An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Immunomodulatory properties of Lactobacillus plantarum and its use as a recombinant vaccine against mite allergy.

BACKGROUND: Selected lactic acid bacteria were reported to prevent atopic dermatitis and experimental asthma but the mechanisms of their immunomodulatory effects are not fully elucidated. In this study, the signaling pathways triggered by Lactobacillus plantarum NCIMB8826 were investigated and the potential use of this strain producing a variant of the mite allergen Der p 1 as live vaccine vehicle was evaluated. METHODS: Mouse bone marrow-derived dendritic cells were stimulated with wild-type or a L. plantarum teichoic acid mutant to evaluate the secretion of cytokines. A recombinant L. plantarum expressing Der p 1 was engineered, its in vitro immunomodulatory properties were characterized and its prophylactic potential was evaluated in a Der p 1-sensitization murine model. RESULTS: Mouse dendritic cells stimulated by L. plantarum triggered the release of interleukin-10 (IL-10), IL-12 p40, IL-12 p70 and tumor necrosis factor-alpha (TNF-alpha). IL-12 p40 secretion was dependent on nuclear factor-kappaB (NF-kappaB), mitogen-activated protein (MAP) kinases, Toll-like receptor 2 (TLR2), TLR9 and on the bacterial teichoic acid composition. Recombinant L. plantarum producing Der p 1 exhibited similar immunostimulatory properties as wild-type. Prophylactic intranasal pretreatment of mice with this recombinant strain prevented the development of the typical Th2-biased allergic response by a drastic reduction of specific IgE and the induction of protective allergen-specific IgG2a antibodies. Moreover, both wild-type or recombinant L. plantarum reduced airway eosinophilia following aerosolized allergen exposure and IL-5 secretion upon allergen restimulation. CONCLUSION: By combining both Th1-type immunostimulatory properties and an efficient allergen delivery capacity, recombinant L. plantarum producing Der p 1 represents a promising vaccine against house dust mite allergy.

In vivo and in vitro immunomodulation of Der p 1 allergen-specific response by Lactobacillus plantarum bacteria.

BACKGROUND: Lactic acid bacteria (LAB) were reported to reduce some allergic manifestations in mice and humans but their impact on the aeroallergen-dependent immune mechanisms is still debated. OBJECTIVE: The potential capacities of Lactobacillus plantarum NCIMB8826 to reduce the allergic response induced by Der p 1, the major house dust mite allergen of Dermatophagoides pteronyssinus, were evaluated in vivo and in vitro. Methods First, the effect of the intranasal co-administration of LAB and purified Der p 1 allergen before a sensitization protocol was evaluated. The allergen-specific antibody and cellular responses as well as airway inflammation were measured. Second, the impact of LAB on the cytokine profile of spleens cells from Der p 1-sensitized mice was assessed. Third, upon stimulation with LAB, the levels of cytokine produced by dendritic cells derived from the bone marrow (BMDCs) of wild-type, Toll-like receptor 2 (TLR2)-, TLR4- and MyD88-KO mice were compared. Results The co-application of L. plantarum and Der p 1 induced a T-helper type 1 (Th1)-biased allergen-specific IgG response, the absence of specific IgE response and favoured the production of INF-gamma upon allergen re-stimulation. Moreover, the previous LAB administration reduced the development of bronchoalveolar lavage eosinophilia usually induced by aerosol exposure. Additionally, the studied LAB strain was shown to modify in vitro the cytokine level produced by Der p 1-sensitized spleen cells mainly towards a Th1 profile. Finally, L. plantarum stimulated high IL-12 and moderate IL-10 production in mouse BMDCs notably through the TLR2-, MyD88-dependent and TLR4-independent pathway. CONCLUSION: In vivo co-administration of probiotic LAB with Der p 1 might prevent the development of the mite allergic response. The probiotic L. plantarum was shown to display in vitro therapeutic potentials for the treatment of allergy and to trigger the immune system by a TLR2- and MyD88-dependent signalling pathway.

Enterococcus villorum sp. nov., an enteroadherent bacterium associated with diarrhoea in piglets.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Polyphasic characterization of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (p(HB-co-HV)) metabolizing and denitrifying Acidovorax sp. strains.

