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Journal of Clinical Microbiology, April 2002, p. 1140-1145, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1140-1145.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. Isolated from Human Clinical Specimens

Gregory J. Tyrrell,^1,2* LeeAnn Turnbull,¹ Lúcia M. Teixeira,³ Johanne Lefebvre,⁴ Maria da Glória S. Carvalho,³ Richard R. Facklam,⁵ and Marguerite Lovgren¹

The National Centre for Streptococcus—Canada, The Provincial Laboratory for Public Health, Northern Alberta,¹ Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta,² Institut Laboratorie de Sante Publique du Quebec, National de Sante Publique du Quebec, Ste. Anne de Bellevue, Quebec, Canada,⁴ Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janerio, RJ, 21941, Brazil ,³ Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333⁵

Received 3 October 2001/ Returned for modification 10 December 2001/ Accepted 14 December 2001

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Light yellow-pigmented (strain PQ1) and yellow-pigmented (strain PQ2), gram-positive, non-spore-forming, nonmotile bacteria consisting of pairs or chains of cocci were isolated from the bile of a patient with cholecystitis (PQ1) and the peritoneal dialysate of another patient with peritonitis (PQ2). Morphologically and biochemically, the organisms phenotypically belonged to the genus Enterococcus. Whole-cell protein (WCP) analysis and sequence analysis of a segment of the 16S rRNA gene suggested that they are new species within the genus Enterococcus. PQ1 and PQ2 displayed less than 70% identities to other enterococcal species by WCP analysis. Sequence analysis showed that PQ1 shared the highest level of sequence similarity with Enterococcus raffinosus and E. malodoratus (sequence similarities of 99.8% to these two species). Sequence analysis of PQ2 showed that it had the highest degrees of sequence identity with the group I enterococci E. malodoratus (98.7%), E. raffinosus (98.6%), E. avium (98.6%), and E. pseudoavium (98.6%). PQ1 and PQ2 can be differentiated from the other Enterococcus spp. in groups II, III, IV, and V by their phenotypic characteristics: PQ1 and PQ2 produce acid from mannitol and sorbose and do not hydrolyze arginine, placing them in group I. The yellow pigmentation differentiates these strains from the other group I enterococci. PQ1 and PQ2 can be differentiated from each other since PQ1 does not produce acid from arabinose, whereas PQ2 does. Also, PQ1 is Enterococcus Accuprobe assay positive and pyrrolidonyl-ß-naphthylamide hydrolysis positive, whereas PQ2 is negative by these assays. The name Enterococcus gilvus sp. nov. is proposed for strain PQ1, and the name Enterococcus pallens sp. nov. is proposed for strain PQ2. Type strains have been deposited in culture collections as E. gilvus ATCC BAA-350 (CCUG 45553) and E. pallens ATCC BAA-351 (CCUG 45554).

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Species identification of enterococci in the clinical laboratory has gained importance in the last decade due primarily to this organism's ability to acquire new antibiotic resistance determinants, including resistance to vancomycin (12). There are five recognized groups of enterococci (groups I to V), which include a total of 21 species (4, 7, 21, 23, 24, 26). The majority of these can be identified to the species level by conventional identification techniques. Classic species identification of enterococci involves assays for a combination of biochemical and morphological characteristics of the unknown organism. Biochemical characteristics refer to the ability of enterococci to utilize approximately 10 or more different substrates, resulting in a characteristic pattern for a particular species (6, 7). This identification scheme also involves assays for two morphological characteristics, motility and yellow pigmentation. There are only two motile enterococci, Enterococcus gallinarum and E. casseliflavus. They can be differentiated from each other on the basis of their pigmentation; E. casseliflavus is yellow and E. gallinarum is not. There are only two yellow-pigmented enterococci of clinical significance to humans, E. mundtii and E. casseliflavus, and they can be distinguished from each other by their motilities. E. flavescens was proposed as a new species of motile yellow-pigmented enterococci; however, further work has suggested that this species is a variant of E. casseliflavus and does not warrant designation as a separate species (17, 19, 23). A third yellow-pigmented enterococcal species is E. sulfureus (14). The environmental niche for this organism appears to be plants, whereas E. mundtii and E. casseliflavus are animal derived (14, 22). Besides E. casseliflavus (E. flavescens), E. mundtii, and E. sulfureus, there have been no reports of other species of yellow-pigmented enterococci.

