1 Laboratory of Microbiology, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
2 BCCM/LMG Bacteria Collection, Ghent University, K. L. Ledeganckstraat 35, Ghent 9000, Belgium
3 Bioinformatics and Evolutionary Genomics, Ghent University/VIB, Technologiepark 927, Ghent 9052, Belgium
Correspondence
Sabri M. Naser
Sabri.Naser{at}Ugent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AJ843476AJ843515, AJ843517AJ843523, AJ843525AJ843534, AJ843537AJ843553, AJ843555AJ843556 and AJ843558AJ843560 (rpoA partial gene sequences), and AJ843373AJ843387, AJ843389AJ843402, AJ843404AJ843410, AJ843412AJ843435, AJ843438AJ843444 and AJ843446AJ843474 (pheS partial gene sequences).
A presentation of the polymorphic sites present in the RpoA and PheS dataset is available as supplementary data with the online version of this paper.
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INTRODUCTION |
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The increased association of enterococci with human disease has raised concern regarding their use as probiotics (Franz et al., 2003). E. faecalis and E. faecium are among the leading causes of nosocomial infections and may cause endocarditis, urinary tract infections and bacteraemia (Ratanasuwan et al., 1999
; Saxena et al., 2003
; Fernandez-Guerrero et al., 2002
). E. faecalis predominates among enterococci isolated from the environment and from human infections (more than 80 %), while E. faecium is associated with the majority of the remaining infections (Jett et al., 1994
). The recent increase in vancomycin-resistant E. faecium (VREF) strains among clinical isolates is a cause of serious concern and has gained clinical significance in the last decade (Michel et al., 1997
; Dzidic & Bedekovic, 2003
; Homan et al., 2002
; Rybak & Coyle, 1999
). Although most human enterococcal infections are caused by E. faecalis and E. faecium, various studies have revealed an increase in infections caused by E. durans, E. hirae, E. gallinarum and E. casseliflavus (Baele et al., 2000
; Kirschner et al., 2001
; Knijff et al., 2001
; Willey et al., 1999
). There is, therefore, a need for rapid and accurate identification of enterococci at species level, as a means of effective infection control.
16S rRNA gene sequencing, DNADNA hybridization and SDS-PAGE of whole-cell proteins are among the most common techniques currently used for Enterococcus species identification (Angeletti et al., 2001; Domig et al., 2003
; Vancanneyt et al., 2001
). However, 16S rRNA gene sequences have limited discriminating power for several closely related enterococcal species, e.g. the E. faecium species group (Devriese et al., 2002
; Poyart et al., 2000
; Vancanneyt et al., 2001
). SDS-PAGE may present problems concerning reproducibility and data portability, and DNADNA hybridization presents several inconveniences, i.e. few laboratories can execute this technique, the method is the slowest and most problematic step in species description and DNADNA data are not cumulative (Stackebrandt, 2003
). rpoB gene sequence analysis (Drancourt et al., 2004
) and multiplex sodA PCR (Jackson et al., 2004
) have been used for identification of several Enterococcus species. So far, these molecular techniques are not yet used for routine identification. The use of protein-coding gene sequence data for the determination of genomic relatedness is emerging as an alternative to overcome these problems (Stackebrandt et al., 2002
; Zeigler, 2003
).
In the present paper, a new approach is applied to discriminate between different species of Enterococcus, multilocus sequence analysis (MLSA). MLSA compares the primary DNA sequences from multiple conserved protein-coding loci for assessing the diversity and relationship of different isolates across related taxa, thereby using an appropriate phylogenetic or cladistic approach. Two studies of complete genomes provided the groundwork for establishing sets of genes useful for MLSA in large numbers of bacterial lineages (Zeigler, 2003; Santos & Ochman, 2004
). Recently, atpA, the gene that encodes the ATP synthase
-subunit has been used as an identification tool for all enterococcal species (Naser et al., 2005
). In the present study, we have investigated the usefulness of the genes that encode the
-subunit of bacterial RNA polymerase (rpoA) and phenylalanyl-tRNA synthase
-subunit (pheS) as alternative identification tools for all enterococci species. We also compared the sequence data of rpoA and pheS genes with the available atpA and 16S rRNA gene sequences.
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METHODS |
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Sequence analysis.
