Enterococcus pseudoavium

Abstract: Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164–2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818–823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116–3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181–1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and Streptococcus iniae, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17 Enterococcus species (Enterococcus asini, Enterococcus rattus, Enterococcus dispar, Enterococcus gallinarum, Enterococcus hirae, Enterococcus durans, Enterococcus cecorum, Enterococcus faecalis, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus avium, Enterococcus pseudoavium, Enterococcus new sp. strain Facklam, and Enterococcus saccharolyticus), and Vagococcus fluvialis, Lactococcus lactis, and Lactococcus garvieae. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731–734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were Enterococcus new sp. strain Facklam (ATCC 700913), 3; E. asini, 1; E. rattus, 4; E. dispar, 2; E. gallinarum, 20; E. hirae, 9; E. durans, 9; E. faecalis, 12; E. mundtii, 3; E. casseliflavus, 8; E. faecium, 25; E. malodoratus, 3; E. raffinosus, 8; E. avium, 4; E. pseudoavium, 1; an unknown Enterococcus clinical isolate, sp. strain R871; Vagococcus fluvialis, 4; Lactococcus garvieae, 3; Lactococcus lactis, 3; Leuconostoc sp., 1; and Pediococcus sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of Enterococcus and related organisms.

Abstract: Accurate species identification of enterococci has become important with the wide prevalence of acquired vancomycin resistance and the presence of less epidemiologically important, inherently vancomycin-resistant enterococci. Using a collection of enterococcal strains, we found that PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes can produce amplicon profiles characteristic of the enterococcus examined. The species examined were group I enterococci (Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus, and Enterococcus pseudoavium), group II enterococci (Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus gallinarum), and group III enterococci (Enterococcus durans and Enterococcus hirae). The enterococcal species in group I, as well as E. faecalis and two strains of E. hirae, were similar and therefore had to be differentiated from each other by Sau3A restriction digests. This produced patterns characteristic of each of these species. The remaining group II and group III enterococcal species produced amplicons characteristic of a particular species except E. gallinarum. The PCR products from E. gallinarum displayed strain-to-strain heterogeneity in the number and size of amplicons. To further test the utility of this technique, 11 phenotypically aberrant strains which had been assigned species identification based on Facklam and Collins-type strain reactions (R.R. Facklam and M.D. Collins, J. Clin. Microbiol. 27:731-734, 1989) were subjected to ITS-PCR. ITS-PCR of the phenotypically aberrant strains identified six strains with reactions consistent with those of type strains. However, five strains were characterized as follows: two strains originally identified as E. mundtii were identified by ITS-PCR as E. casseliflavus, one strain originally identified as E. raffinosus was identified by ITS-PCR as E. durans, one strain originally identified as E. hirae was identified by ITS-PCR as E. faecium [corrected]. We conclude that amplification of the intergenic 23S and 16S rRNA gene regions of enterococci provides a reliable technique for species identification of enterococci.

Abstract: Deoxyribonucleic acid base composition, deoxyribonucleic acid-deoxyribonucleic acid hybridization, and biochemical studies were performed on some enterococci from clinical sources of uncertain taxonomic position. Our results indicate that 6 human strains, a single clinical isolate and a strain from bovine mastitis are genetically distinct from each other and all other previously described Enterococcus species and constitute three new species, for which the names Enterococcus raffinosus, Enterococcus solitarius and Enterococcus pseudoavium are proposed.

Abstract: The elongation factor Tu, encoded by tuf genes, is a GTP binding protein that plays a central role in protein synthesis. One to three tuf genes per genome are present, depending on the bacterial species. Most low-G+C-content gram-positive bacteria carry only one tuf gene. We have designed degenerate PCR primers derived from consensus sequences of the tuf gene to amplify partial tuf sequences from 17 enterococcal species and other phylogenetically related species. The amplified DNA fragments were sequenced either by direct sequencing or by sequencing cloned inserts containing putative amplicons. Two different tuf genes (tufA and tufB) were found in 11 enterococcal species, including Enterococcus avium, Enterococcus casseliflavus, Enterococcus dispar, Enterococcus durans, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, and Enterococcus raffinosus. For the other six enterococcal species (Enterococcus cecorum, Enterococcus columbae, Enterococcus faecalis, Enterococcus sulfureus, Enterococcus saccharolyticus, and Enterococcus solitarius), only the tufA gene was present. Based on 16S rRNA gene sequence analysis, the 11 species having two tuf genes all have a common ancestor, while the six species having only one copy diverged from the enterococcal lineage before that common ancestor. The presence of one or two copies of the tuf gene in enterococci was confirmed by Southern hybridization. Phylogenetic analysis of tuf sequences demonstrated that the enterococcal tufA gene branches with the Bacillus, Listeria, and Staphylococcus genera, while the enterococcal tufB gene clusters with the genera Streptococcus and Lactococcus. Primary structure analysis showed that four amino acid residues encoded within the sequenced regions are conserved and unique to the enterococcal tufB genes and the tuf genes of streptococci and Lactococcus lactis. The data suggest that an ancestral streptococcus or a streptococcus-related species may have horizontally transferred a tuf gene to the common ancestor of the 11 enterococcal species which now carry two tuf genes.