Anaerostipes Caccae Gen. Nov., Sp. Nov., a New Saccharolytic, Acetate-Utilising, Butyrate-Producing Bacterium from Human Faeces

System. Appl. Microbiol. 25, 46Ð51 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Anaerostipes caccae gen. nov., sp. nov., a New Saccharolytic, Acetate-utilising, Butyrate-producing Bacterium from Human Faeces

ANDREAS SCHWIERTZ1, GEORGINA L. HOLD2, SYLVIA H. DUNCAN2, BÄRBEL GRUHL1, MATTHEW D. COLLINS3, PAUL A. LAWSON3, HARRY J. FLINT2 and MICHAEL BLAUT1

1Department of Gastrointestinal Microbiology, German Institute of Human Nutrition, Bergholz-Rehbrücke, Germany 2Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, UK 3School of Food Biosciences, University of Reading, Reading, UK

Received January 28, 2002

Summary

Two strains of a previously undescribed Eubacterium-like bacterium were isolated from human faeces. The strains are Gram-variable, obligately anaerobic, catalase negative, asporogenous rod-shaped cells which produced acetate, butyrate and lactate as the end products of glucose metabolism. The two isolates displayed 99.9% 16S rRNA gene sequence similarity to each other and treeing analysis demonstrat- ed the faecal isolates are far removed from Eubacterium sensu stricto and that they represent a new sub- line within the Clostridium coccoides group of organisms. Based on phenotypic and phylogenetic crite- ria, it is proposed that the two strains from faeces be classified as a new genus and species, Anaerostipes caccae. The type strain of Anaerostipes caccae is NCIMB 13811T (= DSM 14662T).

Key words: Anaerostipes caccae – 16S rRNA – taxonomy – phylogeny – human faeces

Introduction

The human intestinal tract harbours an immense diver- displayed a high degree of relatedness (16S rRNA) to the sity of bacteria and the total number of resident bacteria butyrate-producing strain L1-92 described by BARCENILLA has been estimated to reach 1014 cells (SAVAGE, 1977; SUAU et al. (2000). In this article we report the phenotypic char- et al. 2001). Although the human gut flora has been stud- acteristics of these two isolates and the results of a phylo- ied intensively over several decades using traditional cul- genetic analysis. Based on the findings presented, we pro- ture-based approaches and phenotypic methods of identifi- pose the novel bacterium from human faeces be classified cation, it is now universally recognised that knowledge of as a new genus and species, Anaerostipes caccae. the diversity of species present is far from complete. Cul- ture-independent rRNA-based studies (such as PCR rRNA cloning/sequencing and rDNA TGGE) have revealed that Materials and Methods the majority of the dominant species within the gut have Cultures and cultivation so far eluded taxonomic description (WILSON and BLITCH- Strains L1-92T and P2 were isolated from faecal samples INGTON 1996; ZOETENDAL et al. 1998; SUAU et al. 1999). In from two different healthy volunteers that had not undergone a recent investigation of butyrate-producing bacteria iso- antibiotic therapy in the previous six months. Strains L1-92T was lated from human faeces, using 16S rRNA as a tool to aid isolated as described by BARCENILLA et al. (2000) and deposited identification, numerous novel isolates were recovered in the National Collection of Industrial and Marine Bacteria T within the Clostridium coccoides rRNA lineage which did (UK) under accession number NCIMB 13811 . For the isolation of strain P2, fresh faecal samples were transferred into an anaer- not correspond to recognised species (BARCENILLA et al. obic workstation (MK3; DW Scientific, Shipley, UK) and diluted 2000). One of the organisms isolated was a strictly anaero- 10 serially ten-fold up to 10 in Sorensen buffer (25 mM KH2PO4, bic, non spore-forming, rod-shaped bacterium designated × 33 mM Na2HPO4 12 H2O, 0.04% (v/v), thioglycolic acid, L1-92 (BARCENILLA et al. 2000). In a subsequent and inde- 0.06% (w/v), cysteine, pH 6.8). Aliquots (1 ml) of the dilutions –1 pendent investigation of the human faecal flora, a second were plated onto starch plates containing (l ): 4 g NaHCO3, Eubacterium-like strain designated P2 was isolated which 0.348 g K2HPO4, 0.227 g KH2PO4, 0.5 g NH4Cl, 2.25 g NaCl,

