Identification of Clinically Relevant Enterococcus
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微生物临床杂志 2005年第1期
Division of Neurosurgery, Department of Surgery
Department of Laboratory Medicine, National Taiwan University Hospital
Center for Optoelectronic Biomedicine
School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan
ABSTRACT
We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.
INTRODUCTION
Enterococci are now one of the leading causes of major nosocomial infections (21). The Enterococcus genus comprises more than 21 species. Enterococcus faecalis and Enterococcus faecium are commonly found in patients with bacteremia, surgical wound infections, urinary tract infections, or endocarditis and are responsible for the infections. Other Enterococcus species have also been implicated in human infections (4, 14, 23, 26). It is worth noting that the emergence of vancomycin-resistant enterococci has caused alarm in the global infectious diseases research community (11, 13).
Rapid and correct identification of causative microorganisms is always important in the diagnostic workup of patients with infections. Effective, instead of empirical, antibiotic therapy can be directed toward the known causative organism, leading to more effective management of the patient and may reduce the spread of multidrug-resistant bacteria. However, conventional methods for the identification of microorganisms are based on phenotypic and culture characteristics and may not be able to identify the causative organism correctly when strains with unusual phenotypes, such as nonmotile Enterococcus gallinarum or Enterococcus casseliflavus, are encountered (22). Besides, conventional methods commonly require 2 to 7 days for a definitive result. At this time, in many laboratories, species identification of enterococci relies on automation or rapid kits (7, 15), which are not always reliable. Species identification by the automated identification systems is based on a built-in database and may cause errors which cannot be detected easily. Moreover, the occurrence of atypical phenotypic characteristics in some microorganisms may also lead to misidentifications (28, 34).
To decrease the possibility of misidentification, molecular identification has been considered an alternative to conventional methods (1-3, 5). Among these new methods, small-subunit (16S) ribosomal rRNA gene sequencing is widely accepted as a tool for identifying bacterial isolates. The application of small-subunit (16S) ribosomal rRNA gene sequencing to the identification of aerobic catalase-negative gram-positive cocci was recently evaluated by Bosshard et al. (3). Besides 16S rRNA genes, a variety of other target genes have been used for the identification of bacterial pathogens; for example, the tRNA intergenic spacer, ddl, sod, or tuf gene has been used for the identification of Enterococcus species by many researchers (1, 2, 6, 16, 20, 24, 35, 36). Previously, we determined the full-length sequence of the E. faecalis groESL genes containing groES (282 bp), spacer, and groEL (1,623 bp) and developed a species identification method for E. faecalis based on this sequence instead of 16S rRNA gene sequences (31). In the present study, we determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences.
MATERIALS AND METHODS
Bacterial strains. Thirteen reference strains (from 10 species of Enterococcus and 1 species of Vagococcus) and 74 clinical isolates were included in this study. Enterococcus faecalis ATCC 19433, E. faecalis ATCC 29212, Enterococcus faecium ATCC 19434, E. faecium ATCC 35667, Enterococcus avium ATCC 14025, Enterococcus casseliflavus ATCC 25788, Enterococcus gallinarum ATCC 49573, Enterococcus mundtii ATCC 43186, Enterococcus raffinosus ATCC 49427, Enterococcus hirae ATCC 8043, Enterococcus cecorum ATCC 43198, Enterococcus durans ATCC 19432, and Vagococcus fluvialis ATCC 49525 were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). From May 2002 to April 2003, a total of 114 clinical isolates of Enterococcus from blood culture were collected in the Bacteriology Laboratory of the National Taiwan University Hospital, a 2,000-bed teaching hospital in northern Taiwan. All the clinical isolates in each species of Enterococcus were included in this study if the number of isolates was less than 10. For E. faecalis, 17 isolates were selected randomly from 47 collections; for E. faecium, 26 of 36 isolates were selected. Thus, a total of 74 clinical isolates, including E. faecalis (n = 17), E. faecium (n = 26), E. avium (n = 5), E. casseliflavus (n = 7), E. cecorum (n = 3), E. durans (n = 1), E. gallinarum (n = 5), E. hirae (n = 5), and E. raffinosus (n = 5), were included in this study. The clinical isolates were all reidentified by the Rapid ID 32 STREP system (bioMerieux Vitek, Marcy l'Etoile, France) and molecular methods, including 16S rRNA gene and groESL sequencing.
