Paenibacillus foliorum sp. nov., Paenibacillus phytohabitans sp. nov., Paenibacillus plantarum sp. nov., Paenibacillus planticolens sp. nov., Paenibacillus phytorum sp. nov. and Paenibacillus germinis sp. nov., isolated from the Arabidopsis thaliana phyllosphere.

Six endospore-forming, Gram-stain-positive or variable, motile, rod-shaped, aerobic or facultatively anaerobic bacteria with different MALDI-TOF mass spectra (MS) were isolated from the phyllosphere of Arabidopsis thaliana plants grown in plant chambers after inoculation of surface sterilized seeds with a top soil microbial cell suspension. They were identified as members of the genus Paenibacillus through comparison with a commercial MALDI-TOF MS database and comparative 16S rRNA gene sequencing. Their genome sequences comprised multiple biosynthetic gene clusters and suggested they have unexplored biotechnological potential. Analyses of average nucleotide identity values between these strains and the type strains of their nearest neighbour species demonstrated that they represented a novel Paenibacillus species each. A detailed phenotypic comparison yielded distinctive biochemical characteristics for each of these novel species. We therefore propose to classify that these isolates into six novel species within genus Paenibacillus, for which we propose the names Paenibacillus foliorum sp. nov., Paenibacillus phytohabitans sp. nov., Paenibacillus plantarum sp. nov., Paenibacillus planticolens sp. nov., Paenibacillus phytorum sp. nov. and Paenibacillus germinis sp. nov., with strains LMG 31456T (=R-74617T=CECT 30138T), LMG 31459T (=R-74621T=CECT 30135T), LMG 31461T (=R-74618T=CECT 30133T), LMG 31457T (=R-74619T=CECT 30137T), LMG 31458T (=R-74620T=CECT 30136T) and LMG 31460T (=R-74622T=CECT 30134T) as the type strains, respectively.

Micromonospora fluminis sp. nov., isolated from mountain river sediment.

During a bioprospection of bacteria with antimicrobial activity, the actinomycete strain A38T was isolated from a sediment sample of the Carpintero river located in the Gran Piedra Mountains, Santiago de Cuba province (Cuba). This strain was identified as a member of the genus Micromonospora by means of a polyphasic taxonomy study. Strain A38T was an aerobic Gram-positive filamentous bacterium that produced single spores in a well-developed vegetative mycelium. An aerial mycelium was absent. The cell wall contained meso-diaminopimelic acid and the whole-cell sugars were glucose, mannose, ribose and xylose. The major cellular fatty acids were isoC15:0, 10 methyl C17:0, anteiso-C17:0 and iso-C17:0. The predominant menaquinones were MK-10(H4) and MK-10(H6). Phylogenetic analysis of 16S rRNA gene sequences revealed that this strain was closely related to Micromonospora tulbaghiae DSM 45142T (99.5 %), Micromonospora citrea DSM 43903T (99.4 %), Micromonospora marina DSM 45555T (99.4 %), Micromonospora maritima DSM 45782T (99.3 %), Micromonospora sediminicola DSM 45794T (99.3 %), Micromonospora aurantiaca DSM 43813T (99.2 %) and Micromonospora chaiyaphumensis DSM 45246T (99.2 %). The results of OrthoANIu analysis showed the highest similarity to Micromonospora chalcea DSM 43026T (96.4 %). However, the 16S rRNA and gyrB gene sequence-based phylogeny and phenotypic characteristics provided support to distinguish strain A38T as a novel species. On the basis of the results presented here, we propose to classify strain A38T (=LMG 30467T=CECT 30034T) as the type strain of the novel species Micromonospora fluminis sp. nov.

Proposal of Verticiella gen. nov. as replacement for the illegitimate prokaryotic genus name Verticia Vandamme et al. 2015.

The prokaryotic genus name Verticia Vandamme et al. 2015 is illegitimate because it is a later homonym of the insect genus name Verticia Malloch 1927 [Principle 2, Rule 51b(4) of the Prokaryotic Code (2008 Revision)]. This name is therefore not a correct name (Principle 6), and a replacement genus name must be proposed (Rule 54).

