International Journal of Systematic and Evolutionary (2011), 61, 1962–1967 DOI 10.1099/ijs.0.024232-0

Peptoniphilus methioninivorax sp. nov., a Gram- positive anaerobic isolated from retail ground beef

Alejandro P. Rooney,1 James L. Swezey,1 Ru¨diger Pukall,2 Peter Schumann2 and Stefan Spring2

Correspondence 1Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Alejandro P. Rooney Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, IL [email protected] 61604, USA 2DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany

Strain NRRL B-23883T was isolated from retail ground beef as part of a study on the genetic diversity of Clostridium perfringens. The strain was found to be a strictly anaerobic, Gram-positive coccus that was able to utilize peptone as a sole carbon source. Analysis of the 16S rRNA gene sequence revealed that the strain was closely related to species within the genera and Anaerosphaera, but it was substantially different from the closest recognized species by nearly 10 % sequence divergence. The strain was also found to be closely related (.99 % sequence similarity) to an uncultured bacterial strain that was sequenced from a 16S rRNA gene clone library constructed to characterize the bacterial community of faeces from a captive spotted T hyena. Strain NRRL B-23883 shared the peptidoglycan type A4b, L-Orn–D-Glu with members of the genus Peptoniphilus. Further phenotypic analysis revealed that strain NRRL B-23883T was able to utilize glycyl L-methionine as a sole carbon source, in contrast to other species of the genus Peptoniphilus. Therefore, it is proposed that the isolate represents a novel species, Peptoniphilus methioninivorax sp. nov.; the type strain is NRRL B-23883T (5DSM 22461T).

The genus Peptoniphilus (Ezaki et al., 2001) encompasses strain isolated from retail ground beef was characterized species of Gram-positive anaerobic cocci (Murdoch, 1998) and a novel species, Peptoniphilus methioninivorax that can utilize peptone as a sole carbon source. At the time sp. nov., is proposed. of writing, the genus included seven species: Peptoniphilus Strain NRRL B-23883T was isolated at the National Center asaccharolyticus (type species; Ezaki et al., 2001), for Agricultural Utilization Research (US Department of (Ezaki et al., 2001), Peptoniphilus Agriculture) from retail ground beef as part of a study on indolicus (Ezaki et al., 2001), (Ezaki the genetic variability of Clostridium perfringens (Rooney et al., 2001), Peptoniphilus lacrimalis (Ezaki et al., 2001), et al., 2006). A sample of ground beef (25 g) was Peptoniphilus olsenii (Song et al., 2010) and Peptoniphilus homogenized in 0.5 % (w/v) saline solution and 1 ml was gorbachii (Song et al., 2010). Members of this genus are subsequently added to 9 ml iron milk medium (1 l whole found within Family XI Incertae Sedis (order Clostridiales, milk, 1 g FeSO4 .7H2O, 50 ml double-distilled H2O) and class , phylum ‘’) (Vos et al., 2009) and allowed to incubate anaerobically overnight at 42 uCinan what was referred to by Collins et al. (1994) as ‘Clostridium atmosphere of 5 % H2,5%CO2 and 90 % N2.A1ml Cluster XIII’. Species of the genus Peptoniphilus have been sample was used to create a dilution series to 1026 in sterile found in the gastrointestinal and vaginal tracts of humans, 0.5 % (w/v) saline followed by plating onto tryptose- as well as being associated with human clinical and sulfite-cycloserine (TSC) agar plates (Sigma-Aldrich) con- veterinary wounds, abscesses or other infections taining egg yolk emulsion (50 %); plates were subsequently (Murdoch, 1998; Ezaki et al., 2001; Song et al.,2007).In incubated anaerobically overnight at 37 uC in an atmo- this study, an obligately anaerobic, Gram-positive coccal sphere of 5 % H2,5%CO2 and 90 % N2. TSC-egg yolk emulsion plating is used to presumptively identify C. Abbreviations: MP, maximum-parsimony; NJ, neighbour-joining. perfringens isolates, which form black colonies on this The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene medium; strains that do not turn black must be subject to sequence of strain NRRL B-23883T is GU440754. additional testing to determine their taxonomic identifica- T A supplementary figure is available with the online version of this paper. tion. When it was found that NRRL B-23883 did not form