Determination of the groESL sequences of reference strains. Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.) according to the instructions of the manufacturer. Three sets of primers were used for the amplification and sequencing of the groESL genes. Primers Ent-ES-UP (5'-TTATAAAYAGTGTRRGTTAGCACTC-3', where Y is C or T and R is A or G) and Ent-EL-215-196 (5'-AATTTTGCDCCCATRTTTTC-3') were used to amplify the groES genes and spacer region. Primers EntGroES-4-23 (5'-TTRAARCCNTTRGGNGAYCG-3', where N is A, C, T, or G) and EL-842-817 (5'-TCNCCRAANCCNGGYGCNTTNACNGC-3') and primers EntGroEL-F-481-500 (5'-GCTGAHGCRATGGAAAAAGT-3') and EntGroEL-R-1613-1591 (5'-CCCATNCCCATNGANGGRTCCAT-3') were used to amplify two overlapping fragments of groESL genes by PCR. The PCR was performed in a DNA thermal cycler (MJ Research, Inc., Watertown, Mass.). Thirty cycles of PCR, with 1 cycle consisting of denaturation (30 s at 94°C), annealing (1 min at 50°C), and extension (1.5 min at 72°C), were done. This was followed by a final extension step (7 min at 72°C). Amplification fragments were subsequently sequenced on a genetic analyzer (model ABI PRISM 3100; Applied Biosystems, Foster City, Calif.) with a Taq BigDye-Deoxy Terminator cycle sequencing kit (Applied Biosystems), according to the instructions of the manufacturer.
Intraspecies polymorphism. The intraspecies polymorphism of the groES genes, spacer, and partial groEL genes were evaluated among the clinical isolates of Enterococcus species. Bacterial samples were subjected to PCR amplification of groES and spacer region with primers Ent-ES-UP and Ent-EL-215-196. PCR with primers Ent-GroEL-F-481-500 and Ent-GroEL-1352-1332 (5'-ATTTGACGRAYTGGTTCTTCT-3') was used to amplify the partial groEL fragment. The amplified PCR products were subsequently sequenced.
Similarity between species and phylogeny of the groESL sequences. DNA and deduced amino acid sequences were compared and aligned using the Gene-Works software (IntelliGenetics, Mountain View, Calif.). The phylogenetic relationships among species were analyzed by the neighbor-joining (NJ) method listed in the MEGA2 (molecular evolutionary genetic analysis) analytical package (17). For the NJ analysis, the distance between the sequences was calculated using Kimura's two-parameter model. Levels of similarity between species were determined. Bootstrap values were obtained on 500 randomly generated trees.
Nucleotide sequence accession numbers. The sequences of the enterococcal groESL genes of ATCC reference strains determined in this study have been submitted to the GenBank database. Their accession numbers were listed in Table 1.
RESULTS
Nucleotide sequences of the enterococcal groESL genes. The nearly full-length sequences of the groESL genes were determined from 10 reference species of Enterococcus and a closely related genus, V. fluvialis. The Enterococcus species included phenotype group I (E. avium and E. raffinosus), phenotype group II (E. faecalis, E. faecium, E. casseliflavus, E. mundtii, and E. gallinarum), phenotype group III (E. durans and E. hirae), and phenotype group IV (E. cecorum).
The full-length sequences of groES were obtained from all the reference strains of Enterococcus species studied here. Sequence determination showed that the groES genes of all the Enterococcus species studied here are 285 nucleotides long and contain an unusual putative start codon, GTG. The similarities of the groES sequences among Enterococcus species ranged from 73.0 to 94.1% at the nucleotide sequence level and 73.9 to 98.9% at the amino acid level. The similarities of the deduced amino acid sequence of GroES were highest for E. avium and E. raffinosus (98.9%), followed by E. casseliflavus and E. gallinarum or E. hirae and E. faecium (96.8%). The identities of the groES sequences between V. fluvialis and each of the Enterococcus species ranged from 68.0 to 72.9% (nucleotide sequences) and 60.1 to 66.7% (amino acid sequences).
The overall interspecies similarities of the groEL sequences of the Enterococcus species studied here ranged from 77.9 to 88.0% at the nucleotide sequence level and 85.9 to 98.7% at the amino acid sequence level. The similarities of the deduced amino acid sequences of GroEL was highest (98.7%) for E. faecium and E. durans (or E. hirae), followed by E. avium and E. raffinosus or E. casseliflavus and E. gallinarum (98.1%). The identities of the groEL sequences between V. fluvialis and each of the Enterococcus species ranged from 76.2 to 78.8% (nucleotide sequences) and 81.0 to 83.5% (amino acid sequences).
The lengths of the spacers between groES and groEL ranged from 17 to 57 bp and were species specific (Table 2). Each of the spacers contained a putative ribosome-binding sequence, GGTGA, for the groEL gene.
Phylogenetic trees inferred from groES and 16S rRNA genes. The unrooted phylogenetic trees constructed from the alignment of the nucleotide sequences of the groES genes are shown in Fig. 1A. The 10 Enterococcus species clustered together. Of the Enterococcus species, E. faecium, E. raffinosus, E. hirae, E. cecorum, and E. mundtii clustered together in a group, while E. gallinarum, E. avium, and E. durans clustered together in another group. According to the phylogenetic analysis based on the groES genes, E. cecorum was most closely related to E. mundtii. The phylogeny inferred from the 16S rRNA gene sequences from GenBank is shown in Fig. 1B. The clusters did not coincide completely with the clusters by groES phylogeny. However, species within a genus usually clustered together.