For the purpose of denitrification in small drinking water plants, a bacterial mixed population was isolated from a packed bed column bioreactor with poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P(HB-co-HV)) as a substrate for the denitrification of ground water (10 degrees C). Isolates 2nIII from the mixed culture, with the ability to denitrify and metabolize P(HB-co-HV), were used as starter cultures for the elimination of nitrate in ground water. The strains were characterized by diverse techniques. Classical phenotypic studies lead to rRNA group III of the genus Pseudomonas. Results obtained by molecular techniques demonstrated that the 2nIII strains are members of the Comamonadaceae and shows similarities to the genus Acidovorax. However, an integration of the 2nIII isolates within one of the known Acidovorax species is not possible for the moment. The 2nIII starter cultures clustered close to Av. temperans according to their whole cell proteins and fatty acids, whereas in DNA/DNA hybridization no significant DNA binding (< 25%) was found. In contrast a significant but low degree of DNA/DNA hybridization was found between the 2nIII strains and Av. facilis and Av. delafieldii. Our polyphasic results lead to the conclusion that the 2nIII strains may constitute a separate Acicdovorax species.

A polyphasic approach towards the identification of strains belonging to Lactobacillus acidophilus and related species.

A set of 98 strains belonging to nine species of the Lactobacillus acidophilus rRNA-group have been analysed by SDS-PAGE of cellular proteins, RAPD-PCR and AFLP with fluorescently labeled primers in order to find improved methods for their identification. Strains of the following phenotypically highly similar species were examined: L. acidophilus, L. amylovorus, L. crispatus, L. johnsonii, L. gasseri, L. gallinarum, L. helveticus, L. iners and L. amylolyticus. Although the majority of the species can be differentiated by SDS-PAGE of whole-cell proteins, the latter technique showed poor discrimination between L. gasseri and L. johnsonii strains and between some strains of L. amylovorus and L. gallinarum. However, this study shows that the RAPD-PCR (using at least 3 different primers followed by numerical analysis of the combined patterns) and AFLP are most suitable genomic fingerprinting techniques for the differentiation of all the species listed above, and that databases for identification can be constructed, particularly when commercially available molecular tool-kits are used. The separate species status of the recently described L. amylolyticus and L. iners was fully confirmed.

Polyphasic approach to the classification and identification of Gardnerella vaginalis and unidentified Gardnerella vaginalis-like coryneforms present in bacterial vaginosis.

A taxonomic study of Gardnerella vaginalis and G. vaginalis-like coryneforms was performed in order to clarify the phylogenetic affiliation of these organisms and to improve future identification. We examined 50 strains by performing whole-cell protein and fatty acid analyses, a 16S rRNA sequence analysis, and an extensive phenotypic characterization analysis. The results of both chemotaxonomic techniques which we used divided the organisms into two main clusters, and the 16S rRNA sequence analysis revealed that the clusters represent different genera, which were easily distinguished by the results of classical phenotypic tests. The cluster I strains were identified as G. vaginalis, which was shown to be a close relative of the genus Bifidobacterium. An improved description of G. vaginalis is presented. The cluster II strains belong to or are closely related to Actinomyces turicensis.

Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3.

[Pseudomonas] rubrisubalbicans, a mild plant pathogen. Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates. Our results show that they all belong to the genus Herbaspirillum. In addition to H. seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin. The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities. The type strain of H. rubrisubalbicans is NCPPB 1027 (=LMG 2286); representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321). It has been confirmed that H. rubrisubalbicans is an endophytic diazotroph. It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae. Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine. Oligonucleotide probes based on partial sequences of the 23S rRNA of H. seropedicae and H. rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

Polyphasic taxonomy, a consensus approach to bacterial systematics.

Over the last 25 years, a much broader range of taxonomic studies of bacteria has gradually replaced the former reliance upon morphological, physiological, and biochemical characterization. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them in a consensus type of classification, framed in a general phylogeny derived from 16S rRNA sequence analysis. In some cases, the consensus classification is a compromise containing a minimum of contradictions. It is thought that the more parameters that will become available in the future, the more polyphasic classification will gain stability. In this review, the practice of polyphasic taxonomy is discussed for four groups of bacteria chosen for their relevance, complexity, or both: the genera Xanthomonas and Campylobacter, the lactic acid bacteria, and the family Comamonadaceae. An evaluation of our present insights, the conclusions derived from it, and the perspectives of polyphasic taxonomy are discussed, emphasizing the keystone role of the species. Taxonomists did not succeed in standardizing species delimitation by using percent DNA hybridization values. Together with the absence of another "gold standard" for species definition, this has an enormous repercussion on bacterial taxonomy. This problem is faced in polyphasic taxonomy, which does not depend on a theory, a hypothesis, or a set of rules, presenting a pragmatic approach to a consensus type of taxonomy, integrating all available data maximally. In the future, polyphasic taxonomy will have to cope with (i) enormous amounts of data, (ii) large numbers of strains, and (iii) data fusion (data aggregation), which will demand efficient and centralized data storage. In the future, taxonomic studies will require collaborative efforts by specialized laboratories even more than now is the case. Whether these future developments will guarantee a more stable consensus classification remains an open question.

