Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract and emended description of the genus Cloacibacillus

A novel anaerobic, mesophilic, amino-acid-fermenting bacterium, designated strain CL-84T, was isolated from the swine intestinal tract on mucin-based media. Cells were curved rods (0.8–1.2×3.5–5.0 µm), stained Gram-negative and were non-motile with no evidence of spores. Strain CL-84T produced acetate, propionate, formate and butyrate as the end products of metabolism when grown on serine. Optimum growth occurred at 39 °C and pH 6.5. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH, iso-C17 : 0 and C16 : 0, distinguishing strain CL-84T from closely related species. The DNA G+C content of strain CL-84T was 55.1 mol%. 16S rRNA gene sequence analysis showed that strain CL-84T shared 90–95 % similarity with characterized genera within the phylum Synergistetes, family Synergistaceae. Phylogenetic analysis showed that strain CL-84T was related to, but distinct from, Cloacibacillus evryensis. Based on these findings, we propose that strain CL-84T represents a novel species of the genus Cloacibacillus. We further propose the name Cloacibacillus porcorum sp. nov. be designated for this species. The type strain is CL-84T ( = DSM 25858T = CCUG 62631T). An emended description of the genus Cloacibacillus is provided.

Members of the recently described phylum Synergistetes have been identified in a diverse range of anaerobic environments, including anaerobic digesters (Ganesan et al., 2008) and some infections in the human body (e.g. peritoneal fluid, soft tissues, blood and periodontal pockets) (Vartoukian et al., 2007). Despite their culture-independent identification in a wide range of environments, there are few cultured representatives of this phylum. Synergistes jonesii, the first characterized Synergistetes species, was isolated from a goat rumen. S. jonesii degrades toxic pyridinediols in the animals' diet, and in turn the animal's gut provides required nutrients (Allison et al., 1992). In this paper we describe the isolation and characterization of a mucin-degrading bacterium, strain CL-84 T , from the swine intestine. We suggest that strain CL-84 T represents a novel species of the genus Cloacibacillus, for which the name Cloacibacillus porcorum sp. nov. is proposed. How many taxa may share this trait is unclear, but to our knowledge, this is the first description of a member of the phylum Synergistetes that can use mucin as a sole source of carbon and energy.
Strain CL-84 T was one of eight Synergistetes strains isolated during the characterization of mucosa-associated and mucin-degrading micro-organisms from the swine intestinal tract. The gently rinsed mucosal surface of a pig caecum was scraped with a sterile microscope slide and inoculated into minimal medium containing mucin. A series of three enrichments (10 days each) in broth, containing a basal medium, described below, and 1 % (w/ v) hog gastric mucin (HGM) (Sigma-Aldrich), were used to enhance the growth of mucolytic bacteria before inoculation on solid media. Mucin-degrading bacteria were isolated on solid basal medium supplemented with 1 % (w/v) HGM after incubation at 39 u C for 5 days. Pure cultures were obtained after isolates were streaked for isolation three times. All cultures were inoculated and incubated (39 u C) in a Coy anaerobic chamber inflated with an atmosphere of N 2 (85 %), CO 2 (5 %) and H 2 (10 %). The basal medium contained (per litre) 0.45 g CaCl 2 , 0.45 g MgSO 4 , 2.25 g KH 2 PO 4 , 2.25 g K 2 HPO 4 , 4.5 g NaCl, 4.5 g (NH 4 ) 2 SO 4 , 0.05 % cysteine, 0.05 g haemin, 0.0001 % resazurin and 1.6 % Noble agar.