Intraspecies variations in clinical isolates. The intraspecies variations of the groES, spacer, and groEL sequences in each enterococcal species were investigated. To evaluate the feasibility of identifying species on the basis of the sequences of the groESL genes, we compared the sequences of groES genes (full-length, 285 bp), spacer, and partial groEL genes (577 bp, nucleotide positions 512 to 1088) of 74 clinical isolates and analyzed intraspecies variation. The comparisons of these sequences between clinical isolates and the corresponding reference strains are shown in Table 3. The intraspecies identity of the groES sequences ranged from 97.5 to 100%, while that of groEL ranged from 91.0 to 100%. The least intraspecies similarity of groES was seen among isolates of E. gallinarum (97.5% similarity), followed by E. faecium (98.2% similarity). The least intraspecies similarity of groEL was seen among isolates of E. cecorum (91.0% similarity), followed by E. gallinarum (93.4% similarity) and E. faecium (94.8%). Two distinct types of spacers were found in E. faecium clinical isolates. Type I (the standard or ATCC type), which is identical to the spacers of the two E. faecium ATCC reference strains, was seen in 19 isolates. Type II spacer (the variant type), which had 2 nucleotides changed and 1 nucleotide (G) inserted compared to the ATCC type spacer, was seen in seven isolates of E. faecium (Table 4). Minor variations were also observed in some clinical isolates of E. casseliflavus, E. gallinarum, and E. hirae (Table 4). In clinical isolates of E. faecalis, E. avium, E. raffinosus, E. cecorum, and E. durans, no sequence variations in spacers were observed (Table 3).
Discrepant results of identification of clinical isolates by the Rapid ID 32 STREP system and by sequencing 16S rRNA genes, groES, and spacers. Isolates with discrepant results by the Rapid ID 32 STREP system and by sequencing 16S rRNA genes, groES, and spacers were listed in Table 5. Note that E. cecorum and E. mundtii strains could not be identified by the Rapid ID 32 STREP system.
DISCUSSION
Accurate identification of enterococcal species has become increasing important as acquired vancomycin resistance has become more prevalent (11). Rapid and sensitive DNA-based assays may improve the speed and accuracy in the diagnosis of enterococcal infections. To date, amplification of the target genome by PCR followed by sequencing and phylogenetic analysis has been generally accepted as the technique for classification and identification of microorganisms.
The groESL genes, which encode the 10-kDa and 60-kDa heat shock proteins (cpn10/60, groESL), are ubiquitous and highly conserved among bacteria (12). The cpn60 (groEL) genes have been used for the identification of many microorganisms, such as Staphylococcus (10), Enterococcus (9, 31), Streptococcus (8, 32), Ehrlichia (30), Bartonella (19), Mycobacterium (25, 29), and Rickettsia (18) species. In contrast to groEL genes, groES genes have been used only for the identification of a limited number of species (31, 32).
Previously, we successfully identified the groESL sequences of E. faecalis (31). In this study, we further determined the groESL sequences (full-length sequences of groES, spacer, and nearly full-length sequences of groEL) for a panel of Enterococcus species and evaluated the possibility of differentiating Enterococcus species based on these sequences. We found that the structures of enterococcal groESL genes were similar to those of most bacterial species studied by others (27, 33). The nucleotide and deduced amino acid sequences of GroES or GroEL of Enterococcus species exhibited a high degree of overall identity to those of homologous bacterial GroESL (cpn10/60).
All the groES genes of Enterococcus species studied here are 285 nucleotides long and contain an unusual putative start codon, GTG. It has been suggested that non-AUG initiation codons might limit the expression of genes at the translational level (27). The significance of this unusual start codon in Enterococcus species is not clear. Sequence analysis showed a higher divergence for groES genes (73.0 to 94.1%) than for groEL genes (77.9 to 88.0%) in Enterococcus species; thus, the groES genes could be more discriminative than the groEL genes in the differentiation of closely related Enterococcus species.
The nearly full-length groEL sequences (1,573 bp for most Enterococcus species) determined in this study are in agreement with those published by others and contain the partial sequence (600 bp) of cpn60 which was used for the identification of Enterococcus species by Goh et al. (9). Analysis of the partial or nearly full-length groEL sequences showed a higher similarity among Enterococcus species than between Enterococcus species and other gram-positive bacteria, including V. fluvialis.
Enterococcus species are commonly grouped into five phenotypes. In this study, we showed that species in the same phenotypic group clustered together by phylogenetic analysis based on the groES or groEL sequences. However, some species in different phenotypic groups may also cluster together. For example, E. hirae (phenotype group III) and E. mundtii (phenotype group II) are different in many phenotypic characteristics, but their groES and groEL sequences are 93.6 and 96.8% similar, respectively, at the amino acid level. On the other hand, species with similar phenotypes may fall into different clusters; an example of this is the two yellow-pigmented species, E. mundtii and E. casseliflavus.
