| Vibrio harveyi is a Gram-negative bacterium widely distributed in marine environments and can infectaquatic animals with vary clinical symptoms and high mortality rate in favorable conditions，causingsevere economic losses for the aquaculture industry. While V. harveyi and closely related species, such asV. campbellii, V. parahaemolyticus et al, are considered to have a very high degree of both genetic andphenotypic similarity, leading to a burdensome task for accurately identifying and typing. In addition,researching on the antibiogram type of V. harveyi isolates has a significant value for epidemiologicalinvestigation and practical drug application, which is a focus in mainly current reports. This study usedmultilocus sequence analysis (MLSA) to identify and type V. harveyi strains collected from diseasedmarine fishes cultured along coastal of south China among2007-2012and aimed to investigate thecapability of the MLSA approach for the resolution of the V. harveyi clade and multi-locus genotyping of V.harveyi strains, employing five protein-encoding genes and the16SrRNA gene. In order to further explorethe resistant profiles of V. harveyi isolates, Kirby-Bauer (K-B) method was applied to carry out the drugsusceptibility test utilizing12kinds of antibiotic. The spectral property of resistance of the isolatedrepresentatives was additionally uncovered base on the SPSS cluster analysis. The main results wereshowed as follows:1. Six conserved genes including mreB, rctB, rpoD, topA, toxR (encoding actin-like cytoskeleton protein,replication origin-binding protein, RNApolymerase σ70factor, topoisomerase I, transmembrane regulatoryprotein, respectively) and16S rRNA, were individually hired to evaluate the210clinical strains. Ninetyfive V. harveyi clade and seven V. vulnificus isolates were detected via PCR amplifying and sequencesblasting. Phylogenetic analysis of the gene sequences revealed that all of the genes, except for toxR,appeared to encounter with recombination events in evolutionary process playing a role ofmisidentification of V. harveyi relative strains on phylogenetic tree construction. The ranges ofintraspecific sequence similarity of V. harveyi strains were99.1-100%（16S rRNA）,91.1-100%（mreB）,98.5-100%（rctB）,87.1-100%（rpoD）,86.6-100%（topA）,98.4-100%（toxR）, whereas the rangesof interspecific sequence similarity of V. harveyi clades were98.4-99.9%（16S rRNA, without V. communis）,87.0-98.4%（mreB）,73.2-98.9%（rctB）,80.8-100%（rpoD）,75.9-100%（topA）,24.4-78.9%（toxR）. The discriminative power of the genes gradually reduced from virulent protein coding genes oftoxR, conserved protein coding genes including mreB, rctB, rpoD, topA to16S rRNA.2. The protein coding genes were concatenated for further analysis. Consequences described that theweight of recombination events were minimized and V. harveyi interrelated species were well-defined inphylogenetic trees, yielding monophyletic branches. One approach of that to combine sequence data usedthe two most resolving genes rctB and toxR inferred a coordinated result to cluster V. harveyi interrelatedspecies with all five concatenated protein coding genes (mreB-rctB-rpoD-topA-toxR), branching eightyfour V. harveyi isolates (40.0%), two V. communis isolates (0.95%), two V. parahaemolyticus isolates(0.95%), four V. rotiferianus isolates (1.9%), three V. alginolyticus isolates (1.4%).3. Eighty four V. harveyi isolates were typed into eleven sub-branches of a-k according to secondly studyof phylogenetic trees of five concatenated protein coding genes. Each sub-branch had its owncharacteristics of source distribution. Sub-branches of a-c merely contained one strain which isolated fromdiseased Trachinotus ovatus in Guangdong, Lutjanus erythopterus in Fujian during2010-2011,respectively. Sub-branch of d inculded eighteen isolates, owning21.4%of the total strains. Among these,Grouper isolates were all obtained from Hainan areas, in contrarily, Trachinotus ovatus and Siganusguttatus strains were totally obtained fron Guangdong areas. Six isolates clustered in e sub-branch werecompletely gotted from unhealthy Trachinotus ovatus. Seven isolates falled into f sub-branch were entirelysampled during2011year. Four isolates of g sub-branch were wholely distributed in Guangdong areas.Fifteen strains gathered in h sub-branch sincerely possessed seven kinds of hosts (including four kinds ofGrouper). Sub-branch of i was made up of reference strain of V. harveyi ATCC33868and Lates calcariferstrain of104CCL. Sub-branch of j consisted of two Lutjanus erythopterus isolates, eight Grouper isolates,typed strain of V. harveyi ATCC14126and reference strain of V. harveyi LMG4044. The k sub-branchcontained twenty isolates and occupied23.8%of the sampled strains, including the whole four Guangxiisolates, gathering all the strains isolated during2010year (except for72BKI) and clustering eighteen(owning90.0%) Grouper strains with different investigated aspects. It represented clearly that the samesub-branch was consist of isolates obtained from diverse sample sites, years, even hosts. Contrarily,isolates received from the same sources distributed in different sub-branches. These preliminary resultssuggested the presence of similar genotypes among the inclusive isolates and abundant genotypes withinthe universal ones.4. Results of the twelve antibiotic susceptibility testing indicated that eighty four V. harveyi isolatesformed26antibiogram types, with antibiogram abundance of31.0%. The number of multiple antibioticresistant types (be resistant to more than three antibiotics) of the experimental strains got to13and owned50.0%of the total antibiogram types, prompting the multi-antibiogram abundance rising up to59.1%. Isolates originated from Hainan, Guangdong, Guangxi, Fujian province reflected to possess18,15,3,1antibiogram types and shared the J antibiogram type (FUR/AMO). Antibiogram types of representativesseparated from2007were J and M, adding A and B in2010, expanding to24types including A to X(except for Y, Z) in2011, and appearing split-new Y and Z in the next year. The number of suppressionantimicrobial agents was2,5,7,8, mirroring to the sampled years. Strains obtained from E. awoara, E.fuscogottaus, Plectropomus leopardus, Trachinotus ovatus and E. lanceolatus×E. fuscogottauscontained6to10antibiogram types, comparing to the rest with merely1to3types. The experimentalstrains were firstly clustered into six subgroups (i-vi), and then gathered for GroupI and GroupII. Eachsubgroup contained characteristic antibiogram types, followed by N,P,T,U,Y; R,S; K,P; F,H,O,X,V,W;E,G,Q; C,L,Z. The common antibiogram types were not found between the strains of GroupI and GroupII.Our research demonstrated that V. harveyi isolates owned polymorphism antibiogram type and moderatelyhigh type abundance, and could be effectively typed with similar resistance by utilizing cluster analysis.The resistant patterns of isolates gathered from different sources were diverse.5. Comparison of the antibiogram typing and genotyping illustrated a show of V. harveyi isolates whichsettled into the same antibiogram subgroup, diffused in different monophyletic sub-branch. However,those classified within sub-monophyletic branch included multiple antibiogram types. These results revealthat there was no obvious correction between approach of antibiogram typing and genotyping onanalyzing V. harveyi isolates. |
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