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Journal of Clinical Microbiology, May 2005, p. 2224-2230, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2224-2230.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Phylogeny and Identification of Enterococci by atpA Gene Sequence Analysis

S. Naser,1,2* F. L. Thompson,2 B. Hoste,1,2 D. Gevers,3 K. Vandemeulebroecke,2 I. Cleenwerck,2 C. C. Thompson,1,2 M. Vancanneyt,2 and J. Swings1,2

Laboratory of Microbiology,1 BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium,2 Bioinformatics & Evolutionary Genomics, Ghent University/VIB, Technologiepark 927, Ghent 9052, Belgium3

Received 13 September 2004/ Returned for modification 23 November 2004/ Accepted 6 December 2004


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ABSTRACT
 
The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed >99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.


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INTRODUCTION
 
The genus Enterococcus comprises 30 validly published species (www.bacterio.cict.fr/e/enterococcus.html) of gram-positive, oxidase- and catalase-negative, non-spore-forming, ovoid bacteria that are arranged in single cells, pairs, or chains. The genus Enterococcus was first proposed by Schleifer and Kilpper-Bälz in 1984 (31). Enterococci belong to the lactic acid bacteria, which are part of the clostridial branch of the gram-positive bacteria. The closest phylogenetic neighbors of enterococci are Tetragenococcus, Vagococcus, Carnobacterium, and Aerococcus (16, 20). The classification of the enterococci underwent considerable changes in recent years. Since the recognition of Enterococcus as a separate genus (31), several new species, e.g., Enterococcus canis (4), E. hermanniensis (21), and E. phoeniculicola (22), have been described as a result of improvements of the methods for their classification. In addition, E. porcinus and E. seriolicida were reclassified as E. villorum (4) and Lactococcus garvieae (35), respectively. The phylogenetic relationship of the different species within the genus Enterococcus has been determined by comparative sequence analysis of their 16S rRNA genes. Different species groups can be distinguished on the basis of these data (6, 12, 20).

Several molecular biology-based techniques, such as multilocus sequence typing, randomly amplified polymorphic DNA (RAPD) analysis, 16S rRNA gene sequencing, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis (PFGE), and intergenic ribosomal PCR, have been used to identify enterococci to the species and the strain levels (1, 2, 3, 5, 8, 17, 26). AFLP analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) are among the most reliable techniques currently used for Enterococcus species identification (39). However, AFLP analysis and SDS-PAGE may present problems concerning reproducibility and data portability. 16S rRNA gene sequences have limited discriminating power for several closely related enterococcal species, e.g., members of the E. faecium species group (7, 29, 38). PFGE was found to be superior for interpretation of the interstrain relationships among enterococci but did not result in species-specific discriminative DNA bands (5).

Recent in silico studies based on the whole-genome sequences of different bacterial groups proposed that atpA may be an alternative phylogenetic and identification marker for Enterococcus (14, 15, 23, 40). atpA codes for the {alpha} subunit of the bacterial ATP synthase, which functions in ATP synthesis coupled to proton transport (24). The aim of present study was to analyze the usefulness of atpA gene sequences for the reliable identification of Enterococcus species.


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MATERIALS AND METHODS
 
The strains used in this study are listed in Table 1. The strains were grown on blood agar medium (Columbia agar base) under microaerophilic conditions by using CO2-Gen (Oxoid Co.) at 37°C for 48 h. All strains included in this study are present in the BCCM/LMG Bacteria Collection at Ghent University (Ghent, Belgium). Bacterial genomic DNA was extracted by the methodology described by Gevers et al. (13).


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  		TABLE 1. Enterococcal strains used in this study

The sequences of the primers used for amplification and sequencing of atpA are listed in Table 2. These primers were designed by using 12 atpA gene sequences of lactic acid bacteria, i.e., E. faecalis (V583), Lactobacillus plantarum (WCFS1), Lactococcus lactis subsp. lactis (IL-1403), Streptococcus pneumoniae (TIGR4 and R6), Streptococcus agalactiae (NEM316 and 2603 V/R), Streptococcus pyogenes (MGAS8232, SSI-1, MGAS315, and SF370), and Streptococcus mutans (UA159), which originated from publicly available data from whole-genome sequencing projects.


