4% similarity of the clam isolates, which was higher than that observed between the fish isolate and either clam strain (98.2%). The topology of the maximum parsimony tree, obtained from 2D-PAGE analysis, and the phylogenetic tree, constructed with the maximum likelihood algorithm from concatenated sequences of 16S rRNA gene and five housekeeping genes (atpA, pyrH, recA, rpoA and rpoD), was very similar, confirming the closer relationship FG-4592 ic50 between the two clam isolates. Vibrio species are extensively
distributed in marine environments, associated with a wide range of marine organisms; some of the species are pathogenic to humans (Thompson et al., 2006; Beaz-Hidalgo et al., 2010). Genotyping strategies such as restriction fragment length polymorphism and pulse field gel electrophoresis have been used traditionally for epidemiological analysis of Vibrio isolates (Castro et al., 1997; Romalde et al., 2002). PCR typing methods have also been widely used, including randomly amplified polymorphic DNA analysis and repetitive-sequence-based polymerase chain reaction based on polymorphic, repetitive extragenic palindromic sequences and enterobacterial repetitive
intergenic consensus (Rodríguez et al., 2006). More recently, amplified fragment length polymorphism and multilocus sequence analysis (MLSA) (Maiden, 2006) have allowed a more precise identification of Vibrio species (Beaz-Hidalgo et al., 2008, 2010; and references therein). Proteomics could complement and extend Amobarbital the nucleic acid analytical find more technologies,
being an experimental link between the expressed product and the genome (Lester & Hubbard, 2002; Phillips & Bogyo, 2005; Norbeck et al., 2006; Cash, 2009; Zhang et al., 2010). 2D-PAGE has been successfully applied for the discrimination of closely related isolates (Cash et al., 1995; Dumas et al., 2008), revealing even more variability than with DNA–DNA hybridization, as protein content reflects dynamic changes produced in the cells as a response to changes in the environment (Andersen et al., 1984; Cash, 2009; Zhang et al., 2010). Vibrio tapetis is the causative agent of an epizootic infection in adult clams called brown ring disease (Borrego et al., 1996). The first studies indicated that strains of this pathogen constituted a homogeneous group. However, as new strains were isolated from different hosts, including different mollusk and fish species, some variability on the basis of their antigenic, phenotypic and genotypic characteristics has been demonstrated, leading to the description of three main groups within this species that correlate with the type of host (Castro et al., 1996, 1997; Romalde et al., 2002; Rodríguez et al., 2006). In this work, a proteomic method, 2D-PAGE, was used to study the intraspecific variability of representative strains of the three groups described for V. tapetis, as well as an additional indication of their phylogenetic relationship.