Three strains of a hitherto unknown, Gram-negative, tiny, anaerobic coccus were collected from human clinical samples originating from skin and soft tissues. The three isolates displayed at least 99.9 % identity in their 16S rRNA gene sequences and more than 99.8 % identity in their dnaK gene sequences. The isolates were affiliated to the family Veillonellaceae, the coccobacillus Dialister micraerophilus being the most closely related species, but there was no more than 91.1 % identity in the 16S rRNA gene sequence between this species and the three isolates. Phylogeny based on the 16S rRNA gene confirmed that the three strains represent a novel and robust lineage within the current family Veillonellaceae. A similar genomic structure was demonstrated for the three isolates by PFGE-based analysis. Morphology and metabolic end products, as well as genotypic and phylogenetic data supported the proposal of the novel genus Negativicoccus gen. nov., with the novel species Negativicoccus succinicivorans sp. nov. [type strain ADV 07/08/06-B-1388T (=AIP 149.07T=CIP 109806T=DSM 21255T=CCUG 56017T) as type species]. Phylogenetic analyses based on the 16S rRNA gene sequences of members of the phylum Firmicutes and other phyla indicated that the family Veillonellaceae forms a robust lineage clearly separated from those of the classes ‘Bacilli’, ‘Clostridia’, Thermolithobacteria and ‘Erysipelotrichi’ in the phylum Firmicutes. Therefore, we propose that this family is a class-level taxon in the phylum Firmicutes, for which the name Negativicutes classis nov. is proposed, based on the Gram-negative type of cell wall of its members, with the type order Selenomonadales ord. nov. In this order, a novel family, Acidaminococcaceae fam. nov., is proposed and description of the family Veillonellaceae is emended.

Among the six species characterized within the genus Ochrobactrum, Ochrobactrum anthropi and Ochrobactrum intermedium are currently reported as opportunistic pathogens in humans. Since the species identification is mainly based on 16S rDNA analysis, the aim of this study was to search for other characteristics useful for Ochrobactrum species discrimination. Ribotyping, morphological and biochemical analyses, and antimicrobial susceptibility testing were performed for a panel of 35 clinical isolates, first identified to the species level using 16S rDNA sequencing. Type and reference strains of five Ochrobactrum species were comparatively analysed. Commercial identification systems such as API 20NE and VITEK 2 were tested for their ability to identify Ochrobactrum anthropi and to detect other members of the genus Ochrobactrum. An improved protocol for the identification of Ochrobactrum spp. by routine medical microbiology practices is proposed: isolation of a non-fastidious non-fermenting oxidase-positive Gram-negative rod resistant to all â-lactams except imipenem indicates the genus Ochrobactrum, and the API 20NE system confirms the genus identification for most strains, whereas the VITEK 2 system using ID-GNB cards was less powerful. Urease activity, the mucoidy of the colonies, growth at 45 8C on tryptic soy agar, and susceptibility to colistin, tobramycin and netilmicin should be considered as differential characteristics for identification of O. anthropi and O. intermedium to the species level. However, definitive identification depends on genotyping methods.

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1?43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus Veillonella. These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria. The 16S rDNA sequences of nine O. intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp. available in data banks. Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA. The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule. Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion. Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone. Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion. In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone. Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies. The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies.The genus Ochrobactrum belongs to the alpha 2 subgroup of Proteobacteria and is the closest relative to the genus Brucella. Ochrobactrum anthropi was considered the sole and type species of the genus Ochrobactrum (15) until 1998, when Ochrobactrum intermedium was proposed as a new species of this genus (50). Factors discriminating between O. anthropi and O. intermedium were their low DNA-DNA hybridization, their different 16S ribosomal DNA (rDNA) sequences, the different Western blot profiles of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell proteins, and the resistance of O. intermedium to colistin and polymyxin B (50). In a recent study, Lebuhn et al. (21) performed a polyphasic analysis of a large collection of Ochrobactrum sp. strains isolated from the wheat rhizoplane. They described two new species, Ochrobactrum grignonense and Ochrobactrum tritici and proposed the separation of the Brucella-Ochrobactrum group into five clades with O.

Six clinical isolates of a hitherto unknown, strictly anaerobic, Gram-negative rod showing fastidious growth were subjected to a polyphasic taxonomic study, including phenotypic, genomic and phylogenetic feature analyses. 16S rRNA gene sequenced-based phylogeny revealed that the novel strains represent a homogeneous group distant from any recognized species in the candidate phylum ‘Synergistetes’. The novel isolates were most closely related to species of the genus Dethiosulfovibrio, with 88.2–88.7 % 16S rRNA gene sequence similarity. Large-scale chromosome structure and DNA G+C content also differentiated the novel strains from members of the genus Dethiosulfovibrio. The novel strains were asaccharolytic. Major metabolic end products in trypticase/glucose/yeast extract broth were acetic, lactic, succinic and isovaleric acids and the major cellular fatty acids iso-C15 : 0 and C16 : 0. Based on the data presented here, a new genus, Jonquetella gen. nov., is proposed with one novel species, Jonquetella anthropi sp. nov. J. anthropi is the first characterized species of the candidate phylum ‘Synergistetes’ that includes human isolates. The G+C content of the DNA of the type strain of J. anthropi ADV 126T (=AIP 136.05T=CIP 109408T=CCUG 53819T) is 59.4 mol%.

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Three strains of a hitherto unknown, Gram-negative, anaerobic coccus were isolated from human samples. At the phenotypic level, the isolates displayed all the characteristics of bacteria belonging to the genus Veillonella. Sequence analysis revealed that the three strains shared >99·5 % similarity in 16S rDNA sequence and >98·4 % similarity in dnaK sequence. The three unknown strains formed a separate subclade that was clearly remote from Veillonella species of human and animal origin. Based on these results, the three strains were considered to represent a novel species within the genus Veillonella, for which the name Veillonella montpellierensis is proposed. The type strain of the species is ADV 281.99T (=CIP 107992T=CCUG 48299T).