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  		TABLE 2. Amplification and sequencing primers used in this study

PCR mixtures were composed of 33.5 µl sterile MilliQ water, 5.0 µl PCR buffer (10x), 5.0 µl deoxynucleoside triphosphates (2 mM each), 0.5 µl forward primer (atpA-20-F; 50 µM); 0.5 µl reverse primer (atpA-27-R), 0.5 µl AmpliTaq DNA polymerase (1 U/µl), and 5.0 µl template DNA (0.01 µg/µl). PCR was performed with a GeneAmp PCR system 9600 thermocycler (Applied Biosystems). The thermal program consisted of (i) 5 min at 95°C; (ii) 3 cycles of 1 min at 95°C, 2 min 15 s at 55°C, and 1 min 15 s at 72°C; (iii) 30 cycles of 35 s at 95°C, 1 min 15 s at 55°C, and 1 min 15 s at 72°C; and (iv) a final 7 min at 72°C. An annealing temperature of 58°C was used as an exception for a few strains. Amplifications were qualitatively and quantitatively checked by RESult 1% LE Agarose (Biozym, The Netherlands) gel electrophoresis, with SmartLadder included as a reference. Positive PCR results gave a product with the expected size (ca. 1,100 bp), and the products were purified by using the Nucleofast 96 PCR cleanup membrane system (Macherey-Nagel, Germany). Subsequently, 3.0 µl of the purified and concentration-normalized PCR product was mixed with 1.0 µl ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Mix (version 3.1; Applied Biosystems), 3.0 µl sequencing primer (4 µM), 1.5 µl dilution buffer (5x), and 1.5 µl MilliQ water. The primers listed in Table 2 were used for sequencing. The thermal program consisted of 30 cycles of 15 s at 96°C, 1 s at 35°C, and 4 min at 60°C. Sequencing products were purified with a Montage SEQ96 sequencing reaction cleanup kit (Millipore). Purified products from the sequencing reactions were recovered in 20 µl of injection solution and mixed with 20 µl deionized formamide. Sample preparation was assisted by use of a Tecan Genesis Workstation 200 (Tecan, Switzerland). Subsequently, separation of the DNA fragments was performed in an ABI PRISM 3100 genetic analyzer (Applied Biosystems). Sample injection was performed for 20 s at 1.25 kV. Each run was performed at 50°C for 6,500 s at 0.1 mA and 12.2 kV. Raw sequence data were transferred to Factura 1.2or6 and AutoAssembler software 1.4.0 (Applied Biosystems), with which consensus sequences were determined by using the six reads. Consensus sequences were imported into BioNumerics 3.0 software (Applied Maths, Belgium), with which a similarity matrix and phylogenetic trees were created on the basis of the maximum-parsimony and neighbor-joining method (30). The reliability of the groups was evaluated by bootstrap analysis with 1,000 resamplings. The 16S rRNA gene sequence data were obtained from EMBL. SplitsTree decomposition analysis was done with software available on the web (http://bibiserv.techfak.uni-bielefeld.de/splits/) (18), while the G+C content, the ratio of the mean number of synonymous substitutions per synonymous site/mean number of nonsynonymous substitutions per nonsynonymous site (ds/dn) and Sawyer's test were calculated by using the software package START, which was obtained from (http://pubmlst.org/software/analysis/start/) (19).

Nucleotide sequence accession numbers. The EMBL accession numbers of the atpA gene sequences are AJ843267 to AJ843313, AJ843315 to AJ843325, AJ843329 to AJ843338, AJ843340, AJ843341, AJ843343, and AJ843345 to AJ843372.


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RESULTS AND DISCUSSION
 
Primers atpA-20-F and atpA-27-R enabled the amplification and final comparison of a 1,102-bp atpA fragment of all Enterococcus species, which corresponded to 73.5% of the coding region of this gene. The mean ± standard deviation G+C content of the atpA genes of the 91 enterococcal strains (43% ± 2%) was consistent with the average G+C content for the total genome of enterococci (20, 28). Correlations and regression curves between pairwise similarities of the atpA and the 16S rRNA gene sequences were made by using Pearson's product-moment correlation coefficient. The results showed a significant correlation (R = 0.7) between the results obtained with both genes. The atpA and 16S rRNA genes had a logarithmic relationship and best fit a polynomial regression of the second degree (Fig. 1). Analysis of the regression curve between the atpA and the 16S rRNA gene sequences clearly shows that atpA is a phylogenetic marker for enterococci, but it also revealed that atpA gene sequences are much more discriminatory than 16S rRNA gene sequences. 16S rRNA gene sequence similarities above 97% and 99% corresponded to atpA gene sequence pairwise similarities above 74% and 84%, respectively (Fig. 1). SplitsTree decomposition analysis on the basis of the results for all 91 enterococcal strains resulted in a star-like tree (fit = 56). In addition, Sawyer's test based on the maximum condensed fragments (P = 1) did not show evidence of gene conversion in any of the strains. The ds/dn ratio for atpA for the whole panel of strains was 25.7, suggesting that this locus is under neutral selective pressure. Overall, these analyses did not show evidence of horizontal gene transfer of the atpA gene sequences of the Enterococcus strains.