0723-2020/02/25/01-046 $ 15.00/0 Anaerostipes caccae gen. nov., sp. nov. 47

× × × 0.5 g MgCO3 7 H2O, 0.07 g CaCl2 2H2O, 5 mg FeSO4 mately 2 to 4 cells. In the exponential growth phase cells 7H2O, 0.15 mg NaSeO3, 2 g tryptically digested peptone from stained Gram-positive, while stationary phase cells casein, 2 g yeast extract, 2 g soluble starch, 15 g agar, 1 mg re- stained Gram-negative. Cultures in ST medium had ropy sazurin, 3 ml trace element solution SL 10 (WIDDEL et al., 1983) sediment with little or no turbidity. Both strains formed and 0.5 mg cysteine × HCl, at pH 7.0. Following autoclaving, white-opaque, non-haemolytic colonies on Columbia 1 l medium was supplemented with 20 ml of a filter-sterilised vita- min solution as described by WOLIN et al. (1964). Single colonies blood agar and Wilkens Chalgren agar; the colonies were were isolated and re-streaked until pure cultures were obtained. approximately 1 to 3 mm in diameter, circular, convex, smooth and shiny. Using traditional testing the two Phenotypic characterisation strains produced acid from D-fructose, fructooligasaccha- All incubations were performed using strictly anaerobic con- rides (FOS), D-glucose, D-galactose, inositol, maltose, D- ditions. For morphological and physiological studies both mannose, ribose (weak), soluble starch, sucrose, L-sor- strains were grown on Columbia blood agar (bioMérieux), in bose and sorbitol. Strain L1-92T produced acid (weak) ST medium (SCHWIERTZ et al. 2000) or in a medium used for cul- from salicin whereas strain P2 did not. Aesculin was hy- turing acetogenic bacteria (KAMLAGE et al. 1997) but modified T by adding 0.5 g Proteose-Peptone No.2 (Difco) per litre (HA drolysed only by strain L1-92 . Both organisms failed to medium). The morphology of the isolates was examined by produce acid from L-arabinose, cellobiose, glycerol, in- phase contrast microscopy. Growth was monitored by changes ulin, lactose, lactulose, melibiose, melezitose, L-rham- in pH and optical density, measured at 600 nm. Cells were test- nose, D-trehalose or D-xylose. Using the commercially ed for catalase and oxidase as described by SMIBERT and KRIEG available API 50 system both strains displayed very simi- (1994). Other biochemical features were determined with the lar carbohydrate reactions: acid was produced from API rapid ID32A, API ZYM and API50 CHL systems adonitol, D-arabitol, L-arabitol, D-arabinose, dulcitol, (BioMérieux) according to the manufacturer’s instructions. Ex- erythritol, galactose, D-glucose, D-fructose, inositol, D- periments with resting cells were performed as described by lyxose, maltose, D-mannose, mannitol, melibiose, α- KAMLAGE et al. (1997). Acetate, butyrate, propionate, valerate, iso-valerate and hydrogen were determined by gas chromatogra- methyl-D-glucoside, N-acetyl-glucosamine, D-raffinose phy (HARTMANN et al. 2000; SCHNEIDER et al. 1999). Succinate, (weak), ribose (weak), L-sorbose, sorbitol, sucrose, D- ethanol, formate, acetate, D-lactate, L-lactate and glucose were tagatose, D-turanose and xylitol but not from amygdalin, determined enzymatically (BERGMEYER and GRASSL, 1984). Ac- L-arabinose, cellobiose, D-fucose, β-gentiobiose, glu- etate utilisation was tested on M2GSC medium (MIYAZAKI et al. conate, glycerol, glycogen, inulin, lactose, 5-keto- 1997) and the acetate concentrations determined by capillary gluconate, melezitose, α-methyl-D-mannoside, β-methyl- GC