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

Abstract

Strain NRRL B-23883 T 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 hyena. Strain NRRL B-23883 T shared the peptidoglycan type A4b, L-Orn-D-Glu with members of the genus Peptoniphilus. Further phenotypic analysis revealed that strain NRRL B-23883 T 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-23883 T (5DSM 22461 T ). (Murdoch, 1998;Ezaki et al., 2001;Song et al., 2007). In this study, an obligately anaerobic, Gram-positive coccal strain isolated from retail ground beef was characterized and a novel species, Peptoniphilus methioninivorax sp. nov., is proposed. The genusStrain NRRL B-23883 T was isolated at the National Center for Agricultural Utilization Research (US Department of Agriculture) from retail ground beef as part of a study on the genetic variability of Clostridium perfringens (Rooney et al., 2006). A sample of ground beef (25 g) was homogenized in 0.5 % (w/v) saline solution and 1 ml was subsequently added to 9 ml iron milk medium (1 l whole milk, 1 g FeSO 4 . 7H 2 O, 50 ml double-distilled H 2 O) and allowed to incubate anaerobically overnight at 42 u C in an atmosphere of 5 % H 2 , 5 % CO 2 and 90 % N 2 . A 1 ml sample was used to create a dilution series to 10 26 in sterile 0.5 % (w/v) saline followed by plating onto tryptosesulfite-cycloserine (TSC) agar plates (Sigma-Aldrich) containing egg yolk emulsion (50 %); plates were subsequently incubated anaerobically overnight at 37 u C in an atmosphere of 5 % H 2 , 5 % CO 2 and 90 % N 2 . TSC-egg yolk emulsion plating is used to presumptively identify C. perfringens isolates, which form black colonies on this medium; strains that do not turn black must be subject to additional testing to determine their taxonomic identification. When it was found that NRRL B-23883 T did not form Abbreviations: MP, maximum-parsimony; NJ, neighbour-joining.

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