1962 024232 Printed in Great Britain Peptoniphilus methioninivorax sp. nov. black colonies on TSC-egg yolk emulsion plates, the were conducted using the ABI BigDye version 3.0 sequen- molecular methods described below were used to further cing kit (Applied Biosystems) following the manufacturer’s investigate the identity of the strain. suggested protocol but at one-quarter of the recom- mended volume. The reaction products were purified via Strain NRRL B-23883T was grown in 5 ml liver infusion ethanol precipitation and run on an ABI 3730 genetic broth (Becton Dickinson) and incubated anaerobically at analyser (Applied Biosystems). The resulting DNA 37 uC for 24 h in an atmosphere of 5 % H ,5%CO and 2 2 sequence was edited visually using Sequencher version 90 % N . DNA was extracted using the MasterPure Gram- 2 4.1.2 (Gene Codes). positive DNA purification kit (Epicentre Biotechnologies) following the manufacturer’s protocol. PCR amplifications Results of BLAST searches of the GenBank database with the of the 16S rRNA gene were performed with 1 unit AmpliTaq 16S rRNA gene sequence of NRRL B-23883T indicated that DNA polymerase (Invitrogen Life Technologies), 2.5 mM the sequence shared a relatively high level of similarity MgCl2, 200 mM dNTPs, 16 reaction buffer, 50 ng template (approx. 90 %) with sequences of species of the genus DNA and 1.0 mM each of primers 27f (Lane, 1991) and Peptoniphilus. Thus, the 16S rRNA gene sequences for the 1387r (Marchesi et al., 1998). All PCRs were performed for type strains of these species were downloaded, as well as the 35 cycles, each consisting of a 30 s denaturation step at sequences for the type strains of species from related genera, 94 uC, a 30 s annealing step at 54 uC and a 1 min extension in order to determine the phylogenetic placement of strain step at 72 uC. The amplification product was purified NRRL B-23883T. Sequences were aligned using the com- using Montage PCR Cleanup Filter Plates (Millipore). puter program CLUSTAL X (Thompson et al., 1997) and the Sequencing reactions for both forward and reverse strands resulting alignment was edited manually for errors.

Fig. 1. NJ phylogeny showing the phylogenetic placement of P. methioninivorax sp. nov. GenBank accession numbers are given in parentheses. Bar, branch length as a percentage of total distance. http://ijs.sgmjournals.org 1963 A. P. Rooney and others

Table 1. Growth characteristics, Biolog anaerobe identification test data and total cellular fatty acid contents of strain NRRL B-23883T and type strains of species of the genus Peptoniphilus

Strains: 1, P. methioninivorax sp. nov. NRRL B-23883T;2,P. asaccharolyticus DSM 20463T (5NRRL B-51293T); 3, P. ivorii DSM 10022T (5NRRL B-51295T); 4, P. indolicus DSM 20464T (5NRRL B-51300T); 5, P. harei DSM 10020T (5NRRL B-51297T); 6, P. lacrimalis DSM 7455T (5NRRL T T T T T B-51294 ); 7, P. olsenii DSM 21460 (5NRRL B-51299 ); 8, P. gorbachii DSM 21461 (5NRRL B-51298 ). +, Strong growth/positive; W, weak growth/weak reaction; 2, no growth/negative. All strains were able to grow at 37 uC and utilize pyruvic acid.

Characteristic 1 2 3 4 5 6 7 8

Growth in/at:* 3 % NaCl +++ 22222 5 % NaCl + 2 + 22222 15 uC + 22 2 2 2 22 25 uC +++ + + 2 ++ 37 uC +++ + + + ++ 42 uC ++2222+ 2 Biolog anaerobe identification testsD Alaninamide 22+ 2222+ L-Alanine 22+ 22+ 22 L-Alanyl L-glutamine +++ + + + 2 + L-Alanyl L-histidine +++ + + 22+ L-Alanyl L-threonine + 2 + 2 + 222 L-Asparagine + 2 ++2 + 22 29-Deoxyadenosine +++ + + 22+ Dextrin W + 2 + 2222 L-Glutamic acid +++ 22222 L-Glutamine +++ 2 + 22+ Glycyl L-aspartic acid 222 2 2 2 2W Glycyl L-proline 222 + 2222 Glycyl L-methionine + 22 2 2 2 22 Glyoxylic acid W 2 ++2222 a-Hydroxybutyric acid + 22 2 + 22+ b-Hydroxybutyric acid + 2 +++222 Inosine ++2 ++222 a-Ketobutyric acid + 2 ++22+ 2 a-Ketovaleric acid +++ + W 2 + 2 DL-Lactic acid + 22 +++2 + L-Lactic acid + 22 + 2222 D-Lactic acid methyl ester + 22 + 22++ L-Methionine + 2 + 22++2 L-Phenylalanine 22+ 22222 Pyruvic acid methyl ester + 2 ++2222 L-Serine + 2 + 2 + 222 m-Tartaric acid 222 + 2222 L-Threonine + 2 + 2 + 2 + 2 Thymidine ++2 +++22 TMP ++2 + 2222 Uridine ++2 + 2 + 2 + UMP ++2 + 2222 Urocanic Acid ++2 ++222 L-Valine 22+ 22222 L-Valine plus L-aspartic acid + 2 + 2 + 222 Fatty acid content (%)D

