Faecalicoccus acidiformans gen. nov., sp. nov ...

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Sep 1, 2014 - Celine De Maesschalck1, Filip Van Immerseel1, Venessa Eeckhaut1, Siegrid De Baere2, Margo. 7 ..... Holdemanella (Hol.de.man.el'la.
IJSEM Papers in Press. Published September 1, 2014 as doi:10.1099/ijs.0.064626-0

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Faecalicoccus acidiformans gen. nov., sp. nov. isolated from the chicken

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caecum and reclassification of Streptococcus pleomorphus (Barnes et al.,

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1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato

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et al., 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis

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gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov.,

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respectively, within the family Erysipelotrichaceae

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Celine De Maesschalck , Filip Van Immerseel , Venessa Eeckhaut , Siegrid De Baere , Margo

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Cnockaert , Siska Croubels , Freddy Haesebrouck , Richard Ducatelle and Peter Vandamme

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Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent

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University, Salisburylaan 133, B-9820 Merelbeke, Belgium

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University, Salisburylaan 133, B-9820 Merelbeke, Belgium

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Ghent, Belgium

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Author for correspondence: Filip Van Immerseel, [email protected], tel. +3292647447,

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fax. +3292647789

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Running title: Faecalicoccus acidiformans gen.nov., sp. nov.

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Category: New Taxa – Firmicutes and Related Organisms

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Footnotes: The GenBank accession numbers for the 16S rRNA sequences of strains LMG 27428

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and LMG 27427 are HQ452864 and HQ452862. The GenBank accession numbers for the hsp60

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sequence of strains LMG 27428 , Streptococcus pleomorphus LMG 17756 and LMG 27427 are

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KJ512711, KJ512712 and KJ512713 respectively.

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Non-standard abbreviations: SEM, scanning electron microscopy; hsp60, 60kDa heat-shock protein;

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HPLC-UV, high pressure liquid chromatography-ultra violet; SCFA, short-chain fatty acid

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Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent

Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000

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Abstract

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Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and

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produced butyric, lactic and formic acid as major metabolic end products. The genomic DNA

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G+C content of strain LMG 27428T was 40.4 mol% and 38.8 mol% for LMG 27427. On the

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basis of 16S rRNA gene sequence similarity, both strains were most closely related to the

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generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T

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could be distinguished from S. pleomorphus ATCC 29734T based on higher lactic acid and

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less formic acid production in M2GSC medium, a higher DNA G+C content and absence of

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acid phosphatase, leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine

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arylamidase activity while strain LMG 27428 was biochemically indistinguishable from S.

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pleomorphus. The novel genus Faecalicoccus within the family Erysipelotrichaceae is

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proposed to accommodate strain LMG 27428T = (DSM 26963T) as Faecalicoccus

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acidiformans sp. nov. and strain LMG 27427 (DSM 26962) as Faecalicoccus pleomorphus

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comb. nov.. Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus

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are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA

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sequence similarity to the type strain) and Eubacterium biforme DSM 3989T (92.7 % 16S

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rRNA sequence similarity to the type strain). We present genotypic and phenotypic data that

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allow the differentiation of each of these taxa and formally propose to reclassify these

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generically misnamed Eubacterium species as Faecalitalea cylindroides comb. nov. (DSM

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3983T = ATCC 27803T = JCM 10261T) and Holdemanella biformis comb. nov. (DSM 3989T =

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ATCC 27806T = CCUG 28091T), respectively.

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Main text

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The complex microbiota of the gastrointestinal tract is dominated by microorganisms

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belonging to the phylum Firmicutes (Ley et al., 2008; Stanley et al., 2013). Eeckhaut et al.

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(2011) investigated the diversity and phylogenetic relationships of butyrate-producing

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bacteria isolated from the chicken caeca and observed that butyrate producers belonging to

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Clostridium cluster XVI as determined by Collins et al. (1994) may play a more important role

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in the chicken gut than in the human colon. Members of Clostridium cluster XVI or the

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Erysipelotrichaceae family stain Gram-positive with incoherent cell morphology and include

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the generically misclassified Streptococcus pleomorphus, Eubacterium cylindroides and

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Eubacterium biforme (Collins et al., 1994). The recent isolation of butyrate-producing

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bacteria from the caecal content of a 14-week old Isa Brown layer type pullet yielded strains

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LMG 27428T and LMG 27427 (Eeckhaut et al., 2011). Based on their near-entire 16S rRNA

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gene sequence, these strains appeared to be most closely related to the above mentioned

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members of the Erysipelotrichaceae family. In the present study we describe the

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morphological, biochemical and genotypic characterization of strains LMG 27428T and LMG

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27427 and their nearest phylogenetic neighbours and propose a novel classification for each

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of these taxa.

