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Journal of Clinical Microbiology, October 2008, p. 3473-3476, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00603-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Designation of the Provisional New Enterococcus Species CDC PNS-E2 as Enterococcus sanguinicola sp. nov., Isolated from Human Blood, and Identification of a Strain Previously Named Enterococcus CDC PNS-E1 as Enterococcus italicus Fortina, Ricci, Mora, and Manachini 2004

Maria da Glória S. Carvalho,¹ Arnold G. Steigerwalt,¹ Roger E. Morey,¹ Patricia Lynn Shewmaker,¹ Enevold Falsen,² Richard R. Facklam,¹ and Lucia M. Teixeira^3*

Division of Bacterial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,¹ Culture Collection, Department of Clinical Bacteriology, University of Göteborg, S-413 46 Göteborg, Sweden,² Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941, Brazil³

Received 30 March 2008/ Returned for modification 25 May 2008/ Accepted 22 July 2008

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We have previously characterized two new enterococcal species (provisionally designated CDC PNS-E1 and CDC PNS-E2) recovered from clinically significant specimens associated with invasive infections in humans. In the present report we provide additional data and propose formal denominations for isolates of these two species of Enterococcus. Results of 16S rRNA gene sequencing, sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of whole-cell protein profiles, and DNA-DNA reassociation experiments indicated that the blood isolate ATCC BAA-780 (SS 1728; CDC PNS-E1) corresponds to Enterococcus italicus, whose species epithet was proposed to designate isolates from artisanal Italian cheese. Strain ATCC BAA-781 (CCUG 47861; SS 1729; CDC PNS-E2), a vancomycin-resistant isolate recovered from the blood of a patient in the United States, was found to be highly related at the species level to another blood isolate (SS 1743; CCUG 47884) from Sweden, and for these we propose the designation Enterococcus sanguinicola sp. nov.

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Several new enterococcal species have been described since the recognition of Enterococcus as a separate genus (8), as a result of improvements in identification methods, combined with a growing interest in the role of these microorganisms as opportunistic pathogens (3, 9). Additionally, the enterococci are frequently associated with important antimicrobial resistance markers, and therefore, their presence in the microbiota of humans and animals, as well as in food and environmental sources, may play a significant role in the dissemination of antimicrobial resistance genes. Interestingly, most of the enterococcal species described more recently have been recovered from sources other than humans, or from human patients with invasive infections.

We have previously characterized three new enterococcal species isolated from clinically significant sources that indicate their association with invasive infections in humans (2). Because only a single isolate of each was available, these species were provisionally designated (CDC PNS-E1, CDC PNS-E2, and CDC PNS-E3) in accordance with a recommendation in minute 10 of the July 2002 meeting of the International Committee on Systematics of Prokaryotes, Subcommittee on the Taxonomy of Staphylococci and Streptococci (11). In the present report, we discuss supplementary characteristics and the formal designation of two of these proposed new species of Enterococcus (CDC PNS-E1 and CDC PNS-E2), after comparative analysis with additional isolates. We provide evidence that CDC PNS-E1 corresponds to Enterococcus italicus, whose species epithet was proposed to name a group of isolates recovered from artisanal Italian cheese (4). The name Enterococcus sanguinicola sp. nov. is proposed to designate CDC PNS-E2.

The strains included in this study comprised two of the clinical isolates described by Carvalho et al. (2) as mentioned below, one additional clinical isolate obtained more recently, and the type strain of Enterococcus italicus (DSM 15952^T; SS 1773), a new species that was described by Fortina et al. (4) almost simultaneously with our previous report. The three clinical isolates were recovered from the blood of different patients in diverse locations. One clinical isolate (ATCC BAA-780; SS 1728; previously designated PNS-E1) was isolated from a patient in Evanston, IL, in 1991; another (ATCC BAA-781; SS 1729; previously designated PNS-E2) was isolated from a patient in Los Angeles, CA, in 1997; and the third (CCUG 47884; SS 1743) was isolated from a patient in Sweden. The type strains of all other enterococcal species available to date were included for comparative purposes.

The strains were tested for their phenotypic characteristics by conventional physiological tests and serological grouping as described previously (2, 9). Biochemical tests were also performed using the rapid ID 32 Strep (ID 32) system (version 3.0; bioMerieux, Inc., Durham, NC) according to the manufacturer's instructions. Reactivity with the AccuProbe Enterococcus culture identification test (Gen-Probe, Inc., San Diego, CA) was assayed as directed by the manufacturer.

Extracts were prepared, and whole-cell protein (WCP) profiles were analyzed by 1-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as described by Merquior et al. (6). Coefficients of similarity or Dice indices were determined for each isolate by using the Molecular Analyst Fingerprinting Plus software package, version 1.6 (Bio-Rad Laboratories, Hercules, CA), and a dendrogram was constructed by the unweighted pair-group method with arithmetic averages.