Strain CL-84 T grew optimally on brain heart infusion broth with 0.05 % cysteine and 0.0001 % resazurin, and supplemented with 20 mM arginine and histidine (BHIAH). This medium was used to maintain cultures. After 3 days growth at 39 u C on BHIAH medium, strain CL-84 T reached a terminal OD 620 of 1.2, representing 1.5610 9 c.f.u. ml 21 . The calculated doubling time was 8 h. Cells of strain CL-84 T cultured in BHIAH broth had a curved-rod shape, were nonmotile (determined with motility test medium; Greene et al., 1951) and spores were not seen (Fig. 1a). On BHIAH plates after 5 days growth, strain CL-84 T produced small semitranslucent brown colonies that were 1 mm in diameter. Gram strain was negative. Ultrathin sections were prepared for transmission electron microscopy (TEM) from 4-day-old cultures, stained with uranyl acetate and lead citrate, and examined with a Tecnai 12 G 2 Biotwin microscope (FEI). Micrographs revealed a cell outer envelope structure consistent with Gram-negative cells with a thin peptidoglycan layer surrounded by an outer membrane (Fig. 1b). No spores or inclusion bodies were seen. Cells were variable in size (0.8-1.2 mm 6 3.5-5.0 mm). The TEM appearance and Gram staining characteristics were consistent with the Gramnegative envelope characteristics of other Synergistetes spp.
Genomic DNA was extracted from pelleted cells of strain CL-84 T by following the protocol of Dale & Greenaway (1985). PCR amplification was carried out as described by Downes et al. (2005), with the conserved bacterial primers 8F (Wilmotte et al., 1993) and 1492R (Stackebrandt & Goodfellow, 1991). Purified PCR products were sequenced, yielding nearly full-length (1447 bp) sequences for the 16S rRNA gene of strain CL-84 T . Taxonomic assessments of the 16S rRNA gene sequences were made using the Ribosomal Database project (RDP) web tools (http://rdp.cme.msu. edu/) (Cole et al., 2009), which placed strain CL-84 T within the phylum Synergistetes. The closest matches were to Cloacibacillus evryensis 158 T (95 % sequence similarity) and S. jonesii 78-1 T (90 %). C. evryensis was isolated from a municipal anaerobic waste digester (Ganesan et al., 2008) and S. jonesii was isolated from a goat rumen (Allison et al., 1992). Support for other Synergistetes bacteria being associated with mucus and possibly utilizing mucin can be found from their identification from subgingival plaque samples, and optimized growth (in co-culture) by the addition of mucin to the media (Vartoukian et al., 2010).
Neighbour-joining, maximum-parsimony and maximumlikelihood phylogenetic analyses were performed based on the alignment of the 16S rRNA gene sequence from strain CL-84 T with Silva's SINA web aligner (Pruesse et al., 2007) in the software package ARB (Ludwig et al., 2004). The methods used to construct the tree were ARB neighbourjoining (10 000 bootstrap replicates), maximum-parsimony with DNAPARS v3.6 (1000 bootstraps) and maximumlikelihood with RAxML ('advanced bootstrap+refinement of BS tree' algorithm, GTRGAMMA model, 1000 bootstraps). All three analyses produced trees with the same topology, and therefore only the neighbour-joining tree is presented (Fig. 2). Strain CL-84 T grouped with uncharacterized isolates from infected human blood (GenBank accession numbers GQ258969, EF551162 and EF551160) (Marchandin et al., 2010) and peritoneal fluid samples (DQ412721) (Horz et al., 2006). These sequences were approximately 99 % similar to each other and to that of strain CL-84 T , forming a cluster previously designated OTU cluster 2 by Ganesan et al. (2008). In contrast, the 16S rRNA gene sequences from C. evryensis 158 T and S. jonesii 78-1 T were only 95 and 90 % similar to the sequences in OTU cluster 2, respectively. Strain CL-84 T and related sequences formed a branch distinct from C. evryensis (OTU cluster 1), within the genus Cloacibacillus.