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Strains LMG 27428T and LMG 27427 were isolated from chicken caecal content and grown

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anaerobically for 48 h on solid M2GSC medium pH 6 at 38 °C (Eeckhaut et al., 2011). The

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colonies of both strains were 0.5-1.5 mm in diameter and white in colour. The cell

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morphology was investigated using Gram-staining and scanning electron microscopy (SEM).

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Cells of both strains stained Gram-positive and were observed as cocci-bacilli-shaped pairs

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measuring 1.1-1.2 µm and 0.9-1.0 µm for strains LMG 27428T and LMG 27427respectively

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(Fig. 1). Spore formation was not detected.

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DNA was extracted from strains LMG 27428T and LMG 27427 using an alkaline lysis

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procedure. Universal bacterial primers fD1 and rD1 (Weisburg et al., 1991) and primers H279

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and H280 (Goh et al., 1996) were used to amplify the 16S rRNA gene and part of the 60kDa

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heat shock protein (hsp60) gene, respectively. After purification, the amplicons were

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sequenced by GATC Biotech (GATC Biotech AG, European Genome and Diagnostics

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Centre, Konstanz, Germany) using the same primers for hsp60 and primers pD, Gamma*, 3

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and O* for 16S rRNA (Coenye et al., 1999). For the 16S rRNA sequence, the closest match

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to the deduced sequences was found using the EzTaxon-e server (Kim et al., 2012), while

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for the hsp60 gene sequence, an independent mapping against a reference cpn60 gene

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database was used (Hill et al., 2004). The sequences were aligned with reference 16S rRNA

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gene sequences and hsp60 sequences using the MUSCLE program (Edgar, 2004a, b).

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Phylogenetic trees were constructed using MEGA 6 software (Tamura et al., 2013).

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Clustering was determined with the maximum likelihood method based on the Tamura-Nei

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model (Tamura & Nei, 1993) and bootstrap values were calculated based on 100 replications

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(Fig. 2-3). Strain LMG 27428T showed moderate sequence similarity to Streptococcus

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pleomorphus ATCC 29734T (96.0 %) and lower similarity to Eubacterium cylindroides DSM

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3983T (94.4 %) and Eubacterium biforme DSM 3989T (92.7 %). The 16S rRNA gene

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sequence of strain LMG 27427 was 99.6 % similar to that of S. pleomorphus ATCC 29734T.

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Phylogenetic analysis of protein encoding genes such as the hsp60 gene is commonly used

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as an alternative identification instrument to distinguish between closely related species. The

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analysis shown in Figure 3 demonstrates that S. pleomorphus ATCC 29734T and strain LMG

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27427 have highly similar (98.2 %) hsp60 gene sequences that can be used to differentiate

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them from their nearest neighbour, i.e. strain LMG 27428T.

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Genomic DNA of S. pleomorphus ATCC 29734T and strain LMG 27427 was prepared

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according to Pitcher et al. (1989) and DNA-DNA hybridizations were performed as described

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by Ezaki et al. (1989) with an adapted hybridization temperature of 35 °C. The level of DNA-

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DNA hybridization between strain LMG 27427 and S. pleomorphus ATCC 29734T was 83 %

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(the reciprocal hybridization values were 94 and 71 %), which demonstrated that they indeed

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represent the same species. The mol% G+C content of strains LMG 27428T and LMG 27427

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was determined using a Waters Breeze HPLC system and an XBridge Shield RP18 column

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maintained at 37 °C (Mesbah & Whitman, 1989). The genomic DNA G+C content of strains

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LMG 27428T and LMG 27427 was determined to be 40.4 and 38.8 mol% respectively, which

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is similar to that of S. pleomorphus ATCC 29734T (39.4 mol%) (Barnes et al., 1977).

