Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota.

The evaluation of the impact of probiotics on host health could help to understand how they can be used in the prevention of diseases. On the basis of our previous studies and in vitro assays on PBMC and Caco-2 ccl20:luc reporter system presented in this work, the strain Lactobacillus kefiri CIDCA 8348 was selected and administrated to healthy Swiss mice daily for 21 days. The probiotic treatment increased IgA in feces and reduced expression of proinflammatory mediators in Peyer Patches and mesenteric lymph nodes, where it also increased IL-10. In ileum IL-10, CXCL-1 and mucin 6 genes were upregulated; meanwhile in colon mucin 4 was induced whereas IFN-γ, GM-CSF, and IL-1ß genes were downregulated. Moreover, ileum and colon explants showed the anti-inflammatory effect of L. kefiri since the LPS-induced increment of IL-6 and GM-CSF levels in control mice was significantly attenuated in L. kefiri treated mice. Regarding fecal microbiota, DGGE profiles allowed differentiation of experimental groups in two separated clusters. Quantitative PCR analysis of different bacterial groups revealed only significant changes in Lactobacillus population. In conclusion, L. kefiri is a good candidate to be used in gut inflammatory disorders.

Comparative effects of level of dietary fiber and sanitary conditions on the growth and health of weanling pigs.

There are conflicting results on the growth and health of weanling pigs (Sus scrofa) fed high-fiber diets, and responses may differ according to sanitary conditions. This study was conducted to explore the growth, health, and fecal microbiota of weanling pigs fed either low- or high-fiber diets in 2 different sanitary conditions. Forty-eight pigs weaned at 28 d of age were individually housed in "good" (clean) or "poor" (unclean) sanitary conditions. During 2 consecutive phases, pigs were fed 2 diets containing a low (control) or high level of fiber: 121 or 169 g/kg total dietary fiber (TDF) for Phase I and 146 or 217 g/kg for Phase II, which lasted 15 and 20 d, respectively. This led to 4 experimental treatments in Phase I in a 2 × 2 factorial arrangement (2 sanitary conditions × 2 diets) and 8 experimental treatments in Phase II in a 2 × 2 × 2 factorial arrangement (2 sanitary conditions × 2 diets in Phase I × 2 diets in Phase II). The poor sanitary conditions led to a reduced G:F (0.617 vs. 0.680 for poor and good sanitary conditions, respectively; P = 0.01) over the entire experimental period. The number of pigs with diarrhea in Phase I tended to be greater in the poor sanitary conditions with the high-fiber diet than the control diet (7 vs. 3 pigs, P = 0.07). Enterococcus was prominent in feces of these diarrheic pigs. At 5 wk after weaning, compared with good sanitary conditions, the fecal microbiota of pigs housed in poor sanitary conditions was characterized by more Lactobacillus (9.24 vs. 8.34 log cfu/g, P < 0.001), more Enterobacteria (6.69 vs. 5.58 log cfu/g, P < 0.001), and less anaerobic sulfite bacteria (3.72 vs. 5.87 log cfu/g; P < 0.001). The feces of pigs in poor sanitary conditions contained more total VFA and proportionally more butyrate (9.7 vs. 5.7% for poor and good conditions, respectively, independently of dietary treatment, P < 0.001). At 5 wk after weaning, feces of pigs fed the high-fiber diet during Phase II contained less Enterococcus bacteria than pigs fed the control diet (4.06 vs. 4.56 log cfu/g; P = 0.05), and more total VFA with a decreased proportion of branched-chain fatty acids (5.0 vs. 6.1%; P = 0.006). To conclude, feeding pigs a high-fiber diet in the immediate period after weaning is probably an additional risk factor for slower BW gain, especially in poor sanitary conditions.

Identification of novel proteins secreted by Lactobacillus rhamnosus GG grown in de Mann-Rogosa-Sharpe broth.

