International Journal of Systematic and Evolutionary Microbiology (2000), 50, 979–983 Printed in Great Britain Bulleidia extructa gen. nov., sp. nov., isolated from the oral cavity Julia Downes,1 Bente Olsvik,†2 Sarah J. Hiom,1 David A. Spratt,1 Sarah L. Cheeseman,1 Ingar Olsen,2 Andrew J. Weightman3 and William G. Wade1 Author for correspondence: William G. Wade. Tel: j44 207 955 2849. Fax: j44 207 955 2847. e-mail: william.wade!kcl.ac.uk 1 Oral Microbiology Unit, Five strains of anaerobic non-sporing Gram-positive bacilli isolated from Division of Oral Medicine, advanced periodontitis (four strains) and a dentoalveolar abscess (one strain) Pathology, Microbiology and Immunology, Guy’s, that did not correspond to existing species were subjected to phenotypic and King’s and St Thomas’ genetic characterization. Following 16S rDNA sequence analysis, they were Dental Institute, found to constitute a novel branch of the low GMC Gram-positive division of King’s College London, Guy’s Hospital, London the phylogenetic tree related to Erysipelothrix rhusiopathiae and Holdemania SE1 9RT, UK filiformis. A new genus Bulleidia, and the species Bulleidia extructa, are 2 Institutt for oral biologi, proposed. Growth of B. extructa in broth media was poor but was enhanced by University of Oslo, the addition of fructose, glucose or maltose together with Tween 80. Glucose N-0316 Oslo, Norway and maltose were fermented and arginine was hydrolysed. Acetate, lactate 3 Cardiff School of and trace amounts of succinate were the end products of glucose Biosciences, Cardiff fermentation. The GMC content of the DNA of the type strain is 38 mol%. The University, PO Box 915, T Cardiff CF1 3TL, UK type strain of Bulleidia extructa is DSM 13220 . Keywords: Bulleidia, Eubacterium, taxonomy, phylogeny INTRODUCTION 1990; Olsvik, 1995). Strains GF10, MK1, UM3 and W1219T were isolated from periodontal pockets and W2274 was Anaerobic non-sporing Gram-positive bacilli isolated from a dentoalveolar abscess. Erysipelothrix rhusio- pathiae NCTC 8163T ( l ATCC 19414T) was obtained from currently assigned to the genus Eubacterium are an T extremely diverse group of organisms (Moore & the NCTC and Holdemania filiformis ATCC 51649 from Holdeman Moore, 1986). A number of new species the ATCC. have recently been proposed (Cheeseman et al., 1996; Morphology. Strains were grown at 37 mC on fastidious Poco et al., 1996a, b; Uematsu et al., 1993) and it is anaerobe agar (FAA, LabM) supplemented with 5% horse estimated that there are at least 25 un-named taxa blood under anaerobic conditions (80% N#,10%H#,10% (Wade, 1997). Phylogenetic analysis of 16S rRNA CO#). Colonial morphologies were determined using a plate gene sequence data has placed most of these new microscope after incubation for 7 d. Cellular morphology species in either the Actinobacteria or the low GjC was recorded after Gram-staining of 3 d plate cultures. Gram-positive division of the phylogenetic tree Hanging-drop preparations of 18 h cultures of peptone\ et al et al yeast extract\glucose (PYG) broth supplemented with 0n5% (Cheeseman ., 1996; Wade ., 1999). In this Tween 80 were examined by phase-contrast microscopy for study, a group of isolates from oral infections, which cellular motility. could not be identified as belonging to an existing species, were studied. Ultrastructure. Transmission electron microscopy was used to examine the cell-wall ultrastructure. Cells were fixed in METHODS 2n5% glutaraldehyde in 0n1 M Sorensen’s buffer, then centrifuged and washed in the same buffer. The cells were Bacterial strains. The strains included in the study had been post-fixed in 1% osmium tetroxide, dehydrated by a graded provisionally identified as Eubacterium spp. (Wade et al., series of ethanol, treated with propylene oxide and em- ................................................................................................................................................. bedded in Taab epoxy resin. Ultrathin sections were stained † Sadly, Bente Olsvik passed away in December 1998. with uranyl acetate and lead citrate for transmission electron microscopy. Abbreviation: PRAS, pre-reduced aerobically sterilized. The GenBank accession number for the 16S rDNA sequence of Bulleidia Biochemical and physiological tests. Fermentation tests were extructa is AF220064. performed using pre-reduced, anaerobically sterilized 01180 # 2000 IUMS 979 J. Downes and others (PRAS) sugars according to the methods of Holdeman et al. RESULTS AND DISCUSSION (1977) except that the PRAS media were prepared in an anaerobic workstation using pre-reduced distilled water. Five strains of Gram-positive bacilli were studied. Other biochemical tests were performed as described by They were found to be obligately anaerobic, non- Holdeman