International Journal of Systematic and Evolutionary Microbiology (2000), 50, 179–181 Printed in Great Britain

Luteococcus peritonei sp. nov., isolated from NOTE the human peritoneum

Matthew D. Collins,1 Paul A. Lawson,1 Natalia Nikolaitchouk2,3 and Enevold Falsen2

Author for correspondence: Matthew D. Collins. Tel: j44 118 935 7226. Fax: j44 118 926 7917. e-mail: m.d.collins!reading.ac.uk

1 Department of Food An unusual catalase-positive pleomorphic Gram-positive rod isolated from a Science of Technology, human clinical specimen was subjected to a polyphasic taxonomic analysis. University of Reading, Reading RG6 6AP, UK Comparative 16S rRNA gene sequence analysis revealed the unknown bacterium was a member of the high GMC branch of the Gram-positive bacteria 2 Culture Collection, Department of Clinical (Actinobacteria), and was phylogenetically a member of the family Bacteriology, University of Propionibacteriaceae, with Luteococcus japonicus as its nearest relative. Based Go$ teborg, Go$ teborg, on both phenotypic and phylogenetic evidence, it is proposed that the Sweden unknown bacterium be classified in the genus Luteococcus,asLuteococcus 3 Department of peritonei sp. nov. The type strain of Luteococcus peritonei is CCUG 38120T. Microbiology, Russian People’s Friendship University, Moscow, Russia Keywords: Luteococcus peritonei, taxonomy, phylogeny, 16S rRNA

The genus Luteococcus was proposed by Tamura et al. Strain CCUG 38120T was isolated from human per- (1994) to accommodate two strains of a Gram-positive itoneum during a foetal autopsy and submitted to the coccus-shaped bacterium which were originally iso- Culture Collection of the University of Go$ teborg lated in Japan from soil and from water used for (CCUG) for identification. The unknown strain was brewing ‘miyamizu’ (Oda, 1935). Currently only a biochemically characterized by using the API Rapid single species, Luteococcus japonicus, is recognized. ID32 Strep, API CORYNE and API ZYM systems Phylogenetically Luteococcus japonicus is a member of according to the manufacturer’s instructions (API the family Propionibacteriaceae (Stackebrandt et al., bioMe! rieux). Cells were grown on chocolate agar at 1997) which includes Propionibacterium, Propioniferax 30 mC for cellular fatty acid determination and fatty and Microlunatus. Luteococcus japonicus resembles acids were examined using the MIDI system. The cell most other species of this family in possessing a cell wall composition of the isolate was determined as wall murein based on -diaminopimelic acid (- described by Schleifer & Kandler (1972) and res- Dpm) but differs significantly in synthesizing pre- piratory quinone content as described by Collins dominantly (approx. 90% of total acids) mono- (1985). Phylogenetic analysis was performed by com- unsaturated long-chain cellular fatty acids (Tamura et parative 16S rRNA gene sequence analysis. A large al., 1994). In contrast other members of the Propioni- fragment of the 16S rRNA gene (corresponding to bacteriaceae produce major amounts of iso- and positions 30–1521 of the Escherichia coli 16S rRNA anteiso- methyl-branched cellular fatty acids. During gene) was amplified by PCR using conserved primers the course of a study of unusual Actinobacteria that close to the 3h and 5h ends of the gene. The PCR cause or are associated with human disease, we have products were purified using a Prep-A-Gene kit (Bio- characterized a -Dpm-containing rod-shaped bac- Rad) according to the manufacturer’s instructions and terium which resembles Luteococcus japonicus in pos- directly sequenced using a Taq Dye-Deoxy terminator sessing very high levels of monounsaturated cellular cycle sequencing kit (Applied Biosystems) and an fatty acids. Based on the results of a polyphasic automatic DNA sequencer (model 373A; Applied taxonomic study, we propose the unknown rod-shaped Biosystems). The closest known relatives of the new bacterium be classified in the genus Luteococcus as isolate were determined by performing sequence data- Luteococcus peritonei sp. nov. base searches. These sequences and those of other known related strains were retrieved from the Gen- ...... Bank or Ribosomal Database Project (RDP) data- Abbreviation: LL-Dpm, LL-diaminopimelic acid. bases and aligned with the newly determined sequence The GenBank accession number for the 16S rRNA gene sequence of strain using the program  (Devereux et al., 1984). The CCUG 38120T is AJ132334. resulting multiple sequence alignment was corrected

01172 # 2000 IUMS 179 M. D. Collins and others

...... Fig. 1. Unrooted tree based on 16S rRNA showing the phylogenetic relationships of Luteococcus peritonei sp. nov. Branch lengths in the tree are additive and the figures shown at the nodes correspond to bootstrap values. Bar, 1% sequence divergence.