We report on the isolation and identification of two new yellow-pigmented Enterococcus species that we have designated Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Bacterial strains. The unknown enterococci (strains PQ1 and PQ2) were referred to the National Centre for Streptococcus (Edmonton, Alberta, Canada) for species identification. PQ1 was cultured from the bile of a patient with cholecystitis, and PQ2 was cultured from a peritoneal dialysate of another patient who developed peritonitis from a perforated intestine. PQ1 and PQ2 have been deposited with the American Type Culture Collection (ATCC) and the Culture Collection of the University of Göteborg (CCUG). Type strains (obtained from ATCC) representing the enterococcal species that were physiologically most related to the two isolates were also included in the study. These were E. avium ATCC 14025^T, E. casseliflavus ATCC 25788^T, E. faecalis ATCC 19433^T, E. malodoratus ATCC 43197^T, E. mundtii ATCC 43186^T, E. raffinosus ATCC 49427^T, E. pseudoavium ATCC 49372^T, E. saccharolyticus ATCC 43076^T, and E. sulfureus ATCC 49903^T.

Morphological and biochemical analysis. Strains PQ1 and PQ2 were grown on Trypticase soy sheep blood agar (TSA-SB) incubated at 35°C without CO₂. Cellular morphology was observed after Gram staining of a smear prepared from a culture grown in thioglycolate broth, air dried, and fixed with methanol. Grouping was done by the Lancefield hot acid extraction method (9). Lancefield acid extracts were examined for lines of identity with group-specific antisera. Phenotypic characterization was carried out by conventional tests (6, 7). These included catalase production; susceptibility to vancomycin as determined with a 30-µg vancomycin disk; pyrrolidonyl-ß-naphthylamide (PYR) and leucine-ß-naphthylamide (LAP) hydrolysis; hydrolysis of esculin in the presence of 40% bile; arginine hydrolysis; esculin hydrolysis; growth at 10 and 45°C; growth in 2.0, 4.0, and 6.5% NaCl; motility; pigment production; pyruvate utilization; hippurate hydrolysis; reduction of tetrazolium; growth on potassium tellurite agar; and acid production from the carbohydrates listed in Table 1. Pigment production was visually assayed by growing the bacteria on Luria-Bertani (LB) agar or TSA-SB for 24 h and scraping off the growth with a white cotton swab (20). Biochemical reactions were read after 7 days of incubation; however, bile esculin hydrolysis and hippurate hydrolysis tests were read at 48 h, and the test for the production of acid from methyl--D-glucopyranoside was read at 72 h. Motility was assayed with 0.03% semisolid agar with tetrazolium chloride at 30°C for 7 days. Tests for growth in 2.0, 4.0, and 6.5% NaCl and at 10 and 45°C were performed in duplicate in heart infusion broth with 0.1% bromocresol purple indicator. The results were recorded after 24 h, 48 h, and 7 days. The Enterococcus Accuprobe assay (Gen-Probe, San Diego, Calif.) was carried out according to the instructions of the manufacturer. The Enterococcus Accuprobe assay is a nonsubjective method for the identification of Enterococcus species. The assay uses a single-stranded DNA probe with a chemiluminescent label that is complementary to specific rRNA sequences unique to Enterococcus.

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TABLE 1. Differential characteristics of PQ1 and PQ2 and comparison with those of other pigmented enterococci and related group I enterococcal speciesa

Long-chain fatty acid analysis. Isolates were grown on brain heart infusion agar at 35°C for 24 h. The organisms were harvested into 1 ml of NaOH in aqueous methanol and were incubated at 100°C for 0.5 h. The tubes were then cooled, and 2 ml of HCl in aqueous methanol was added. This mixture was then incubated at 80°C for 10 min and cooled rapidly. The fatty acids were extracted into hexane and methyl-tert-butyl ether and washed with dilute NaOH. The extracts were run on a Hewlett-Packard model HP5890 Series II gas chromatograph (MIDI, Inc., Newark, Del.). The MIDI Microbial Identification System consists of the Hewlett-Packard gas chromatograph linked to a computer system with Sherlock and ChemStation software (MIDI, Inc.).

Whole-cell protein preparation and analysis. Preparation of whole-cell extracts and analysis of profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were performed as described previously (11, 15), with a few modifications. Briefly, the strains were grown on plates containing TSA-SB. Bacterial cells were removed from the surface of the plate with an inoculating loop and suspended in 5 ml of sterile saline solution in order to obtain a turbidity equal to that of a no. 8 McFarland density standard, centrifuged, and resuspended in 0.25 ml of an aqueous lysozyme solution (10 mg/ml). The protein profiles of the type strains of the different species were compared according to their percentages of similarity, estimated by use of the Dice coefficient and clustered by the unweighted pair group method with averages by using the Molecular Analyst Fingerprinting Plus software package, version 1.12, of the Image Analysis System (Bio-Rad Laboratories, Hercules, Calif.).

