In their classical paper 20 years ago, Collins et al. (1994) already noted that C. difficile and its close relatives C. paradoxum and C. sticklandii, along with Peptostreptococcus anaerobius, belong to a distinct family-level group (cluster XI or Family 13). Accordingly, the recent edition of Bergey's reclassified these 13 species into the family Peptostreptococcaceae (Ludwig et al., 2009). Unfortunately, the names of these organisms have not been changed. As a result, many biologists remain unaware that C. difficile is substantially distinct from Clostridium butyricum (the type species of the genus) and its close relatives, such as C. botulinum, C. perfringens and C. tetani. This distance is manifested, for example in the sporulation gene patterns: most C. difficile genomes lack such genes as spoIVFB, bofA, cotS, cotM, ydhD, gerA and gerC, which are widespread among Clostridium sensu stricto (Xiao et al., 2011; Galperin et al., 2012), but encode certain proteins that are not found in other clostridia (Lawley et al., 2009). The current versions of the NCBI Taxonomy database and SILVA database display C. difficile and its relatives as [Clostridium] spp., which is somewhat better but hardly resolves the confusion. We suggest renaming these organisms Peptoclostridium spp. Complete genomes of numerous isolates of C. difficile (Peptoclostridium difficile) and of C. sticklandii (Peptoclostridium sticklandii) strain DSM 519 are already available, and sequencing of other representatives of this group is currently under way. Analysis of their genome sequences may suggest that these organisms represent as many as four genera as has been suggested by Collins et al. (1994).
In the 2nd edition of Bergey's (Ludwig et al., 2009), Clostridium symbiosum and 31 other Clostridium species that fall within clusters XIVa and XIVb of Collins et al. (1994) have been transferred to the family Lachnospiraceae, in full agreement with the assignments of SILVA and RDP databases. One of these species, Clostridium lentocellum, has been renamed Cellulosilyticum lentocellum (Cai and Dong, 2010), whereas, as far as we could see, the rest still retain the Clostridium name. Several well-known species from clusters XIVa and XIVb, such as C. sphenoides or C. piliforme, have not been mentioned by Ludwig et al. (2009) or Rainey et al. (2009), creating further confusion as to which Clostridium spp. belong to the family Lachnospiraceae and which should stay in Clostridiaceae. Thus, the web site http://www.broadinstitute.org/annotation/genome/clostridium_group/ of the Broad Institute, which has obtained draft genomic sequences of several organisms from this group (C. aldenense, C. bolteae, C. citroniae, C. clostridioforme, C. hathewayi and C. symbiosum), lists them all as Clostridium spp. Again, protein trees supported 16S rRNA-based assignments, placing C. phytofermentans and C. symbiosum in a tight cluster with Butyrivibrio proteoclasticus, Roseburia hominis and other members of Lachnospiraceae. We propose tentatively assigning all cluster XIVa organisms that are still listed as Clostridium spp. to the new genus Lachnoclostridium. For the cluster XIVb organisms, which include C. piliforme, the causative agent of Tyzzer's disease, we propose the new genus Tyzzerella (see below).
Clostridium ramosum and C. spiroforme, members of the cluster XVIII (Collins et al. (1994) along with two other Clostridium species, have been transferred by Ludwig et al. (2009) to the family Erysipelotrichaceae in the class Erysipelotrichi. Our data confirm their close relationship to Erysipelothrix rhusiopathiae, as well as clustering with mollicutes (Marchandin et al., 2010; Ogawa et al., 2011), which are currently assigned to the separate phylum, the Tenericutes. We propose assigning C. ramosum, C. spiroforme and three related species, to the new genus Erysipelatoclostridium.
Proposed new genera
The above suggestions are based primarily on the results from ribosomal protein-based phylogenetic trees (Fig. 1) and therefore miss those Clostridium spp. for which sequence information has been unavailable or insufficient. However, the excellent agreement of our protein trees with 16S rRNA-based classification presented in the SILVA and RDP databases indicates that, at least in the case of Clostridia, the assignments of these databases could be used to build a fairly reliable phylogeny-based taxonomy. Hence, based on the assignments in Bergey's, RDP and SILVA, as well as results of 16S rRNA similarity searches, we propose the following new genera (Table 2; see Table S8).
