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Molecular taxonomy of Vibrio isolated from coastal environments of Grenada

Page history last edited by SKotelnikova 8 years, 7 months ago


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    By S. Kotelnikova, G. Penny, S. James and R. Naraine



    We study fundamental aspects of the biology of Vibrio with the long-term goal of understanding their physiology, life cycle and evolution.  A better understanding of these characteristics will facilitate the development of novel therapeutic strategies to prevent Vibrio infections of humans and marine animals, as well as providing essential insights into Vibrio biology that will help our comprehension of future viral threats to human and animal health.


    These microorganisms are the most often cultured organisms from the seawater and many of them are associated with multiple marine animals. They display polymorphism, produce different types of flagellation and have large genomes consisting of two unevenly sized circular chromosomes. Vibrios have been extensively studied by microbiologists for almost three decades.  Despite this intensive study there are still gaps in our understanding of their diversity and evolution.


    Vibrios, based on 16S rDNA analysis, are gram-negative Gammaproteobacteria. They are usually motile rods that are mesophilic and chemoorganotrophic, and have a facultatively fermentative metabolism (Brenner et al., 2005). Many species of Vibrionaceae have polar flagella when grown in liquid media, but synthesize peritrichous flagella when grown on solid media (Baumann and Baumann, 1981).They are generally able to grow on marine agar and on the selective medium thiosulfate-citrate- bile salt-sucrose agar and are mostly oxidase positive. Some Vibrios grow on MacConkey and TCBS Agar. Many species are also very susceptible to the vibriostatic compound O129 (2,4-diamino-6,7–diisopropyl-pteridine phosphate). Some member species within this genus also exhibits bioluminescence in e.g. V. fischeri & V. natriegens (Dworkin et al, 2006).


    The genus Vibrio is one of the oldest and most dynamically growing bacterial genera. It was once a taxonomically obscure group, but is now a well defined-genus due to the removal of certain groups such as “non-fermentive vibrios” and “aerobic vibrios” to other genera (Dworkin et al, 2006). The family Vibrionaceae, as of 2005 comprised the genera Enterovibrio (2 species), Grimontia (1 species), Photobacterium (8 species), Salinivibrio (1 species), and Vibrio (64 species) (Farmer et al., 2005). An increased number of 68 type strains were recorded in the genus of Vibrio  in 2007, according to Garrity et al (2007). However, the Vibrio genus is rapidly expanding as new species are isolated and varying species numbers have been reported. Euzeby (1997) classifies 95 species, while the DSMZ reported 94 (DSMZ, 2009).There are 99 species currently affiliated with genus Vibrio (Brenner et al., 2005; Thompson et al, 2003, 2007, DSMZ, October, 2010).


    The family Vibrionaceae contains a variety of very important organisms. The family Vibrionaceae includes several species that cause intestinal tract and extraintestinal infections in both humans and animals. One of these species include the Vibrio cholerae, an organism that has killed millions of people during numerous devastating epidemics of cholera that terrorized most parts of the world.

    Other Vibrio species such as V. parahaemolyticus, V. vulnificus, V. damsela, V. alginolyticus, V. mimicus and V. fluvialis are also notable pathogens known for causing diarrhea and infections in humans (Dworkin et al, 2006).


    Species of Vibrionaceae have also been widely used in physiological, biochemical, molecular biology, and pathogenicity studies (Baumann and Baumann, 1981; Baumann and Schubert, 1984; Hastings and Nealson, 1981, Cano Gomez et al., 2009). Many of the species of Vibrionaceae are widely distributed in the marine environment, where they contribute to the cycling of organic and inorganic compounds. These bacteria are found abundantly in aquatic habitats and in association with eukaryotes. Associations established by vibrios range from mutualistic, e.g., V. fischeri-bobtail squid, V. nereis-sea nymph (Harwood et al., 1981), V. mediterranei in sea plankton (Gomez-Gil et al, 2004), V. gallaecicusin clams (Beaz-Hidalgo et al., 2009) to pathogenic, e.g.V. celticus in clams (Beaz-Hidalgo et al., 2010). Probiotic Vibrio strains for fish and shellfish have also been documented (Verschuere et al., 2000).


