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International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3538–3545 DOI 10.1099/ijs.0.058941-0

Peptoniphilus stercorisuis sp. nov., isolated from a swine manure storage tank and description of Peptoniphilaceae fam. nov.

Crystal N. Johnson,1 Terence R. Whitehead,2 Michael A. Cotta,2 Robert E. Rhoades1 and Paul A. Lawson1,3

Correspondence 1Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Paul A. Lawson OK 73019 USA [email protected] 2Bioenergy Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA 3Ecology and Evolutionary Biology Program, University of Oklahoma, Norman, OK 73019 USA

A species of a previously unknown Gram-positive-staining, anaerobic, coccus-shaped bacterium recovered from a swine manure storage tank was characterized using phenotypic, chemotaxonomic, and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies and biochemical characteristics demonstrated that this organism is genotypically and phenotypically distinct, and represents a previously unknown sub-line within the order Clostridiales, within the phylum . Pairwise sequence analysis demonstrated that the novel organism clustered within the genus , most closely related to Peptoniphilus methioninivorax sharing a 16S rRNA gene sequence similarity of 95.5 %. The major long-chain

fatty acids were found to be C14 : 0 (22.4 %), C16 : 0 (15.6 %), C16 : 1v7c (11.3 %) and C16 : 0 ALDE (10.1 %) and the DNA G +C content was 31.8 mol%. Based upon the phenotypic and phylogenetic findings presented, a novel species Peptoniphilus stercorisuis sp. nov. is proposed. The type strain is SF-S1T (5DSM 27563T5NBRC 109839T). In addition, it is proposed to accommodate the genera Peptoniphilus, Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Helcococcus, Murdochiella and Parvimonas in a new family of the order Clostridiales, for which the name Peptoniphilaceae fam. nov. is proposed; the type genus of the family is Peptoniphilus.

At the time of writing, the genus Peptoniphilus consists of from human clinical specimens (Ezaki et al., 2001; Madsen 12 species with validly published names, including Pepto- et al., 1991); while Peptoniphilus methioninivorax was isolated niphilus asaccharolyticus (Ezaki et al., 2001), Peptoniphilus from retail ground beef (Rooney et al., 2011). coxii (Citron et al., 2012), Peptoniphilus duerdenii (Ulger- The exact relationship of this group of organisms with Toprak et al., 2012), Peptoniphilus harei (Ezaki et al., 2001), other close members of the Firmicutes is somewhat uncertain, Peptoniphilus indolicus (Ezaki et al., 2001), Peptoniphilus and this is reflected in their placement in the Family XI ivorii (Ezaki et al., 2001), Peptoniphilus koenoeneniae (Ulger- Incertae Sedis (order Clostridiales,classClostridia,phylum Toprak et al., 2012), Peptoniphilus lacrimalis (Ezaki et al., Firmicutes) in the current edition of Bergey’s Manual of 2001), Peptoniphilus methioninivorax (Rooney et al., 2011), Systematic Bacteriology (De Vos et al., 2009). Our studies Peptoniphilus olsenii (Song et al., 2007), Peptoniphilus demonstrate that the genus Peptoniphilus shares a close gorbachii (Song et al., 2007) and Peptoniphilus tyrrelliae relationship with the genera Anaerococcus, Anaerosphaera, (Citron et al., 2012). Almost all species have been isolated Helcococcus , Finegoldia, Gallicola, Murdochiella and Parvi- from human clinical material. However, Peptoniphilus monas and that these organisms share consistent biochemical indolicus can be isolated from cattle, swine and occasionally and chemotaxonomic traits which support the proposal to also be recovered from insects associated with cattle and create a family to embrace these genera. In this study, we report the isolation and characterization of a novel species The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene recovered from stored swine manure. The production of sequence of strain SF-S1T is KF705042. odorous chemicals that include ammonia, organic acids Two supplementary figures and a supplementary table are available with and alcohols, and sulphides by micro-organisms, and an the online version of this paper. understanding of the underlying processes of the production