The intergenic spacers of the 10 Enterococcus species studied here are between 17 and 57 nucleotides long, and each contains a putative ribosome-binding sequence, GGTGA, for the groEL gene. In a previous study, we found that the spacers of 10 viridans group streptococci are species specific either in length (15 to 111 bp) or sequence (32). In the present study, the spacers of the 10 Enterococcus species are also species specific. The length and sequence of spacers of strains within the same species are usually conserved. Sequence variation of spacers was found in E. casseliflavus, E. faecium, E. gallinarum, and E. hirae. For E. faecium isolates, there were two distinct types of spacers. The spacers differ in length (50 or 51 bp) by one nucleotide, and their sequences differed by two other nucleotides (a total of three nucleotide differences). It is possible that the variation in sequence within a species might increase if more isolates were studied. However, our present data indicate that the sequence of the spacer can provide useful information for the differentiation of Enterococcus species with similar phenotypes and 16S rRNA gene sequences, such as E. avium and E. raffinosus, E. durans and E. hirae, or E. casseliflavus and E. gallinarum. Sequences of the spacer region between other genes have also been used to differentiate between species of other bacteria. For example, Tyrrell et al. (35) found that PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes produced amplicon profiles characteristic of several Enterococcus species.
It is important to distinguish clinically significant E. faecalis or E. faecium from other Enterococcus species. At this time, most identification methods perform reasonably well for the identification of E. faecalis and E. faecium but may have problems with the identification of other enterococcal species. The phenotypic differentiation between E. faecium and E. gallinarum usually relies on the motility test. However, the results of the motility test are not always easy to interpret. Moreover, some strains of E. gallinarum are nonmotile and are difficult to differentiate from E. faecium unless extensive biochemical testing is performed. It has also been reported that strains of E. faecium with atypical phenotypes were misidentified as E. durans or E. hirae by commercial systems alone (28). Differentiation between these three species is also difficult by either 16S or 23S rRNA gene sequences alone. The 16S rRNA gene sequences of E. faecium, E. durans, and E. hirae are highly similar (99.7 to 99.8% identity) (22). Taking all the data together, the groES sequences are more discriminative than the 16S rRNA gene sequences in differentiating Enterococcus species. In this study, isolates with a doubtful result identified by the Rapid ID 32 STREP system or isolates with discrepant data by 16S rRNA gene sequences and Rapid ID 32 STREP system could be identified on the basis of groES and spacer region sequences.
For E. cecorum isolates, identification based on the groES sequences (with 99.3 to 99.6% intraspecies identity) or spacer sequences (with 100% intraspecies identity) may be more accurate than that based on the groEL sequences, since the intraspecies variation of groEL among E. cecorum isolates was relatively high (91.0 to 95.9% identity). The groEL sequences of E. cecorum strains (one reference strain and three clinical isolates examined) are 6 nucleotides (nucleotide positions 1291 to 1296) longer than those of other Enterococcus species. Of the 577-bp sequence of the partial groEL fragment examined, there were 50 nucleotide differences between the sequences of the two clinical isolates and the E. cecorum reference strain. E. cecorum has been recognized as a significant pathogen in humans (14). Phenotypically, it is possible to differentiate E. cecorum from other Enterococcus species by its characteristics of negative pyrrolidonyl-arylamidase activity and inability to grow on 6.5% NaCl medium. However, E. cecorum is easily misidentified as Streptococcus species or other related gram-positive cocci by routine identification methods, because many commercial systems do not have E. cecorum data in their database. In this study, we showed that E. cecorum can be correctly identified on the basis of its groES or spacer sequence.
The genotypic methods, such as PCR and direct sequencing of groES and spacer used in this study, may not be able to replace the phenotypic methods currently in use because of their cost and lack of automation. However, the genotypic methods are more rapid and may provide additional information for bacterial identification. We understand that bacterial identification on the basis of these small parts of the microbial genome could be limited by the cost and the availability of known sequences in database. However, the costs of PCR and direct sequencing are decreasing as these methods are getting more and more popular. We believe that the accumulation of more and more nucleotide sequences in the database will solve the other problems in the near future.
In conclusion, our results indicate that the groES and spacer sequences are useful for identification of 10 clinically relevant Enterococcus species. The groES and spacer sequences may become useful alternative genetic targets for the identification of Enterococcus species, especially when the biochemical profiles are ambiguous or when species identification by using the 16S rRNA gene sequences is inconclusive. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing, and may provide important information for the differentiation of closely related species of Enterococcus.
ACKNOWLEDGMENTS
This work was supported in part by grant NSC 90-2320-B-002-188 from the National Science Council of Taiwan.