16S rRNA gene sequencing. The methodologies for extraction of chromosomal DNA and performance of the PCR are described in detail elsewhere (18, 25). Oligonucleotides were purchased from Gibco BRL (Burlington, Ontario, Canada). PCR amplifications were performed with a model 9600 thermocycler (Perkin-Elmer, Norwalk, Conn.). The oligonucleotides used as primers for PCR and for sequencing were as described previously (9). The DNA sequences of the 16S rRNA gene (rDNA) amplicons were determined directly by use of the oligonucleotide primers as sequencing primers, the DYEnamic terminator cycle sequencing premix kit (Amersham Pharmacia Biotech Inc., Cleveland, Ohio), and an ABI 373 sequencer. The amplicons were sequenced in both directions.

Sequence and phylogenetic analyses. The sequences obtained were compared with the sequences of strains belonging to other related enterococcal species retrieved from the GenBank database by the CLUSTAL method with the Expert Sequences Analysis software of the DNASTAR program (DNASTAR Inc., Madison, Wis.). Consensus sequences were determined and then grouped into clusters according to the sequence distances between all pairs. Clusters were aligned as pairs and then collectively as sequence groups to produce the overall alignment. After the multiple-sequence alignment was completed, the neighbor-joining method was used to construct a dendrogram showing the phylogenetic relationships (8).

Nucleotide sequence accession numbers. The partial 16S rDNA sequences of strains PQ1 and PQ2 have been submitted to GenBank and can be found under accession numbers AY033814 and AY033815, respectively.

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Initial isolation and cultural characteristics. Strain PQ1 was isolated from the bile of a patient with cholecystitis . E. faecium and E. casseliflavus were also isolated from the same specimen. Strain PQ2 was isolated from peritoneal dialysate fluid of another patient. Initial characterization placed PQ1 and PQ2 in the genus Enterococcus. When grown on TSA-SB or LB agar or in broth, the cells of PQ1 and PQ2 were gram positive and spherical, occurred in chains, and were nonmotile. PQ1 produced a light yellow pigment, and PQ2 produced a bright yellow pigment. Both strains grew in 6.5% NaCl and at 10, 35, and 45°C. PQ1 and PQ2 were catalase negative, reacted with group D antiserum, and were positive for hydrolysis of esculin and esculin with 40% bile. PQ1 and PQ2 were LAP hydrolysis positive and utilized pyruvate. PQ1 was Enterococcus Accuprobe assay positive and PYR hydrolysis positive, whereas PQ2 was Accuprobe assay negative and PYR negative. Currently, only three species of enterococci are Accuprobe assay negative and PYR hydrolysis negative: E. cecorum, E. columbae, and E. saccharolyticus (7). Both PQ1 and PQ2 were sensitive to vancomycin by the 30-µg vancomycin disk assay, and both strains were tellurite negative. Both strains produced acid from mannitol and sorbose and were arginine negative, suggesting that they are members of the group I enterococci (Table 1) (7). PQ1 and PQ2 also produced acid from glycerol, lactose, melibose, raffinose, ribose, salicin, sorbitol, sucrose, and trehalose. They did not produce acid from inulin, methyl--D-glucopyranoside, or xylose. Also, PQ1 did not produce acid from arabinose or hydrolyze hippurate, whereas PQ2 did. PQ1 reduced tetrazolium, whereas PQ2 did not. Biochemically, PQ1 most closely resembles E. malodoratus. However, unlike E. malodoratus, PQ1 was pigmented yellow, although the color was duller than the bright yellow pigment typical of E. casseliflavus or E. mundtii. PQ2 most closely resembles E. raffinosus; however, PQ2 is pigmented a bright yellow, whereas E. raffinosus is not. Also, PQ2 does not hydrolyze PYR, unlike E. raffinosus. Pigment production was easily visualized when the organisms were grown on LB agar or a blood agar plate and then scraped off with a cotton swab. The pigmentation phenotype was preserved upon subculture.

Three enterococci are currently classified as having yellow pigmentation. These are E. casseliflavus (group II), E. mundtii (group II), and E. sulfureus (Group IV).