Description of Peptoclostridium gen. nov.
Peptoclostridium [Pep.to.clos.tri'di.um. Gr. v. peptô, digest; N.L. neut. dim. n. Clostridium, a bacterial genus name (from Gr. n. klôstêr, a spindle); N.L. neut. dim. n. Peptoclostridium, the digesting clostridium].
Gram-staining-positive, motile, spore-forming rods 0.3–1.5 μm × 1.5–20 μm. Obligate anaerobes, no microaerophilic or aerobic growth. Strains are mesophilic or thermophilic (temperature range from 20°C to 63°C) and grow in neutral to alkaline pH (some strains up to pH 11). Chemoorganotrophs. Oxidase and catalase negative. Peptone may serve as nitrogen source. Yeast extract can be used as the sole carbon and energy source. Several members require 1.5% NaCl for growth. Some mono- and disaccharides can be fermented; acetate is produced as a major end product. Sulfate is not reduced. The G+C content of the genomic DNA ranges from 25 to 32 mol%. The type species is Peptoclostridium difficile (formerly C. difficile); the type strain is ATCC 9689 = DSM 1296.
The newly proposed genus Peptoclostridium is equivalent to genus Clostridium XI in the RDP and the Peptostreptococcaceae genus Incertae Sedis in SILVA [see the 16S rRNA trees in Song et al. 2004) and Pikuta et al. (2009)]. It includes 11 validly described species that have been transferred to the family Peptostreptococcaceae in the recent edition of Bergey's, as well as C. mayombei and C. thermoalcaliphilum (Table 2). In addition, we propose that the genus include Eubacterium tenue, Eubacterium yurii and the following species that have not been validly described but whose 16S rRNA sequences are available in GenBank: C. maritimum (GenBank accession number EU089965), C. metallolevans (DQ133569), C. ruminantium (EU089964), C. venationis (EU089966) and the misnamed C. hungatei strain mc (JX073559; other C. hungatei strains go to Ruminiclostridium, see below). Two more members of the family Peptostreptococcaceae, Clostridium sticklandii and C. litorale, have been tentatively assigned to the genus Peptoclostridium to resolve the naming conundrum but might deserve to be put into a separate genus (or genera) [see Fig. 1B and Pikuta et al. (2009)]. Sporacetigenium mesophilum falls within the diversity of the new genus but is left as is because of its unusual metabolic properties (Chen et al., 2006).
Description of Gottschalkia gen. nov.
Gottschalkia (Gott.shal'ki.a. N.L. fem. n., named after Gerhard Gottschalk, in recognition of his contributions to the studies of various anaerobic bacteria, including clostridia).
Obligately anaerobic purinolytic spore-forming rods that, in the presence of 0.1% yeast extract, are capable of utilizing uric acid as sole carbon and energy source. Gram-staining is variable; motility is by peritrichous flagella. Optimal growth is at 19–37°C and pH 7.3–8.1. No utilization of carbohydrates, no reduction of nitrate and no production of H2. The DNA G+C content is 28–29 mol%.
The proposed genus has been first suggested by Collins et al. (1994); it is equivalent to Clostridiales Family XI Incertae Sedis genus Incertae Sedis in SILVA and includes two validly described organisms: Clostridium acidurici and C. purinilyticum [see Hartwich et al. (2012) and references therein]. Based on the similar 16S rRNA sequence and metabolic properties, Eubacterium angustum could be assigned to the same genus, despite its inability to form spores and higher G+C content (Beuscher and Andreesen, 1984). The type species is Gottschalkia acidurici (formerly Clostridium acidurici); the type strain is ATCC 7906 = DSM 604.
Description of Ruminiclostridium gen. nov.