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Department of Laboratory Medicine, National Taiwan University Hospital
Center for Optoelectronic Biomedicine
School of Medical Technology, National Taiwan University College of Medicine, Taipei, Taiwan
ABSTRACT
We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.
INTRODUCTION
Enterococci are now one of the leading causes of major nosocomial infections (21). The Enterococcus genus comprises more than 21 species. Enterococcus faecalis and Enterococcus faecium are commonly found in patients with bacteremia, surgical wound infections, urinary tract infections, or endocarditis and are responsible for the infections. Other Enterococcus species have also been implicated in human infections (4, 14, 23, 26). It is worth noting that the emergence of vancomycin-resistant enterococci has caused alarm in the global infectious diseases research community (11, 13).
Rapid and correct identification of causative microorganisms is always important in the diagnostic workup of patients with infections. Effective, instead of empirical, antibiotic therapy can be directed toward the known causative organism, leading to more effective management of the patient and may reduce the spread of multidrug-resistant bacteria. However, conventional methods for the identification of microorganisms are based on phenotypic and culture characteristics and may not be able to identify the causative organism correctly when strains with unusual phenotypes, such as nonmotile Enterococcus gallinarum or Enterococcus casseliflavus, are encountered (22). Besides, conventional methods commonly require 2 to 7 days for a definitive result. At this time, in many laboratories, species identification of enterococci relies on automation or rapid kits (7, 15), which are not always reliable. Species identification by the automated identification systems is based on a built-in database and may cause errors which cannot be detected easily. Moreover, the occurrence of atypical phenotypic characteristics in some microorganisms may also lead to misidentifications (28, 34).
To decrease the possibility of misidentification, molecular identification has been considered an alternative to conventional methods (1-3, 5). Among these new methods, small-subunit (16S) ribosomal rRNA gene sequencing is widely accepted as a tool for identifying bacterial isolates. The application of small-subunit (16S) ribosomal rRNA gene sequencing to the identification of aerobic catalase-negative gram-positive cocci was recently evaluated by Bosshard et al. (3). Besides 16S rRNA genes, a variety of other target genes have been used for the identification of bacterial pathogens; for example, the tRNA intergenic spacer, ddl, sod, or tuf gene has been used for the identification of Enterococcus species by many researchers (1, 2, 6, 16, 20, 24, 35, 36). Previously, we determined the full-length sequence of the E. faecalis groESL genes containing groES (282 bp), spacer, and groEL (1,623 bp) and developed a species identification method for E. faecalis based on this sequence instead of 16S rRNA gene sequences (31). In the present study, we determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences.
MATERIALS AND METHODS
Bacterial strains. Thirteen reference strains (from 10 species of Enterococcus and 1 species of Vagococcus) and 74 clinical isolates were included in this study. Enterococcus faecalis ATCC 19433, E. faecalis ATCC 29212, Enterococcus faecium ATCC 19434, E. faecium ATCC 35667, Enterococcus avium ATCC 14025, Enterococcus casseliflavus ATCC 25788, Enterococcus gallinarum ATCC 49573, Enterococcus mundtii ATCC 43186, Enterococcus raffinosus ATCC 49427, Enterococcus hirae ATCC 8043, Enterococcus cecorum ATCC 43198, Enterococcus durans ATCC 19432, and Vagococcus fluvialis ATCC 49525 were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). From May 2002 to April 2003, a total of 114 clinical isolates of Enterococcus from blood culture were collected in the Bacteriology Laboratory of the National Taiwan University Hospital, a 2,000-bed teaching hospital in northern Taiwan. All the clinical isolates in each species of Enterococcus were included in this study if the number of isolates was less than 10. For E. faecalis, 17 isolates were selected randomly from 47 collections; for E. faecium, 26 of 36 isolates were selected. Thus, a total of 74 clinical isolates, including E. faecalis (n = 17), E. faecium (n = 26), E. avium (n = 5), E. casseliflavus (n = 7), E. cecorum (n = 3), E. durans (n = 1), E. gallinarum (n = 5), E. hirae (n = 5), and E. raffinosus (n = 5), were included in this study. The clinical isolates were all reidentified by the Rapid ID 32 STREP system (bioMerieux Vitek, Marcy l'Etoile, France) and molecular methods, including 16S rRNA gene and groESL sequencing.