3538 058941 Printed in Great Britain Peptoniphilus stercorisuis sp. nov. of these compounds is the focus of ongoing research on negative for nitrate reduction and indole production. The swine manure management (Miller, 2001). The Gram- novel strain was also asaccharolytic and did not produce positive, obligately anaerobic, coccal-shaped organism iso- acid from the carbohydrates tested. lated from stored swine manure was characterized using The Rapid ID 32A profile for strain SF-S1T was determined biochemical, chemotaxonomic and phylogenetic methods, to be 2000053705, which corresponded to positive reac- and based upon these findings presented here, a novel species tions for arginine dihydrolase, arginine arylamidase, of the genus Peptoniphilus is proposed. phenylalanine arylamidase, leucine arylamidase, tyrosine Strain SF-S1T was isolated from a manure storage tank arylamidase, alanine arylamidase, glycine arylamidase, located at a pig production facility in central Oklahoma, histidine arylamidase and serine arylamidase, and a weakly USA. A sample of pig slurry was collected from a 3.0 m positive reaction for leucyl glycine arylamidase. Using the depth using a tank sampler (NASCO); the environmental API ZYM test system, positive reactions were obtained for conditions of the sample site were 28 uC, pH 7.3 and when leucine arylamidase, cystine arylamidase and phosphohy- allowed to settle, the particulate matter was found to be drolase, with weak reactions obtained for a-galactosidase approximately 50 % (v/v). The sample was transferred to a and a-mannosidase. All other API ZYM tests were negative. nitrogen-flushed sterile bottle that was then sealed to For chemotaxonomic analysis of cellular fatty acids, biomass maintain an anaerobic environment, and transported back of strain SF-S1T was collected from BHI Chocolate Agar to the laboratory on ice. A 10 % inoculum was used for (Oxoid) after 72 h anaerobic growth at 37 uC and the enrichment in Brain Heart Infusion (BHI; Oxoid) broth at analysis performed at the Center for Microbial Identification pH 7.0 and was incubated at room temperature with and (University of Oklahoma). Fatty acid methyl H :N :CO headspace (5 : 10 : 85) at 20 p.s.i. Isolation was 2 2 2 esters were extracted using the Sherlock Microbial Identi- achieved through serial plate dilutions and sub-cultures on fication System (MIDI) version 6.1 as described previously Tryptic Soy Agar (Becton, Dickinson and Company) (Sasser, 1990; Ka¨mpfer & Kroppenstedt, 1996). Analysis was amended with 5 % defibrinated sheep blood, and incubated carried out with an Agilent Technologies 6890N gas in anoxic jars containing H :N :CO headspace (5 : 10 : 85) 2 2 2 chromatograph equipped with a phenyl methyl silicone at 37 uC. Colonies were picked and sub-cultured onto fresh fused silica capillary column (HP-2 25 m 6 0.2 mm 6 media until well-isolated. For phenotypic analysis, cells that 0.33 mm film thickness) and a flame-ionization detector. had been grown anaerobically for 5 days at 37 uCon Hydrogen was used as the carrier gas. The temperature Brucella blood agar (Oxoid) were used for all tests unless program was initiated at 170 uC and increased at 5 uC min21 otherwise stated. Cells were examined with an Olympus to a final temperature of 270 uC. The relative amount of each CX41 microscope using phase-contrast at 61000 mag- fatty acid was expressed in terms of the percentage of total nification. For biochemical characterization, conventional fatty acids. The quantitative fatty acid data of strain SF-S1T tests and Rapid ID 32A and API ZYM test systems was C (22.4 %), C v8c (8.7 %). C (15.6 %), (bioMe´rieux) were employed with all tests performed in 14 : 0 15 : 1 16 : 0 C v7c (11.3 %), C ALDE (10.1 %) with minor duplicate (Jousimies-Somer et al., 2002; Tindall et al., 16 : 1 16 : 0 amounts of C (2.2 %), C (3.2 %), anteiso-C 2007). For additional tests, except where stated, the strain 12 : 0 13 : 1 15 : 0 (1.7 %), C v5c, (2.9 %), C v7c (1.8 %), C v8c was incubated in Hungate tubes containing anoxic peptone- 16 : 1 17 : 1 17 : 1 (1.9 %), C v9c (1.1 %), C (3.0 %) and cyclo C / yeast extract (PY) medium (Holdeman et al., 1977) supple- 17 : 1 17 : 0 17 : 0 C (2.9 %). Trace amounts of C (0.8 %), C 2-OH mented with 0.1 % cysteine sulfide. Fermentation tests were 18 : 0 13 : 0 14 : 0 (0.9 %) and iso-C H/C 3-OH (0.9 %) were also performed using fructose, mannose, glucose, sucrose, xylose 15 : 1 13 : 1 detected. In addition a number of unknown products were and cellobiose using pre-reduced, anaerobically sterilized detected with estimated carbon lengths of C (0.8 %), peptone/yeast/carbohydrate broth tubes (Anaerobe Systems). 18 : 3 C (2.2 %) and C (5.8 %). Table 1 shows the fatty acid Growth was tested at temperatures of 4, 10, 15, 20, 25, 30, 37, 16 : 3 13 : 8 composition of strain SF-S1T as well as some other type 43, 47 and 60 uC; pH values of 5, 5.5, 6, 6.5, 7, 7.3, 7.75, 8, 8.5 strains of the genus Peptoniphilus. The major products of and 9; and NaCl concentrations of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8 C and isomers of C are consistent with the genus and 9 %. Optimum growth conditions were determined 14 : 0 16 : 0 description, however C ALDE is produced in strain SF- using a spectrophotometer at 600 nm (Spectronic 20D; 16 : 0 S1T but is lacking or only present in minor proportions in Milton Roy). All tests were performed in duplicate. some species (Peptoniphilus asaccharolyticus, Peptoniphilus The novel strain grew anaerobically but could not grow in harei and Peptoniphilus olsenii). Strain SF-S1T also has an air. Cells were Gram-stain positive, coccus-shaped (Fig. S1, elevated level of C16 : 1v7c that is seen in Peptoniphilus available in the online Supplementary Material) with a methioninivorax NRRL B-23883T but not in other species typical colony being circular, grey, convex, entire and of the genus. In addition, fatty acid analyses were per- opaque with a diameter of 1–2 mm on Brucella base agar formed on strain SF-S1T using a number of different (Oxoid) plus defibrinated sheep blood after 5 days incu- media [and a direct comparison with Peptoniphilus meth- bation at 37 uC. Tested by antibiotic discs, the organism ioninivorax NRRL B-23883T grown on Columbia agar was susceptible to penicillin, chloramphenicol and baci- (Sigma-Aldrich)]; the profiles obtained were remarkably tracin, but resistant to trimethoprim and ciprofloxacin. stable with C14 : 0,C16 : 0 and C16 : 1v7c always forming the Strain SF-S1T was catalase- and urease- negative and major products (Table S1). http://ijs.sgmjournals.org 3539 C. N. Johnson and others

