Study On Class â…  Antibiotic Resistance Integrons Of Vibrios And Multilocus Sequence Typing Of Vibrio Parahaemolyticus

Vibrio is a genus of Gram-negative bacteria possessing a curved rod shape, which has polar flagellum and do not form spores. It is one of the most common bacterial groups in the ocean and widely distributed in the near-shore, estuary area and organisms. For its pathogenicity and host’s physiological status, environmental conditions together with other factors are closely related; vibrios are designated as conditional pathogens. Once vibriosis is outbreak in the large area, the sustainable development of mariculture industry would be threatened. Vibrio parahaemolyticus, an important zoonotic pathogen, is a main cause of the food poisoning in the coastal areas in summer and autumn. In this study,214vibrio strains were isolated from aquaculture water and sick animals in Chinese districts including Zhejiang, Hainan, Jiangsu and Fujian province. They were then identified by TCBS medium combined with molecular methods. These vibrios were composed of82strains of V. parahaemolyticus,47strains of V. alginolyticus,45strains of V. harveyi,27strains of V. vulnificus and13strains of other V. species.Firstly, antibiotic resistance analysis.214strains of vibrio were divided into9categories contain15kinds of antibiotics sensitivity. The results showed isolates to penicillin and novobiocin sensitivity were low; the lowest is only6.5%to penicillin. To the rifampicin, class of macrolide erythromycin and aminoglycoside kanamycin and streptomycin sensitivity is lower than17%, while the sulfonamides compound sulfamethoxazole and cephalosporin ceftriaxone sodium are more sensitive. Norfloxacin sensitivity is higher than that of enrofloxacin in fluoroquinolones and tetracycline, doxycycline and oxytetracycline show sensitive rate is higher than99%. All strains are sensitive to chloramphenicol class of florfenicol and also reached98.1%on chloramphenicol sensitivity. Multidrug resistance analysis showed that, resistant to3kinds of antibiotics or more strains accounted for49.1%, resistant to4or more accounted for27.1%, resistant to5or more accounted for12.1%and resistant to6accounted for3.3%. All strains were resistant up to6kinds of antibiotics. Actually,6resistance (strains which resistant to6antibiotics) appeared2strains that drug-resistant spectrum type are "NV/P/RD/E/ENR/TE",5resistant to "NV/P/RD/E/ENR" spectrum are the most common type,4resistant and3resistant to most antibiotics are "NV/P/RD/E" and "P/RD/E" type. Separation from the geographical point of view, resistance of isolates from different provinces showed some geographical variation.Secondly, detection of typical class I antibiotic resistance integrons.In this part, class I integron integrase gene,3’end of the conserved region of qacE△1and Sul I gene and the antibiotic resistance gene cassette in variable regions were identified. The results showed that6strains out of214vibrio isolates were positive for integrase, just conservative region of3’end of one strain of V. vulnificus was positive. The strain amplification of a variable region acquired resistance gene cassette arr-3-dfrA27structure are mediated by rifampicin and trimethoprim resistance. Analysis of drug resistance and integrons correlation showed that there were correlation between vibrio integrons and antibiotic resistance phenotype, but not absolute correlation. This paper first defined the existence of class I antibiotic resistance integron in vibrio and found class I integron-gene cassettes typically in V. vulnificus and also submitted new sequence data about class I integron in vibrio vulnificus to GenBank for the first time.Thirdly, molecular sequence typing (MLST) of V. parahaemolyticus.82strains of V. parahaemolyticus were identified by the method of Multilocus sequence typing (MLST). PCR amplification of virulence genes including TDH, TRH, T3SS1(4genes) and T3SS2(2genes) showed that82strains of the test results of TDH,TRH and T3SS2were negative, while2-4virulence genes within T3SS1(except for4strains) were positive. MLST analysis showed that32new alleles were found by allele comparison and82V. parahaemolyticus strains were divided into44different types of ST (38STs were new). ST cluster analysis showed that only7ST types could be divided into3groups and the rest were unique. So V. parahaemolyticus strains isolated from mariculture showed highly genetic diversity.