Determination of the groESL sequences of reference strains. Genomic DNA was isolated using the Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.) according to the instructions of the manufacturer. Three sets of primers were used for the amplification and sequencing of the groESL genes. Primers Ent-ES-UP (5'-TTATAAAYAGTGTRRGTTAGCACTC-3', where Y is C or T and R is A or G) and Ent-EL-215-196 (5'-AATTTTGCDCCCATRTTTTC-3') were used to amplify the groES genes and spacer region. Primers EntGroES-4-23 (5'-TTRAARCCNTTRGGNGAYCG-3', where N is A, C, T, or G) and EL-842-817 (5'-TCNCCRAANCCNGGYGCNTTNACNGC-3') and primers EntGroEL-F-481-500 (5'-GCTGAHGCRATGGAAAAAGT-3') and EntGroEL-R-1613-1591 (5'-CCCATNCCCATNGANGGRTCCAT-3') were used to amplify two overlapping fragments of groESL genes by PCR. The PCR was performed in a DNA thermal cycler (MJ Research, Inc., Watertown, Mass.). Thirty cycles of PCR, with 1 cycle consisting of denaturation (30 s at 94°C), annealing (1 min at 50°C), and extension (1.5 min at 72°C), were done. This was followed by a final extension step (7 min at 72°C). Amplification fragments were subsequently sequenced on a genetic analyzer (model ABI PRISM 3100; Applied Biosystems, Foster City, Calif.) with a Taq BigDye-Deoxy Terminator cycle sequencing kit (Applied Biosystems), according to the instructions of the manufacturer.
Intraspecies polymorphism. The intraspecies polymorphism of the groES genes, spacer, and partial groEL genes were evaluated among the clinical isolates of Enterococcus species. Bacterial samples were subjected to PCR amplification of groES and spacer region with primers Ent-ES-UP and Ent-EL-215-196. PCR with primers Ent-GroEL-F-481-500 and Ent-GroEL-1352-1332 (5'-ATTTGACGRAYTGGTTCTTCT-3') was used to amplify the partial groEL fragment. The amplified PCR products were subsequently sequenced.
Similarity between species and phylogeny of the groESL sequences. DNA and deduced amino acid sequences were compared and aligned using the Gene-Works software (IntelliGenetics, Mountain View, Calif.). The phylogenetic relationships among species were analyzed by the neighbor-joining (NJ) method listed in the MEGA2 (molecular evolutionary genetic analysis) analytical package (17). For the NJ analysis, the distance between the sequences was calculated using Kimura's two-parameter model. Levels of similarity between species were determined. Bootstrap values were obtained on 500 randomly generated trees.
Nucleotide sequence accession numbers. The sequences of the enterococcal groESL genes of ATCC reference strains determined in this study have been submitted to the GenBank database. Their accession numbers were listed in Table 1.
RESULTS
Nucleotide sequences of the enterococcal groESL genes. The nearly full-length sequences of the groESL genes were determined from 10 reference species of Enterococcus and a closely related genus, V. fluvialis. The Enterococcus species included phenotype group I (E. avium and E. raffinosus), phenotype group II (E. faecalis, E. faecium, E. casseliflavus, E. mundtii, and E. gallinarum), phenotype group III (E. durans and E. hirae), and phenotype group IV (E. cecorum).
The full-length sequences of groES were obtained from all the reference strains of Enterococcus species studied here. Sequence determination showed that the groES genes of all the Enterococcus species studied here are 285 nucleotides long and contain an unusual putative start codon, GTG. The similarities of the groES sequences among Enterococcus species ranged from 73.0 to 94.1% at the nucleotide sequence level and 73.9 to 98.9% at the amino acid level. The similarities of the deduced amino acid sequence of GroES were highest for E. avium and E. raffinosus (98.9%), followed by E. casseliflavus and E. gallinarum or E. hirae and E. faecium (96.8%). The identities of the groES sequences between V. fluvialis and each of the Enterococcus species ranged from 68.0 to 72.9% (nucleotide sequences) and 60.1 to 66.7% (amino acid sequences).
The overall interspecies similarities of the groEL sequences of the Enterococcus species studied here ranged from 77.9 to 88.0% at the nucleotide sequence level and 85.9 to 98.7% at the amino acid sequence level. The similarities of the deduced amino acid sequences of GroEL was highest (98.7%) for E. faecium and E. durans (or E. hirae), followed by E. avium and E. raffinosus or E. casseliflavus and E. gallinarum (98.1%). The identities of the groEL sequences between V. fluvialis and each of the Enterococcus species ranged from 76.2 to 78.8% (nucleotide sequences) and 81.0 to 83.5% (amino acid sequences).
The lengths of the spacers between groES and groEL ranged from 17 to 57 bp and were species specific (Table 2). Each of the spacers contained a putative ribosome-binding sequence, GGTGA, for the groEL gene.
Phylogenetic trees inferred from groES and 16S rRNA genes. The unrooted phylogenetic trees constructed from the alignment of the nucleotide sequences of the groES genes are shown in Fig. 1A. The 10 Enterococcus species clustered together. Of the Enterococcus species, E. faecium, E. raffinosus, E. hirae, E. cecorum, and E. mundtii clustered together in a group, while E. gallinarum, E. avium, and E. durans clustered together in another group. According to the phylogenetic analysis based on the groES genes, E. cecorum was most closely related to E. mundtii. The phylogeny inferred from the 16S rRNA gene sequences from GenBank is shown in Fig. 1B. The clusters did not coincide completely with the clusters by groES phylogeny. However, species within a genus usually clustered together.