Classification of Pseudomonas diminuta Leifson and Hugh 1954 and Pseudomonas vesicularis Büsing, Döll, and Freytag 1953 in Brevundimonas gen. nov. as Brevundimonas diminuta comb. nov. and Brevundimonas vesicularis comb. nov., respectively.

The taxonomic positions of strains previously assigned to Pseudomonas diminuta and Pseudomonas vesicularis were investigated by a polyphasic approach. The results of DNA-rRNA hybridization studies indicated that these two species belong to a separate genus in the alpha subclass (rRNA superfamily IV) of the Proteobacteria, for which the name Brevundimonas is proposed. Genus delineation and species delineation were determined by comparing the results of numerical analyses of whole-cell protein patterns, fatty acid compositions, and phenotypic characteristics and by measuring DNA base ratios and degrees of DNA relatedness. Taxonomic characteristics of Brevundimonas diminuta and Brevundimonas vesicularis strains were compared with characteristics of reference strains belonging to the following phylogenetically related taxa: a group of organisms gathered in Enevold Falsen group 21, the genera Sphingomonas and Rhizomonas, and the generically misclassified organisms [Pseudomonas] echinoides and "[Pseudomonas] riboflavina."

Identification of lactobacilli from sourdough and description of Lactobacillus pontis sp. nov.

The microflora of sourdough preparations was investigated by examining the physiological characteristics, whole-cell protein patterns, and 16S rRNA sequences of Lactobacillus isolates. Strains isolated from sourdough were placed in the species Lactobacillus brevis, Lactobacillus sanfrancisco, and Lactobacillus reuteri. 16S rRNA sequences were determined for L. brevis, Lactobacillus fructivorans, Lactobacillus fermentum, L. sanfrancisco, and L. reuteri, and oligonucleotide probes for fast specific identification of these sourdough lactobacilli were deduced. The physiological characteristics, protein patterns, and 16S rRNA sequences of these organisms were compared with data for other sourdough lactobacilli and additional reference strains. Strains of a Lactobacillus species were isolated from rye sourdough; these strains may account for most of the flora in sourdough made from wheat or rye. These organisms were differentiated from other sourdough lactobacilli by their protein pattern, 16S rRNA sequence, G + C content, and physiological properties. The 16S rRNA sequence of this species was determined, and we constructed a phylogenetic tree which reflected the relationships of this species to other lactobacilli. This organism is closely related to L. reuteri. A new Lactobacillus species, Lactobacillus pontis, is proposed. The type strain is L. pontis LTH 2587 (= DSM 8475 = LMG 14187). We describe a general strategy in which a polyphasic approach was used to characterize a new species.

Polyphasic taxonomic study of the emended genus Arcobacter with Arcobacter butzleri comb. nov. and Arcobacter skirrowii sp. nov., an aerotolerant bacterium isolated from veterinary specimens.

The relationships of 77 aerotolerant Arcobacter strains that were originally identified as Campylobacter cryaerophila (now Arcobacter cryaerophilus [P. Vandamme, E. Falsen, R. Rossau, B. Hoste, P. Segers, R. Tytgat, and J. De Ley, Int. J. Syst. Bacteriol. 41:88-103, 1991]) and 6 reference strains belonging to the taxa Arcobacter nitrofigilis, Arcobacter cryaerophilus, and "Campylobacter butzleri" were studied by using a polyphasic approach, in which we performed DNA-rRNA hybridizations, DNA-DNA hybridizations, a numerical analysis of whole-cell protein patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an analysis of cellular fatty acid compositions, and a phenotypic analysis and determined DNA base ratios. Our results indicate that "C. butzleri" should be transferred to the genus Arcobacter as Arcobacter butzleri comb. nov., as was suggested by Kiehlbauch and coworkers (J. A. Kiehlbauch, D. J. Brenner, M. A. Nicholson, C. N. Baker, C. M. Patton, A. G. Steigerwalt, and I. K. Wachsmuth, J. Clin. Microbiol. 29:376-385, 1991). A rapid screening of all strains in which we used the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique revealed five major groups, which were identified by using DNA-DNA hybridization data as A. cryaerophilus (two distinct electrophoretic subgroups), A. butzleri, A. nitrofigilis, and a new species, for which we propose the name Arcobacter skirrowii. The phylogenetic position within rRNA superfamily VI was established for each species. A. butzleri strains and strains belonging to one of the electrophoretic subgroups of A. cryaerophilus had similar fatty acid contents. An analysis of fatty acid compositions allowed clear-cut differentiation of all of the other groups. All of the species could be distinguished by using classical phenotypic tests, although erroneous identifications due to a shortage of clear-cut differentiating tests could occur.