Harvesting and lysis of the bacterial cells to obtain DNA for reassociation experiments were carried out as previously described (10). Extraction, purification of DNA, and determination of DNA relatedness were performed using the hydroxyapatite hybridization method as recommended by Brenner et al. (1). DNA hybridization experiments were performed at 55°C for optimal DNA reassociation and at 70°C for stringent conditions. The G+C content was determined according to the procedures proposed by Mandel et al. (5).

Sequencing of the 16S rRNA gene and phylogenetic analysis of the isolates were performed by the methods described by Carvalho et al. (2). The final phylogenetic tree was rooted with Vagococcus fluvialis as an outgroup, and bootstrap values were displayed as percentages.

All the isolates were nonmotile, catalase-negative, gram-positive cocci, occurring singly, in pairs, or in short chains. Colonies in blood agar were circular and gray, with alpha-hemolysis. They were positive by the Enterococcus AccuProbe assay, the leucine aminopeptidase (LAPase) and pyrrolidonyl arylamidase (PYRase) activity tests, and the Voges-Proskauer test. They also hydrolyzed esculin in the presence of bile; grew in broth containing 6.5% NaCl and at 10°C and 45°C; produced acids from lactose, maltose, and sucrose; and reacted with group D antiserum. All strains were negative for pigment production, production of gas in MRS broth, motility, hippurate hydrolysis, and production of acids from arabinose, inulin, melibiose, raffinose, sorbitol, and sorbose. Additional physiological characteristics of the isolates are shown in Table 1. The ID 32 system generated identical profiles (30331701150, categorized by the system as a "doubtful profile") for strains ATCC BAA-781^T (SS 1279; CDC PNS-2) and CCUG 47884 (SS 1743). The ID 32 profile (00011101121) obtained for strain ATCC BAA-780 (SS 1728; CDC PNS-E1) was categorized as an "unacceptable profile," while the profile (00231101101) generated for strain DSM 15952^T (SS 1773; E. italicus) was categorized as a "doubtful profile."

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TABLE 1. Characteristics of the enterococcal strains included in the present studya

Comparative analysis showed that the WCP profile of strain ATCC BAA-780 (CDC PNS-E1) was highly related to that of the E. italicus type strain DSM 5952^T, while the WCP profile of strain ATCC BAA-781 (CDC PNS-E2) was highly related to the profile of strain CCUG 47884 (SS 1743) (Fig. 1). In contrast, these profiles were distinct from the protein profiles of the type strains of all the other species of Enterococcus.

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FIG. 1. SDS-PAGE profiles of whole-cell protein extracts of Enterococcus sanguinicola sp. nov. (strains ATCC BAA-781T and CCUG 47884) and Enterococcus italicus (strains DSM 15952T and ATCC BAA-780). A dendrogram resulting from computer-assisted analysis of the protein profiles is shown on the left. MM, molecular mass marker (in kilodaltons).

The results of DNA-DNA relatedness experiments performed with strain CCUG 47884 (SS 1743) showed 84% homology with strain ATCC BAA-781 (CDC PNS-E2) at the optimum temperature and 83% homology at the stringent temperature, with 0.5% divergence, indicating that these strains are a single taxon. DNA-DNA reassociation data also showed a species relationship between strains ATCC BAA-780 (CDC PNS-E1) and DSM 5952^T: they had 82% homology at the optimum temperature and 78% homology at the stringent temperature, with 1.5% divergence. Relative binding ratios, expressing the percentages of homology at 55°C of these strains with the type strains of all other enterococcal species tested, ranged from 6 to 41.

Analysis of 16S rRNA gene sequences (approximately 1,480 bp) showed species-level relationships between strains ATCC BAA-780 (CDC PNS-E1) and DSM 15952^T (E. italicus) and between strains ATCC BAA-781 (CDC PNS-E2) and CCUG 47884 (SS 1743). A multiple-sequence-alignment analysis trimmed to consensus, comparing these 16S rRNA gene sequences with those of selected Enterococcus species, is shown in Fig. 2.

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FIG. 2. Distance matrix tree based on 16S rRNA gene sequence comparisons showing the phylogenetic relationships among Enterococcus italicus, Enterococcus sanguinicola, and some of the most closely related species of Enterococcus. The Vagococcus fluvialis sequence was used as an outgroup. Bootstrap probability values (percentages of 1,000 tree replications) are given at branch points. Sequence accession numbers are given in parentheses after strain names.

Overall, the results of 16S rRNA gene sequencing, SDS-PAGE analysis of WCP profiles, and DNA-DNA reassociation experiments indicated that strain ATCC BAA-780 (CDC PNS-E1) corresponds to Enterococcus italicus, a new species that was proposed to designate a group of isolates recovered from artisanal Italian cheese (4). Comparative analysis of the 16S rRNA gene sequence of our isolate ATCC BAA-780 also showed species-level similarity with the sequence of Enterococcus saccharominimus, confirming the recent finding reported by Naser et al. (7) that E. italicus and E. saccharominimus (also represented by isolates obtained from dairy products) are a single species. Therefore, to our knowledge, ATCC BAA-780 is the only E. italicus isolate reported to have been recovered from a clinically significant human source (blood) to date.