Table 1. Comparison of fatty acid profile of strain SF-S1T with other members of the genus Peptoniphilus

Strains: 1, SF-S1T;2,Peptoniphilus asaccharolyticus CCUG 9988T;3,Peptoniphilus duerdenii WAL 18896T;4,Peptoniphilus koenoeneniae WAL 18898T;5,Peptoniphilus gorbachii CCUG 53341T;6,Peptoniphilus harei CCUG 38491T;7,Peptoniphilus indolicus CCUG 17639T;8,Peptoniphilus ivorii CCUG 38492T;9,Peptoniphilus lacrimalis CCUG 31350T; 10, Peptoniphilus methioninivorax NRRL B-23883T; 11, Peptoniphilus olsenii CCUG 53342T. Data from Ulger-Toprak et al. (2012); Rooney et al. (2011); CCUG (http://www.ccug.se). Predominant products are shown in bold, values ,1 % are not shown. Profiles for Peptoniphilus coxii and have not been performed.

Fatty acid 1 2 3 4 5 6 7 8 9 10* 11

C10 : 0 2.8 1.1 0.3 C12 : 0 2.2 2.3 1.3 1.5 1.2 2.0 isoC13 : 0 2-OH 2.5 3.4 C14 : 0 22.4 5.4 4.4 3.1 2.9 3.6 12.5 4.4 1.5 10.9 8.2 iso-C15 : 0 2.6 anteiso-C15 : 0 1.7 C15 : 1v8c 8.7 1.0 C16 : 0 15.6 14.4 33.0 22.6 24.0 18.8 11.3 29.5 20.2 25.1 13.1 C16 : 0 ALDE 10.1 0.7 1.8 0.7 C16 : 1v5c 2.9 1.5 1.1 C16 : 1v7c 11.3 3.9 2.7 3.3 5.3 1.0 3.4 14.1 4.8 C16 : 1v7c DMA 1.5 C16 : 1v9c 3.3 3.1 3.4 1.3 C17 : 1v9c 1.1 3.0

iso-C17 : 1v5c 3.0 2.6 18.5 C17 : 0 DMA 3.0 iso-C17 : 1/C16 : 0 DMA 4.3 5.4 4.5 anteiso-C17 : 0 1.6 3.5 1.2 3.8 1.9 1.6 iso-C17 : 0 3-OH 3.3 4.9 7.6 7.7 9.2 9.1 Cyclo C17 : 0/C18 : 0 2.9 C18 : 0 9.4 16.2 12.1 8.1 4.0 4.8 12.3 3.0 6.3 C18 : 1v7c DMA 1.1 C18 : 1v7c/12t/9t 1.4 2.3 C18 : 1v9c 20.2 22.6 24.4 22.6 17.6 2.8 11.4 31.2 5.1 12.0 C18 : 1v9c DMA 6.6 6.3 4.4 7.3 2.6 3.1 10.3 C18 : 2v6,9c/anteisoC18 : 0 22.0 21.1 20.6 26.8 19.8 9.7 24.0 25.7 8.5 12.2 iso-C19 : 1 1.5 1.0 Unknown C13 : 835 5.8 Unknown C16 : 315 2.2 Unknown C18 : 177 5.1 8.9 1.9 Unknown C16 : 754 1.4 2.1

*Data from Rooney et al. (2011), the organism was grown on Columbia blood agar.

