Lactobacillus ceti sp. nov., isolated from beaked whales (Ziphius cavirostris)

The genus Lactobacillus represents a large group within the Gram-positive bacteria and is presently classified within the order ‘Lactobacillales’ according to the latest release of the Taxonomic Outline of the Prokaryotes (Garrity et al., 2004). The genus Lactobacillus has undergone considerable expansion in the past decade and, at the time of writing, over 114 species and 16 subspecies (Euzéby, 1997; Felis & Dellaglio, 2007) are recognized and novel taxa are continuously arising within this group. Lactobacilli have been isolated from a wide range of environments and can be found on plants or material of plant origin, in manure and man-made habitats such as sewage, and in fermenting or spoiled food. Lactobacilli have also been associated with the intestinal tract and mucous membranes of man and many animals (Wibowo et al., 1985; Hammes et al., 1992; Aguirre & Collins, 1993; Roos et al., 2000). During an investigation into the microbiota of stranded beaked whales (Ziphius cavirostris), three unidentified Gram-positive, rod-shaped organisms were recovered from the liver (strain 159-2) and lungs (strains 142-2^T and 160-1) of two animals. Neither of these two animals showed clinical signs of disease and no lesions were apparent after post-mortem examination. Strains were isolated on Columbia blood agar plates (bioMérieux) after incubation at 37 °C for 24 h under aerobic and anaerobic conditions. On the basis of the phenotypic and phylogenetic results, a novel species of the genus Lactobacillus is proposed.

Gram determination was performed by Gram staining (Merck). The isolates were tested for a number of key characteristics by using standard procedures (Barrow & Feltham, 1993). Growth in brain heart infusion broth was assessed at 4 and 15 °C for up to 14 days, at 22 °C for up to 7 days and at 30, 37 and 42 °C for 48 h. Growth in the presence of 3, 4.5 and 6.5 % NaCl (w/v) and under anaerobic (with 4–10 % CO₂) and microaerobic (with 5–15 % O₂ and 5–12 % CO₂) conditions using GasPak Plus and CampyPak Plus systems (BBL), respectively, was assessed at 37 °C for 48 h. Growth on MRS agar (bioMérieux) was tested at 37 °C for 48 h. The strains were biochemically characterized using the API Coryne, API 50 CH and API ZYM systems (bioMérieux) according to the manufacturer's instructions. The API 50 CH strips were read after 7 days incubation at 37 °C. Using the miniaturized biochemical kits, the novel isolates displayed identical phenotypic profiles. The lactate isomer was determined enzymically using the dl-lactate test kit (Boehringer Mannheim). A detailed description of the morphological, physiological and biochemical characteristics of the isolates is given in the species description and in Table 1⇓.

Analysis of the peptidoglycan structure of one isolate (strain 142-2^T) was performed as described by Schleifer (1985) and Schleifer & Kandler (1972) with the modification that TLC on cellulose was used instead of paper chromatography. Analysis of the cell-wall composition of strain 142-2^T revealed the presence of lysine and serine, indicating the presence of the A3α l-Lys–d-Ser peptidoglycan type (DSMZ, 2001).

To establish the phylogenetic affinities of the novel isolates, the 16S rRNA gene sequences were determined as described previously (Vela et al., 2005) and were subjected to a comparative analysis. The almost complete sequences (>1400 nucleotides) of the three novel isolates were determined and pairwise analysis revealed that the isolates were phylogenetically identical (100 % gene sequence similarity). Sequence searches of GenBank using the fasta program (Pearson, 1994) revealed that the novel isolates had <95 % gene sequence similarity with any recognized species. The novel bacteria were phylogenetically closely related to Lactobacillus satsumensis DSM 16230^T (94.2 % 16S rRNA gene sequence similarity), Lactobacillus salivarius JCM 1047 (94.0 %), Lactobacillus nagelii ATCC 700692^T (94 %) and Lactobacillus saerimneri DSM 16049^T (93.8 %). These sequences, and those of other known related strains, were retrieved from GenBank and aligned with the newly determined sequences using the DNAtools program (Rasmussen, 1995). Phylogenetic trees were constructed according to three different methods, a neighbour-joining algorithm (Saitou & Nei, 1987), performed with the DNAtools and treeview (Page, 1996) programs, maximum-likelihood analysis conducted using phyml software (Guindon & Gascuel, 2003) and the maximum-parsimony method carried out using the mega (Molecular Evolutionary Genetics Analysis) version 3.1 software package (Kumar et al., 2004). Genetic distances for the neighbour-joining and maximum-likelihood algorithms were calculated by the Kimura two-parameter method (Kimura, 1980) and close-neighbour-interchange (search level=2, random additions=100) was applied in maximum-parsimony analysis. The stability of the groupings was estimated by bootstrap analysis (1000 replications). Phylogenetic trees obtained by using the neighbour-joining (Fig. 1⇓) and the other two methods revealed a clear affiliation of the novel strains (as exemplified by strain 142-2^T) to the genus Lactobacillus, as they were positioned as a separate branch close to L. saerimneri DSM 16049^T, Lactobacillus aviarius ATCC 43234^T, L. salivarius JCM 1047and Lactobacillus acidipiscis JCM 10692^T. Although bootstrap resampling analysis did not reveal the affiliation between strain 142-2^T and the aforementioned species to be statistically significant (50 % value), the branching position of strain 142-2^T within this clade was relatively stable according to the three tree-making algorithms used in this study (Fig. 1⇓). These data together with 16S rRNA gene sequence divergence values of >4 % between the novel isolates and other species of the genus Lactobacillus suggest they represent a separate species (Stackebrandt & Goebel, 1994). In order to differentiate the three novel isolates, pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI (MBI Fermentas) was performed as described previously (Vela et al., 2003). Strains 159-2 and 142-2^T isolated from different whales generated distinguishable PFGE fingerprint profiles that revealed their genotypic differences at the strain level. On the other hand, strains 160-1 and 159-2, isolated from different organs of the same animal, exhibited undistinguishable PFGE profiles (data not shown).

Overall, the results of the present study show that the novel isolates from whales constitute a distinct branch and do not display a close relationship with any recognized organism (Fig. 1⇑). Moreover, the novel isolates could be distinguished from their closely phylogenetic relatives on the basis of phenotypic characteristics (Table 1⇑). Therefore, based on both phylogenetic and phenotypic criteria, it is evident that the new isolates merit classification as a novel species of the genus Lactobacillus, for which the name Lactobacillus ceti sp. nov. is proposed. Strains 142-2^T, 159-2 and 160-1 showed identical phenotypic and physiological properties; these are given in the species description. Characteristics that are useful in differentiating Lactobacillus ceti sp. nov. from other members of the L. salivarius group of lactobacilli are summarized in Table 1⇑.