as described in (RICHARDSON et al. 1989). D-xyloside, rhamnose, trehalose, D-xylose or L-xylose. Different results were obtained for arbutin, salicin and DNA base composition T The mol%G + C content of DNA was determined by HPLC 2-keto-gluconate: strain L1-92 produced acid from ar- according to MESBAH et al. (1989) except that the methanol con- butin and salicin but not from 2-keto-gluconate, whereas tent of the chromatographic buffer was decreased to 8% and the strain P2 failed to produce acid from the former two sub- temperature was increased to 37 °C. strates but produced acid albeit weakly from 2-keto- gluconate. In addition strain L1-92T hydrolysed aesculin 16S rRNA gene sequencing and phylogenetic analysis whereas strain P2 showed a weak reaction. The 16S rRNA genes of the isolates were amplified by PCR The enzyme profiles of the two strains were identical and directly sequenced using a Taq DyeDeoxy terminator cycle using the API ZYM and API rapid ID32A systems. Using sequencing kit (Applied Biosystems, Foster City, USA) and an the API ZYM kit, both strains gave positive reactions only automatic DNA sequencer (model 377; Applied Biosystems). The closest known relatives of the new isolates were determined by for acid phosphatase and phosphoamidase, with all other performing database searches. These sequences and those of the tests being negative. Using the API rapid ID32A system known related strains were retrieved from the GenBank or the Ri- both isolates displayed activity for arginine dihydrolase bosomal Database Project (RDP) databases and aligned with the and α-galactosidase and were weakly nitrate reductase newly determined sequences using the program DNATools (RAS- positive. To ascertain further information about the MUSSEN, 1995). The resulting multiple sequence alignment was catabolic potentials of the two iolates, resting cells in corrected manually using the program GeneDoc (NICHOLAS et al. 50 mM anoxic potassium phosphate buffer (pH 7.0, 1997) and a distance matrix was calculated using the program 1 mg of resazurin per litre, 5 mM dithioerythritol) were DNADIST (using Kimura’s two-parameter correction) (FELSEN- incubated with glucose under N /CO (80/20) and the STEIN, 1989). A phylogenetic tree was constructed according to 2 2 the neighbour-joining method with the program NEIGHBOR and degradation products analysed. The fermentation balance the stability of the groupings was estimated by bootstrap analysis was as follows (CO2 content was calculated from redox (500 replications) using the programs DNABOOT, DNADIST, balance): 1 glucose → 0.2 acetate + 0.9 lactate + 0.4 bu- NEIGHBOR and CONSENSE FELSENSTEIN, 1989). tyrate + 1.2 H2 + 1 CO2. The carbon recovery in this ex- periment was 95%. Growth of L1-92T and P2 on anaerobic M2GSC revealed that both strains were net acetate Results and Discussion utilisers in this medium (utilising 6.29 and 6.41 mM respectively). The DNA base composition values for the two The two faecal isolates (L1-92T and P2) were non faecal isolates L1-92T and P2 were 46.0 and 45.4 mol% G spore-forming, non-motile, strictly anaerobic, rod-shaped + C, respectively. The genotypic relatedness of the isolates organisms. Cells were 0.5–0.6 µm wide × 2.0–4.3 µm was further investigated by comparative 16S rRNA gene long and occurred sometimes in short chains of approxi- sequence analysis. Almost complete sequences of L1-92T 48 A. SCHWIERTZ et al.