T The DNA G+C content of strain SF-S1 was determined reconstructions were performed in MEGA software (version according to the method of Mesbah et al. (1989) and found 4) (Tamura et al., 2007) using the neighbour-joining to be 31.8 mol%. For phylogenetic analyses, methods for method (Saitou & Nei, 1987), applying evolutionary DNA extraction, primers and PCR conditions were as genetic distances that had been calculated by Kimura’s previously described (Ulger-Toprak et al., 2012). The two-parameter model (Kimura, 1980). Results of the DNA closest known relatives of the novel isolate based on the database searches showed the novel isolate to be a member 16S rRNA gene sequence were determined by performing of the phylum Firmicutes and within the genus Peptoni- searches of EMBL/GenBank databases using the EzTaxon-e philus. Pairwise comparisons revealed that strain SF-S1T server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). was most closely related to Peptoniphilus methioninivorax The most related sequences were then selected for the NRRL-23883T sharing a 16S rRNA gene sequence similarity calculation of pairwise sequence similarity using a global value of 95.5 %, with all other species displaying16S rRNA alignment algorithm (Myers & Miller, 1988). These gene sequence similarity values of between 88.0 and 91.0 %. sequences and those of other related strains were aligned These values are well-below the generally recognized with the sequence derived from strain SF-S1T using the divergence value of 3 % used to facilitate the assignment of program CLUSTAL W (Thompson et al., 1994). Phylogenetic novel species (Stackebrandt & Goebel, 1994). The novel strain

3540 International Journal of Systematic and Evolutionary Microbiology 64 Peptoniphilus stercorisuis sp. nov.

98 Peptoniphilus gorbachii WAL 10418T (DQ911241) 96 Peptoniphilus harei DSM 10020T (Y07839) 99 Peptoniphilus tyrrelliae CCUG 59621T (GU938835) 98 Peptoniphilus olsenii WAL 12922T (DQ911242) CCUG 31350T (AF542230) 96 Peptoniphilus lacrimalis Peptoniphilus koenoeneniae WAL 18898T (EU526291) Peptoniphilus duerdenii WAL 18896T (EU562290) Anaerosphaera aminiphila JCM 15094T (AB298735) Peptoniphilus asaccharolyticus CCUG 9988T (AF542228) 100 Peptoniphilaceae Peptoniphilus indolicus CCUG 17639T (AY153430) 98 Peptoniphilus methioninivorax NRRL-23883T (GU440754) 100 Peptoniphilus stercorisuis SF-S1T (KF705042) Peptoniphilus coxii CCUG 59622T (GU938836) 96 100 Peptoniphilus ivorii DSM 10022T (Y07840) 99 Finegoldia magna CCUG 17636T (AF542227) 98 Gallicola barnesae ATCC 49795T (AB038361) Parvimonas micra ATCC 33270T (AF542231) Anaerococcus prevotii ATCC 9321T (D14139) Helcococcus kunzii NCIMB 702900T (x69837) ATCC BAA-1631T (EU483153) ------Murdochiella asaccharolytica------Soehngenia saccharolytica ATCC BAA-502T (AY353956) Tepdimicrobium ferriphilum SB91T (AY656718) Lup 33T (AF358114) Sporanaerobacter acetigenes Family XI Incertae sedis 91 89 Tissierella praeacuta ATCC 25539T (X80833) ZF2T (AJ404680) ------Sedimentibacter saalensis ------ anaerobius ATCC 27337T (AY326462) Peptostreptococcaceae ------Fusibacter paucivorans SEBR 4211T (AF050099) Natronicola histidinovorans DSM 11416T (Y16716) 100 Tindallia magadiensis DSM 10318T (Y15626) Oxobacter pfennigii DSM 3222T (X77838) Sporosalibacterium faouarense JCM 15487T (EU567322) Caloranaerobacter azorensis DSM 13643T (AJ272422) Clostridiaceace 100 Thermobrachium celere DSM 8682T (X99238) 99 Caloramator fervidus ATCC 43204T (L09187) Clostridium butyricum ATCC 19398T (AB07568) 100 Anaerobacter polyendosporus DSM 5272T (Y18189) JCM 14817T (DQ852338) ------Proteiniclasticum ruminis ------90 Eubacterium eligens ATCC 27750T (L34420) Lachnospiracea 100 Anaerostipes caccae NCIMB 13811T (AJ270487) ATCC 27755T (L34619) ------Dorea formicigenerans ------Ruminococcus flavefaciens ATCC 19208T (x85097) 100 Anaerofilum agile DSM 4272T (x97852) Ruminococcaceae 100 Subdoligranulum variabile CCUG 47106T (AJ518869) 99 ATCC 27768T (AJ413954) ------Faecalibacterium prausnitzii ------Megasphaera elsdenii ATCC 25940T (U95027) 100 Veillonella parvula DSM 2008T (X84500) Dialister pneumosintes ATCC 33048T (X82500) 100 Sporomusa paucivorans DSM 3697T (M59117) 100 Acidaminococcus fermentans ATCC 25085T (X65935) Veillonellaceae 95 Phascolarctobacterium faecium ACM 3679T (X72865) 96 Succiniclasticum ruminis SE10T (X81137) Atopobium parvulum ATCC 33793T (AF292372)