Strain ATCC BAA-781 (CDC PNS-E2), a vancomycin resistant blood isolate (2), was found to be related at the species level to CCUG 47884 (SS 1743), another blood isolate, recovered from a patient in Sweden. For these we propose the designation Enterococcus sanguinicola sp. nov. In contrast to the first E. sanguinicola isolate, CCUG 47884 (SS 1743) was susceptible to vancomycin.

In conclusion, the present report provides a proposal for formal designation and additional information on the characteristics of two enterococcal species previously provisionally named CDC PNS-E1 (now E. italicus) and CDC PNS-E2 (now E. sanguinicola). The isolates were obtained from clinically significant human specimens, indicating that these species can cause invasive infections in humans. The fact that one of the isolates carried the vancomycin resistance gene vanA is of particular importance because it calls attention to one additional enterococcal species capable of acquiring this important resistance marker. Considering the high transmissibility of the vanA gene, clinicians and microbiologists should be able to promptly recognize and identify glycopeptide-resistant isolates of this new enterococcal species.

After the present work was completed, one additional E. sanguinicola isolate was identified at the CDC Streptococcus laboratory. The isolate (DS 483-07) had phenotypic characteristics identical to those shown in Table 1 for the other two E. sanguinicola isolates and an ID 32 profile (30337701151) categorized as a "doubtful profile." Sequencing of the 16S rRNA gene showed a species-level relationship with the E. sanguinicola type strain. As attention is directed to these newly recognized species and accurate procedures are incorporated into the identification schemes to properly detect and characterize these microorganisms in the clinical setting, more information will become available on their occurrence and characteristics and on their role as agents of infections.

Considering the physiological profile of the single E. italicus isolate obtained from a human source, we recommend that the species description of E. italicus be emended to indicate that growth in broth containing 6.5% NaCl and production of acid from trehalose may be strain-variable traits. The description of E. sanguinicola is presented below.

Description of Enterococcus sanguinicola sp. nov. Enterococcus sanguinicola (san.gui.ni'co.la. L. n. sanguis, -inis, blood; L. suffix n. -cola [from incola], inhabitant; N.L. masc./fem. n. sanguinicola, inhabitant of blood [nominative in apposition]).

Cells are gram-positive cocci occurring as short chains, in pairs, or singly. Nonpigmented, alpha-hemolytic, small colonies up to 0.5 mm in diameter are formed in sheep blood agar at 37°C and are unaffected by the absence or presence of 5% CO₂. The organism is catalase negative and nonmotile. Growth occurs at 10°C, at 45°C, and in broth containing 6.5% NaCl. The organism is positive for PYRase activity, LAPase activity, hydrolysis of esculin in the presence of bile, and hydrolysis of arginine; it is also positive by the Voges-Proskauer test. Acid is produced from glycerol, lactose, maltose, mannitol, ribose, sucrose, and trehalose. Acid is not produced from arabinose, inulin, melibiose, methyl--D-glucopyranoside, raffinose, sorbitol, or sorbose. The organism is negative for production of gas in MRS broth, hydrolysis of hippurate, and pyruvate utilization. It reacts with the AccuProbe Enterococcus genetic probe, and Lancefield extracts react with CDC group D antiserum. The type strain is SS-1729^T (ATCC BAA-781^T; CCUG 47861^T), a vancomycin-resistant isolate (with a MIC of >256 µg/ml, and harboring the vanA gene) recovered from the blood of a patient in Los Angeles, CA. The G+C content of the type strain is 39.0 mol%.

Nucleotide sequence accession number. The GenBank accession number for the 16S rRNA gene sequence of Enterococcus sanguinicola strain SS-1729^T (ATCC BAA-781^T; CCUG 47861^T) is AY321376.

 
L.M.T. received support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Ministério da Ciência e Tecnologia (MCT/PRONEX), Brazil.

We are grateful to Hans Trüper for assisting with the definition of the species epithet.

 
* Corresponding author. Mailing address: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Cidade Universitária Rio de Janeiro, RJ 21941-590, Brazil. Phone: 55 21 2260 4193. Fax: 55 21 2560 8344. E-mail: lmt2{at}micro.ufrj.br

Published ahead of print on 30 July 2008.

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Journal of Clinical Microbiology, October 2008, p. 3473-3476, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00603-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Carvalho, M. d. G. S. Articles by Teixeira, L. M. Search for Related Content PubMed PubMed Citation Articles by Carvalho, M. d. G. S. Articles by Teixeira, L. M.