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The whole cell fatty acid methyl ester (FAME) composition was determined for strains LMG

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27428T, LMG 27427, ATCC 29734T, DSM 3983T and DSM 3989T using an Agilent

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Technologies 6890N gas chromatograph (Santa Clara, CA, USA). Fatty acids extraction and

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analysis of the fatty acid methyl esters were performed according to the recommendations of

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the Microbial Identification System, Sherlock version 3.10 (MIDI, Hewlett Packard, Newark,

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DE, USA). Fatty acids were extracted from cultures grown in M2GSC for 24 h at 38°C under

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anaerobic conditions. The peaks of the profiles were identified using the TSBA50

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identification library version 5.0 (MIDI, Hewlett Packard, Newark, DE, USA). The

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predominant fatty acid for strain LMG 27428T was iso-C19:1 (17.6 %), while for the closest

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phylogenetic neighbours it was C16:0 (13.5 % - 24.5 %) (Table 1). Also other fatty acids were

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present in lower percentages (above 1%) for the 5 strains: C12:0 (3.4 %- 7.3 %), C14:0 (4.4 %-

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9.0 %), C18:0 (4.6 %- 20.3%), C18:1 ω9c (8.0 %- 13.2%) and C18:1 ω7c (2.6 %- 6.8 %).

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The fermentation pattern of the two strains and their closest phylogenetic neighbours was

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analysed using HPLC-UV (De Baere et al., 2013). After 24 h growth in M2GSC broth, strains

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LMG 27428T, LMG 27427 and S. pleomorphus ATCC 29734T produced 7 - 8.5 mM lactic

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acid, 2.4 - 3.5 mM butyric acid and 1.5-6.5 mM formic acid (Table 1). Strains LMG 27428T

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and LMG 27427 consumed 2.8-3.7 mM acetic acid and 0.8-1.1 mM propionic acid. Both

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Eubacterium strains DSM 3983T and DSM 3989T produced acids in the range of 0.3-4.0 mM

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except for lactic acid of which the concentration was much higher for Eubacterium

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cylindroides DSM 3983T (13.6 mM). Substrate utilization properties of strains LMG 27428T

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and LMG 27427 were compared to those of their nearest phylogenetic neighbour species

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using the API 20 A, rapid ID 32A and API ZYM systems (bioMérieux) according to the

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manufacturer’s instructions except that the incubation was performed anaerobically for API

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ZYM. S. pleomorphus ATCC 29734T exhibited enzymatic activity for acid phosphatase and

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different arylamidases like leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and

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histidine and differed from strain LMG 27427 only in alanine arylamidase and in gelatin

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hydrolysis. Strain LMG 27428T fermented D-glucose and D-mannose, but not D-mannitol, D-

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lactose, D-saccharose, D-maltose, salicin, D-xylose, L-arabinose, glycerol, D-cellobiose, D-

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melezitose, D-raffinose, D-sorbitol, L-rhamnose and D-trehalose. Hydrolysis of gelatin and

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aesculin was not detected. Strain LMG 27428T did not exhibit arylamidase or acid

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phosphatase activity and could thus easily be distinguished from S. pleomorphus ATCC

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29734T. E. cylindroides DSM 3983T only fermented D-saccharose and D-raffinose, while E.

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biforme DSM 3989T fermented D-raffinose, D-mannitol, salicin, D-xylose and L-arabinose.

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Gelatin hydrolysis was observed for E. biforme DSM 3989T, while E. cylindroides DSM 3983T

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hydrolysed aesculin and exhibited esterase, ester lipase and α-glucosidase activity, hence

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allowing a straightforward differentiation of strain LMG 27428T and its nearest phylogenetic

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neighbours (Table 2).

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In conclusion, the high degree of phenotypic similarity together with the DNA-DNA

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hybridisation value demonstrate that strain LMG 27427 should be classified within the

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generically misclassified S. pleomorphus ATCC 29734T (Fig. 1-2). In addition, strain LMG

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27428T is the nearest phylogenetic neighbour of the latter but can be distinguished from it by

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a considerable 16S rRNA divergence (4.0 %), hsp60 sequence analysis, a higher lactic acid

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production but lower formic acid production, a higher DNA G+C content and the absence of

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acid phosphatase and various arylamidases activities. S. pleomorphus, strain LMG 27428T,

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and the generically misclassified E. cylindroides and E. biforme all belong to a single line of

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descent within the family Erysipelotrichaceae and can be distinguished by both genotypic

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and phenotypic characteristics (Table 1). On the basis of these polyphasic taxonomic data

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we propose to classify strain LMG 27428T into the new genus Faecalicoccus, as

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Faecalicoccus acidiformans sp. nov., and to reclassify the generically misnamed S.