Ruminiclostridium [Ru.mi.ni.clos.tri'di.um. L. n. rumen -inis, the rumen; N.L. neut. dim. n Clostridium, a bacterial genus name (from Gr. n. klôstêr, a spindle); N.L. neut. dim. n. Ruminiclostridium, clostridia-like bacteria in the family Ruminococcaceae].
Obligately anaerobic, mesophilic or moderately thermophilic, spore-forming, straight or slightly curved rods 0.5–1.5 μm × 1.5–8 μm. The cells have a typical Gram-positive cell wall, although often stain Gram-negative. Produce spherical or oblong terminal spores, which results in swollen cells. Most species are motile and have polar, subpolar or peritrichous flagella. The temperature range for various species is from 20°C to 70°C with Topt between 33 and 65°C. Optimal pH values are between 7 and 9 (some members can grow at pH as low as 5.9 or as high as 10.2). Oxidase and catalase are not produced. Yeast extract or vitamins are usually required for anabolic purposes. All known members can use cellulose, xylan and/or cellobiose as substrates, fermenting them primarily to acetate, ethanol, H2 and CO2, as well as lactate, propionate, butyrate or other end products. The ability to ferment other carbohydrates varies between species. Several species are capable of fixing N2. Sulfate is not reduced. The G+C content of the genomic DNA is typically 39–41.5%, but ranges from 27 to 51 mol% [while two species, C. alkalicellulosi and C. papyrosolvens, have been initially reported to have the G+C content of 29.9–30.0%, the genomic sequence of C. papyrosolvens DSM 2782 showed G+C content of 36.9% (Hemme et al., 2010)]. The type species is Ruminiclostridium thermocellum (formerly Clostridium thermocellum); the type strain is ATCC 27405 = DSM 1237.
The proposed genus Ruminiclostridium includes organisms from clostridial cluster III of Collins et al. (1994) and is equivalent to the genus Clostridium III in the RDP and the Ruminococcaceae genus Incertae Sedis in SILVA [see the 16S rRNA trees in Shiratori et al. (2009) and Izquierdo et al. (2012)]. It includes 15 validly described species, 12 of which have been transferred to the family Ruminococcaceae in the recent edition of Bergey's, as well as C. caenicola, C. clariflavum and C. sufflavum (Table 2). The Clostridium strain Rt51.B1 (GenBank: L09175), misnamed as C. sporogenes, and Clostridium sp. BNL1100 also belong to this genus.
Several Clostridium spp. that fall within the family Ruminococcaceae have been tentatively assigned to the genus Ruminiclostridium but will have to be reclassified and renamed after their phylogenetic status is better resolved. These include five members of the Collins et al. (1994) cluster IV (and genus Clostridium IV in the RDP): Clostridium leptum, C. cellulosi, C. methylpentosum, C. sporosphaeroides and C. viride. One more member of Ruminococcaceae, Clostridium orbiscindens, has been recently reclassified as Flavonifractor plautii (Carlier et al., 2010). In addition to former Clostridium spp., Bacteroides cellulosolvens, Eubacterium desmolans and Eubacterium siraeum fall within the proposed new genus. However, in future, some of its members might have to be reassigned to Acetanaerobacterium, Acetivibrio, Flavonifractor, Oscillibacter, Ruminococcus and/or new genera of Ruminococcaceae.
Description of Lachnoclostridium gen. nov.
Lachnoclostridium [Lach.no.clos.tri'di.um. Gr. n. lachnos, wool; N.L. neut. dim. n. Clostridium, a bacterial genus name (from Gr. n. klôstêr, a spindle); N.L. neut. dim. n. Lachnoclostridium, the clostridia within the family Lachnospiraceae].
Gram-positive, motile, obligately anaerobic spore-forming rods 0.3–1.5 μm × 1.5–20 μm. Strains are mesophilic or thermophilic (temperature range from 20°C to 63°C) and grow in neutral to alkaline pH (some up to pH 11). Chemoorganotrophs. Oxidase and catalase are not produced. Some mono- and disaccharides can be fermented; acetate is produced as a major end product. Sulfate is not reduced. The G+C content of the genomic DNA ranges from 25.6 to 32 mol%. The type species is Lachnoclostridium phytofermentans (formerly C. phytofermentans); the type strain is ATCC 700394 = DSM 18823.