manually and approximately 100 bases at the 5h end of 21n4%;C"':",17n5%;C"(:",37n5%;C"):",6n4%). The the rRNA were omitted from further analyses because presence of such high levels ("80%) of mono- of alignment ambiguities. A continuous stretch of 1320 unsaturated fatty acids is very unusual but has been bases was used for distance matrix analysis. A distance previously reported in Luteococcus japonicus (Tamura matrix was calculated using the programs  and et al., 1994). Determination of the fatty acid com-  (using the Kimura 2-correction parameter) of position of Luteococcus japonicus CCUG 38731T, the  package (Felsenstein, 1989), and a tree was grown under similar conditions as the unknown constructed by the neighbour-joining method with the organism, revealed a remarkably similar profile (com- program  of the same package. The stability position: C"%:!,0n8%;C"&:!,8n6%;C"':!,5n4%;C"(:!, of the groupings was estimated by bootstrap analysis 3n9%; C"):!,0n9%; C"&:",20n2%;C"':",14n2%; C"(:", (500 replications) using the programs , - 44%; C"):", 2%). To investigate the phylogenetic ,  and  (Felsenstein, 1989). position of the unknown isolate its almost complete 16S rRNA gene sequence was determined and sub- The unknown isolate consisted of Gram-positive jected to a comparative analysis. Sequence database pleomorphic rod-shaped cells which were non-motile. searches revealed the unknown bacterium was phylo- The strain was facultatively anaerobic and catalase- genetically a member of the Actinobacteria and dis- positive. Using commercially available API kits the played a specific association with members of the organism produced acid from glucose, lactose, man- Propionibacteriaceae. Highest sequence similarity was nitol, methyl β--glucopyranoside and sucrose, but shown with Luteococcus japonicus (94%; 82 mis- not from -arabinose, -arabitol, cyclodextrin, gly- matches, two unmatched; 1395 bp), with Propioni- cogen, melibiose, melezitose, pullulan, ribose, raffin- ferax innocua (91n5%; 114 mismatches, five un- ose, sorbitol, trehalose, tagatose or -xylose. The matched; 1380 bp), Microlunatus phosphovorus organism hydrolysed aesculin but not gelatin or (91n2%; 123 mismatches, four unmatched; 1397 bp), hippurate. Activities for α-galactosidase, β-galacto- Friedmanniella antarctica (93n4%; 91 mismatches, two sidase, β-galacturonidase, α-glucosidase, β-glucosid- unmatched; 1395 bp) and Propionibacterium spp. ase, β-glucuronidase, leucine arylamidase and pyrazin- (90–91n5%) also displaying high levels of relatedness. amidase were detected but not for arginine dihydro- A tree constructed by the neighbour-joining method lase, chymotrypsin, α-fucosidase, glycyl-tryptophan depicting the phylogenetic relationships of the un- arylamidase, N-acetylglucosaminidase, lipase C14, α- known isolate is shown in Fig. 1 and demonstrates that mannosidase, β-mannosidase, pyroglutamic acid aryl- it represents a new subline, adjacent to Luteococcus amidase, trypsin, valine arylamidase or urease. The japonicus, within the Propionibacteriaceae. The bran- organism reduced nitrate to nitrite and was Voges– ching point of the unknown strain CCUG 38120T and Proskauer-negative. Analysis of the cell wall hydro- Luteococcus japonicus was supported by 93% recovery lysates of the unknown isolate revealed -Dpm as the in the bootstrap analysis. dibasic amino acid, whilst MK-9(II, III, H%) was found to constitute the major respiratory menaquinone. This The polyphasic taxonomic analysis has shown the latter combination of chemical features is found in a unknown clinical isolate CCUG 38120T originating number of Actinobacteria including members of the from a foetal autopsy represents a hitherto un- family Propionibacteriaceae. An examination of the recognized species within the Propionibacteriaceae. long-chain cellular fatty acids of the unknown bac- Phylogenetically the organism forms a distinct subline terium was, therefore, performed and revealed pre- and has a statistically significant affinity with Lute- dominantly acids of the monounsaturated types (com- ococcus japonicus. This association between the un- position: C"&:!, 8%; C"':!, 5%; C"(:!,4n2%; C"&:", identified rod and Luteococcus japonicus is strongly