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pleomorphus (Kawamura et al., 1995; Ludwig et al., 1988) into this novel genus as

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Faecalicoccus pleomorphus comb. nov.. Furthermore, there is a growing consensus that the

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genus Eubacterium sensu stricto should be restricted to the type species, Eubacterium

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limosum ATCC 8486T, and its closest phylogenetic relatives, and that the majority of

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Eubacterium species therefore needs reclassification (Kageyama et al., 1999; Moore et al.,

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1976; Nakazawa & Hoshino, 1994; Willems & Collins, 1996). The considerable phylogenetic

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divergence between E. biforme DSM 3989T, E. cylindroides DSM 3983T and their nearest

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phylogenetic neighbours, and the difference in mol% G+C content, in lactic acid production

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and in various other biochemical characteristics (Fig. 1-3 and Table 1), together warrant the

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reclassification of E. biforme and E. cylindroides into two new genera, as Holdemanella

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biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov.,

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respectively.

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DESCRIPTION OF FAECALICOCCUS GEN. NOV.

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Faecalicoccus (Fa.e.ca.li.coc’cus. N.L. adj. faecalis (from L.n. faex faecis), pertaining to

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feces; N.L. masc. n. coccus (from Gr. masc. n. kokkus, a grain, seed), a coccus; N.L. masc.

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n. Faecalicoccus, coccoid bacteria that are isolated from faecal material.).

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The cells are obligate anaerobic non-spore-forming cocci that stain Gram-positive. The

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bacteria have lactic acid as major fermentation product from glucose and mannose, and

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exhibit naphthol-A,S-BI-phosphohydrolase activity. The major fatty acid (> 10 % of total fatty

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acids) is C16:0. The DNA G+C content is 39-41 mol%. Strains have been isolated from avian

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caecum. The type species is Faecalicoccus acidiformans.

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DESCRIPTION OF FAECALICOCCUS ACIDIFORMANS SP. NOV.

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Faecalicoccus acidiformans (a.ci.di.for’mans. N.L. n. acidum (from L. adj. acidus, sour), an

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acid; L. part. adj. formans, forming; N.L. part. adj. acidiformans, acid-forming bacteria.).

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The cells are anaerobic, non-motile, Gram-positive non-sporulating cocci. Individual cells are

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about 1.1-1.2 µm long and occur in pairs or short chains. On M2GSC agar plates, they form

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minute colonies, white in colour and measure 1-1.5 mm in diameter after 48 h growth at 38

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°C in an anaerobic workstation. The major fatty acids (> 10 % of total fatty acids) are C16:0,

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C18:1 ω9c and iso-C19:1. Test for acid production using API 20 A shows positive reaction with

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D-glucose and D-mannose, but not with D-mannitol, D-lactose, D-saccharose, D-maltose,

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salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-raffinose, D-sorbitol, L-

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rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin and gelatin

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observed in the API 20 A test. Test for enzyme activities by use of API ZYM shows positive

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reaction with naphthol-AS-BI-phosphohydrolase, but not with acid phosphatase, alkaline

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phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine

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arylamidase,

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galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase,

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α-mannosidase and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows

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no positive reaction. High amounts of lactic acid are produced as main fermentation product

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in M2GSC broth, in addition to moderate amounts of butyric acid and low amounts of formic

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acid. The G+C content of the DNA is 40.4 mol%.

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The type strain LMG 27428T (DSM 26963T) was isolated from the caecal content of a 14-

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week old Isa Brown layer chicken in Ghent (Belgium) in 2006.

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DESCRIPTION OF FAECALICOCCUS PLEOMORPHUS COMB. NOV.

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Faecalicoccus pleomorphus (ple.o.mor’phus. N.L. masc. adj. pleomorphus (from Gr. adj.

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pleos, full, and Gr. n. morphê, form, shape), pleomorphic, different forms for the bacteria.).

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Basonym: Streptococcus pleomorphus Barnes et al., 1977 (Approved Lists 1980).

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The description of Faecalicoccus pleomorphus is as given for Streptococcus pleomorphus by

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Barnes et al. (1977), with the following additions. The major fatty acids (> 10 % of total fatty

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acids) are C16:0 and C18:1 ω9c. Test for acid production by use of API 20 A shows positive

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reaction with D-glucose and D-mannose, but not with D-mannitol, D-lactose, D-saccharose,

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D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-raffinose, D-sorbitol,

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L-rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin while

cysteine

arylamidase,

trypsin,

α-chymotrypsin,

α-galactosidase,

β-

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some strains shows the hydrolysis of gelatin by the use of API 20 A. Test for enzyme

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activities by use of API ZYM shows positive reaction with acid phosphatase, leucine

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arylamidase and naphthol-AS-BI-phosphohydrolase, but not with alkaline phosphatase,

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esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cysteine arylamidase,

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trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase,

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β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Test for

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enzyme activities by use of rapid ID 32 A shows positive reaction with leucine arylamidase,

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arginine arylamidase, pyroglutamic acid arylamidase, glycine arylamidase, histidine

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arylamidase and alanine arylamidase for some strains but not with arginine dihydrolase, α-

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galactosidase,

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glucosidase, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid

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decarboxylase, α-fucosidase, alkaline phosphatase, proline arylamidase, phenylalanine

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arylamidase, tyrosine arylamidase, serine arylamidase and glutamyl glutamic acid

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arylamidase.. On M2GSC agar, the species grows in white colonies of 1.0-1.5 mm after 48 h

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incubation in anaerobic conditions at 38 °C. Overnight growth in M2GSC broth results in

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moderate amounts of butyric acid and formic acid.