Reclassification of Achromobacter spiritinus Vandamme et al. 2013 as a later heterotypic synonym of Achromobacter marplatensis Gomila et al. 2011.

A repeat multi-locus sequence analysis (MLSA) of concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequences of strains classified as Achromobacter marplatensis was performed. The results revealed that earlier reported sequence data of the proposed type strain were erroneous, and that the corrected concatenated sequence divergence between the A. marplatensis LMG 26219T (=CCUG 56371T) sequence type and that of strains of Achromobacter spiritinus was well below the 2.1% threshold value that delineates species of the genus Achromobacter. These results therefore demonstrated that strains which were classified as A. spiritinus should be reclassified as A. marplatensis and that the name Achromobacter spiritinus should no longer be used. An emendation of the description of Achromobacter marplatensis is warranted.

Taxonomic dissection of Achromobacter denitrificans Coenye et al. 2003 and proposal of Achromobacter agilis sp. nov., nom. rev., Achromobacter pestifer sp. nov., nom. rev., Achromobacter kerstersii sp. nov. and Achromobacter deleyi sp. nov.

The phenotypic and genotypic characteristics of a historical collection of strains identified as Achromobacter denitrificanswere examined. Sequence analysis of a 765 bp nrdA gene fragment revealed that eight of these strains belonged to the recently described Achromobacter aegrifaciens, Achromobacter mucicolens, and Achromobacter insolitus, and that one strain belonged to Achromobacter xylosoxidans. The analysis also suggested the presence of four novel species of the genus Achromobacter among the remaining strains. The latter was confirmed by multilocus sequence analysis of concatenated nusA, eno, rpoB, gltB, lepA, nuoL andnrdA gene fragments and extensive genotypic and phenotypic characterization. We propose to name these novel species as Achromobacter agilis sp. nov., nom. rev. (type strain LMG 3411T=CCUG 62454T), Achromobacter pestifer sp. nov., nom. rev. (type strain LMG 3431T=CCUG 61959T) , Achromobacter kerstersii sp. nov. (type strain LMG 3441T=CCUG 62449T) and Achromobacter deleyi sp. nov. (type strain LMG 3458T=CCUG 62433T).

Bordetella bronchialis sp. nov., Bordetella flabilis sp. nov. and Bordetella sputigena sp. nov., isolated from human respiratory specimens, and reclassification of Achromobacter sediminum Zhang et al. 2014 as Verticia sediminum gen. nov., comb. nov.

The phenotypic and genotypic characteristics of four Bordetella hinzii-like strains from human respiratory specimens and representing nrdA gene sequence based genogroups 3, 14 and 15 were examined. In a 16S rRNA gene sequence based phylogenetic tree, the four strains consistently formed a single coherent lineage but their assignment to the genus Bordetella was equivocal. The respiratory quinone, polar lipid and fatty acid profiles generally conformed to those of species of the genus Bordetella and were characterized by the presence of ubiquinone 8, of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several aminolipids, and of high percentages of C16 : 0, cyclo-C17 : 0 and summed feature 2, as major chemotaxonomic marker molecules, respectively. The DNA G+C content was about 66 mol%, which corresponded with that of the high-percentage DNA G+C content genera of the family Alcaligenaceae including the genus Bordetella. DNA­DNA hybridization experiments revealed the presence of three distinct genomospecies and thus confirmed phenotypic differences as revealed by means of extensive biochemical characterization. We therefore propose to formally classify Bordetella genogroups 3, 14 and 15 as Bordetella bronchialis sp. nov. (type strain LMG 28640T = AU3182T = CCUG 56828T), Bordetella sputigena sp. nov. (type strain LMG 28641T = CCUG 56478T) and Bordetella flabilis sp. nov. (type strain LMG 28642T = AU10664T = CCUG 56827T). In addition, we propose to reclassify Achromobacter sediminum into the novel genus Verticia, as Verticia sediminum, gen. nov., comb. nov., on the basis of its unique phylogenetic position, its marine origin and its distinctive phenotypic, fatty acid and polar lipid profile.

Burkholderia dilworthii sp. nov., isolated from Lebeckia ambigua root nodules.