(1455 bp) and P2 (1506bp) were determined. Pair-wise rRNA cluster XIVa, [COLLINS et al., 1994]). The novel analysis showed that the two strains had almost identical bacterium (as exemplified by isolate L1-92T) formed a dis- 16S rRNA gene sequences (99.9% sequence similarity) tinct sub-line within this cluster, but did not display a par- and searches of GenBank and RDP databases revealed ticularly close nor statistically significant association (as that the isolates were closely related to members of the shown by bootstrap re-sampling) with any recognised Clostridium coccoides group of organisms (Clostridium species of the Clostridium coccoides group (Fig. 1).

Fig. 1. Unrooted phylogenetic tree showing the phylogenetic relationships of Anaerostipes caccae gen. nov., sp. nov. within the Clostridium coccoides rRNA group or organisms. The tree constructed using the neighbor-joining method was based on a comparison of approximately 1300 nucleotides. Bootstrap values expressed as a percentage of 500 replications are given at the branching points. Anaerostipes caccae gen. nov., sp. nov. 49

Table 1. Characteristics which are useful in differentiating Anaerostipes caccae from some miss-classified Eubacterium species of the Clostridium coccoides rRNA group.

Test/Species 1a 2345678910111213

Acid end Ab, B, A, B, A, E, F A, E, F A, E, F A, E, F A, B, A, B, F A, B, L A, E, F A, B, F A, B, F A, B, F products L L L L But, F L L L L, S Motility – vc –+v–––v– –+ – Acid from: – v – – – –––vnd–nd– Amygdalin L-Arabinose – – + – v v––++ –– + Cellobiose – v v + – – – + + nd – + + Dulcitol + – – – – – nd nd – nd nd nd – Fructose + – + + + ++++v +– v Galactose + – + – + + + nd + + + – – Glucose + + + + + +++++ +– + Inulin – – – – – – nd nd + nd – nd – Lactose – v v + – v + + v + – – – Maltose + + + – + + + v + + + – + Melezitose – – – – – –––+nd–nd– Melibiose + – v – – – – + + v – – – Raffinose + – + – – – – + + nd – nd – Ribose + – + – v v–––nd–nd– Starch + – – – – –––vndvnd+ Sucrose + + + – + –v–+ndvnd– Trehalose – v – – + –––vnd–nd– Xylose – – v – v v––++ –– v a1 – Anaerostipes caccae; 2 – E. cellulosolvens; 3 – E. contortum; 4 – E. eligens; 5 – E. fissicatena; 6 – E. formicigenerans; 7 – E. hallii; 8 – E. ramulus; 9 – E. rectale; 10 – E. uniforme; 11 – E. ventriosum; 12 – E. xylanophilum; 13 – E. ruminantium. b A – Acetic acid; B – butyric acid; But – butanol; E – ethanol; F – formic acid; L – lactic acid. c v – variable; nd – not determined.

It is evident from the results of the taxonomic investi- as a new species, its generic placement is problematic. gation that the two faecal isolates belong to a hitherto Phylogenetically the novel bacterium is only distantly re- undescribed species. As a strictly anaerobic non spore- lated to recognised species and genera within the forming rod, the unidentified bacterium somewhat re- Clostridium coccoides group (displaying divergence val- sembles the genus Eubacerium. It is now recognised ues of greater than 10%). The unidentified bacterium is however that the genus Eubacterium is phenotypically also phenotypically incompatible with all currently and phylogenetically diverse and should be restricted to named genera within this rRNA cluster. For example the Eubacterium limosum, the type species of the genus, and new rod-shaped bacterium can be distinguished from its close relatives (viz: E. barkeri, E. callanderi and E. ag- Clostridium spp. and Sporobacterium in not producing gregans; WILLEMS and COLLINS, 1996). The human iso- endospores, from Coprococcus, Lachnospira and Ru- lates described here are phylogenetically only remotely minococcus by cellular morphology and end-products of related to E. limosum and close relatives (i.e. Eubacteri- glucose metabolism, and from Butyrivibrio and Rose- um sensu stricto, WILLEMS and COLLINS, 1996), with the buria in being non-motile and end-products of glucose latter species forming a different cluster (rRNA cluster metabolism. The human faecal bacterium can also be XV, COLLINS et al. 1994) and displaying approximately biochemically readily distinguished from miss-classified 20% sequence divergence with the unidentified rod- Eubacterium species (such as E. hallii) within the C. coc- shaped bacterium. Hence the new feacal bacterium can- coides cluster (see Table 1). These miss-classified eubac- not be assigned to the genus Eubacterium sensu stricto. terial species invariably display 10% or more sequence Phylogenetically the novel rod-shaped bacterium belongs divergence with the unidentified bacterium and therefore to the C. coccoides rRNA group of organisms. The cannot be considered members of the same genus. Thus Clostridium coccoides group is a phylogenetically diverse it is clear that the novel faecal bacterium reported here, is supra-generic grouping embracing organisms bearing a both phylogenetically and phenotypically incompatible variety of generic names (e.g. Butyrivibrio, Clostridium, with Eubacterium sensu stricto and all recognised genera Coprococcus, Lachnospira, Roseburia, Ruminococcus, within the C. coccoides rRNA cluster, and merits classifi- Sporobacterium, Syntrophococcus) and includes several cation in a separate genus. Therefore based on the pre- miss-classified Eubacterium species. Although tree topol- sented findings we propose that the unknown rod- ogy and 16S rRNA sequence divergence values clearly shaped organism be classified as a new genus and show the unidentified bacterium warrants classification species, Anaerostipes caccae. 50 A. SCHWIERTZ et al.