AIMS: To identify novel proteins secreted by the probiotic bacterium Lactobacillus rhamnosus GG after growth in de Mann-Rogosa-Sharpe broth (MRS), a complex medium often used for the culture of Lactobacillus. METHODS AND RESULTS: The proteins secreted by L. rhamnosus GG strain were precipitated using a trichloroacetic acid-based protocol, resolved by SDS-PAGE, and identified by tandem mass spectrometry (MS/MS). Among the proteins secreted by this bacterium, a leukocyte elastase inhibitor, already present in the MRS broth, was identified. Other proteins such as cell wall hydrolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, and an extracellular transcriptional regulator have been also identified. CONCLUSIONS: Lactobacillus rhamnosus GG secretes several proteins during its growth in MRS, some of them with assigned functions in the prevention of the molecular mechanisms that lead to damage in the epithelial barrier (cell wall hydrolase) and in adhesion (GAPDH). The rest of the proteins require further genetic analysis in order to establish their precise roles. None of the proteins bound to mucin or fibronectin. SIGNIFICANCE AND IMPACT OF THE STUDY: Some of these secreted proteins could be involved in the probiotic effects exerted by L. rhamnosus GG strain, their identification being the first step towards in depth functional studies.

Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin.

Several Bacillus strains isolated from commercial probiotic preparations were identified at the species level, and their adhesion capabilities to three different model intestinal surfaces (mucin, Matrigel and Caco-2 cells) were assessed. In general, adhesion of spores was higher than that of vegetative cells to the three matrices, and overall strain Bacillus cereus(CH) displayed the best adhesion. Different biochemical treatments revealed that surface proteins of B. cereus(CH) were involved in the adhesion properties of the strain. Surface-associated proteins from vegetative cells and spores of B. cereus(CH) were extracted and identified, and some proteins such as S-layer components, flagellin and cell-bound proteases were found to bind to mucin or fibronectin. These facts suggest that those proteins might play important roles in the interaction of this probiotic Bacillus strain within the human gastrointestinal tract.

Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae.

AIMS: To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers. METHODS AND RESULTS: EPSs synthesized by Idiomarina fontislapidosi F32(T), Idiomarina ramblicola R22(T) and Alteromonas hispanica F23(T) were collected and analysed under optimum conditions: MY medium supplemented with 7.5% (w/v) salts; 32 degrees C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1.5 g l(-1). The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals. CONCLUSIONS: The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica, which may prove to be a viable source of xylo-oligosaccharides.

The safety of Bacillus subtilis and Bacillus indicus as food probiotics.

AIMS: To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. METHODS AND RESULTS: Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The Natto strain of B. subtilis was shown to be able to invade and lyse cells. Neither species was able to adhere significantly to any cell line. The Natto strain was also shown to form biofilms. No strain produced any of the known Bacillus enterotoxins. Disc-diffusion assays using a panel of antibiotics listed by the European Food Safety Authority (EFSA) showed that only Bacillus indicus carried resistance to clindamycin at a level above the minimum inhibitory concentration breakpoints set by the EFSA. In vivo assessments of acute and chronic dosing in guinea pigs and rabbits were made. No toxicity was observed in animals under these conditions. CONCLUSIONS: Bacillus indicus and B. subtilis should be considered safe for oral use although the resistance of B. indicus to clindamycin requires further study. SIGNIFICANCE AND IMPACT OF THE STUDY: The results support the use of B. subtilis and B. indicus strains as food supplements.

Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds.

AIMS: The ability of 31 Lactobacillus plantarum strains to adhere to biological matrixes was evaluated, and the molecules involved in adherence were studied. METHODS AND RESULTS: Mucin, basement membrane proteins and Caco-2 cells were used in adhesion tests. These in vitro assays, together with a yeast agglutination test, were found to be discriminative for screening Lact. plantarum strains for adhesion. Some strains, such as 299v, CBE, BMCM12, Col4S and T25, were shown to possess interesting adhesion properties in at least two models. The adhesion of these strains was strongly inhibited when the bacterial cells were pretreated with trypsin. Lithium chloride and methyl-alpha-D-mannoside also inhibited adhesion to a lower extent. CONCLUSIONS: The adhesion of Lact. plantarum depends on both the model and the strain used. The chemical and enzymatic pretreatments applied to the bacterial cells suggested that lectin-like adhesins and other proteinaceous cell-surface structures are involved in adhesion of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We found a great diversity in the adhesion properties between Lact. plantarum strains. Based upon the adhesive property of these strains interesting candidates were identified, that will undergo further study as potential probiotics.