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The type strain is LMG 17756T (= ATCC 29734T, DSM 20574T).

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DESCRIPTION OF HOLDEMANELLA GEN. NOV.

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Holdemanella (Hol.de.man.el’la. N.L. fem. dim. n. Holdemanella, named in honor of Lillian V.

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Holdeman Moore, a contemporary American microbiologist, for her outstanding contribution

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to the bacteriology of anaerobes).

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The members of the genus are strictly anaerobic. The cells stain Gram-positive, are coccus-

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shaped and often form pairs or chains. They are non-motile and non-spore-forming. D-

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glucose, D-mannitol, salicin, D-xylose, L-arabinose, D-mannose and D-raffinose are

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fermented. The major fatty acids (> 10 % of total fatty acids) are C16:0 and C18:0. The DNA

β-galactosidase,

β-galactosidase

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phophatase,

α-glucosidase,

β-

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G+C content is 32-34 mol%. Strains have been isolated from human faeces. The type

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species is Holdemanella biformis.

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DESCRIPTION OF HOLDEMANELLA BIFORMIS COMB. NOV.

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Holdemanella biformis (bi.for’mis L. fem. adj. biformis, two-shaped, two-formed (pertaining to

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cellular morphology)).

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Basonym: Eubacterium biforme (Eggerth, 1935) Prévot 1938 (Approved Lists 1980).

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The description of Holdemanella biformis is as given for Eubacterium biforme by Moore and

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Holdeman (1974), with the following additions. The major fatty acids (> 10 % of total fatty

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acids) are C16:0 and C18:0. Test for acid production by use of API 20 A shows positive reaction

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with D-glucose, D-mannitol, salicin, D-xylose, L-arabinose, D-raffinose and D-mannose, but

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not with D-lactose, D-saccharose, D-maltose, D-cellobiose, D-melezitose, D-sorbitol, L-

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rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin but gelatine

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is hydrolyzed. Test for enzyme activities by use of API ZYM shows positive reaction with acid

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phosphatase, alkaline phosphatase and naphthol-AS-BI-phosphohydrolase, but not with

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esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase

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cysteine

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glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase

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and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows no positive

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reaction.

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propionic acid are produced.

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The type strain is DSM 3989T (= ATCC 27806T, CCUG 28091T), and has been isolated from

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human faeces.

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DESCRIPTION OF FAECALITALEA GEN. NOV.

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Faecalitalea (Fa.e.ca.li.ta’le.a N.L. adj. faecalis (from L. n. faex faecis), pertaining to faeces;

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L.fem. n. talea, a rod; N.L. fem. n. Faecalitalea, rods isolated from faeces.)

arylamidase,

trypsin,

α-chymotrypsin,

α-galactosidase,

β-galactosidase,

β-

In M2GSC broth, moderate amounts of butyric acid, acetic acid, lactic acid and

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The members of the genus are strictly anaerobic. The cells stain Gram-positive and are

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pleomorphic rods which occur single, in pairs or in short chains. Filamentous forms are often

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seen. They are non-motile and non-spore-forming. D-glucose, D-sucrose, D-mannose and D-

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raffinose are fermented. The major end products of metabolism are lactic and butyric acid.

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The major fatty acids (> 10% of total fatty acids) are C16:0 and C18:1 ω9c. The DNA G+C

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content is 26-35 mol%. Strains have been isolated from the human, chicken and pig gut. The

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type species is Faecalitalea cylindroides.

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DESCRIPTION OF FAECALITALEA CYLINDROIDES COMB. NOV.

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Faecalitalea cylindroides (cy.lin.dro’i.des. Gr. n. kulindros, a cylinder; L. suff. –oides (from Gr.

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suff. eides, from Gr. N. eidos, which is seen, form, shape, figure), resembling, similar; N.L.

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fem. adj. cylindroides, cylinder-shaped.) .

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Basonym: Eubacterium cylindroides (Rocchi 1908) Holdeman and Moore 1970 (Approved

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Lists 1980).