Long-chain fatty acid analysis. The following long-chain fatty acids were detected in strain PQ1: 12:0, 14:0, 15:0, 16:0, 17:0, 18:0, 18:19c, 18:17c, and cyclo-C₁₉ (Table 2). Other major fatty acids which were present in PQ1 (but which could not be quantitated owing to the poor resolution of the chromatogram) were 16:17c and i15:0 2-OH, 18:26,9c and 18:0, and 19:0 cyclo 10c and 196. The following long-chain fatty acids were detected in strain PQ2: 14:0, 15:0, 16:0, 17:0, 18:0, 18:19c, 18:17c, and cyclo-C₁₉ (Table 2). Other major fatty acids which were present in PQ2 (but which could not be quantitated owing to the poor resolution of the chromatogram) were 16:17c/il15:0 2-OH, 18:26, 9c/18:0, and 19:0 cyclo 10c/196. The long-chain fatty acid profiles of PQ1 and PQ2 do not match those of any of the group I enterococci. However, the long-chain fatty acid profile of PQ1 does most closely resemble that of E. raffinosus, suggesting that it is most related to this species, and the long-chain fatty acid profile of PQ2 most closely resembles that of E. avium.

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TABLE 2. Long-chain fatty acid compositions of E. gilvus, and E. pallens, and related group I enterococcal species

Whole-cell protein analysis. SDS-PAGE analysis of whole-cell proteins showed that neither PQ1 nor PQ2 had an identity of 70% of greater to any of the enterococcal species examined, including those to which they were most physiologically related (Fig. 1), suggesting that PQ1 and PQ2 are distinct species of enterococci. The whole-cell protein profiles of PQ1 and PQ2 were easily distinguishable from each other. PQ1 most closely resembles E. raffinosus, E. casseliflavus, and E. mundtii (60% identity); and PQ2 most closely resembles E. faecalis (64% identity).

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FIG. 1. (A) SDS-PAGE profiles of whole-cell protein extracts of E. gilvus, E. pallens, and strains belonging to related enterococcal species. Lanes 1 and 13, molecular mass markers; lane 2, E. avium ATCC 14025T; lane 3, E. malodoratus ATCC 43197T; lane 4, E. pseudoavium ATCC 49372T; lane 5, E. saccharolyticus ATCC 43076T; lane 6, E. raffinosus ATCC 49427T; lane 7, E. pallens (PQ2); lane 8, E. gilvus (PQ1); lane 9, E. casseliflavus ATCC 25788T; lane 10, E. mundtii ATCC 43186T; lane 11, E. sulfureus ATCC 49903T; lane 12, E. faecalis ATCC 19433T. (B) Dendrogram resulting from computer-assisted analysis of the protein profiles in panel A. The scale represents average percent similarities. A 0.6% tolerance value was used to construct the dendrogram.

16S rDNA sequence analysis. To assess the genealogical affinities between PQ1 and PQ2 and their relationship with other enterococci, comparative 16S rRNA gene sequencing analysis was performed. Amplification of the 16S rRNA gene sequences yielded the expected PCR products. PCR of the 16S rDNAs of isolates PQ1 and PQ2 generated sequences of 1,295 and 1,294 bp, respectively. The first base corresponds to position 7 and the last base corresponds to position 1342 of the Escherichia coli 16S rRNA gene (5). The sequenced DNA was compared to those of the other enterococcal species obtained from GenBank or published previously (17). The accession numbers of these sequences are shown in Fig. 2.

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FIG. 2. Phylogenetic relationships of E. gilvus, E. pallens, and related enterococcal species based on 16S rRNA gene sequencing analysis. The dendrogram is based on the sequence identities of 1,294 nucleotides of the 16S rRNA gene. The dendrogram was constructed by the CLUSTAL method with the Expert Sequences Analysis software of the DNASTAR program. The neighbor-joining method was used to construct a dendrogram showing the phylogenetic relationships. The scale units indicate the distance between sequence pairs. The ATCC strain numbers and the GenBank accession numbers of the 16S rRNA gene sequences (in parentheses) are indicated for each species used to construct the dendrogram.

Sequence searches of the GenBank and Ribosomal Database Project data libraries revealed that PQ1 and PQ2 are phylogenetically most closely associated with species of the genus Enterococcus. A tree depicting the phylogenetic affinity of PQ1 and PQ2 with other enterococci is shown in Fig. 2.

The PQ1 sequence analyzed most closely matched those of E. malodoratus and E. raffinosus. PQ1 is 99.8% similar to E. raffinosus and 99.8% similar to E. malodoratus. There are 3 nucleotide differences between E. raffinosus and PQ1 and between E. malodoratus and PQ1 in the 1,295 bp of the 16S rDNA sequenced. This suggests that these three species are very closely related from an evolutionary perspective. The next most closely related species are E. pseudoavium (99.5% identity) and E. avium (99.5% identity).