Adherence and colonization properties of Lactobacillus rhamnosus TB1, a broiler chicken isolate.

AIMS: Selected lactic acid bacteria (LAB) isolated from intestinal tract of chicken have been studied in order to investigate their ability to adhere in vitro to Basement Membrane Matrigel (BMM). A selected strain showing a good adherence in BMM test was used for in vivo colonization assays. METHODS AND RESULTS: In vitro assessment of adhesion of broiler chicken isolates was performed using BMM assay. Among LAB strains tested, Lactobacillus rhamnosus TB1 showed a good adherence that was comparable to the one of an Escherichia coli EPEC strain used as positive control. For in vivo colonization assays this strain was fluorescently stained with the carboxyfluorescein diacetate succinimidyl ester (cFDA-SE) thus allowing its detection in different layers of intestinal tract after inoculation in broiler chicken. Further, stained L. rhamnosus were found with a highest value in rectum, jejunum and ileum both 3 and 24 h after administration. CONCLUSIONS: BMM assay is a quick method to test in vitro adhesion properties of bacterial strains and cFDA-SE-stained bacteria may be considered as an alternative method to test in vivo adhesion and colonization properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus rhamnosus TB1 was therefore showed to be able to adhere strongly in vitro to BMM and in vivo to intestinal epithelial cells of chicken and may be considered as a potential probiotic for chicken.

Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum.

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.

Genetic diversity and involvement in bread spoilage of Bacillus strains isolated from flour and ropy bread.

AIMS: To study Bacillus contamination of wheat flour and ropy bread, to analyse genetic diversity of isolated strains and to evaluate the ability of these strains to produce ropy bread. METHODS AND RESULTS: Classical and molecular methods [16S rDNA sequencing and random amplified polymorphic DNA (RAPD)-PCR] were used to identify and type-isolated strains. The predominant species isolated were Bacillus subtilis and B. licheniformis. RAPD analysis demonstrated that the same sample may harbor different strains. Ten of 15 strains of B. subtilis and four of six strains of B. licheniformis were able to cause rope spoilage of the laboratory-baked bread. CONCLUSION: RAPD typing can be useful in the tracking of Bacillus strains during bakery processing and in the understanding of the role of different Bacillus strains in the rope spoilage of bread. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate the variability of Bacillus strains isolated from flour and responsible for rope spoilage of bread.

Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum.

AIMS: The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. METHODS AND RESULTS: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugar-modified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. CONCLUSIONS: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum.

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.

In vitro anti-Helicobacter pylori activity of the probiotic strain Bacillus subtilis 3 is due to secretion of antibiotics.

A limited number of antibiotics can be used against Helicobacter pylori infection, and resistance jeopardizes the success of treatment. Therefore, a search for new agents is warranted. The use of probiotics to enhance gastrointestinal health has been proposed for many years, but the scientific basis of the prophylactic and therapeutic actions of probiotics has not yet been clearly delineated. Probiotic strain Bacillus subtilis 3, whose safety has previously been demonstrated, is known to have antagonistic properties against species of the family Enterobacteriaceae. In the present study, it was also found to inhibit H. pylori. The anti-H. pylori activity present in the cell-free supernatant was not related to pH or organic acid concentration. It was heat stable and protease insensitive. At least two antibiotics, detected by thin-layer chromatography (R(f) values, 0.47 and 0.85, respectively) and confirmed by high-performance liquid chromatographic analysis, were found to be responsible for this anti-H. pylori activity. All H. pylori strains tested were sensitive to both compounds. One of these compounds was identified as amicoumacin A, an antibiotic with anti-inflammatory properties. MICs for H. pylori determined in solid and liquid media ranged between 1.7 and 6.8 microg/ml and 0.75 and 2.5 microg/ml, respectively. The underestimation of MICs determined in solid medium may be due to physicochemical instability of the antibiotic under these test conditions. An additive effect between amicoumacin A and the nonamicoumacin antibiotic against H. pylori was demonstrated.

Identification by in situ hybridization of segmented filamentous bacteria in the intestine of diarrheic rainbow trout (Oncorhynchus mykiss).

