International Journal of Systematic and Evolutionary Microbiology (2011), 61, 1962–1967 DOI 10.1099/ijs.0.024232-0 Peptoniphilus methioninivorax sp. nov., a Gram- positive anaerobic coccus 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 Peptoniphilus 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 Peptoniphilus harei (Ezaki et al., 2001), Peptoniphilus Agriculture) from retail ground beef as part of a study on indolicus (Ezaki et al., 2001), Peptoniphilus ivorii (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 Clostridia, phylum ‘Firmicutes’) (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.