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  		FIG. 1. Regression curve between atpA and 16S rRNA pairwise gene sequence similarity.

The neighbor-joining tree created by use of the atpA gene sequences revealed distinct clusters within the genus Enterococcus (Fig. 2). Members of the E. avium, E. faecalis, E. casseliflavus, E. cecorum, and, with the exception of E. canis, E. faecium species groups (20) cluster together in the phylogenetic trees created from both the atpA and the 16S rRNA gene sequences. However, the 16S rRNA gene sequences were much less discriminatory for differentiating closely related enterococcal species within the various species groups. Different topologies between the atpA and 16S rRNA phylogenetic trees between different species groups were often observed. Maximum values of atpA sequence similarity of 92% were observed among the species groups.



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  		FIG. 2. Neighbor-joining tree based on the atpA gene sequences of 91 enterococcal strains. Bootstrap values after 1,000 repetitions are indicated. Carnobacterium piscicola, Carnobacterium divergens, Lactococcus plantarum, Lactococcus lactis subsp. lactis, Streptococcus pneumoniae, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus salivarius subsp. salivarius, Bacillus cereus, and Listeria monocytogenes were included as outgroups.

Seven members of the E. faecium species group (E. faecium, E. canis, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed >98.8% 16S rRNA gene sequence similarity, but highest values of only 89.9% atpA gene sequence similarity were observed. The atpA gene sequence analysis did not cluster E. canis as a member of this species group but showed that it occupied a distinct branch with 80.2% similarity in its atpA gene sequence compared to those of E. asini 18727T and E. dispar 13521T. In order to evaluate the intraspecies atpA gene sequence similarity, multiple strains of each species and, in particular, 16 well-characterized strains of E. faecium were included. Some of the strains were previously extensively studied by AFLP analysis, RAPD analysis, PFGE, and DNA-DNA hybridization studies (37). The phylogenetic tree of the atpA gene revealed two subclusters within the E. faecium strains with 0.9% and 3.7% sequence divergence, respectively. The two subclusters obtained did not correspond to the two genomic groups, delineated on the basis of AFLP and RAPD analyses by Vancanneyt et al. (37). Homan et al. (17) confirmed an intraspecies atpA sequence similarity of about 97% in E. faecium. It has also been concluded that horizontal gene transfer plays a role in the long-term evolution of E. faecium (17). SplitsTree decomposition analysis of our 16 E. faecium strains showed a net-like tree (fit = 78), suggesting that recombination may indeed be an important mechanism in the evolution of the species E. faecium.

Within the E. faecalis species group, the tree of the atpA gene revealed two subclusters, i.e., E. faecalis and E. moraviensis-E. haemoperoxidus. E. faecalis was more distantly related to E. moraviensis (85%) and E. haemoperoxidus (83.3%), and these results confirm the 16S rRNA gene sequencing data. It is worth mentioning that E. haemoperoxidus LMG 19487T and E. moraviensis LMG 19486T have 99.4% 16S rRNA gene sequence similarities but only 92% atpA gene sequence similarity. This result further emphasizes the discriminatory power of atpA gene sequence analysis.

Analogous congruence between the atpA gene- and 16S rRNA gene-based phylogenetic trees was obtained for the other species groups, although the topology of the species within the groups may have been different. Within the E. avium species group, all species occupied distinct positions with at a maximum of 89.5% atpA gene sequence similarity. The closest neighbors of E. avium were E. malodoratus (89.5%), E. gilvus (87.6%), E. pseudoavium (86.9%), E. hermanniensis (86.4%), E. raffinosus (85.1%), and E. pallens (80.5%). Two distinct subclusters were found within the E. casseliflavus species group, i.e., E. gallinarum and E. casseliflavus-E. flavescens. E. casseliflavus LMG 10745T and E. flavescens LMG 13518T were highly related to each other (98.9% atpA sequence similarity). The corresponding 16S rRNA (27), sodAint (29), ddl (25), and vanC (9) gene sequences of the E. casseliflavus and E. flavescens type strains were almost identical (98.8%, 98%, 99.5%, and 96% sequence similarities, respectively). This confirms that E. flavescens is not a separate species but, in fact, should be classified as E. casseliflavus, in accordance with the findings of previous studies (5, 36). The two species had at a maximum 85.7% gene sequence similarity to E. gallinarum. The E. cecorum species group comprises E. cecorum and E. columbae. The type strains of both species had at a maximum 88.1% atpA gene sequence similarity.

