province. Antimicrobial susceptibilty tests for 13 antimicrobials and infection assay for Litopenaeus vannamei were performed to determine the phenotype of antimicrobial resistance and pathogenicity of VA-76, respectivly. To explore the genotype of antimicrobial resistance and pathogenicity, the isolate was also analyzed by PC R detection for virulence genes, integron-related genes and antimircobial resistance genes. In artificial infection assay, death occurred in all 4 groups of Litopenaeus vannamei infected with 3.5×107cfu, 3.5×106cfu, 3.5×105cfu, and 3.5×104cfu, and the death rates were 100%, 100%, 80%, and 65%, respectively. The virulence-related genes such as tdh, tlh, tox S, collagenase, fla A, omp W, asp A, and fur were identified in V-76.The results of antimicrobial susceptibility tests showed that this bacteria was resistant to multiple antimicribals, including florfenicol(32μg.m L-1), doxycycline(32μg.m L-1), ciprofloxacin(>64μg.m L-1), chloramphenicol(128μg.m L-1), streptomycin(>512μg.m L-1), erythromycin(256μg.m L-1), sulfamethoxazole(>2048μg.m L-1), TMP/SMZ(>128/2432μg.m L-1), and rifampin(>64μg.m L-1). Especially, V-76 exhibited high- level resistance to the latter 7 antimicrobials, and had relatively high MIC for neomycine(>32μg.m L-1). Furthermore, integron 1 and SXT was also detected in this isolate, and the gene cassete arrangement arr2-dfr A27 was identified inside this class 1 integron. Besides, insertion sequence common region 1(ISCR1), containing a chloramphenicol acetyltransferase gene cat2, was also detected. Several other antimicrobial resistance genes, cluding str A, str B, flo R and qnr VC, were also found in V-76. However, no mutation was identified in gyr A and Par C genes of V-76, which were usually involved in fluroquinolones resistance. This study revealed that this Vibrio alginolyticus V-76 could cause the death of Litopenaeus vannamei, exhibit resistance to multiple antimicrobials, and contain multiple virulence and antimicrobial resistance genes, indicating that prudent use of antimicrobial agents in aquaculture should be emphasized to control the development and distribution of antimicrobial resistant Vibrio alginolyticus. |
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