Multilocus Sequence Analysis Reveals that Vibrio harveyi and V. campbellii Are Distinct Species

Abstract

Identification and classification of Vibrio species have relied upon band pattern methods (e.g., amplified fragment length polymorphism) and DNA-DNA hybridization. However, data generated by these methods cannot be used to build an online electronic taxonomy. In order to overcome these limitations, we developed the first standard multilocus sequence scheme focused on the ubiquitous and pathogenic Vibrio harveyi species group (i.e., V. harveyi, V. campbellii, V. rotiferianus, and a new as yet unnamed species). We examined a collection of 104 isolates from different geographical regions and hosts using segments of seven housekeeping genes. These two species formed separated clusters on the basis of topA, pyrH, ftsZ, and mreB gene sequences. The phylogenetic picture obtained by the other three loci, i.e., gyrB, recA, and gapA, was more complex though. V. campbellii appeared nested within V. harveyi in the recA trees, whereas V. harveyi formed a tight nested cluster within V. campbellii by gapA. The gyrB gene had no taxonomic resolution and grouped the two species together. The fuzziness observed in these three genes seems not be related to recombination but to low divergence due to the accumulation of only a few substitutions. In spite of this, the concatenated sequences provided evidence that the two species form two separated clusters. These clusters did not arise by recombination but by accumulation of point mutations. V. harveyi and V. campbellii isolates can be readily identified through the open database resource developed in this study (http://www.taxvibrio.lncc.br/). We argue that the species should be defined by evolutionary criteria. Strains of the same species will share at least 95% concatenated sequence similarity using the seven loci, and, most importantly, cospecific strains will form cohesive readily recognizable phylogenetic clades.The species Vibrio harveyi and V. campbellii are widespread in the marine environment and among the main species responsible for disease in many wild and reared aquatic organisms, most notably peneid shrimp, several fish species, and mollusks (2). Luminous vibrios related to V. harveyi have been implicated principally in disease outbreaks in shrimp larviculture facilities and in grow-out ponds worldwide. More recently V. harveyi has been associated with infections in corals (20). Indeed recent studies have shown that vibrios, including the V. harveyi group, are abundant in the mucus of corals and may cause infections during periods of environmental imbalances (15).In the past, the identification of V. harveyi and related species isolated from the marine environment has been imprecise and represents hard work as it involves performing many biochemical and/or physiological tests (14). Presumptive V. harveyi isolates grow on thiosulfate-citrate-bile saltssucrose agar are motile, ferment glucose, and are oxidase positive and sensitive to the vibriostatic agent 0/129 at 150 g. They are arginine dihydrolase negative and lysine and ornithine decarboxylase positive. Most presump...

Identification of Vibrio campbellii isolated from diseased farm-shrimps from south India and establishment of its pathogenic potential in an Artemia model

“…To this end, 28 isolates of diverse origins within the shrimp farm ponds were randomly selected and characterized by different molecular tools. Thompson et al (2005Thompson et al ( , 2007 analysed a series of housekeeping genes and concluded that there is no single gene which can clearly differentiate all species of vibrios. Further, it was reported that V. harveyi and V. campbellii have less than 97 % pyrH gene sequence similarity and it is possible to differentiate a series of V. campbellii and V. harveyi species on the basis of this gene sequence (Thompson et al, 2007).…”

“…Genomic DNA was prepared according to the method of Ausubel et al (2002). Partial sequencing of the 16S rRNA and uridylate kinase (pyrH) genes was done for all 28 strains as described previously (Haldar et al, 2007;Thompson et al, 2007). The sequence of the pyrH gene from all experimental strains was compared with published sequence data in GenBank using BLAST (http://www.ncbi.nih.gov/BLAST).…”

“…Further, the wellstudied V. harveyi strain BB120 was recently proved to be V. campbellii by microarray-based comparative genomic hybridization (Lin et al, 2010). However, based on comparison of a series of housekeeping genes (pyrH, topA, ftsZ and mreB) a few researchers have successfully identified and differentiated V. campbellii from V. harveyi (Thompson et al, 2007).…”

“…Further, the wellstudied V. harveyi strain BB120 was recently proved to be V. campbellii by microarray-based comparative genomic hybridization (Lin et al, 2010). However, based on comparison of a series of housekeeping genes (pyrH, topA, ftsZ and mreB) a few researchers have successfully identified and differentiated V. campbellii from V. harveyi (Thompson et al, 2007).In addition to problems of accurate identification, a second challenge is to understand the virulence potential of marine vibrios, which is still difficult due to the lack of simple and reliable animal models. Nauplii of the brine shrimp Artemia have been used in many studies because this shrimp can be cultured easily in gnotobiotic conditions (Austin et al, 2005).…”

Phylogeographic patterns in the Philippine archipelago influence symbiont diversity in the bobtail squid–Vibrio mutualism

“…Isolated DNA extracted from each V. fischeri isolate was used to amplify a portion of the glyceraldehyde phosphate dehydrogenase ( gapA ) locus (~900 bp) by PCR, using previously described Vibrio ‐specific primers (Table 2; (Jones et al., 2006; Nishiguchi & Nair, 2003). The gapA locus has been used reliably to estimate deep phylogenetic connections between bacterial families (Nelson, Whittam & Selander, 1991) within the Vibrionaceae (Thompson, Gomez‐Gil, Vasconcelos & Sawabe, 2007) as well local population structure of mutualist V. fischeri (Jones et al., 2006; Nishiguchi & Nair, 2003). …”