The recently described species E. saccharominimus LMG 21727T and E. italicus LMG 22039T were highly related, having about 100% 16S rRNA gene sequence similarity and an analogous atpA sequence similarity, which suggests that E. saccharominimus and E. italicus might be synonymous species (10, 39). The 16S rRNA gene-based phylogenetic tree showed that E. saccharolyticus LMG 11427T, E. sulfureus LMG 13084T, E. saccharominimus LMG 21727T, and E. italicus LMG 22039T group in one species group (data not shown), whereas on the basis of the atpA gene-based tree, E. saccharolyticus LMG 11427T and E. sulfureus LMG 13084T occupied distinct branches. Also, E. phoeniculicola LMG 22471T and E. solitarius LMG 12890T constituted distinct branches. E. solitarius is phylogenetically more closely related to Tetragenococcus than to the other enterococci (11, 20).

We report in this study on the identification and phylogenetic positioning of all enterococcal species, which are not clearly distinguishable by their 16S rRNA gene sequences. The 16S rRNA gene is very useful for discriminating the main groups of enterococci, i.e., the E. avium, E. casseliflavus, E. cecorum, E. faecalis, and E. faecium species groups; but it fails to discriminate closely related species, i.e., the members of E. faecalis and E. faecium species groups. Consequently, all currently known Enterococcus species were clearly differentiated on the basis of their atpA sequences (Fig. 2). At the interspecies level, the atpA gene sequence similarity was always a maximum of 92% for all species. In order to evaluate the atpA gene sequence variation at the intraspecies level, we included several representative strains of each Enterococcus species. These strains were selected on the basis of AFLP analysis, SDS-PAGE of whole-cell proteins, and, if they were available, other polyphasic data and represent the known heterogeneity of Enterococcus species. With the exception of the E. faecium strains, strains of the same species had 98.6 to 100% atpA gene sequence similarity; the E. faecium strains had 96.3% atpA gene sequence similarity. We may therefore conclude that strains of a single species will have at least 96.3% atpA sequence similarity. The use of protein-coding gene sequence data for the determination of genomic relatedness at the intra- and interspecies levels has recently been advocated because of its advantages over the banding pattern techniques, i.e., reproducibility and portability (33, 40). For determination of relatedness at the interspecies level, DNA-DNA hybridization presents several inconveniences; i.e., few laboratories can execute this technique, the method is the slowest and the most problematic for the description of species, and the DNA-DNA hybridization data are not cumulative (34). AFLP analysis randomly samples the whole genome and better differentiates closely related strains. At the strain level, atpA gene sequence analysis is less discriminatory than AFLP analysis; strains with identical atpA sequences had 70 to 93% AFLP pattern similarities. Although it is not valuable for species differentiation, PFGE appeared to be superior for interpretation of intraspecies relationships (5). We conclude that atpA sequence analysis can be used as an alternative to currently used techniques for the identification and phylogenetic analysis of clinically important enterococcal species. In addition, our data may be useful for the rapid detection of Enterococcus by using, e.g., real-time PCR.


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ACKNOWLEDGMENTS
 
S.N. acknowledges a Ph.D. scholarship from the Palestinian Ministry of Higher Education. F.L.T. acknowledges a postdoctoral fellowship from BCCM/LMG Bacteria Collection. D.G. acknowledges financial support from BOF (project no. 01110803). J.S. acknowledges grants from the Fund for Scientific Research (FWO), Belgium.

We thank Cindy Snauwaert, An Beckers, and Marjan De Wachter for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium. Phone: 32-9-264 5238. Fax: 32-9-264 5092. E-mail: Sabri.Naser{at}Ugent.be. Back


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Journal of Clinical Microbiology, May 2005, p. 2224-2230, Vol. 43, No. 5
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.5.2224-2230.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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