Fig. 1. Phylogenetic dendrogram of 16S rRNA gene sequences indicating the position of Peptoniphilus stercorisuis sp. nov. The tree was reconstructed using the neighbour-joining algorithm in the MEGA software package (version 4) with Atopobium parvulum as the outgroup. Bootstrap values are displayed on their relative branches. Scale Bar represents 1%. http://ijs.sgmjournals.org 3541 C. N. Johnson and others from the swine manure storage tank represents a new sub-line Moreover, with the internal structure present within this within the genus Peptoniphilus sharing the closest relationship rRNA lineage it is likely that further taxonomic revisions with Peptoniphilus methioninivorax DSM 22461T which was may be necessary in the future. supported by a 100 % bootstrap value (Fig. 1) and was In addition to its unique 16S rRNA gene sequence, traits confirmed by the maximum-parsimony method (Fitch, 1971) useful in distinguishing the novel species from other species using MEGA software (version 4) (Fig. S2). It is interesting to of the genus Peptoniphilus are shown in Table 1, Table 2, note that Peptoniphilus methioninivorax is the only other Table S1 and the species description. Based on phenotypic, species of the genus Peptoniphilus to date that has not been genotypic and chemotaxonomic evidence, strain SF-S1T recovered from human clinical material but is associated with isolated from a swine manure storage tank represents a novel animals, albeit from ground beef. species of the genus Peptoniphilus, for which the name In the current edition of Bergey’s Manual of Systematic Peptoniphilus stercorisuis sp. nov. is proposed. Bacteriology (De Vos et al., 2009) the exact relationship of The genera Anaerosphaera, Anaerococcus, Helcococcus, the genus Peptoniphilus with other close members within Finegoldia, Gallicola, Murdochiella, Parvimonas and Pepto- the order Clostridiales is uncertain and placed in the Family niphilus share a phylogenetic ancestry and form a robust XI Incertae sedis along with the genera Anaerosphaera, group. Based on phylogenetic, biochemical and chemo- Anaerococcus, Helcococcus, Finegoldia, Gallicola, Murdo- taxonomic information we propose the designation of chiella and Parvimonas. From the phylogenetic data pre- Peptoniphilaceae fam. nov. to accommodate these genera. sented in Fig. 1 and Fig. S2, it is clear that these genera Peripheral to this group of organisms are members belong- form an rRNA gene super-cluster that share consistent ing to the genera Sedimentibacter, Soehngenia, Sporanaerobac- biochemical and chemotaxonomic traits (Table 2). It is ter, Tepidimicrobium and Tissierella that can be differentiated pertinent to note that the genus Peptoniphilus is not mono- on the basis of their rod-shaped cell morphology and other phyletic, with Anaerosphaera located within this group; the features shown in Table 3. genus Anaerosphaera was originally circumscribed on the basis of the presence of lysine rather than ornithine as the Description of Peptoniphilus stercorisuis sp. nov. diagnostic diamino acid of the cell-wall peptidoglycan and the presence of thermotolerant cells. In addition, the type Peptoniphilus stercorisuis (ster.co.ri.su9is. L. masc. n. stercus, and only species of this genus, Anaerosphaera aminiphila, -oris faeces, manure; L. gen. n. suis of a pig; N.L. gen. n. was phylogenetically located on the periphery of the stercorisuis from pig faeces/manure, referring to the Peptoniphilus cluster of organisms, but with the subsequent isolation of the organism from pig faeces/manure). description of a number of species of the genus Pepto- Cells are anaerobic, Gram-stain-positive cocci occurring niphilus, Anaerosphaera aminiphila is now phylogenetically singly or in pairs. Growth is strictly anaerobic. Cells are located within the centre of this cluster of organisms. non-motile, non-spore-forming and non-haemolytic.