Three strains of Gram-stain-negative, rod-shaped bacteria were isolated from Lebeckia ambigua root nodules and authenticated on this host. Based on the 16S rRNA gene sequence phylogeny, they were shown to belong to the genus Burkholderia, with the representative strain WSM3556(T) being most closely related to Burkholderia caledonica LMG 23644(T) (98.70 % 16S rRNA gene sequence similarity) and Burkholderia rhynchosiae WSM3937(T) (98.50 %). Additionally, these strains formed a distinct group in phylogenetic trees of the housekeeping genes gyrB and recA. Chemotaxonomic data, including fatty acid profiles and analysis of respiratory quinones, supported the assignment of our strains to the genus Burkholderia. Results of DNA-DNA hybridizations, MALDI-TOF MS analysis and physiological and biochemical tests allowed genotypic and phenotypic differentiation of our strains from their nearest neighbour species. Therefore, these strains represent a novel species, for which the name Burkholderia dilworthii sp. nov. is proposed, with the type strain WSM3556(T) ( = LMG 27173(T) = HAMBI 3353(T)).

Growth in Stewart's medium is a simple, rapid and inexpensive screening tool for the identification of Burkholderia cepacia complex.

Ninety-one percent of Burkholderia cepacia complex reference strains (66 out of 72) displayed a yellow slope-green butt colour reaction after growth in Stewart's medium indicating the oxidation of glucose and the absence of an arginine dihydrolase system. This same colour reaction was observed for Burkholderia gladioli and several Ralstonia species, but not for Pseudomonas aeruginosa, Stenotrophomonas, Achromobacter, Pandoraea and several other Gram-negative non-fermenting bacilli. We therefore consider growth in Stewart's medium a useful, simple, rapid and inexpensive screening test to reduce the number of false positive isolates from B. cepacia complex selective media.

Evaluation of restriction fragment length polymorphism analysis of 16S rDNA as a tool for genomovar characterisation within the Burkholderia cepacia complex.

A total of 154 Burkholderia cepacia complex strains, isolated from cystic fibrosis and non-cystic fibrosis patients and the environment, representing all nine genomovars and a putative tenth, were analysed by 16S rDNA-restriction fragment length polymorphism using the restriction enzymes AluI, CfoI and DdeI. Examining this diverse strain collection resulted in very diverse restriction patterns. Only B. cepacia genomovar VI could be identified unambiguously. The same restriction patterns were observed for B. cepacia genomovars I and III and approximately half of the Burkholderia ambifaria, B. anthina and B. pyrrocinia strains. Burkholderia vietnamiensis and B. ubonensis, a putative tenth B. cepacia complex genomovar, shared identical restriction profiles. The majority of Burkholderia multivorans and B. stabilis isolates generated a unique restriction pattern, but two strains of each showed divergent restriction profiles which were also observed in other genomovars.

Enumeration of viable anaerobic bacteria by solid phase cytometry under aerobic conditions.

Classical enumeration methods for anaerobes are time-consuming and require special conditions. Solid phase cytometry (SPC) is a recent laser scanning technique for the quantitative detection of fluorescently labelled bacteria on a membrane filter that eliminates the need for a growth phase. Fluorescent labelling of cells results from the cleavage by intracellular esterases of a fluorescein type ester to yield a free fluorescein derivative, which is retained only in cells with an intact cytoplasmic membrane. However, as the standard labelling procedure is carried out under the conditions of aerobiosis, labelling of anaerobic bacteria does not appear to be obvious. We have labelled eight strains of vegetative anaerobic bacteria (i.e. Bacteroides thetaiotaomicron, Clostridium bifermentans, C. butyricum, C. perfringens, Fusobacterium nucleatum, Porphyromonas canoris, P. gingivalis, Propionibacterium acnes) and two strains of spores (C. butyricum, C. perfringens,) within 4 h under aerobic conditions. However, anaerobiosis remained necessary for spores of C. sordellii, C. sporogenes, C. tyrobutyricum. For vegetative cells of all strains, plots of SPC versus plate counts were linear with slopes exceeding 1.0, indicating that SPC consistently yielded higher numbers of bacteria.

