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1 Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mail Stop C-02, Atlanta, GA 30333, USA
2 Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941, Brazil
Correspondence
Maria da Glória S. Carvalho
MCarvalho{at}cdc.gov
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), several novel species have been described as a result of improvements in the methods used for their identification combined with a growing interest in their role as opportunistic pathogens (Teixeira & Facklam, 2003). The differentiation of some of the species belonging to the genus Enterococcus remains problematic because of the overlap of phenotypic characteristics, and questions might arise as to their precise identification (Devriese et al., 2002; Facklam et al., 2002; Teixeira et al., 1995). In order to accomplish the precise identification of these micro-organisms, we systematically applied analysis of whole-cell-protein profiles and 16S rRNA gene sequencing and DNA–DNA reassociation experiments, in conjunction with conventional physiological tests (Merquior et al., 1994; Teixeira et al., 1995; Carvalho et al., 2004). In the present study, these techniques were used to characterize two enterococcus-like isolates recovered from stool samples obtained from a healthy individual in Portland, OR, USA, on different occasions in July 2000, during routine outpatient surveillance carried out by the National Antimicrobial Resistance Monitoring System Laboratory at the Centers for Disease Control and Prevention (Atlanta, GA, USA), and were referred to the CDC Streptococcus Laboratory for further identification.
The strains were characterized phenotypically using conventional biochemical tests, as previously described (Facklam et al., 2002; Teixeira & Facklam, 2003), and the API Rapid ID 32 Strep system (bioMérieux). The AccuProbe Enterococcus identification test (Gen-Probe) was performed according to the manufacturer's instructions and was positive for both strains. The phenotypic identification scheme initially used subdivides the enterococcal species into five groups, as described previously (Facklam et al., 2002; Teixeira & Facklam, 2003). Using this scheme, both stool strains were placed into group IV, which includes Enterococcus asini, Enterococcus cecorum, Enterococcus phoeniculicola and Enterococcus sulfureus. However, their biochemical profiles did not match those of any enterococcal species with validly published names. The results of physiological tests are presented in the species description below. Table 1 shows the phenotypic tests used to differentiate the stool strains from the phylogenetically closest relatives (Enterococcus moraviensis, Enterococcus haemoperoxidus and Enterococcus faecalis) and the species included in phenotypic group IV.
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Extract preparation and whole-cell-protein profile analysis using one-dimensional SDS-PAGE were performed as described previously (Merquior et al., 1994). Dice indices were determined for each isolate by using the Molecular Analyst Fingerprint Plus software package, version 1.6 (Bio-Rad), and a dendrogram was constructed by the unweighted pair-group method with arithmetic averages. Fig. 1 shows the SDS-PAGE profiles of whole-cell-protein extracts of the stool isolates and their separate position relative to the phylogenetically closest relatives, E. moraviensis, E. haemoperoxidus and E. faecalis.
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. The sequences obtained for stool isolates 2215-02T and 826-03 and reference sequences obtained from GenBank were aligned with CLUSTAL in the program MEGA, version 3.0, before trimming to a 1409 bp consensus. A neighbour-joining tree was created using the Kimura two-parameter model, with bootstrapping based on 1000 replications (Kumar et al., 2004). The resulting phylogram (Fig. 2) was confirmed with the interior branch test and Tajima's test (Kumar et al., 2004).
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). Extraction, purification of the DNA and determination of DNA relatedness by the hydroxyapatite hybridization method were performed as described by Brenner et al. (1982). DNA hybridization experiments were performed at 55 °C for optimal DNA reassociation conditions and at 70 °C for stringent conditions. DNA of type strain 2215-02T was hybridized with the phenotypically similar isolate, 826-03, and with the type strains of the most closely related species of Enterococcus (Table 2). The relative binding ratio at the optimal reassociation temperature for the two stool isolates was greater than 70 % and the divergence was less than 1 %. On the basis of these results, these two strains met the criteria for the definition of species relatedness (Wayne et al., 1987). In addition, when strain 2215-02T was hybridized against the type strains of the species of Enterococcus most closely related phylogenetically, the highest level of DNA–DNA relatedness found was 32 % (for E. moraviensis and E. haemoperoxidus), the value for E. faecalis being 9 % (Table 2). These data, in combination with the phenotypic results and the origin of the isolates, indicate that the two isolates could represent one strain that is clearly genetically distinct from the other enterococcal species (Stackebrandt et al., 2002; Wayne et al., 1987), representing a novel taxon, for which the name Enterococcus caccae sp. nov. is proposed.