    Many of the Vibrio species are opportunistic fish or shellfish pathogens that are common to marine and estuarine environments. They have been identified as the main cause of vibriosis, a potentially fatal septicemia that affects fish and shellfish in marine aquaculture, which results in economic losses.V. harveyi, V. anguillarum, V. splendidus, V. salmonicida, V. logei, V. halioticoli, V. scophthalmi, V. trachuri, V. ordalii, V. pectenicida, V. wadanis, and V. tubiashii are frequently associated with disease in different species of fish worldwide, while the group V. damselacomprises species that are potentially pathogenic to tropical fish. Due to the economic implications of marine Vibrio infections, there is considerable interest in methods to identify, type and track Vibrio related populations associated with marine reared animals. Identification of marine Vibrio strains can be a challenging task since species within the clade (V. harveyi, V.campbellii, V.alginolyticus, V.rotiferianus, V.parahaemolyticus, V.mytili and V. natriegens) have a very high degree of both genetic and phenotypic similarity.

    There are several sources within the marine environment of Grenada, where Vibrio-related organisms can be isolatd. These include, sponges (Craine et al., 2007; Jaimiesson, 2008), bottom biofilms (Caputo and Kotelnikova, 2005; Caputo et al., 2008; Kotelnikova et al., 2006), clams (Rodriguez, 2010) and oyster (Rodriguez et al., 2010). The identification has been based on a combination of molecular and phenotypic studies. The differentiation power of 16S rRNA gene and FAME has been shown to be low for this particular group of organisms. The reproducibility of identification can be limited by changing phenotypes and genotypes in individual strains over time (genomic plasticity). Variable genetic events may be a part of the evolution might be responsible for the changing phenotypes in members of the marine Vibrio group including such as horizontal gene transfer (Tagomori et al., 2002). chromosomal rearrangements (Makino et al., 2003), point mutations (Thompson et al., 2004a), duplication (Zhang et al., 2001), and transduction (Owens and Busico-Salcedo, 2006). The relationship between the presence of virulent genes, their expression, and their virulence to different hosts would have to be demonstrated by a combination of molecular methods and traditional diagnostic methods. Therefore, we are currently adapting a number of molecular methods for identification and typing of Vibrio related species isolated in Grenada at the Department of Microbiology. The major tools that we have used included Multilocus Sequence Analysis (MLSA).


    MLSA is based on the core genes that were shown earlier to provide good resolution while remaining specific for the genus of Vibrio. The targeted genes are present in a single copy and include the following housekeeping genes: Recombinase A, recA; rpoD; thermostable direct hemolysin, tdh; 16S rDNA, regulator of chromosome II replication, rctB; and urydilate kinase,  pyrH. Our review discusses prospects and challenges for developing molecular methods for direct detection of marine Vibrios in complex samples (http//sgugenetics.vibrio). The analysis includes 9 unknown organisms isolated from shellfish, sea sponges, and  bottom biofilms along with 6 control type strains representing 6 species of marine Vibrio and Photobacterium.

    Phylogenetic tree Vibrio 2011.pdf


    In the future, we will use QT PCR that will allow us to quantify single cells of V, parahaemolyticus based on gyrB gene which is present in a single copy and has variable sequences that are specific to only V. parahemolyticus. Vibrio parahaemolyticus is currently the most common pathogen contributing to seafood originating GI cases  in the USA (CDC, 2010). The recent outbreak of cholera in Caribbean region Western Haiti exemplify the importance of our project as it indicates the need for advanced diagnostics of Vibrio for the prevention and tracking of epidemics.


    Bacterial typing systems therefore form the basis for the integration of bacterial taxonomy and epidemiology. Pathogen tracking is relevant for epidemiological studies concerned with the ecology and natural history of a disease; or with planning, monitoring and assessment of disease control programs. Methods for pathogen tracking include identification and typing methods as well as methods for direct detection and quantification of the relevant organism in environmental samples.





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