The proposed genus Lachnoclostridium includes organisms from clostridial cluster XIVa of Collins et al. (Collins et al., 1994), the genus Clostridium XIVa in the RDP and the Lachnospiraceae genus Incertae Sedis in SILVA [see the 16S rRNA trees in Warren et al. (2006) and Domingo et al. (2009)]. It includes 30 validly described species, most of which have been assigned to the family Lachnospiraceae in the recent edition of Bergey's (Table 2). It also includes the following species that have not been validly described but whose 16S rRNA sequences are available in GenBank: C. boliviensis (AY943862), C. fusiformis (AB702934), C. sulfatireducens (AY943861) and a misnamed strain of C. leptum (AF262239; C. leptum type strain DSM 753 goes to Ruminiclostridium; see Fig. 1B). Desulfotomaculum guttoideum, Eubacterium contortum, Eubacterium fissicatena and Ruminococcus torques also belong to this genus.
Description of Tyzzerella gen. nov.
Tyzzerella [Ti.ze.rel'la] N.L. fem. n. Tyzzerella, named after Ernest Tyzzer, an American pathologist who isolated and described ‘Bacillus piliformis’, the causative agent of Tyzzer's disease.
A closely related cluster of organisms in the family Lachnospiraceae includes six Clostridium spp. that warrant assignment to a separate genus. The description of the new genus is essentially the same as that of Lachnoclostridium (see earlier), although some of its members are non-motile and non-spore-forming and have higher G+C contents, from 40% in C. nexile to 46.8% in C. colinum. The genus is named after Ernest Edward Tyzzer (1875–1965), who in 1917 characterized ‘Bacillus piliformis’, the causative agent of an infectious diarrhoea of laboratory mice, which was later found in a variety of animals and became known as ‘Tyzzer's disease’. Unfortunately, no Tyzzerella piliformis (formerly Clostridium piliforme) strains have been deposited in public culture collections so far (deposition is currently in progress). Accordingly, Tyzzerella nexilis [formerly Clostridium nexile (Holdeman and Moore, 1974)] is selected as the type species and ATCC 27757 = DSM 1787 as the type strain.
Description of Erysipelatoclostridium gen. nov.
Erysipelatoclostridium [E.ri.si.pe.la.to.clos.tri'di.um]. Gr. n. erusipelas -pelatos, erysipelas; N.L. masc. n. Clostridium, a bacterial genus name (from Gr. n. klôstêr, a spindle); N.L. neut. dim. n. Erysipelatoclostridium, Clostridium-like members of the order Erysipelotrichales.
Gram-positively staining, non-motile, obligately anaerobic straight or helically curved rods 0.3–1.0 μm × 2–4 μm. Spore formation is rare or absent. The G+C content of the genomic DNA is 27–33 mol%. Ferment glucose, fructose and sucrose [see Kaneuchi et al. (1979) for a detailed comparison]. The type species is Erysipelatoclostridium ramosum (formerly Clostridium ramosum); the type strain is ATCC 25582 = DSM 1402.
The newly proposed genus Erysipelatoclostridium is equivalent to the Clostridium XVIII genus in RDP and Erysipelotrichaceae genus Incertae Sedis in SILVA [see the 16S rRNA trees in Clavel et al. (2007) and Ogawa et al. (2011)]. It includes four validly described species: Clostridium cocleatum, C. ramosum, C. saccharogumia and C. spiroforme. In addition, Clostridium innocuum, which is more distantly related to the rest of the group and has G+C content of 43–44%, is tentatively assigned to this species but might have to be reclassified in the near future. The ability of C. cocleatum, C. ramosum and C. spiroforme to form spores contradicts the current description of the family Erysipelotrichaceae, which is why the proposed genus Erysipelatoclostridium should either be placed in the order Erysipelotrichales outside the family Erysipelotrichaceae or the description of the family be emended.