180 International Journal of Systematic and Evolutionary Microbiology 50 Luteococcus peritonei sp. nov. supported by cellular fatty acid data. Both taxa are acids. Habitat is not known. Isolated from human highly unusual in synthesizing predominantly mono- peritoneum. Pathogenic potential is not known. The unsaturated long-chain fatty acids. All other recog- GjC content of DNA is 65 mol%. The type strain is nized members of the Propionibacteriaceae (viz.: CCUG 38120T. Friedmanniella, Microlunatus, Propioniferax and Pro- Luteococcus peritonei Lute pionibacterium) synthesize major amounts of methyl- can be distinguished from - ococcus japonicus branched-chain fatty acids (e.g. Pitcher & Collins, by its pleomorphic rod-shaped mor- 1991; Yokata et al., 1994; Nakamura et al., 1995; phology. Additionally, using API systems and ident- Luteococcus peritonei Schumann et al., 1997). Under other circumstances we ical test conditions, differs from Luteococcus japonicus would consider differences in cellular morphology in producing acid from lactose combined with a 16S rRNA gene sequence divergence and methyl β--glucopyranoside, producing β-glucu- of 6% between the unknown rod and Luteococcus ronidase, failing to produce pyrrolidonyl arylamidase, japonicus to possibly be indicative of different genera. and by reducing nitrate. However, the cellular fatty acid composition of Luteo- coccus japonicus is so unusual, that the occurrence of a Acknowledgements virtually identical profile within the unknown rod, is in our opinion, sufficiently compelling to outweigh the We are grateful to Professor Hans Tru$ per (Bonn, Germany) aforementioned considerations. Therefore, based on for coining the species name. molecular genetic and molecular chemical evidence, T we propose the unknown isolate CCUG 38120 be References classified in the genus Luteococcus,asLuteococcus peritonei sp. nov. Collins, M. D. (1985). Analysis of isoprenoid quinones. Methods Microbiol 18, 329–366. Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive Emended description of the genus Luteococcus set of sequence analysis programs for the VAX. Nucleic Acids Tamura, Takeuchi and Yokota Res 12, 387–395. The description of the genus Luteococcus (Tamura et Felsenstein, J. (1989).  – phylogeny inference package al., 1994) should be emended to include the following: (version 3.2). Cladistics 5, 164–166. some cells consist of Gram-positive pleomorphic rods. Nakamura, K., Hiraishi, J., Yoshimi, Y., Kawaharasaki, M., Nitrate may or may not be reduced to nitrite. Masuda, K. & Kamagata, Y. (1995). Microlunatus phosphovorus gen. nov., sp. nov., a new Gram-positive polyphosphate- accumulating bacterium isolated from activated sludge. Int J Description of Luteococcus peritonei sp. nov. Syst Bacteriol 45, 17–22. Luteococcus peritonei (pe.ri.to.nehi. L. n. peritoneum Oda, M. (1935). Bacteriological studies on water used for peritoneum; L. gen. neut. n. peritonei of the per- brewing sake (part 6). I. Bacteriological studies on ‘‘miyamizu’’ itoneum). Micrococcus and Actinomyces isolated from ‘‘miyamizu’’. Jozogaku Zasshi 13, 1202–1228. Cells consist of pleomorphic Gram-positive rods. Cells Pitcher, D. G. & Collins, M. D. (1991). Phylogenetic analysis of are non-pigmented. Facultatively anaerobic and cata- some -diaminopimelic acid-containing coryneform bacteria lase-positive. Acid is produced from glucose, lactose, from human skin: description of Propionibacterium innocuum sucrose, mannitol and methyl β--glucopyranoside. sp. nov. FEMS Microbiol Lett 84, 295–300. Acid may or may not be produced from maltose. Acid Schleifer, K. H. & Kandler, O. (1972). Peptidoglycan types of is not produced from -arabinose, -arabitol, cyclo- bacterial cells walls and their taxonomic implications. Bacteriol dextrin, glycogen, melibiose, melezitose, pullulan, Rev 36, 407–477. ribose, raffinose, sorbitol, trehalose, tagatose or - Schumann, P., Prauser, H., Rainey, F. A., Stackebrandt, E. & xylose. Aesculin is hydrolysed but not gelatin or Hirsch, P. (1997). Friedmanniella antarctica gen. nov., sp. nov., hippurate. α-Galactosidase, β-galactosidase, β-galact- an -diaminopimelic acid-containing actinomycete from Ant- uronidase, α-glucosidase, β-glucosidase, β-glucuron- arctic sandstone. Int J Syst Bacteriol 47, 278–283. idase, leucine arylamidase and pyrazinamidase are Stackebrandt, E., Rainey, F. A. & Ward-Rainey, N. L. (1997). produced. Arginine dihydrolase, lipase C14, chymo- Proposal for a new hierarchic classification system, Actino- trypsin, α-fucosidase, glycyl-tryptophan arylamidase, bacteria classis nov. Int J Syst Bacteriol 47, 479–491. N-acetylglucosaminidase, α-mannosidase, β-mannos- Tamura, T., Takeuchi, M. & Yokota, A. (1994). Luteococcus idase, pyroglutamic acid arylamidase, trypsin, valine japonicus gen. nov., sp. nov., a new Gram-positive coccus with arylamidase and urease are not produced. Activity for -diaminopimelic acid in the cell wall. Int J Syst Bacteriol 44, alkaline phosphatase and acid phosphatase may or 348–356. may not be detected. Voges–Proskauer-negative. Ni- Yokota, A., Tamura, T., Takeuchi, M., Weiss, N. & Stackebrandt, E. trate is reduced to nitrite. Cell wall contains -Dpm. (1994). Transfer of Propionibacterium innocuum Pitcher and MK-9(H%) is the predominant menaquinone. Synth- Collins 1991 to Propioniferax gen. nov. as Propioniferax innocua esizes predominantly monounsaturated cellular fatty comb nov. Int J Syst Bacteriol 44, 579–582.

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