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. The samples were analysed at least three times; DNA from Escherichia coli K-12 was used as a control. The DNA G+C content of strain 2215-02T was 32.5 mol%.
Description of Enterococcus caccae sp. nov.
Enterococcus caccae (cac'cae. Gr. n. kakke faeces; N.L. gen. n. caccae of faeces).
Cells consist of Gram-positive cocci occurring as short chains, in pairs and as single cells. Non-pigmented, 
-haemolytic, small colonies up to 0.5 mm in diameter are formed on sheep-blood agar at 37 °C and are unaffected by the absence or presence of 5 % CO2. Strains are catalase-negative, non-motile and susceptible to vancomycin. Growth occurs at 45 °C and in broth containing 6.5 % NaCl. Strains are positive for pyrrolidonyl arylamidase activity, leucine aminopeptidase activity, hydrolysis of aesculin in the presence of bile, hippurate hydrolysis, pyruvate utilization and in the Voges–Proskauer test. Acid is produced from glycerol, maltose, methyl -D-glucopyranoside, ribose, sucrose and trehalose. Acid is not produced from arabinose, inulin, lactose, mannitol, melibiose, raffinose, sorbitol or sorbose. Reactions are negative for production of gas in MRS broth, hydrolysis of arginine and tolerance of 0.04 % tellurite. In the API Rapid ID 32 Strep system, acid is produced from maltose, methyl 
-D-glucopyranoside, ribose, sucrose, tagatose and trehalose. The enzymes -glucosidase, glycyl-tryptophan arylamidase, -mannosidase, pyroglutamic acid arylamidase and N-acetyl--glucosaminidase are produced. Arginine dihydrolase, -galactosidase (both substrates), -glucuronidase, -galactosidase, alkaline phosphatase, alanine-phenylalanine-proline arylamidase and urease are not produced. Acetoin is produced and hippurate is not hydrolysed. Acid is not produced from L-arabinose, D-arabitol, cyclodextrin, glycogen, lactose, mannitol, melezitose, melibiose, pullulan, raffinose or sorbitol. The species is distinguished by whole-cell-protein profiling and 16S rRNA gene sequencing. The AccuProbe Enterococcus genetic probe result is positive and Lancefield extracts react with Centers for Disease Control and Prevention group D antiserum. The DNA G+C content of the type strain is 32.5 mol%.
The type strain, strain 2215-02T (=SS-1777T=ATCC BAA-1240T=CCUG 51564T), was isolated from human stool samples, in Portland, OR, USA.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Carvalho, M. G. S., Steigerwalt, A. G., Morey, R. E., Shewmaker, P. L., Teixeira, L. M. & Facklam, R. R. (2004). Characterization of three new enterococcal species, Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, isolated from human clinical specimens. J Clin Microbiol 42, 1192–1198.
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Law-Brown, J. & Meyers, P. R. (2003). Enterococcus phoeniculicola sp. nov., a novel member of the enterococci isolated from the uropygial gland of the Red-billed Woodhoopoe, Phoeniculus purpureus. Int J Syst Evol Microbiol 53, 683–685.
Mandel, M., Igambi, L., Bergendahl, J., Dodson, M. L., Jr & Scheltgen, E. (1970). Correlation of melting temperature and cesium chloride buoyant density of bacterial deoxyribonucleic acid. J Bacteriol 101, 333–338.
Martinez-Murcia, A. J. & Collins, M. D. (1991). Enterococcus sulfureus, a new yellow-pigmented Enterococcus species. FEMS Microbiol Lett 1, 69–74.
Merquior, V. L. C., Peralta, J. M., Facklam, R. R. & Teixeira, L. M. (1994). Analysis of electrophoretic whole-cell protein profiles as a tool for characterization of Enterococcus species. Curr Microbiol 28, 149–153.[CrossRef]
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Shewmaker, P. L., Steigerwalt, A. G., Morey, R. E., Carvalho, M. G. S., Elliott, J. A., Joyce, K., Barrett, T. J., Teixeira, L. M. & Facklam, R. R. (2004). Vagococcus carniphilus sp. nov., isolated from ground beef. Int J Syst Evol Microbiol 54, 1505–1510.
Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]
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Teixeira, L. M. & Facklam, R. R. (2003). Enterococcus. In Manual of Clinical Microbiology, 8th edn, pp. 422–433. Edited by P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller & R. H. Yolken. Washington, DC: American Society for Microbiology.
Teixeira, L. M., Facklam, R. R., Steigerwalt, A. G., Pigott, N. E., Merquior, V. L. C. & Brenner, D. J. (1995). Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains. J Clin Microbiol 33, 1520–1523.[Abstract]
Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.
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