Intraspecies variations in clinical isolates. The intraspecies variations of the groES, spacer, and groEL sequences in each enterococcal species were investigated. To evaluate the feasibility of identifying species on the basis of the sequences of the groESL genes, we compared the sequences of groES genes (full-length, 285 bp), spacer, and partial groEL genes (577 bp, nucleotide positions 512 to 1088) of 74 clinical isolates and analyzed intraspecies variation. The comparisons of these sequences between clinical isolates and the corresponding reference strains are shown in Table 3. The intraspecies identity of the groES sequences ranged from 97.5 to 100%, while that of groEL ranged from 91.0 to 100%. The least intraspecies similarity of groES was seen among isolates of E. gallinarum (97.5% similarity), followed by E. faecium (98.2% similarity). The least intraspecies similarity of groEL was seen among isolates of E. cecorum (91.0% similarity), followed by E. gallinarum (93.4% similarity) and E. faecium (94.8%). Two distinct types of spacers were found in E. faecium clinical isolates. Type I (the standard or ATCC type), which is identical to the spacers of the two E. faecium ATCC reference strains, was seen in 19 isolates. Type II spacer (the variant type), which had 2 nucleotides changed and 1 nucleotide (G) inserted compared to the ATCC type spacer, was seen in seven isolates of E. faecium (Table 4). Minor variations were also observed in some clinical isolates of E. casseliflavus, E. gallinarum, and E. hirae (Table 4). In clinical isolates of E. faecalis, E. avium, E. raffinosus, E. cecorum, and E. durans, no sequence variations in spacers were observed (Table 3).
Discrepant results of identification of clinical isolates by the Rapid ID 32 STREP system and by sequencing 16S rRNA genes, groES, and spacers. Isolates with discrepant results by the Rapid ID 32 STREP system and by sequencing 16S rRNA genes, groES, and spacers were listed in Table 5. Note that E. cecorum and E. mundtii strains could not be identified by the Rapid ID 32 STREP system.
DISCUSSION
Accurate identification of enterococcal species has become increasing important as acquired vancomycin resistance has become more prevalent (11). Rapid and sensitive DNA-based assays may improve the speed and accuracy in the diagnosis of enterococcal infections. To date, amplification of the target genome by PCR followed by sequencing and phylogenetic analysis has been generally accepted as the technique for classification and identification of microorganisms.
The groESL genes, which encode the 10-kDa and 60-kDa heat shock proteins (cpn10/60, groESL), are ubiquitous and highly conserved among bacteria (12). The cpn60 (groEL) genes have been used for the identification of many microorganisms, such as Staphylococcus (10), Enterococcus (9, 31), Streptococcus (8, 32), Ehrlichia (30), Bartonella (19), Mycobacterium (25, 29), and Rickettsia (18) species. In contrast to groEL genes, groES genes have been used only for the identification of a limited number of species (31, 32).
Previously, we successfully identified the groESL sequences of E. faecalis (31). In this study, we further determined the groESL sequences (full-length sequences of groES, spacer, and nearly full-length sequences of groEL) for a panel of Enterococcus species and evaluated the possibility of differentiating Enterococcus species based on these sequences. We found that the structures of enterococcal groESL genes were similar to those of most bacterial species studied by others (27, 33). The nucleotide and deduced amino acid sequences of GroES or GroEL of Enterococcus species exhibited a high degree of overall identity to those of homologous bacterial GroESL (cpn10/60).
All the groES genes of Enterococcus species studied here are 285 nucleotides long and contain an unusual putative start codon, GTG. It has been suggested that non-AUG initiation codons might limit the expression of genes at the translational level (27). The significance of this unusual start codon in Enterococcus species is not clear. Sequence analysis showed a higher divergence for groES genes (73.0 to 94.1%) than for groEL genes (77.9 to 88.0%) in Enterococcus species; thus, the groES genes could be more discriminative than the groEL genes in the differentiation of closely related Enterococcus species.
The nearly full-length groEL sequences (1,573 bp for most Enterococcus species) determined in this study are in agreement with those published by others and contain the partial sequence (600 bp) of cpn60 which was used for the identification of Enterococcus species by Goh et al. (9). Analysis of the partial or nearly full-length groEL sequences showed a higher similarity among Enterococcus species than between Enterococcus species and other gram-positive bacteria, including V. fluvialis.
Enterococcus species are commonly grouped into five phenotypes. In this study, we showed that species in the same phenotypic group clustered together by phylogenetic analysis based on the groES or groEL sequences. However, some species in different phenotypic groups may also cluster together. For example, E. hirae (phenotype group III) and E. mundtii (phenotype group II) are different in many phenotypic characteristics, but their groES and groEL sequences are 93.6 and 96.8% similar, respectively, at the amino acid level. On the other hand, species with similar phenotypes may fall into different clusters; an example of this is the two yellow-pigmented species, E. mundtii and E. casseliflavus.