C12 : 0 1.2 3.2 0.5 1.2 0.5 0.8 3.1 0.6 C14 : 0 10.9 20.2 4.4 12.6 4.4 2.9 16.4 5.6 C16 : 1v9c 3.4 4.9 2 3.6 222.5 2 C16 : 0 25.1 24.9 29.5 19.4 32.1 27.7 22.1 34.8 anteiso-C17 : 0 1.9 1.3 3.8 2.6 4.2 1.8 3.9 3.2 C18 : 1v7c 5.9 4.0 2.6 3.5 1.9 3.5 4.5 2.6

1964 International Journal of Systematic and Evolutionary Microbiology 61 Peptoniphilus methioninivorax sp. nov.

Table 1. cont.

Characteristic 1 2 3 4 5 6 7 8

C18 : 1v9c 5.1 8.6 11.4 6.2 17.0 25.7 5.2 13.4 C18 : 0 3.0 3.5 4.8 2.5 7.2 11.2 3.5 6.3 iso-C17 : 0 3-OH 9.2 2 7.7 4.8 6.0 3.0 5.4 4.6 C16 : 1v7c 14.1 6.5 1.0 7.0 1.0 3.2 7.5 1.2 iso-C17 : 1 and/or ai-C17 : 1 4.5 2.3 5.3 10.3 3.8 1.8 7.7 4.4 C18 : 2v6,9c 8.5 17.5 24.0 13.0 17.0 13.6 7.1 19.9

*Growth experiments for salt tolerance and temperature range were conducted in liver infusion liquid media at 37 uC for 48 h using NRRL type strains. DDSM type strains were used for Biolog and fatty acid analyses.