Description of Anaerostipes dase, histidine arylamidase, leucine arylamidase, leucyl- glycine arylamidase, lipase C14, α-mannosidase, N-acetyl- Anaerostipes (an. ae. ro. sti’pes) Gr. pref. an not; Gr. n. α-glucosaminidase, phenylalanine arylamidase, pyroglu- aer air; anaero not living in air; L. masc. n. stipes club of tamic acid arylamidase, proline arylamidase, serine ary- stick; Anaerostipes, a stick not living in air) consists of lamidase, trypsin, tyrosine arylamidase, valine arylamidase non-motile, non spore-forming rod-shaped cells. Cells are or urease. Gelatin is not hydrolysed and indole is not pro- Gram-positive, but in older cultures may stain Gram-neg- duced. The DNA G + C content is 45.5–46.0 mol%. Iso- ative. Strictly anaerobic and catalase and oxidase nega- lated from human faeces. The type strain is L1-92T (= DSM tive. Non-haemolytic. Glucose and some other sugars 14662T = NCIMB 13811T). The GenBank accession num- may be fermented. Butyrate, acetate and lactate are the ber of the 16S rRNA sequence of the type strain is major products of glucose metabolism. Acetate is utilised. AJ270487. Arginine dihydrolase, phosphoamidase, and α-galactosidase are produced. Gelatin and urease are not hydrolysed. Indole is not produced. Nitrate is reduced. The Acknowledgments This work has been carried out with financial support from DNA G + C content is 45.5–46.0 mol%. The type species the Commission of the European Communities, specific RTD is Anaerostipes caccae. programme “Quality of Life and Management of Living Re- sources”, QLK1-2000-108, “Microbe Diagnostics”. It does not necessarily reflect its views and in no way anticipates the Com- Description of Anaerostipes caccae sp. nov. mission’s future policy in this area. Work at Rowett Research In- stitute was supported by the Scottish Executive Environment Anaerostipes caccae (cac’cae, pronounced kak’ka Gr. and Rural Affairs Department (SEERAD). n. kakke faeces: N.L. gen. n. caccae of faeces) cells consist of non-motile, non spore-forming rods. Individual cells are 0.5–0.6 µm in width × 2.0–4.0 µm in length and References occur in chains of up to 4 cells. Cells stain Gram-positive but older cultures (>16 h) may stain Gram-negative. On BARCENILLA, A., PRYDE, S. E., MARTIN, J. C., DUNCAN, S. H., Columbia blood agar, white-opaque colonies are formed STEWART, C. S., HENDERSON, C., FLINT, H. J.: Phylogenetic re- that are 1–3 mm in diameter, circular, convex, smooth, lationships of butyrate-producing bacteria from the human shiny and sticky, which are non haemolytic. The strains gut. Appl. Environ. Microbiol. 66, 1654–1661 (2000). ERGMEYER, J., GRASSEL, M.: Methods of enzymatic analysis, are strictly anaerobic and catalase and oxidase negative. B 3ed, vol. 6. Germany: Weinheim Verlag Chemie (1984). Butyrate, acetate and lactate are the major products of COLLINS, M. D., LAWSON, P. A., WILLEMS, A., CORDOBA, J. J. glucose metabolism. Acetate is utilised. Using conven- FERNANDEZ-GARAYZABAL, J., GARCIA, P., CAI, J., HIPPE, H., tional tests acid is produced from D-fructose, FOS, FARROW, J. A.: The phylogeny of the genus Clostridium: pro- D-glucose, D-galactose, inositol, maltose, D-mannose, posal of five new genera and eleven new species combina- ribose (weak), soluble starch, sucrose, L-sorbose and sor- tions. Int. J. Syst. Bacteriol. 