Nonculturable segmented filamentous bacteria (SFB) have been described in the gut of rats, mice and chickens, and 16S rRNA sequences for these organisms are available. These organisms, peripherically related to Clostridium phylogenetic group I, have been provisionally named 'Candidatus Arthromitus'. This work reports the observation of similar bacteria in the intestinal content of the distal intestine, preferentially, in the adult rainbow trout (Oncorhynchus mykiss) that exhibited episodic acute diarrhea, usually during the summer. Abdominal distension, intestinal fluid-mucus content and epithelium detachment were observed in trout. The demonstration that the observed microorganisms are bacteria and belong in the 'Candidatus Arthromitus' group was achieved by in situ hybridization with, respectively, a eubacterial probe and an oligonucleotide probe designed to react specifically with SFB 16S rRNA (encoded by the rrs gene) sequences. The sequenced rrs gene was compared with published sequences and found to be closely related to (although distinct from) other SFB sequences. Implication of these bacteria in trout diarrheic illness remains hypothetical.

Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I(4).

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I(4) has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42-50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I(4). Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI(4) DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI(4) when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.

Fingerprinting of Flavobacterium psychrophilum isolates by ribotyping and plasmid profiling.

Flavobacterium psychrophilum is the agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. Ribosomal RNA gene restriction patterns (ribotypes) and plasmid profiles were determined on a collection of 85 strains isolated from different countries and fish species. Several ribotypes were obtained by using the restriction endonucleases Hinc II and Pvu II. Computer analysis of the ribotypes revealed that some of them were clearly associated with the fish species from which the strains were isolated, whereas no correlation with the geographical origin was found. Most of the strains harboured at least one plasmid and several different plasmid profiles were observed, even among strains sharing the same ribotype. These methods, used alone or in combination with other typing techniques, can be considered powerful tools for the epidemiological tracing of F. psychrophilum infections.

Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.

A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum.

Coagulin, a bacteriocin-like inhibitory substance produced by Bacillus coagulans I4.

A protease-sensitive antibacterial substance produced by Bacillus coagulans I4 strain, isolated from cattle faeces, was classified as a bacteriocin-like inhibitory substance and named coagulin. The inhibitory spectrum included B. coagulans and unrelated bacteria such as Enterococcus, Leuconostoc, Oenococcus, Listeria and Pediococcus. Coagulin was stable at 60 degrees C for 90 min, at a pH ranging from 4 to 8 and appeared to be unaffected by alpha-amylase, lipase or organic solvents (10% v/v). Coagulin exhibited a bactericidal and a bacteriolytic mode of action against indicator cells. The apparent molecular mass was estimated to be about 3-4 kDa by SDS-PAGE. The B. coagulans I4 strain harbours a plasmid, pI4, approximately 14 kb in size. Novobiocin curing experiments yielded two derivatives that no longer produced the bacteriocin-like inhibitory substance. Plasmid content of these two derivatives showed that one had lost pI4, whereas the second harboured a deleted form of this plasmid, thus suggesting a plasmid location for the genes for coagulin production.

DNA probe and PCR-specific reaction for Lactobacillus plantarum.

A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.

Vibrio navarrensis sp. nov., a species from sewage.

A group of 11 strains, mostly isolated from sewage water in the Province of Navarra, Spain, were found to constitute a DNA relatedness group which is 2 to 39% related to 23 species of the genus Vibrio and 2 to 3% related to two Aeromonas species. Phenotypically, these strains have all of the properties that define the genus Vibrio. However, they differ from the previously described species by three or more properties. The strains are negative for arginine, ornithine, and lysine decarboxylase activities and the Voges-Proskauer test and are unable to utilize putrescine, gluconate, glucuronate, and histidine. They utilize and produce acid from sucrose and grow at 40 degrees C. All strains grow in the presence of 0.5% (wt/vol) NaCl, and seven strains grow weakly in peptone water lacking NaCl. The group of strains which we studied can also be differentiated from other Vibrio species by fatty acid content. The G+C ratio of the DNA is 45 to 47 mol%. The name Vibrio navarrensis sp. nov. is proposed for these strains; strain 1397-6 (= CIP 103381) is the type strain.