Analysis of the sequenced segment of the 16S rDNA of PQ2 showed that PQ2 is 98.7% similar to E. malodoratus and 98.6% similar to E. raffinosus, E. avium, and E. pseudoavium. In the 1,294 bp examined, there were 20 nucleotide differences between PQ2 and E. malodoratus, 21 nucleotide differences between PQ2 and E. raffinosus, 22 nucleotide differences between PQ2 and E. avium, and 25 nucleotide differences between PQ2 and E. pseudoavium.

The 16S rDNA sequence differences together with the production of yellow pigment, the biochemical utilization patterns, and the results of whole-cell protein and long-chain fatty acid analyses strongly suggest that PQ1 and PQ2 are new species of the genus Enterococcus for which the names Enterococcus gilvus and Enterococcus pallens, respectively, are proposed.

Strains PQ1 and PQ2 are the only yellow-pigmented enterococci in group I. This is the first report of pigmented group I enterococci. It is possible that these bacteria have mistakenly been identified in the past as one of the other yellow-pigmented enterococci and have therefore gone unrecognized as new species. Studies in the early 1970s indicated that the yellow pigment in enterococci was the result of carotenoid production by the organism (13, 22). Carotenoids are naturally occurring pigments found in a wide variety of bacteria, cyanobacteria, algae, fungi, higher plants, crustaceans, insects, fish, and birds (1). In bacteria, carotenoids act as quenchers of potentially toxic oxygen radicals and as light-gathering pigments in photosynthesis (1, 3, 13, 16).

Also, while strain PQ1 (E. gilvus) was isolated from the bile of a patient suffering from cholecystitis and PQ2 (E. pallens) was isolated from the peritoneal dialysate of another patient with peritonitis, it is unclear what pathogenic role E. gilvus and/or E. pallens has in causing infection in humans.

Description of E. gilvus sp. nov. Enterococcus gilvus (gil.vus". L. adj., pale yellow, referring to the pale yellow pigmentation of the bacterium). Cells are gram-positive cocci and spherical and mostly occur in short chains. The cells are pigmented a light yellow color in comparison to the other pigmented enterococci, E. casseliflavus, E. mundtii, and E. sulfureus. The organism is nonmotile and catalase negative. It is Lancefield group D antigen positive. It grows at 10 and 35°C and has delayed growth at 45°C. It grows in 2.0, 4.0, and 6.5% NaCl. It produces acid from glycerol, lactose, mannitol, melibose, raffinose, ribose, salicin, sorbitol, sorbose, sucrose, and trehalose. It does not produce acid from arabinose, inulin, methyl--D-glucopyranoside, or xylose. It is positive for esculin hydrolysis in the presence of 40% bile. Black colonies were not produced when grown on tellurite-containing media. It is positive for pyruvate utilization and reduction of tetrazolium. The strain is positive for LAP hydrolysis and PYR hydrolysis and negative for arginine dihydrolase hydrolysis and hippurate hydrolysis. It is Enterococcus Accuprobe assay positive. The strain can also be differentiated from other enterococcal species by its unique whole-cell protein profile and the sequence of its 16S rRNA gene. The type strain is ATCC BAA-350 (CCUG 45553; PQ1).

Description of E. pallens sp. nov. Enterococcus pallens (pall. ens'. L. adj., yellowish, referring to the yellow pigmentation of the bacterium). Cells are gram-positive cocci and spherical and mostly occur in short chains. The bacterium is pigmented a bright yellow color. The organism is nonmotile and catalase negative. It is Lancefield group D antigen positive. It grows at 10, 35, and 45°C. It grows in 2.0, 4.0, and 6.5% NaCl. It produces acid from arabinose, glycerol, lactose, mannitol, melibose, raffinose, ribose, salicin, sorbitol, sorbose, sucrose, and trehalose. It does not produce acid from inulin, methyl--D-glucopyranoside, or xylose. It is positive for esculin hydrolysis in the presence of 40% bile and for hippurate hydrolysis. It is also positive for pyruvate utilization and negative for reduction of tetrazolium. The strain is positive for LAP hydrolysis and negative for both PYR hydrolysis and arginine hydrolysis. Testing by the Enterococcus Accuprobe assay is negative. The strain can also be differentiated from the other species of Enterococcus by its unique whole-cell protein profile and the sequence of its 16S rRNA gene. The type strain is ATCC BAA-351 (CCUG 45554; PQ2).

 
* Corresponding author. Mailing address: 2B3.13 Walter Mackenzie Centre, 8440-112 St., Edmonton, Alberta, Canada T6G 2J2. Phone: (780) 407-8949. Fax: (780) 407-3864. E-mail: g.tyrrell{at}provlab.ab.ca.

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Journal of Clinical Microbiology, April 2002, p. 1140-1145, Vol. 40, No. 4
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.4.1140-1145.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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