et al. (1977) and Summanen et al. (1993). motile, non-spore-forming short bacilli (0n5i0n8– Sensitivity to bile was determined by comparing growth of 2 µm). Colonies on FAA were 0n8 mm in diameter, strains on FAA with and without 2% oxgall after 5 d circular, entire, low convex and opaque after 7 d incubation. Spore formation was assessed by both direct incubation. Growth in peptone\yeast extract broth microscopic examination and culture following killing of was poor but was enhanced by the addition of 0n5% vegetative cells by either heating to 80 mC for 10 min or Tween 80 together with 1% glucose, maltose or treatment with 95% ethanol for 30 min. Temperature fructose. Glucose and maltose were fermented, with optima were determined by incubation of PYGj0n5% Tween 80 broth cultures at 30, 37 and 42 mC. acetate, lactate and trace amounts of succinate pro- duced as the end products of metabolism. Cells grew in Metabolic-end-product analysis. Bacterial strains were grown PYGj0n5% Tween 80 at 30 and 37 mC but not 42 mC. in PYG broth supplemented with 0n5% Tween 80 and short- chain volatile and non-volatile fatty acids extracted by Transmission electron microscopic examination standard methods (Holdeman et al., 1977). Analysis was revealed a typical Gram-positive cell wall composed of performed by GC with a capillary column coated with CP- a single layer of peptidoglycan approximately 55 nm Wax 58 (free fatty acid) solid phase. thick surrounding the cytoplasmic membrane (Fig. 1). Protein profiles. Protein profiles of whole-cell proteins were All strains hydrolysed arginine. Catalase, indole and generated by SDS-PAGE using 10–15% gradient gels H S were not produced and nitrate was not reduced. and the PhastSystem (Pharmacia) as described previously # (Slayne et al., 1990) except that the cells were pre-treated Aesculin and urea were not hydrolysed and gelatin was " with lysozyme (50 µgml− ) for 3 h at 37 mC and vortexed for not liquefied. All strains were positive for arginine 6 min with 1\10 vol. 100 µm diameter glass beads before dihydrolase, arginine arylamidase and leucine aryl- boiling. amidase in the Rapid ID 32A panel. The GjC content of the DNA of strain W1219T was 38 mol%. Enzyme profiles. Enzyme profiles were generated with the Rapid ID32A anaerobe identification kit (bioMe! rieux) 16S rRNA gene PCR products obtained from strain according to the manufacturer’s instructions. Bacteria were W1219T with primers 27F and 1525R were fully harvested from blood agar plates [Blood Agar Base No. 2 sequenced. Approximately 550 bases of 16S rDNA (LabM) supplemented with 5% horse blood] incubated were sequenced for strains GF10, MK1 and UM3 anaerobically at 37 mC for 72 h. between bases 357 and 907 (Escherichia coli 16S rRNA gene PCR and sequencing. DNA was isolated from numbering). This stretch of sequence includes the the bacteria by standard methods. The 16S rRNA gene was variable regions V3–5 (Neefs et al., 1993). Over 545 amplified by PCR using primers 27F and 1525R (Lane, aligned bases, each strain had "99% sequence simi- 1991). PCR amplification was performed with an Uno II larity with each other strain. Phylogenetic analysis of Thermocycler (Biometra) using PCR buffer (Bioline) con- the sequence data assigned W1219T to the clostridial taining 1n5 mM MgCl#, 200 µM dNTPs, 1 mM of each subphylum within the low GjC Gram-positive bac- oligonucleotide primer, 1 U Taq DNA polymerase (Bioline) and template DNA (100 ng) in a total volume of 100 µl. Thirty amplification cycles were performed with denaturing at 94 mC for 1 min, annealing at 54 mC for 1 min and elongation at 72 mC for 2 min. PCR products were sequenced directly using a dye terminator cycle sequencing kit with AmpliTaq FS (Perkin Elmer) and 60 ng template DNA, according to the manufacturer’s instructions. Sequencing was performed using an automated sequencer (ABI 377; Perkin Elmer) with primers 27F, 342R, 357F, 519R, 907R, 926F, 1100R, 1114F, 1392R and 1525R (Lane, 1991). Sequence analysis. Sequences were connected using (Hitachi) and then submitted to the Ribosomal Database Project via the World Wide Web for provisional identi- fication using the SimilarityIRank program. From the phylogenetic position indicated by SimilarityIRank, related sequences were selected and aligned by means of (Thompson et al., 1994). Further analysis was performed using the suite of programs (Felsenstein, 1993). Specifically, was used to compare sequences by the Jukes–Cantor algorithm and was used for neighbour-joining cluster analysis. ................................................................................................................................................. Estimation of mol% GjC content of DNA. This was done by Fig. 1. Transmission electron micrograph of Bulleidia