16S rRNA Sequence Determination for Members of the Genus Carnobacterium and Related Lactic Acid Bacteria and Description of Vagococcus salmoninarum sp. nov.

Abstract

The phylogenetic interrelationships of members of the genus Carnobacterium and some atypical lactobacilli isolated from diseased salmonid fish were investigated by using reverse transcriptase sequencing of 16s rRNA. The four species Carnobacterium piscicola, Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile exhibited a high degree of sequence similarity with each other (ca. 96 to 98%) and formed a phylogenetically coherent group that was quite distinct from all other lactic acid bacteria. The sequence data clearly demonstrated that carnobacteria are phylogenetically closer to the genera Enterococcus and Vagococcus than to members of the genus Lactobacillus. The strains from fish were found to be phylogenetically related to the genus Vagococcus and represent a new species, Vagococcus salmoninarum. The type strain of Vagococcus salmoninarum is strain NCFB 2777.The genus Carnobacterium was proposed by Collins et al. (4) to accommodate the species Lactobacillus piscicola (17) and Lactobacillus divergens (18) and some so-called atypical lactobacilli isolated from poultry meat (24). Currently, four species, Carnobacterium piscicola (type species), Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile are recognized in this genus (4). Although these four species form a phenotypically coherent group (4, 9), the separateness of the genus Carnobacterium (in particular its relationship to the genus Lactobacillus) remains unclear. Members of the genus Carnobacterium differ from Lactobacillus species by their inability to grow on acetate medium (4, 9) and by their synthesis of oleic acid (c18:1A9,10) instead of cis-vaccenic acid (c18:1A11,12), which is produced by lactobacilli (4). At present these are the only criteria which distinguish the two genera.Sequencing of 16s rRNA by reverse transcriptase (20, 21) is currently the most rapid and powerful technique for elucidating the natural relationships of microorganisms (26). This method produces long stretches of sequence (ca. 95% of the total sequence), which enables precise phylogenetic relationships to be determined (26). In this study we determined 16s rRNA primary structures for members of the genus Carnobacterium by using reverse transcription in an attempt to clarify the relationship of these organisms to the genus Lactobacillus and other lactic acid bacteria. In addition, the phylogenetic position of two representative strains belonging to a group of atypical lactobacilli isolated from salmonid fish was also determined. MATERIALS AND METHODSCultures and cultivation. The test strains which we used are shown in Table 1. Strains of carnobacteria, enterococci, lactococci, leuconostocs, pediococci, and streptococci and fish isolates OS1-68T (T = type strain) and Rangen 128-81 were grown in YGPB broth (12) at 30°C. Strains OS1-68T and Rangen 128-81 were isolated by R.A. Holt, Oregon Department of Fish and Wildlife; strain OS1-68T was isolated in 1968 from an adult rainbow trout at the Oak Spring Hatchery * Corresponding...

Characterization of Carnobacterium species by pyrolysis mass spectrometry

“…This phenomenon is further supported by the work of Wallbanks et al (1990) in which C. gallinarum and C. pzscicola were found to show 98.7% 1 6 s rRNA homology whilst C. gallinarum and C . divergens exhibited only 97% similarity.…”

Lactosphaera gen. nov., a New Genus of Lactic Acid Bacteria, and Transfer of Ruminococcus pasteurii Schink 1984 to Lactosphaera pasteurii comb. nov.

“…Different treeing methods and different data sets were used to reconstruct the phylogenetic relationships of strain K o T~~~. Strain K o T~~~ is only distantly related to members of the genus Ruminococcus (4,6,7), but is closely related to members of the genus Curnobacterium (9,11,34). The overall levels of sequence similarity for strain K o T~~~, members of the genus Curnobucterium, selected lactic acid bacteria, and other bacteria are shown in Table 1.…”

Molecular Fingerprinting of Fish-Pathogenic Lactococcus garvieae Strains by Random Amplified Polymorphic DNA Analysis

“…The identification schemes for L. garvieae, based on biochemical and antigenic characterists (13,17), can barely differentiate this microorganism from other gram-positive cocci such as the human pathogen L. lactis subsp. lactis (13,14) or the Enterococcus-like strains isolated from diseased fish (12,32,36). Studies involving the phenotypic characterization of L. garvieae strains, collected from different species and countries, have been conducted using conventional methods and miniaturized systems and have given variable results (12,13,28,31,35).…”