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The description of Faecalitalea cylindroides is as given for Eubacterium cylindroides by Cato

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et al. (1974), with the following additions. The major fatty acids (> 10 % of total fatty acids)

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are C16:0 and C18:1 ω9c. Test for acid production by use of API 20 A shows positive reaction

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with D-glucose, D-saccharose, D-raffinose and D-mannose, but not with D-mannitol, D-

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lactose, D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-sorbitol, L-

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rhamnose and D-trehalose. There is no urease activity, no hydrolysis of gelatin but aesculine

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is hydrolyzed. Test for enzyme activities by use of API ZYM shows positive reaction with acid

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phosphatase, alkaline phosphatase, esterase (C4), esterase lipase (C8), α-glucosidase and

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naphthol-AS-BI-phosphohydrolase, but not with lipase (C14), leucine arylamidase, valine

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arylamidase,

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galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase

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and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows no positive

cysteine

arylamidase,

trypsin,

α-chymotrypsin,

α-galactosidase,

β-

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reaction. High amounts of lactic acid are produced as main fermentation product in M2GSC

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broth, in addition to moderate amounts of butyric acid.

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The type strain is DSM 3983T (= ATCC 27803T, JCM 10261T) and has been isolated from

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clinically normal human faeces.

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Acknowledgements

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We thank Wim Van Den Broeck, Jurgen De Craene and Bart De Pauw of the Department of

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Morphology, Faculty of Veterinary Medicine, Ghent University, for scanning electron

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microscopy. Dr. J.P. Euzéby is acknowledged for his expert nomenclatural advice.

289

References

290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339

Barnes, E. M., Impey, C. S., Stevens, B. J. & Peel, J. L. (1977). Streptococcus pleomorphus sp.nov.: an anaerobic streptococcus isolated mainly from the caeca of birds. J Gen Microbiol 102, 45-53. Cato, E. P., Salmon, C. W. & Holdeman, L. V. (1974). Eubacterium-Cylindroides (Rocchi) Holdeman and Moore - Emended Description and Designation of Neotype Strain. Int J Syst Bacteriol 24, 256-259. Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J.R.W., Kersters, F & Vandamme, P. (1999). Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp.nov. Int J Syst Bacteriol 49, 405-413 Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812-826. De Baere, S., Eeckhaut, V., Steppe, M., De Maesschalck, C., De Backer, P., Van Immerseel, F. & Croubels, S. (2013). Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. J Pharm Biomed Anal 80, 107-115. Edgar, R. C. (2004a). MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113. Edgar, R. C. (2004b). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792-1797. Eeckhaut, V., Van Immerseel, F., Croubels, S., De Baere, S., Haesebrouck, F., Ducatelle, R., Louis, P. & Vandamme, P. (2011). Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum. Microb Biotechnol 4, 503-512. Eggerth, A. H. (1935). The Gram-positive Non-spore-bearing Anaerobic Bacilli of Human Feces. J Bacteriol 30, 277-299. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric Deoxyribonucleic Acid Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane-Filter Hybridization in Which Radioisotopes Are Used to Determine Genetic Relatedness among Bacterial Strains. Int J Syst Bacteriol 39, 224-229. Goh, S. H., Potter, S., Wood, J. O., Hemmingsen, S. M., Reynolds, R. P. & Chow, A. W. (1996). HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol 34, 818-823. Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H. & Hemmingsen, S. M. (2004). cpnDB: a chaperonin sequence database. Genome research 14, 1669-1675. Holdeman, L. V. and Moore, W. E. C. (1970) Eubacterium. In Elizabeth P. Cato, C. S. Cummins, Lilian V. Holdeman, J. L. Johnoson, W. E. C. Moore, R. M. Smibert, and L. DS. Smith. Outline of clinical methods in anaerobic bacteriology, 2nd rev. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, Va. Kageyama, A., Benno, Y. & Nakase, T. (1999). Phylogenetic and phenotypic evidence for the transfer of Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov. Int J Syst Bacteriol 49, 557-565. Kawamura, Y., Hou, X. G., Sultana, F., Miura, H. & Ezaki, T. (1995). Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45, 406408. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Bacteriol 62, 716721.