44, 812–826 (1994). bitol. Acid may or may not be produced from salicin. Acid FELSENSTEIN, J.: PHYLIP – Phylogeny inference package (version is not produced from L-arabinose, cellobiose, glycerol, 3.2). Cladistics 5, 164–166 (1989). inulin, lactose, lactulose, melibiose, melezitose, L-rham- HARTMANN, L., TARAS, D., KAMLAGE, B., BLAUT, M.: A new tech- nose, D-trehalose or D-xylose. Using the miniaturised nique to determine hydrogen excreted by gnotobiotic rats. Lab. Anim. 34, 162–170 (2000). API 50 system acid is produced from adonitol, D-ara- KAMLAGE, B., GRUHL, B., BLAUT, M.: Isolation and characteriza- bitol, L-arabitol, D-arabinose, dulcitol, erythritol, galac- tion of two new homoacetogenic hydrogen-utilizing bacteria tose, D-glucose, gluconate, D-fructose, inositol, D-lyxose, from the human intestinal tract that are closely related to maltose, D-mannose, mannitol, melibiose, α-methyl- Clostridium coccoides. Appl. Environ. Microbiol. 63, D-glucoside, N-acetyl-glucosamine, D-raffinose (weak), 1732–1738 (1997). ribose (weak), L-sorbose, sorbitol, sucrose, D-tagatose, MESBAH, M., PREMACHANDRAN, U., WHITMAN, W. B.: Precise D-turanose and xylitol but not from amygdalin, L-arabi- measurement of the G + C content of deoxyribonucleic acid nose, cellobiose, D-fucose, β-gentiobiose, glycerol, by high performance liquid chromatography. Int. J. Syst. Bac- glycogen, inulin, lactose, 5-keto-gluconate, melezitose, teriol. 39, 159–167 (1989). α β MIYAZAKI, K., MARTIN, J. C., MARINSEK,LOGAR, R., FLINT, H. J.; -methyl-D-mannoside, -methyl-D-xyloside, rhamnose, Degradation and utilization of xylans by the rumen anaerobe trehalose, D-xylose or L-xylose. Acid may or may not be Prevotella bryantii (formerly Prevotella ruminicola subsp. produced from arbutin, salicin and 2-keto-gluconate. brevis) B14. Anaerobe 3, 373–381 (1977). Aesculin hydrolysis is variable. Using API ZYM and API NICHOLAS, K. B., NICHOLAS, H. B. JR., DEERFIELD, D. W. II: rapid ID32A systems activity is detected for acid phos- GeneDoc: Analysis and visualization of genetic variation, phatase and phosphoamidase, and arginine dihydrolase, EMBNEW. News 4, 14 (1997). α-galactosidase and nitrate reductase (weak reaction) re- RASMUSSEN, S. W.: DNATools, A software package for DNA se- spectively. No activity is detected for alanine arylamidase, quence analysis. Carlsberg Laboratory, Copenhagen (1995). alkaline, phosphatase, arginine arylamidase, α-arabinosi- RICHARDSON, A. J., CALDER, A. G., STEWART, C. S., SMITH, A.: Si- multaneous determination of volatile and non-volatile acidic dase, chymotrypsin, cystine arylamidase, esterase C4, ester α β β fermentation products of anaerobes by capillary gas chro- lipase C8, -fucosidase, -galactosidase, -galactosidase- matography. Lett Appl Microbiol 9, 5–8 (1989). α β β 6-phosphate, -glucosidase, -glucosidase, -glucuronid- SAVAGE, D. C.: Microbial ecology of the gastrointestinal tract. ase, glycine arylamidase, glutamylglutamic acid arylami- Ann. Rev. Microbiol. 31, 107–133 (1977). Anaerostipes caccae gen. nov., sp. nov. 51

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