Table 2. Phenotypic characteristics useful in the differentiation of strain SF-S1T from other members of the genus Peptoniphilus

Strains: 1, SF-S1T;2,Peptoniphilus asaccharolyticus CCUG 9988T;3,Peptoniphilus coxii CCUG 59622T;4,Peptoniphilus duerdenii WAL 18896T;5, Peptoniphilus gorbachii CCUG 53341T 6, Peptoniphilus harei CCUG 38491T;7,Peptoniphilus koenoeneniae WAL 18898T;8,Peptoniphilus indolicus CCUG 17639T;9,Peptoniphilus ivorii CCUG 38492T; 10, Peptoniphilus lacrimalis CCUG 31350T; 11, Peptoniphilus methioninivorax NRRL B-23883T 12, Peptoniphilus olsenii CCUG 53342T 13, CCUG 59621T. Data from Ulger-Toprak et al. (2012); Rooney et al. (2011); Citron et al. (2012). +, Positive; 2, negative; W, weak; ND, not determined.

Characteristic 1234 5 6 78910111213

Alanine arylamidase + 222 2 2 222+ W + 2 Alkaline phosphate 2222 2 W 2 + 22 ++2 Arginine dihydrolase ++2 W 22+ 22 2 + 2 _ Catalase 2222 2+ 2 + 22 ND 2 + Glutamyl glutamic acid arylamidase 2 W 22+ 2222+ 222 Glycine arylamidase + 222+ 2 W 22 + W + 2 Indole 2222+ 22+ 22 2 ++ Leucine arylamidase ++2 ++2 + 22 ++2 + Phenylalanine arylamidase ++22 W 2 + 22 + W + 2 Proline arylamindase 2 W + 222W 2 + 2 + 22 Pyroglutamic acid arylamidase 2 + 2 + 22+ 22 2 2 2 2 Serine arylamidase ++22+ 2 + 22 + W + 2 Tyrosine arylamidase ++22+ 2 + 22 ++++

3542 International Journal of Systematic and Evolutionary Microbiology 64 http://ijs.sgmjournals.org

Table 3. Morphological, biochemical and chemotaxonomic properties of Peptoniphilaceae fam. nov. (genera 1–8)and close relatives (genera 9–13)

Genera: 1, Peptoniphilus;2,Anaerococcus;3,Anaerosphaera;4,Finegoldia;5,Gallicola;6,Helcococcus;7,Murdochiella;8,Parvimonas;9,Sedimentibacter; 10, Soehngenia; 11, Sporanaerobacter; 12, Tepidimicrobium; 13, Tissierella. Data from De Vos et al. (2009); Ezaki et al. (2001); Rooney et al. (2011) and Ulger-Toprak et al. (2012). +, Positive; 2, negative; ND, no data; A, Acetate; B, Butyrate; L, Lactate; IV, Isovaleric; meso-Dpm, meso-diaminopimelic acid.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13

Gram stain + + + +++++ +/2 ++++/2 Cell Cocci Cocci Cocci Cocci Cocci Cocci Cocci Cocci Rods Rods Rods Rods Rods morphology Spore 222222222++++2 formation Metabolism Obligate Obligate Obligate Obligate Obligate Facultative Obligate Obligate Obligate Anaerobe/ Obligate Obligate Obligate anaerobe anaerobe anaerobe anaerobe anaerobe anaerobe anaerobe anaerobe anaerobe aerotolerant anaerobe anaerobe anaerobe End products B, A B A, B A A, B A, L L A A, B F, H2,CO2, A, H2,CO2 A, E, B, A, B, IV from PYG A, E H2,CO2 Major fatty C18 : 1 ,C16 : 0 , C18 : 1 ,C16 : 1 , C17 : 1 v8, C18 : 1 , C18 : 1 , ND C14 : 0, C18 : 0 /C18 : 2, C14 : 0 , ND ND ND iso-C15 : 0 , acids C18 : 0 ,C16 : 1 C18 : 0 , C18 : 1 v7 C16 : 1 , C16 : 1 , C16 : 0, C18 : 1, C16 : 1 v9 C16 : 1 , C16 : 0 DMA, C16 : 0 C18 : 0 , C18 : 0 , C18 : 1 C16 : 0, C16 : 0 , C18 : 0 ,C16 : 0 C16 : 0 C16 : 0 C16 : 1 Diamino acid Orn, D-Glu Lys, D-Glu Lys Lys, D-Asp D-Asp D-Asp ND Lys L-Lys ND Lys ND meso-DAP/ in Cell-wall D-Orn murein DNA G+C 30–34 30–35 32.5 32–34 27–34 ND ND 27–28 34–35.5 43 32.2 33–36.2 28–32 content (mol%) Source Human intestine Human Methanogenic Vagina, Chicken Skin, human Human Oral cavity Methanogenic Anaerobic Upflow Freshwater Human and clinical intestine, cattle manure oral faeces clinical clinical and freshwater digester anaerobic hot spring, clinical material, clinical reactor cavity and material, material human pond sludge sludge anaerobic material, vaginal material, other sheep abscesses sediment, blanket digester human discharges, vaginal bodily freshwater thermophilic blood and ovarian, discharges, abscesses river sludge faeces,