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Ley, R. E., Hamady, M., Lozupone, C., Turnbaugh, P. J., Ramey, R. R., Bircher, J. S., Schlegel, M. L., Tucker, T. A., Schrenzel, M. D. & other authors (2008). Evolution of mammals and their gut microbes. Science 320, 1647-1651. Ludwig, W., Weizenegger, M., Kilpperbalz, R. & Schleifer, K. H. (1988). PhylogeneticRelationships of Anaerobic Streptococci. Int J Syst Bacteriol y 38, 15-18. Mesbah, M. & Whitman, W. B. (1989). Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. Journal of chromatography 479, 297-306. Moore, W. E. & Holdeman, L. V. (1974). Human fecal flora: the normal flora of 20 JapaneseHawaiians. Appl Microbiol 27, 961-979. Moore, W. E. C., Johnson, J. L. & Holdeman, L. V. (1976). Emendation of Bacteroidaceae and Butyrivibrio and Descriptions of Desulfomonas Gen-Nov and 10 New Species in Genera Desulfomonas, Butyrivibrio, Eubacterium, Clostridium, and Ruminococcus. Int J Syst Bacteriol 26, 238-252. Nakazawa, F. & Hoshino, E. (1994). Genetic-Relationships among Eubacterium Species. Int J Syst Bacteriol 44, 787-790. Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid Extraction of Bacterial Genomic DNA with Guanidium Thiocyanate. Letters in Applied Microbiology 8, 151-156. Prévot, A. R. (1938) études de systematique bactérienne. III. Invalidité du genre Bacteroides Castellani et Chalmers. Démembrement et réclassification. Ann. Inst. Pasteur 60: 292 Rocchi, G. (1908) Lo stato attuale delle nostre cognizioni sui germi anaerobi. Tossi-infezioni putirde e gangrenose. Capitolo II. Bull Sci. Med. 8, 457-479 Stanley, D., Geier, M. S., Denman, S. E., Haring, V. R., Crowley, T. M., Hughes, R. J. & Moore, R. J. (2013). Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed. Vet Microbiol 164, 85-92. Tamura, K. & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution 10, 512-526. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 30, 27252729. Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697-703. Willems, A. & Collins, M. D. (1996). Phylogenetic relationships of the genera Acetobacterium and Eubacterium sensu stricto and reclassification of Eubacterium alactolyticum as Pseudoramibacter alactolyticus gen nov, comb nov. Int J Syst Bacteriol 46, 1083-1087.

376

Figures T

377

Fig. 1: Scanning electron micrograph of cells of Faecalicoccus acidiformans LMG 27428 . (scale bar = 1µm).

378

Fig. 2: Rooted tree showing the 16S rRNA phylogenetic relationship of Faecalicoccus pleomorphus gen. nov.,

379

comb. nov. and Faecalicoccus acidiformans sp. nov. with some other members of the Erysipelotrichaceae family.

380

The tree was constructed using the maximum likelihood method based on the Tamura-Nei model and based on a

381

comparison of approximately 1300 nucleotides. Percentage bootstrap values, based on 100 replications, are

382

shown at branch points. The accession numbers of reference organisms are included. Bar, 0.02 substitutions per

383

nucleotide position. Erysipelothrix rhusiopathiae ATCC19414 was used as outgroup. The evolutionary analyses

384

were conducted using MEGA 6 software.

385

Fig. 3: Phylogenetic tree based on the hsp60 (heat-shock protein 60kDa) gene sequences of the two unknown

386

and three reference strains. The hsp60 gene sequence of two reference strains DSM 3989 and DSM 3983 was

387

taken from the cpn60 database while for the two unknown strains and the reference strain LMG 17756 we

388

sequenced them in this study. The tree, constructed using the maximum likelihood method based on the Tamura-

389

Nei model, showed a comparison of two unknown and three reference strains of family Erysipelotrichaceae.

390

Numbers at the nodes indicate the percentages of bootstrap sampling, derived from 100 samples, supporting the

391

internal branches. The accession numbers of the peptide GenBank are included between brackets. Scale bar:

392

0.05 substitutions per nucleotide position. The evolutionary analyses were conducted using MEGA 6 software.

393

T

T

T

T

394

Tables

395

Table 1: Cellular fatty acids profiles of two strains LMG 27428 and LMG 27427 and their closer phylogenetic

396

neighbours of the family Erysipelotrichaceae (determined in this study). Strains: 1, Faecalicoccus acidiformans

397

LMG 27428 ; 2, F. pleomorphus LMG 27427; 3, F. pleomorphus ATCC 29734 ; 4, Faecalitalea cylindroides

398

ATCC 27803 ; 5, Holdemanella biformis DSM 3989 . Major differences are highlighted in bold. ND, not detected