Phylogenetic analyses were conducted using both the suspended to a specific density (63 % transmission) in the neighbour-joining (NJ) method (Saitou & Nei, 1987) and AN inoculating fluid. Samples (100 ml) of the bacterial maximum-parsimony (MP) methods as implemented in the solution were dispensed into each well of the MicroPlate computer program MEGA4 (Tamura et al., 2007). The former and subsequently incubated in an atmosphere of 5 % H2, used maximum composite likelihood distances (Tamura 5% CO2 and 90 % N2 at 37 uC for 48 h. The group et al., 2007) with the pairwise deletion option. The latter was containing P. asaccharolyticus, P. ivorii and strain NRRL T conducted using the closest neighbour interchange heuristic B-23883 was capable of utilizing L-glutamic acid as a sole search algorithm with a search factor of 3 and 50 random carbon source, whereas the group containing the remaining addition sequence replicates. The statistical reliability of currently recognized species of the genus Peptoniphilus internal branches for both NJ and MP analyses was assessed (P. gorbachii, P. harei, P. indolicus, P. lacrimalis and from 1500 bootstrap pseudoreplicates. P. olsenii) was not. Data for utilization of different carbon sources are summarized in Table 1. One question Both NJ (Fig. 1) and MP (Supplementary Figure S1, T available in IJSEM Online) analyses indicate that the genus concerning strain NRRL B-23883 was whether it pos- Peptoniphilus is not monophyletic and, therefore, in need sessed any uniquely defining phenotypic traits that set it of future revision. The 16S rRNA gene sequence of strain apart from other species of the genus Peptoniphilus.Itwas T found using the Omnilog system (Biolog) that NRRL NRRL B-23883 was most similar (P-distance50.004) to T the sequence obtained from a clone isolated from a 16S B-23883 was able to metabolize glycyl-L-methionine, rRNA gene library made to characterize the bacterial whereas the type strains of P. asaccharolyticus, P. gorbachii, community present in the faeces of a captive spotted hyena P. harei, P. indolicus, P. ivorii, P. lacrimalis and P. olsenii (Crocuta crocuta). The results of phylogenetic analyses could not. This result was confirmed through the presented in Fig. 1 showed that Anaerosphaera aminiphila observation of growth at 37 uC in mineral salts medium WN036T (Ueki et al., 2009) was the most closely related supplemented with 2 % (w/v) glycyl-L-methionine member of a formally described species and indeed this (1.0 g KH2PO4, 0.2 g MgSO4 .7H2O, 0.1 g KCl, 20.0 g strain showed a P-distance value of 0.094 in comparison to glycyl-L-methionine, 1 l double-distilled H2O; pH 6.5); T strain NRRL B-23883T. In contrast, members of the genus only strain NRRL B-23883 was capable of growth in this T Peptoniphilus showed more distant levels of 16S rRNA gene liquid medium (Table 1). In addition, NRRL B-23883 was sequence similarity when compared to NRRL B-23883T able to grow at 15 uC in liver infusion broth, whereas the (P-distance range of 0.096–0.133), although the P-distance aforementioned Peptoniphilus type strains were not of P. asaccharolyticus was nearly equal to that of observed to do so (Table 1). A. aminiphila (P50.096 versus P50.094, respectively). Biomass for peptidoglycan analysis was obtained by cultiva- Therefore, in order to further investigate whether strain T tion in pre-reduced peptone-yeast extract-glucose (PYG) NRRL B-23883 should be classified within the genus medium (Holdeman et al., 1977) under strictly anaerobic Peptoniphilus, phenotypic and biochemical analyses were conditions in an atmosphere of 5 % H ,5%CO and 90 % performed as described below. 2 2 N2, incubated at 37 uC. The peptidoglycan was isolated after Enzyme activities of strain NRRL B-23883T were examined disruption of the cells by shaking with glass beads and using the API ZYM system (bioMe´rieux). Positive reac- subsequent trypsin digestion, according to the method of tions were obtained for leucine arylamidase and acid Schleifer & Seidl (1985). The amino acids and peptides in the phosphatase; weak reactions were obtained for esterase, cell wall hydrolysates were analysed by two-dimensional esterase lipase and cystine arylamidase. Utilization of ascending TLC on cellulose plates by using previously different carbon compounds was tested by using AN described solvent systems (Schleifer & Seidl, 1985). The MicroPlates (Biolog). Cells were swabbed from the surface identity of amino acids was confirmed and their molar ratio of Columbia blood agar (Becton Dickinson) plates and was estimated by GC (14A; Shimadzu) and GC-MS (320-MS http://ijs.sgmjournals.org 1965 A. P. Rooney and others