peritoneal and ovarian, sediment, sugar- stercorisuis Peptoniphilus other bodily peritoneal human blood refinery abscesses, cattle and other wastewater mastitis and bodily swine manure abscesses, skin, and nasal passage p nov. sp. 3543 C. N. Johnson and others

Growth occurs at 20 uC and 37 uC (optimum 30 uC), at References pH 6.0 and 9.0 (optimum pH 7.75), and with 0–5 % (w/v) NaCl. Growth is not observed at 15 uCor43uCorat Citron, D. M. D., Tyrrell, K. L. K. & Goldstein, E. J. C. E. (2012). Peptoniphilus coxii sp. nov. and Peptoniphilus tyrrelliae sp. nov. pH 5.5 or 9.5. Colonies are small (1–2 mm diameter), grey, isolated from human clinical infections. Anaerobe 18, 244–248. circular, and convex on Brucella blood agar plates after De Vos, P., Garrity, G. M., Jones, D., Krieg, N. R., Ludwig, W., Rainey, u 5 days incubation at 30 C, while cells in liquid broths F. A., Schleifer, K.-H. & Whitman, W. B. (editors) (2009). Family XI. (PYG) aggregate into large floccules, visible by eye. Cells Incertae Sedis.InBergey’s Manual of Systematic Bacteriology 2nd edn, are both catalase- and urease-negative. Nitrate is not pp. 1130–1149. Edited by The Editorial Board as the authors. New reduced and indole is not produced. Carbohydrate is not York, NY: Springer. utilized. Using the API Rapid 32A kit, positive reactions are Ezaki, T., Kawamura, Y., Li, N., Li, Z. Y., Zhao, L. & Shu, S. (2001). observed for arginine dihydrolase, arginine arylamidase, Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. phenylalanine arylamidase, leucine arylamidase, tyrosine and Gallicola gen. nov. for members of the genus Peptostreptococcus. arylamidase, alanine arylamidase, glycine arylamidase, Int J Syst Evol Microbiol 51, 1521–1528. histidine arylamidase and serine arylamidase, and a weakly Fitch, W. M. (1971). Toward defining the course of evolution: positive reaction is observed for leucyl glycine arylamidase. minimum change for a specific tree topology. Syst Zool 20, 406–416. Negative reactions are observed for a-galactosidase, b- Jousimies-Somer, H., Summanen, P., Citron, D. M., Baron, E. J., galactosidase, b-galactosidase 6-phosphate, a-glucosidase, Wexler, H. M. & Finegold, S. M. (2002). Wadsworth-KTL Anaerobic Bacteriology Manual, 6th edn. Belmont, CA: Star Publishing. b-glucosidase, a-arabinosidase, b-glucuronidase, N-acetyl-b- glucosaminidase, raffinose and mannose fermentation, glu- Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Laboratory Manual, 4th edn. Blacksburg, Virginia: Virginia Polytechnic tamic acid decarboxylase, a-fucosidase, alkaline phosphatase, Institute and State University.. proline arylamidase, pyroglutamic acid arylamidase and Ka¨ mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of glutamyl glutamic acid arylamidase. Using the API ZYM test fatty acid patterns of coryneform and related taxa. Can J system, positive reactions are obtained for leucine arylami- Microbiol 42, 989–1005. dase, cystine arylamidase and phosphohydrolase; weak Kim, O.-S., Cho, Y.-J., Lee, K., Yoon, S.-H., Kim, M., Na, H., Park, S.-C., reactions are obtained for a-galactosidase and a-mannosidase. Jeon, Y. S., Lee, J.-H. & other authors (2012). Introducing EzTaxon-e: Major fatty acids include C14 : 0,C16 : 0,C16 : 1v7c and a prokaryotic 16S rRNA gene sequence database with phylotypes that C16 : 0ALDE. Sensitive to penicillin, chloramphenicol and represent uncultured species. Int J Syst Evol Microbiol 62, 716–721. bacitracin, but resistant to trimethoprim and ciprofloxacin. Kimura, M. (1980). A simple method for estimating evolutionary rates T T of base substitutions through comparative studies of nucleotide The type strain is SF-S1 (5DSM 27563 5NBRC sequences. J Mol Evol 16, 111–120. 