The intergenic spacers of the 10 Enterococcus species studied here are between 17 and 57 nucleotides long, and each contains a putative ribosome-binding sequence, GGTGA, for the groEL gene. In a previous study, we found that the spacers of 10 viridans group streptococci are species specific either in length (15 to 111 bp) or sequence (32). In the present study, the spacers of the 10 Enterococcus species are also species specific. The length and sequence of spacers of strains within the same species are usually conserved. Sequence variation of spacers was found in E. casseliflavus, E. faecium, E. gallinarum, and E. hirae. For E. faecium isolates, there were two distinct types of spacers. The spacers differ in length (50 or 51 bp) by one nucleotide, and their sequences differed by two other nucleotides (a total of three nucleotide differences). It is possible that the variation in sequence within a species might increase if more isolates were studied. However, our present data indicate that the sequence of the spacer can provide useful information for the differentiation of Enterococcus species with similar phenotypes and 16S rRNA gene sequences, such as E. avium and E. raffinosus, E. durans and E. hirae, or E. casseliflavus and E. gallinarum. Sequences of the spacer region between other genes have also been used to differentiate between species of other bacteria. For example, Tyrrell et al. (35) found that PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes produced amplicon profiles characteristic of several Enterococcus species.
It is important to distinguish clinically significant E. faecalis or E. faecium from other Enterococcus species. At this time, most identification methods perform reasonably well for the identification of E. faecalis and E. faecium but may have problems with the identification of other enterococcal species. The phenotypic differentiation between E. faecium and E. gallinarum usually relies on the motility test. However, the results of the motility test are not always easy to interpret. Moreover, some strains of E. gallinarum are nonmotile and are difficult to differentiate from E. faecium unless extensive biochemical testing is performed. It has also been reported that strains of E. faecium with atypical phenotypes were misidentified as E. durans or E. hirae by commercial systems alone (28). Differentiation between these three species is also difficult by either 16S or 23S rRNA gene sequences alone. The 16S rRNA gene sequences of E. faecium, E. durans, and E. hirae are highly similar (99.7 to 99.8% identity) (22). Taking all the data together, the groES sequences are more discriminative than the 16S rRNA gene sequences in differentiating Enterococcus species. In this study, isolates with a doubtful result identified by the Rapid ID 32 STREP system or isolates with discrepant data by 16S rRNA gene sequences and Rapid ID 32 STREP system could be identified on the basis of groES and spacer region sequences.
For E. cecorum isolates, identification based on the groES sequences (with 99.3 to 99.6% intraspecies identity) or spacer sequences (with 100% intraspecies identity) may be more accurate than that based on the groEL sequences, since the intraspecies variation of groEL among E. cecorum isolates was relatively high (91.0 to 95.9% identity). The groEL sequences of E. cecorum strains (one reference strain and three clinical isolates examined) are 6 nucleotides (nucleotide positions 1291 to 1296) longer than those of other Enterococcus species. Of the 577-bp sequence of the partial groEL fragment examined, there were 50 nucleotide differences between the sequences of the two clinical isolates and the E. cecorum reference strain. E. cecorum has been recognized as a significant pathogen in humans (14). Phenotypically, it is possible to differentiate E. cecorum from other Enterococcus species by its characteristics of negative pyrrolidonyl-arylamidase activity and inability to grow on 6.5% NaCl medium. However, E. cecorum is easily misidentified as Streptococcus species or other related gram-positive cocci by routine identification methods, because many commercial systems do not have E. cecorum data in their database. In this study, we showed that E. cecorum can be correctly identified on the basis of its groES or spacer sequence.
The genotypic methods, such as PCR and direct sequencing of groES and spacer used in this study, may not be able to replace the phenotypic methods currently in use because of their cost and lack of automation. However, the genotypic methods are more rapid and may provide additional information for bacterial identification. We understand that bacterial identification on the basis of these small parts of the microbial genome could be limited by the cost and the availability of known sequences in database. However, the costs of PCR and direct sequencing are decreasing as these methods are getting more and more popular. We believe that the accumulation of more and more nucleotide sequences in the database will solve the other problems in the near future.
In conclusion, our results indicate that the groES and spacer sequences are useful for identification of 10 clinically relevant Enterococcus species. The groES and spacer sequences may become useful alternative genetic targets for the identification of Enterococcus species, especially when the biochemical profiles are ambiguous or when species identification by using the 16S rRNA gene sequences is inconclusive. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing, and may provide important information for the differentiation of closely related species of Enterococcus.
ACKNOWLEDGMENTS
This work was supported in part by grant NSC 90-2320-B-002-188 from the National Science Council of Taiwan.
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