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A Gram-negative, rod-shaped, non-spore-forming bacterium was isolated from a nitrifying inoculum. On the basis of 16S rRNA gene sequence similarity, LMG 22862 T was shown to belong to the family Sphingobacteriaceae, related to Pedobacter africanus (98?0 %) and Pedobacter heparinus (97?6 %). The nearly complete 16S rRNA gene sequence of LMG 22862 T (AJ786798) was determined as described below. DNA was extracted according to Pitcher et al. (1989) as modified by Heyndrickx et al. (1996). PCR amplification was performed as described previously (Heyrman & Swings, 2001). PCR-amplified 16S rRNA gene products were purified using the Nucleofast 96 PCR system (Millipore). For each sequence reaction a mixture of 1 ml purified product, 1 ml of BigDye Termination RR mix version 3.1 (Perkin Elmer), 1?5 ml BigDye buffer (56) and 3 ml (20 ng ml 21) of one of the six sequencing primers was used. MilliQ water was added to a total volume of 10 ml. The primers and PCR program were previously described (Heyrman & Swings, 2001). Sequence analysis was performed using an Applied Biosystems 3100 DNA Sequencer following the protocols of the manufacturer (Perkin-Elmer). The sequence was assembled with the Kodon version 2.0 software package (Applied Maths). FASTA analysis at the EMBL database (Pearson & Lipman, 1988) indicated that LMG 22862 T was most closely related to species of the genus Pedobacter. Phylogenetic analysis using Kodon version 2.0 showed that LMG 22862 T was most closely related to P. africanus (98?0 %) and P. heparinus (97?6 %) (Fig. 1).The DNA G+C content of LMG 22862 T , determined by HPLC (Mesbah et al., 1989) according to the further specifications given by Logan et al. (2000), was 42?7 mol%. This is within the range of 36-45 mol% given in the genus description (Steyn et al., 1998 For SDS-PAGE of whole-cell proteins, isolates were grown on phosphate-buffered nutrient agar (NA) (pH 6?8) and incubated aerobically at 28 uC for 48 h. The preparation of whole-cell proteins and SDS-PAGE were performed as described by Pot et al. (1994). Densitometric analysis, normalization and interpolation of the protein profiles were performed using the GelCompar 4.2 software package (Applied Maths). UPGMA clustering of the protein profiles (data not shown) revealed 80 % similarity of LMG 22862 T to the type strains of P. heparinus and P. africanus. This observation further supports the conclusion that LMG 22862 T belongs to a novel species within the genus Pedobacter.Cell morphology was investigated by light microscopy at 61000 of cells grown on TSA for 48 h at 28 u C. Cells were Gram stained and examined for catalase and oxidase activity. Utilization of carbon sources and enzyme production (Table 2) were tested using API 20 NE, API 50 CH and API ZYM strips (bioMérieux) according to the manufacturer's instructions. Antimicrobial susceptibilities (Table 2) were determined with the disc diffusion method using a ST6090 Disc Dispenser (Oxoid). Antibiograms were determined according to the conventional KirbyBauer method (Bauer et al., 1966...

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Aims: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. Methods and Results: Six feed concentrate samples from ®ve different farms were analysed. High levels of spores (up to 10 6 spores g ±1 ) were found. Identi®cation of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, ampli®ed ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identi®ed to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80°C were identi®ed as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100°C, were identi®ed as members of the B. subtilis group (®ve isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. Conclusions: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. Signi®cance and Impact of the Study: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.

Sixty-two strains of thermophilic aerobic endospore-forming bacteria were subjected to polyphasic taxonomic study including 16S rRNA gene sequence analysis, polar lipid and fatty acid analysis, phenotypic characterization, and DNA-DNA hybridization experiments. Distinct clusters of the species Geobacillus stearothermophilus, Geobacillus thermodenitrificans, Geobacillus toebii and Geobacillus thermoglucosidasius were formed, allowing their descriptions to be emended, and the distinctnesses of the poorly represented species Geobacillus jurassicus, Geobacillus subterraneus and Geobacillus caldoxylosilyticus were confirmed. It is proposed that the name Geobacillus thermoglucosidasius be corrected to G. thermoglucosidans. Bacillus thermantarcticus clustered between Geobacillus species on the basis of 16S rRNA gene sequence analysis, and its transfer to Geobacillus is proposed. The above-mentioned species, together with Geobacillus thermoleovorans and Geobacillus thermocatenulatus, form a monophyletic cluster representing the genus Geobacillus. The distinctness of 'Geobacillus caldoxylosilyticus' was confirmed and it is proposed that it be accommodated, along with Geobacillus tepidamans, in Anoxybacillus. The type strain of Geobacillus debilis was not closely related to any members of Anoxybacillus and Geobacillus, and it is proposed that this species be placed in the new genus Caldibacillus

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Twelve strains named Bacillus pantothenticus, a t least 29 Bacillus strains representing 16 species belonging to rRNA groups 1 and 2, one Bacillus dipsosauri strain, and 38 strains of Amphibacillus, Aneurinibacillus, Bre vibacillus, Ha lobacillus, Pa en ibacillus, Sporosarcina and Marinococcus, were characterized genotypically using amplified rDNA restriction analysis (ARDRA), and phenotypically using routine diagnostic characters comprising 61 biochemical tests in the API System and 15 observations of vegetative cell and sporangial morphology. The B. pantothenticus strains were also characterized by fatty acid methyl ester analysis and SDS-PAGE of whole-cell proteins. ARDRA revealed that strains of B. pantothenticus formed a cluster quite separate from other species in rRNA group 1, supporting the recognition of the former as a separate genus, for which the name Virgibacillus is proposed. The polyphasic data also indicate the presence of an as yet undescribed new species within this genus. The species Virgibacillus pantothenticus and related organisms comprising this new genus can be distinguished from members of Bacillus rRNA group 1 (Bacillus sensu stricto), and from members of Paenibacillus and other aerobic endospore-forming bacteria by routine phenotypic tests.Keywords : amplified rDNA restriction analysis, Virgibacillus pan tothen ticus comb.nov., Bacillus, API, fatty acid methyl ester INTRODUCTIONBacillus pantothenticus was described by Proom & Knight (21) following a nutritional analysis of over 300 mesophilic soil isolates of Bacillus species. Eleven strains requiring pantothenic acid were isolated from eleven different soil samples, taken from widely separated localities in Southern England, by incubating soil suspensions in 4 % NaCl broth and plating on nutrient agar at 37 "C. They considered them as members of a species most closely resembling Bacillus circulans, but distinct from it, and subsequent studies have confirmed the validity of the species (3, 9, 17). Later isolations have been made from antacids (3), food, water, bile and soil.Abbreviations: ARDRA, amplified rDNA restriction analysis; FAME, fatty acid methyl ester; UPGMA, unweighted pair group method using arithmetic averages.