Quadrupole; Varian) of N-heptafluorobutyryl amino acid ravenous, voracious, consuming; N.L. masc. adj. methioni- isobutyl esters (MacKenzie, 1987; Groth et al., 1996). nivorax glycyl L-methionine-consuming). Peptidoglycan hydrolysates (4 M HCl, 100 uC, 16 h) Cells are coccus-shaped, 0.6–1.0 mm in diameter. Gram- contained ornithine, alanine and glutamic acid in an type positive. Growth is strictly anaerobic. Growth approximate molar ratio of 1.0 : 2.3 : 3.0. The peptides temperature range is between 15 and 42 uC, with optimal L-Ala–D-Glu and L-Orn–D-Ala were detected in the partial growth at 37 uC. Colonies are small (1–2 mm diameter), hydrolysate of the peptidoglycan (4 M HCl, 100 uC, smooth, translucent and circular on liver infusion agar 0.75 h). Dinitrophenylation according to Schleifer & Seidl (1985) revealed that glutamic acid represented the N plates after 48 h of growth. Growth is observed in liver terminus of the inter-peptide bridge. Taking into account infusion liquid media supplemented with 5 % NaCl after that the high ratio of glutamic acid to ornithine might be 48 h. Growth on mineral salts liquid medium supplemented due to residual protein contamination, the analytical data with glycyl L-methionine (2 %, w/v) as the sole carbon suggest that strain NRRL B-23883T shares the peptidogly- source is observed as a biofilm (surface pellicle) after 48 h. Sugar is not utilized. The fermentation products produced can type A4b, L-Orn–D-Glu with other members of the genus Peptoniphilus (Ezaki et al., 2001). This result sets it include acetate (major product), butyrate and propionate apart from the genus Anaerosphaera as currently described, (trace amounts). In addition to the properties indicated in which possesses lysine as the diagnostic diamino acid in the Table 1, cannot utilize any other compounds provided in the cell-wall peptidoglycan (Ueki et al., 2009). anaerobe identification test panel (http://www.biolog.com/ pdf/AnbBrochure.pdf). Positive reactions are obtained for Short-chain volatile fatty acids were examined following leucine arylamidase and acid phosphatase using the API the GC-based method of Holdeman et al. (1977) as ZYM system; weak reactions are obtained for esterase, modified by Steer et al. (2001). The cells were cultivated in esterase lipase and cystine arylamidase. The peptidoglycan is pre-reduced PYG medium under strictly anaerobic condi- of type A4b, L-Orn–D-Glu. tions in an atmosphere of 5 % H ,5%CO and 90 % N 2 2 2 The type strain is NRRL B-23883T (5DSM 22461T), and incubated at 37 uC. The metabolic end products isolated from a retail sample of ground beef. The genomic produced by NRRL B-23883T were determined to be DNA G+C content of the type strain is 32 mol% using the acetate, butyrate and propionate. Major amounts of acetic thermal denaturation method (Marmur & Doty, 1962). acid (nearly twice those of butyric acid), moderate amounts of butyric acid and trace amounts of propionic acid were detectable. These results are consistent with Acknowledgements observations reported in other studies regarding volatile short-chain fatty acid metabolic end products generated by We thank E. Moore from the Culture Collection of the University of ¨ members of the genus Peptoniphilus (e.g. Ezaki et al., 2001; Goteborg for providing type strains of Peptoniphilus species and H. Kline for technical assistance. We also thank J. P. Euze´by for help with Song et al., 2007). Latin nomenclature. The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Finally, the cellular fatty acid profiles of strain NRRL T Department of Agriculture over other firms or similar products not B-23883 and other type strains of members of the genus mentioned. Peptoniphilus (cultivated on Columbia agar at 37 uC for 1–4 days in order to achieve an equal growth stage) were analysed by using the Sherlock Microbial Identification References system (MIDI) as described by Ka¨mpfer & Kroppenstedt (1996). Summed feature components (except for the Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez- Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. E. (1994). co-eluting anteiso- and iso-isomers of C17 : 1) were The phylogeny of the genus Clostridium: proposal of five new genera identified by GC-MS (320-MS Quadrupole; Varian). The and eleven new species combinations. Int J Syst Bacteriol 44, results of fatty acid methyl ester analyses are listed in Table 812–826. T 1; strain NRRL B-23883 showed remarkably higher Ezaki, T., Kawamura, Y., Li, N., Li, Z. Y., Zhao, L. & Shu, S. (2001). amounts of C16 : 1v7c and iso-C17 : 0 3-OH than the other Proposal of the genera gen. nov., Peptoniphilus gen. nov. species of the genus Peptoniphilus examined. and Gallicola gen. nov. for members of the genus . Int J Syst Evol Microbiol 51, 1521–1528. Based on the results of the above phylogenetic and Groth, I., Schumann, P., Weiss, N., Martin, K. & Rainey, F. A. (1996). phenotypic analyses, it is proposed that strain NRRL Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes T B-23883 represents a novel species, Peptoniphilus methio- with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46, ninivorax sp. nov. 234–239. Holdeman, L. V., Cato, E. P. & Moore, W. E. C. (1977). Anaerobe Description of Peptoniphilus methioninivorax Laboratory Manual, 4th edn. Blacksburg, VA: Virginia Polytechnic sp. nov. Institute and State University. Ka¨ mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of Peptoniphilus methioninivorax (me.thi.o.ni.ni.vo9rax. N.L. fatty acid patterns of coryneform and related taxa. Can J n. methioninum methionine; L. adj. vorax devouring, Microbiol 42, 989–1005.

1966 International Journal of Systematic and Evolutionary Microbiology 61 Peptoniphilus methioninivorax sp. nov.

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