As part of a larger survey of swine intestinal microbes, the genome of strain CL-84 T was sequenced using a 454 titanium pyrosequencing platform (454 Life Sciences). The gyrase B gene sequence of strain CL-84 T (GenBank accession no. JX443487) was obtained from the genome sequence and compared with that of C. evryensis, obtained from the finished genome sequence (IMG/GEBA database: 6858). The gyrase B nucleotide sequence from strain CL-84 T showed only 90 % similarity to C. evryensis, which supports the conclusion that these are different species. Sequence analysis revealed that strain CL-84 T has a DNA G+C content of 55.1 mol%.
Mucins are glycoproteins with carbohydrate side chains connected to a protein backbone by O-glycosidic links (Nataro, 2005) and are the major component of mucus.
The carbohydrate side groups are made up of the sugars galactose, fucose, N-acetyl-D-galactosamine, N-acetyl-Dglucosamine, sialic acid and mannose (Allen et al., 1998). Mucolytic bacteria use proteases and glycosidases to degrade host mucin at polypeptide and glycosidic bonds, respectively (Bradshaw et al., 1994). Growth on different mucin components was examined by preparing basal medium containing 0.2 % (w/v) yeast extract with 0.5 % (w/v) of each of the following: chondroitin sulfate sodium salt, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, hyaluronan biotin sodium salt, mannose, N-acetylneuraminic acid, D-galactose or L-fucose. Strain CL-84 T was further evaluated for its ability to degrade mucin O-linked glycans, purified from the HGM, as described by Martens et al. (2008). Growth on a variety of amino acids was examined by supplementing basal medium containing 0.2 % (w/v) yeast extract with 10 mM of each of the following: arginine, histidine, lysine, serine, tryptophan, alanine, glutamate, aspartate, proline, glycine, cysteine, phenylalanine, isoleucine, leucine, valine, threonine, methionine, glutamine, asparagine or tyrosine. Strain CL-84 T grew on BHI and Casamino acids broth (both used for growth of C. evryensis), but grew faster on BHIAH. Growth was observed on a limited number of amino acids and whole mucin and mucin components. Growth on whole mucin and mucin O-linked glycans was also tested and observed with C. evryensis (OD 620 of 0.8 and 0.06, respectively). Growth results for strain CL-84 T are summarized in Table 1 as OD 620 values.
The supernatants from cultures on each substrate were analysed for fermentation acids by GC of butyl esters (Salanitro & Muirhead, 1975;Stanton & Lebo, 1988). Major fermentation products were formate, acetate or propionate; however, growth on serine, both alone and with threonine and proline, also produced buyrate (Table 1). Strain CL-84 T contained butyryl-CoA : acetate CoA-transferase (E.C. 2.8.3.8) activity [specific activity59.88±0.45 mmol min 21 (mg protein) 21 ] as determined using cells cultured on basal medium with serine. The assay for transferase activity was performed on French press extracts of strain CL-84 T according to Buckel et al. (1981) except that it was evaluated at 412 nm, 39 u C, Table 1. Fermentation products from substrates that support growth of strain CL-84 T in basal medium No growth was observed on methionine, aspartate, valine, proline, isoleucine, lysine, alanine, phenylalanine, leucine, glucose, glutamine, glycine, asparagine, glutamate or tyrosine.

Substrate
Formic acid* Acetic acid* Propionic acid* Butyric acid* OD 620 D  with 0.1 mM butyryl-CoA and 0.5 units (8.8 nkat) of citrate synthase. The most prevalent amino acids in mucin are serine and threonine (Schrager, 1970). The ability of strain CL-84 T to produce butyrate when grown on serine may reflect its role in the gut, as butyrate has been shown to stimulate mucin synthesis in human colonic cell lines and animal mucosa (Finnie et al., 1995;Hatayama et al., 2007).
Additional biochemical characterizations were performed using Rapid ID 32 A and API ZYM test strips (bioMérieux). Strain CL-84 T showed positive arginine dihydrolase and glutamic acid decarboxylase activities and weak alkaline phosphatase and proline arylamidase activities. Additionally, the API ZYM test showed that strain CL-84 T had acid phosphatase, naphthol-AS-BIphosphohydrolase, alkaline phosphatase, esterase (C4), esterase lipase (C8) and leucine arylamidase activities.
