ACCEPTED. JCM Revised

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1 JCM Accepts, published online ahead of print on 30 July 2008 J. Clin. Microbiol. doi: /jcm Copyright 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved JCM Revised Enterococcus sanguinicola sp. nov., Isolated from Human Blood, Comprising the Provisional New Species of Enterococcus CDC PNS-E2, and Recognition of Strain Previously Described as Enterococcus CDC PNS-E1 as Enterococcus italicus Fortina et al 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, USA 1 ; Culture Collection, Department of Clinical Bacteriology, University of Göteborg, S Göteborg, Sweden, 2 and Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941, Brazil 3 Running Title: Enterococcus sanguinicola sp. nov. *Corresponding author Mailing address: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Cidade Universitária Rio de Janeiro, RJ , Brazil Phone: Fax: lmt2@micro.ufrj.br

2 Abstract We have previously characterized two new enterococcal species (provisionally designated as CDC PNS-E1 and CDC PNS-E2) recovered from clinically significant specimens that indicate their association with invasive infections in humans. In the present report we provide additional data and propose formal denomination for isolates of these two species of Enterococcus. Results of 16S ribosomal DNA sequencing, SDS-PAGE 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, a species epithet that was proposed to designate isolates from artisanal Italian cheese. Strain ATCC BAA-781 (CCUG 47861, SS 1729, CDC PNS-E2), which is 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 46 designation Enterococcus sanguinicola sp. nov.

3 Several new enterococcal species have been described since the recognition of the 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). Since only a single isolate of each was available, they were provisionally designated (as CDC PNS-E1, CDC PNS-E2, and CDC PNS-E3) in attention to 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 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, a species epithet that 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.

4 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 more recently obtained, and the type strain of Enterococcus italicus (DSM T, SS 1773) a new species that was described by Fortina et al. (4) almost simultaneously with our previous report. The 3 clinical isolates were recovered from blood of different patients in diverse locations: one (ATCC BAA-780, SS 1728, previously designed as PNS-E1) was isolated from a patient in Evanston, Ill., in 1991; another (ATCC BAA-781, SS 1729, previously designed as PNS-E2) was isolated from a patient in Los Angeles, California, USA, 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 V3.0 (biomerieux, Inc., Durham, N.C.) according to the manufacturer s instructions. Reactivity with the AccuProbe Enterococcus culture identification test (Gen-Probe, Inc., San Diego, Calif.) was assayed as directed by the manufacturer. Preparation of extracts and analysis of whole-cell protein (WCP) profiles by onedimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) were performed as described by Merquior et al. (7). Coefficients of similarity or Dice indexes were determined for each isolate by using the Molecular Analyst Fingerprinting Plus software package, version 1.6 (Bio-Rad Laboratories,

5 Hercules, Calif.) and a dendrogram was constructed by the unweighted pair-group method with arithmetic averages (UPGMA). 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 the 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 ºC for optimal DNA reassociation and at 70 ºC for stringent conditions. The G+C content was determined according to the procedures proposed by Mandel et al. (6). Sequencing of the 16S ribosomal DNA (16S rrna gene) and phylogenetic analysis of the isolates was performed according to 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 non-motile, catalase-negative, Gram-positive cocci, occurring in single, pairs or short chains. Colonies in blood agar were circular and grey, with alpha-hemolysis. They were positive for the Enterococcus Accuprobe assay, leucine aminopeptidase (LAPase) and pyrrolidonyl arylamidase (PYRase) activity tests and for the Voges-Proskauer test. They also hydrolysed esculin in the presence of bile (BE), grew in broth containing 6.5% NaCl and at 10 ºC and 45 ºC, 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 physiologic characteristics of the isolates are shown in Table 1. The ID 32 system generated

6 identical profiles ( , categorized by the system as doubtful profile ) for strains ATCC BAA-781 T (SS 1279, CDC PNS-2) and CCUG (SS 1743). The ID 32 profile ( ) obtained for strain ATCC BAA-780 (SS 1728, CDC PNS-E1) was categorized as unacceptable profile, while the profile ( ) generated for strain DSM T (SS 1773, E. italicus) was categorized as doubtful profile. Comparative analysis showed that WCP profile of strain ATCC BAA-780 (CDC PNS-E1) was highly related to the E. italicus type strain DSM 5952 T profile, while the WCP profile of strain ATCC BAA-781 (CDC PNS-E2) was highly related to the profile of strain CCUG (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. The results of DNA-DNA relatedness experiments performed with strain CCUG (SS 1743) showed that percentages of homology with strain ATCC BAA-781 (CDC PNS-E2) were 84% at the optimum temperature (OT) and 83% at stringent temperature (ST), with % of divergence of 0.5, indicating that they 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 a percentage of homology of 82% at the OT and of 78% at ST, with % of divergence of 1.5. Relative binding ratios (RBRs) expressing the percentages of homology at 55 ºC of these strains with the type strains of all other enterococcal species tested ranged from 6 to 41. Analysis of 16S rdna sequences (approximately 1480 bp) showed species level relationship between strains ATCC BAA-780 (CDC PNS-E1) and DSM