109839T) isolated from a swine manure storage tank + Madsen, M., Høi Sørensen, G. & Nielsen, S. A. (1991). Studies on the possi- located in the state of Oklahoma, USA. The DNA G C bleroleofcattlenuisance flies, especially Hydrotaea irritans,inthetrans- content of the type strain is 31.8 mol% (HPLC). mission of summer mastitis in Denmark. Med Vet Entomol 5, 421–429. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise + Description of Peptoniphilaceae fam. nov. measurement of the G C content of deoxyribonucleic acid by high- performance liquid chromatography. Int J Syst Bacteriol 39, 159–167. Peptoniphilaceae (Pep.to.ni.phi.la.ce9ae. N.L. masc. n. Miller, D. N. (2001). Accumulation and consumption of odorous Peptoniphilus type genus of the family; L. suff. –aceae compounds in feedlot soils under aerobic, fermentative, and ending to denote a family; N.L. fem. pl. n. Peptoniphilaceae anaerobic respiratory conditions. J Anim Sci 79, 2503–2512. the family of the genus Peptoniphilus). Myers, E. W. & Miller, W. (1988). Optimal alignments in linear space. Comput Appl Biosci 4, 11–17. Falls within the order Clostridiales and the delineation of Rooney, A. P., Swezey, J. L., Pukall, R., Schumann, P. & Spring, S. the family is primarily on basis of 16S rRNA gene sequence (2011). Peptoniphilus methioninivorax sp. nov., a Gram-positive phylogeny and is supported by morphological, biochemical anaerobic coccus isolated from retail ground beef. Int J Syst Evol and chemotaxonomic characteristics. The family includes Microbiol 61, 1962–1967. the genera Anaerococcus, Anaerosphaera, Finegoldia, Gallicola, Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new Helcococcus, Murdochiella, Parvimonas and Peptoniphilus. method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. Cells are Gram-stain-positive, strictly anaerobic cocci and Sasser, M. (1990). Identification of bacteria by gas chromatography of are negative for both motility and spore formation. cellular fatty acids, MIDI Technical Note 101. Newark, DE: MIDI, Inc. Carbohydrates are not normally utilized. Peptone and Song, Y., Liu, C. & Finegold, S. M. (2007). Peptoniphilus gorbachii sp. amino acids are metabolized, yielding butyrate, acetate nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. and lactate as major end products. Predominant fatty nov. isolated from clinical specimens of human origin. J Clin Microbiol 45, 1746–1752. acids include C16 : 0,C16 : 1,C18 : 0 and C18 : 1. The cell wall may contain the diamino acids alanine, aspartate, lysine, Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place for ornithine or glutamic acid. Isolated from animal and bird DNA-DNA reassociation and 16S rRNA sequence analysis in the present faeces or human clinical samples. The DNA G+C content species definition in bacteriology. Int J Syst Bacteriol 44,846–849. is between 27–35 mol%. Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol The type genus is Peptoniphilus Ezaki et al. 2001, 1524VP. Evol 24, 1596–1599.

3544 International Journal of Systematic and Evolutionary Microbiology 64 Peptoniphilus stercorisuis sp. nov.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: 330–393. Edited by C. A. Reddy, T. J. Beveridge, J. A. Breznak, improving the sensitivity of progressive multiple sequence alignment G. A. Marzluf, T. M. Schmidt & R. L. Snyder. Washington, D. C.: through sequence weighting, position-specific gap penalties and American Society for Microbiology. weight matrix choice. Nucleic Acids Res 22, 4673–4680. Ulger-Toprak, N., Lawson, P. A., Summanen, P., O’Neal, L. & Tindall, B. J., Sikorski, J., Smibert, R. A. & Krieg, N. R. (2007). Finegold, S. M. (2012). Peptoniphilus duerdenii sp. nov. and Phenotypic characterization and the principles of comparative Peptoniphilus koenoeneniae sp. nov., isolated from human clinical systematics. In Methods for General and Molecular Microbiology, pp. specimens. Int J Syst Evol Microbiol 62, 2336–2341.

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