Strain CL-84 T tested negative for catalase.
Heat, pH and oxygen tolerance were tested in BHIAH medium. The pH range for growth of strain CL-84 T was pH 4-8, with optimal growth (reaching an OD 620 of .1.0) at pH 6-7. Growth between 20 and 45 u C was observed. No viable cells remained after heating cultures to 80 u C for 30 min. Growth of CL-84 T was inhibited by NaCl concentrations above 1.4 % (w/v), with optimal growth at 0.6-0.8 % (w/v) NaCl. Strain CL-84 T cells were aerotolerant and still viable after 24 h of exposure to oxygen. Growth of CL-84 T was not observed on BHIAH plates, exposed to 1.0 % oxygen, after 1 week of incubation.
The closest related type strain to CL-84 T , C. evryensis 158 T , was obtained from the DSM culture collection (accession no.19522). Both strain CL-84 T and C. evryensis 158 T were grown in 10 ml BHIAH medium to an OD 620 of 1.0, and cell pellets were used for cellular fatty acid analysis. The cellular fatty acid composition was determined by Microbial ID Inc. with the GC-based MIDI Sherlock Microbial Identification System. The major cellular fatty acids of strain CL-84 T were iso-C 15 : 0 (27.1 %), iso-C 15 : 0 3-OH (15.0 %), iso-C 17 : 0 (11.7 %) and C 16 : 0 (9.7 %). The profile for strain CL-84 T differed from that of C. evryensis 158 T in both the fatty acid types and the proportions of each component. The top three fatty acids for strain CL-84 T were iso-C 15 : 0 , iso-C 15 : 0 3-OH and iso-C 17 : 0 (27.1, 15 and 11.7 %, respectively), while the top three for C. evryensis 158 T were iso-C 15 : 0 , iso-C 15 : 0 3-OH and C 17 : 1 v11c (14.2, 14 and 12.4 %, respectively) ( Table 2).
Emended description of the genus Cloacibacillus Ganesan et al. 2008 The description of the genus Cloacibacillus follows that of Ganesan et al. (2008) with the following modifications and additions. The ability to grow on mucin and mucin components, as a sole carbon source, is a trait shared by members of this genus. Cells are glucose non-fermenters, but can grow on fucose. The predominant fatty acids include iso-C 15 : 0 and iso-C 15 : 0 . Differential characteristics between strain CL-84 T and C. evryensis 158 T and S. jonesii 78-1 T are given in Table 3.
Cells are obligately anaerobic, non-motile and curved-rod shaped. Cells ferment amino acids, and can use mucin and mucin components as a sole carbon source. Fermentation products are acetate, propionate and formate but only butyrate is produced from serine. This bacterium is an intestinal commensal of swine. Cells have a Gram-negative cell-wall structure and range in size from 0.8 to 1.2 mm wide and 3.5 to 5.0 mm long. Strong growth is obtained in BHI medium, and growth is enhanced by the addition of histidine and arginine, but not glucose. After 7 days incubation, colonies are 1 mm in diameter, circular, shiny, brown and semi-translucent. The main cellular fatty acids are iso-C 15 : 0 , iso-C 15 : 0 3-OH, iso-C 17 : 0 and C 16 : 0 . Cells are resistant to vancomycin and sulfathiazole but are susceptible to tylosin, lincomycin, chlortetracycline, penicillin, florphenicol, ceftiofur and carbadox. Optimal growth occurs at 39 u C and pH 6.5.
The type strain, CL-84 T (5DSM 25858 T 5CCUG 62631 T ), was isolated from the mucosal lining of a pig caecum in Ames, Iowa, USA. The DNA G+C content of the type strain is 55.1 mol%.  Ganesan et al. (2008). DData from Allison et al. (1992).