T

T

T

T

Cellular fatty acid Saturated fatty acid C10:0 C11:0 C12:0 C13:0 C14:0 C16:0 C18:0 C19:0 Unsaturated fatty acids C15:1 ω6c C16:1 ω9c C17:1 ω8c C17:1 ω6c C18:1 ω9c C18:1 ω7c C18:1 ω6c C20:1 ω9c Branched fatty acids iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:0 iso-C17:0 anteiso-C17:0 iso-C18:1 H iso-C19:1 I Hydroxyl fatty acids C16:0 3-OH Summed feature 1 Summed feature 2 Summed feature 3 Summed feature 4 Summed feature 5 Summed feature 6 Summed feature 7

T

1

2

3

4

5

0.9 0.8 5.3 1.0 7.5 12.6 4.6 ND

0.7 0.2 3.4 1.2 6.3 13.5 7.1 0.4

0.6 ND 4.0 1.7 9.0 15.5 10.6 ND

1.2 ND 4.5 1.7 9.0 16.1 7.0 ND

1.0 ND 7.3 ND 4.4 24.5 20.3 ND

ND 1.5 3.5 ND 10.7 5.3 ND 0.5

0.9 0.8 6.3 1.3 12.8 5.8 ND ND

1.4 1.0 4.1 0.8 13.2 6.8 ND ND

1.1 1.8 6.4 1.0 12. 3.7 ND ND

ND ND ND ND 8.0 2.6 3.5 ND

ND 0.5 1.0 ND ND ND 2.3 17.3

0.1 0.5 0.7 0.3 0.4 ND 3.2 10.7

ND ND 1.1 ND ND ND 1.6 8.7

ND ND 0.8 ND ND ND 1.7 6.5

ND 1.1 2.5 0.9 ND 1.3 ND 2.1

ND 0.4 ND 4.4 11.2 2.6 1.1 ND

0.4 0.3 0.7 5.4 9.6 2.3 1.9 0.2

0.6 ND ND 6.0 7.0 2.1 1.3 ND

ND 1.1 1.3 7.3 9.0 2.0 0.7 ND

1.4 ND ND 0.4 8.7 7.1 ND ND

399 400 401 402 403 404

T

Table 2: Comparison between the two strains LMG 27428 and LMG 27427 and their closest phylogenetic T

neighbours of the family Erysipelotrichaceae. Strains: 1, Faecalicoccus acidiformans LMG 27428 ; 2, F. T

T

Holdemanella biformis DSM 3989 . All data arefrom this study unless indicated otherwise. +, Positive; -, Negative; ®

®

ND, not detected (§ api 20A; ‖ api ZYM; ¶ rapid ID 32A)

Characteristic DNA G+C content (%mol) Fermentation acids (mM) Butyric acid Acetic acid Propionic acid Lactic acid Formic acid Acid production from (API system): § D-Mannitol § D-Saccharose § Salicin § D-Xylose § L-Arabinose § D-Raffinose Hydrolysis of: § Gelatin § Aesculin Production of (API system): ‖ Alkaline phosphatase ‖ Esterase (C4) ‖ Ester lipase (C8) ‖ Acid phosphatase ‖¶ α-Glucosidase ¶ Arginine arylamidase ¶ Leucyl glycine arylamidase ¶ Leucine arylamidase‖ ¶ Pyroglutamic acid arylamidase ¶ Alanine arylamidase ¶ Glycine arylamidase ¶ Histidine arylamidase

405 406 407

T

pleomorphus LMG 27427; 3, F. pleomorphus LMG 17756 ; 4, Faecalitalea cylindroides ATCC 27803 ; 5,

1

2

3

40.4

38.8

39.4

3.5 ± 0.2 -3.7 ± 0.6 -0.8 ± 0.1 8.5 ± 0.2 1.5 ± 0.1

2.4 ± 0.3 -2.8 ± 0.3 -1.1 ± 0.1 7.9 ± 0.2 3.8 ± 0.1

-

4 *

5 †



31.0

33.8

3.2 ± 0.3 N.D N.D 7.0 ± 0.3 6.5 ± 0.3

2.6 ± 0.6 1.0 ± 0.3 1.3 ± 0.4 13.6 ± 2.1 0.7 ± 0.2

3.2 ± 0.8 2.4 ± 0.5 3.9 ± 0.2 2.1 ± 0.4 0.3 ± 0.1

-

-

+ +

+ + + + +

-

+ -

-

+

+ -

-

+ + + + + + + +

+ + + + + + +

+ + + + + -

+ + -

DNA G+C content data were taken from: *, (Barnes et al., 1977); †,(Cato et al., 1974); ‡, (Eggerth 1935)

408

409

410 411

412