Chryseobacterium vrystaatense sp. nov., isolated from raw chicken in a chicken-processing plant.

Yellow-pigmented, Gram-negative organisms isolated from raw chicken were investigated by means of a polyphasic taxonomic approach and were shown to represent a novel species in the genus Chryseobacterium, for which the name Chryseobacterium vrystaatense sp. nov. is proposed. Its nearest phylogenetic neighbours were Chryseobacterium joostei, Chryseobacterium indologenes and Chryseobacterium gleum, which showed 16S rRNA gene sequence similarity levels of 96.9, 97.1 and 96.1%, respectively. Levels of DNA-DNA hybridization between strains of C. vrystaatense and Chryseobacterium reference species were below 46%. Strain LMG 22846(T) (=CCUG 50970(T)) was chosen as the type strain and has a DNA G+C content of 37.1 mol%.

Identification of distinct Campylobacter lari genogroups by amplified fragment length polymorphism and protein electrophoretic profiles.

Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters (NARTC) and the biochemical C. lari variants, including the urease-positive campylobacters (UPTC), the nalidixic acid-susceptible campylobacters (NASC), and the urease-producing nalidixic acid-susceptible campylobacters. To study the taxonomic and epidemiological relationships among strains of the C. lari variants, amplified fragment length polymorphism (AFLP) profiling and whole-cell protein profile analysis were performed with 55 C. lari strains. Great genetic heterogeneity in AFLP and protein profiles was observed. Numerical analysis of AFLP profiles and of partial protein profiles allowed discrimination of four distinct genogroups. AFLP cluster I included nearly homogeneous patterns for C. lari NARTC strains (genogroup I). UPTC strains together with non-urease-producing NASC strains produced highly diverse patterns and were placed in genogroup II. The genogroup III strains had the NASC phenotype and produced more homogeneous patterns. Finally, genogroup IV strains had the classical NARTC phenotype and produced AFLP patterns that were very distinct from those of other genogroups. One UPTC strain had aberrant patterns and clustered separately, which may indicate that there is an additional genogroup. Preliminary DNA-DNA hybridization experiments suggested that genogroups I and III represent a single genomic species and that genogroup IV represents a distinct species. The detection of moderate levels of DNA-DNA hybridization between a genogroup II reference strain and genogroup I and III reference strains highlights the need for further DNA-DNA hybridization experiments to clarify the taxonomic status of the former group. No correlation of genogroups with different sources of strains was identified. These data show that UPTC strains are genetically diverse and distinct from NARTC strains. In addition, they indicate that the classical NARTC phenotype encompasses at least two genogroups.

Differentiation of Campylobacter species by AFLP fingerprinting.

The fluorescent amplified fragment length polymorphism (AFLP) fingerprinting method was tested for its ability to identify and subtype the most important Campylobacter species found in veterinary infections. Sixty-nine reference strains and 19 clinical isolates of Campylobacter jejuni subsp. jejuni, Campylobacter jejuni subsp. doylei, Campylobacter upsaliensis, Campylobacter coli, Campylobacter lari, Campylobacter fetus subsp. fetus, C. fetus subsp. venerealis, Campylobacter hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, Campylobacter mucosalis, Campylobacter helveticus and Campylobacter sputorum were subjected to analysis. The topology of the dendrogram obtained by numerical analysis of the AFLP profiles did not reflect the phylogenetic relationships as derived from 16S rDNA sequence comparison. However, except for C. lari, AFLP analysis grouped the strains that belonged to the same genomic species into distinct clusters. C. lari strains were separated into two distinct AFLP groups, which corresponded with nalidixic-acid-sensitive and -resistant variants of C. lari. These results correlated with data from whole-cell protein profiling. Within C. jejuni, C. hyointestinalis and C. fetus, strains could be identified at the subspecies level. AFLP analysis also allowed the subtyping of most species at the strain level. It is concluded that AFLP analysis is a valuable tool for concurrent identification of campylobacters at the species, subspecies and strain levels. In addition, the data confirm and extend previous reports showing that C. lari is a heterogeneous species that may comprise multiple taxa.