7 T (E. italicus); and between strains ATCC BAA-781 (CDC PNS-E2) and CCUG (SS 1743). A multiple sequence alignment analysis trimmed to consensus comparing these 16S rrna gene sequences, with sequences of selected species of Enterococcus is shown in Fig. 2. Overall, the results of 16S ribosomal DNA (rdna) sequencing, SDS-PAGE analysis of whole-cell protein 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 16S rdna sequence of our isolate ATCC BAA-780 also showed species level similarity with the sequence of E. saccharominimus, confirming the recent finding reported by Naser et al. (5) 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 be 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 (SS 1743), another blood isolate recovered from a patient in Sweden. For these we propose the designation Enterococcus sanguinicola sp. nov. Differently from the first E. sanguinicola isolate, CCUG (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 named provisionally as CDC PNS-E1 (now E. italicus) and CDC PNS- E2 (now E. sanguinicola). The isolates were obtained from clinically significant

8 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 peculiar importance because calls the attention to one additional enterococcal species capable of acquiring such an important resistance marker. Considering the high transmissibility of the vana gene, clinicians and microbiologists should be aware for promptly recognize and identify glycopeptide- resistant isolates of this new enterococcal species. After the present work was completed, one additional S. sanguinicola isolate was identified at the CDC Streptococcus laboratory. The isolate (DS ) had phenotypic characteristics identical to those shown in Table 1 for the other two S. sanguinicola isolates and a ID 32 profile ( ) categorized as doubtful profile. Sequencing of the 16 S gene showed 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 obtained from a human source, we recommend that the species description of E. italicus be emended to indicate that growth in both containing 6.5% NaCl and production of acid from trehalose may be strain variable traits. The description of S. sanguinicola is presented below

9 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, and singly. Nonpigmented, alpha-hemolytic, small colonies of 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% CO 2. It is catalase-negative, nonmotile. Growth occurs at 10 C, at 45 C, and in broth containing 6.5% NaCl. It is positive for pyrrolidonyl arylamidase activity, leucine aminopeptidase activity, hydrolysis of esculin in the presence of bile, hydrolysis of arginine, and Voges-Proskauer test. Acid is produced from glycerol, lactose, maltose, mannitol, ribose, sucrose, and trehalose. Acid is not produced from arabinose, inulin, melibiose, MGP, raffinose, sorbitol and sorbose. It 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 T ), a vancomycin-resistant isolate (with MIC >256 µg/ml; and harboring the vana gene) recovered from the blood of a patient in Los Angeles, California, USA. The G+C content of the type strain is 39.0 mol %. The GenBank accession number for the 16S rdna sequence of Enterococcus sanguinicola strain SS-1729 T (= ATCC BAA-781 T = CCUG T ) is AY

10 LMT received support from 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. epithet. We are grateful to Dr. Hans Trüper for assisting with definition of the species

11 REFERENCES Brenner, D. J., A. C. McWhorter, J. K. Knutson, and A. G. Steigerwalt, Escherichia vulneris: a new species of Enterobacteriaceae associated with human wounds. J. Clin. Microbiol. 15: Carvalho, M. G. S., A. G. Steigerwalt, R. E. Morey, P. L. Shewmaker, L. M. Teixeira, and R. R. Facklam 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: Facklam, R. R., M. G. S. Carvalho, and L. M. Teixeira History, taxonomy, biochemical characteristics, and antibiotic susceptibility testing of enterococci, p In M. S. Gilmore, D. B. Clewell, P. Courvalin, G. M. Dunny, B. E. Murray, and L. B. Rice (ed.), The enterococci: pathogenesis, molecular biology, and antibiotic resistance. American Society for Microbiology, Washington, D.C Fortina, M.G., G. Ricci, D. Mora, and P. L. Manachini Molecular analysis of artisanal Italian cheeses reveals Enterococcus italicus sp. nov. Int. J. Syst. Evol. Microbiol. 54:

12 Naser, S. M., M. Vancanneyt, B. Hoste, C. Snauwaert, K. Vandemeulebroecke, and J. Swings Reclassification of Enterococcus flavescens Pompei et al as a later synonym of Enterococcus casseliflavus (ex Vaughan et al. 1979) Collins et al and Enterococcus saccharominimus Vancanneyt et al as a later synonym of Enterococcus italicus Fortina et al Int. J. Syst. Evol. Microbiol. 56: Mandel, M., L. Igambi, J. Bergendahl, M. L Dodson Jr., and E. Scheltgen Correlation of melting temperature and cesium chloride buoyant density of bacterial deoxyribonucleic acid. J. Bacteriol. 101: Merquior, V. L. C., J. M. Peralta, R. R.Facklam, and L. M. Teixeira Analysis of electrophoretic whole-cell protein profiles as a tool for characterization of Enterococcus species. Curr. Microbiol. 28: Schleifer, K. H., and R. Kilpper-Balz Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb. nov. Int. J. Syst. Bacteriol. 34: Teixeira, L. M., M.G.S. Carvalho, and R. R. Facklam Enterococcus, p In P. R. Murray, E. J. Baron, J. H. Jorgensen, M.L. Landry, and M. A. Pfaller (ed.), Manual of clinical microbiology, 9 th ed. ASM, Washington, D.C. 264

13 Teixeira, L. M., R. R Facklam, A. G. Steigerwalt, N. E. Pigott, V. L. C. Merquior, and D. J. Brenner Correlation between phenotypic characteristics and DNA relatedness within Enterococcus faecium strains. J. Clin. Microbiol. 33: Whiley, R. A., and M. Kilian International Committee on Systematics of Prokaryotes subcommittee on the taxonomy of staphylococci and streptococci: minutes of the closed meeting, 31 July 2002, Paris, France. Int. J. Syst. Evol. Microbiol. 53:

14 TABLE 1. Characteristics a study of the enterococcal strains included in the present Test or Characteristic E. italicus DSM T E. italicus (CDC PNS-E1) ATCC BAA-780 E. sanguinicola (CDC PNS-2) ATCC BAA-781 T E. sanguinicola CCUG Hydrolysis of arginine Susceptibility to vancomycin S S R S Pyruvate utilization Acid production from: Glycerol Mannitol MGP b Ribose Trehalose a All the isolates were positive for: Enterococcus Accuprobe assay, leucine aminopeptidase 280 (LAPase) and pyrrolidonyl arylamidase (PYRase) activity tests, hydrolysis of esculin in the 281 presence of bile BE, Voges-Proskauer test, growth in broth containing 6.5% NaCl, growth at ºC and at 45 ºC, production of acids from lactose, maltose, and sucrose, and reactivity 283 with group D antiserum. All the isolates were negative for: pigment production, production of 284 gas in MRS broth, motility, hippurate hydrolysis and production of acids from arabinose, 285 inulin, melibiose, raffinose, sorbitol, and sorbose. 286 b MGP, Methyl-α-D-glucopyranoside.

15 FIG.1. SDS-PAGE profiles of whole-cell protein extracts of Enterococcus sanguinicola sp. nov. (strains ATCC BAA-781 T and CCUG 47884) and Enterococcus italicus (strains DSM T and ATCC BAA-780) and corresponding dendrogram resulting from computer-assisted analysis of the protein profiles. % Similarity E. italicus ATCC BAA-780 (CDC PNS-E1) E. italicus DSM T E. sanguinicola ATCC BAA-781 T (CDC PNS-E2) E. sanguinicola CCUG MM

16 FIG. 2. Distance matrix tree based in 16SrDNA sequence comparisons showing the phylogenetic relationship of Enterococcus italicus, Enterococcus sanguinicola, and some of the most closely related species of Enterococcus. Vagococcus fluvialis sequence was used as outgroup. Bootstrap probability values (percentage of 1000 tree replications) are indicated at branch-points. Sequences accession numbers are shown in parentheses E. pseudoavium ATCC T (DQ411809) E. sp CDC PNS-E3 ATCC BAA-782 T (AY321377) E. gilvus ATCC BAA-350 T (DQ411810) E. avium ATCC T (DQ411811) E. pallens ATCC BAA-351 T (DQ411812) 98 E. sanguinicola CCUG (DQ411817) E. sanguinicola ATCC BAA-781 T (AY321376) E. faecium ATCC T (DQ411813) E. italicus ATCC BAA-780 (AY321375) E. italicus DSM T (AJ582753) E. sulfureus ATCC T (DQ411815) E. saccharolyticus ATCC T (DQ411816) E. faecalis ATCC T (DQ411814) Vagococcus fluvialis NCDO 2497 T (X54258)