Raw sequence data were transferred to Factura 1.2.Or6 and AutoAssembler software 1.4.0 (Applied Biosystems) or GeneBuilder (Applied Maths) where consensus sequences were determined using two reads for each rpoA and pheS gene, respectively. Consensus sequences were imported into BioNumerics 3.5 software (Applied Maths), where a similarity matrix and phylogenetic trees were created based on the maximum-parsimony and neighbour-joining methods (Saitou & Nei, 1987). The reliability of the groups was evaluated by bootstrap with 500 resamplings. 16S rRNA gene sequence data were obtained from EMBL. Splits decomposition tree analysis was done using software available on the web (http://bibiserv.techfak.uni-bielefeld.de/splits/) (Huson, 1998
), while the GC content, the ratio of mean synonymous substitutions per synonymous site/mean non-synonymous substitutions per non-synonymous site (dS/dN) and Sawyer's test were calculated using the software package START obtained from http://pubmlst.org/software/analysis/start/ (Jolley et al., 2001
).
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RESULTS AND DISCUSSION |
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Interspecies and intraspecies heterogeneity of rpoA gene sequences
On the basis of rpoA gene sequences, all Enterococcus species were clearly differentiated, forming distinct branches (Fig. 1). At the interspecies level, the rpoA gene sequence similarity was at maximum 97 % for all species. Strains of the same species had at least 99 % rpoA gene sequence similarity. Members of the E. avium, E. casseliflavus, E. faecalis, E. cecorum and E. faecium (except for E. canis) species groups clustered together as in the 16S rRNA based phylogeny, although the closest neighbours were not the same as in the 16S rRNA analysis. Within the E. faecium species group, all species occupied distinct positions with at maximum 97 % rpoA gene sequence similarity. The closest neighbours of E. faecium were E. villorum (97 % rpoA gene sequence similarity), E. durans and E. hirae (96 %), E. mundtii (95 %) and E. ratti (94 %). The rpoA gene sequence tree revealed two subclusters within the E. faecalis species group, i.e. E. faecalis and E. moraviensis/E. haemoperoxidus. E. faecalis was more distantly related to E. moraviensis (90 %) and E. haemoperoxidus (89 %). Within the E. casseliflavus species group, E. casseliflavus LMG 10745T and E. flavescens LMG 13518T were highly related to each other having >99 % rpoA gene sequence similarity. Both species shared at maximum 94 % similarity with E. gallinarum, the other member of this species group. Similarly, E. saccharominimus LMG 21727T (Vancanneyt et al., 2004
), E. italicus LMG 22039T (Fortina et al., 2004
) and Enterococcus CDC PNS-E1 (=LMG 22681T) (Carvalho et al., 2004
) were highly related, having about 100 % rpoA gene sequence similarity. All species within the E. avium species group occupy distinct positions. The closest neighbours of E. avium were E. malodoratus, E. gilvus, E. raffinosus (97 %), E. pseudoavium (96 %), E. hermanniensis (95 %) and E. pallens (90 %). The E. cecorum species group consists of E. cecorum and E. columbae. The type strains of both species had at maximum 87 % rpoA gene sequence similarity.
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The aim of this study was to focus particularly on tools for rapid, reliable and inexpensive identification for discrimination among different species of enterococci, and not for typing or phylogeny purposes, and to that end our data convincingly prove that the partial sequences of pheS and rpoA gene sequences perfectly fulfil this aim and show high resolution for differentiating all enterococcal species, even better than 16S rRNA gene sequences. At species level, the bootstrap values for rpoA and pheS gene sequences are always 100 %, proving that both rpoA and pheS genes are reliable genomic markers for species differentiation within the genus Enterococcus (Figs 1 and 2). Our choice for using partial sequences neither gave enough resolution for typing at the intraspecies level nor provided sufficient evidence at deeper phylogenetic branches. To fulfil these approaches, the inclusion of more loci and full sequences would be essential, but this is beyond the scope of this paper. The full gene sequences can be obtained by gene cloning, but the amount of work and costs will be increased and this is not favourable in comparison with MLSA, i.e. rapid, reliable and inexpensive identification. The 16S rRNA gene is very useful for discriminating the main groups of enterococci, i.e. E. avium, E. casseliflavus, E. cecorum, E. faecalis and E. faecium species groups, but it fails to discriminate closely related species. One example is the members of the E. faecium species group, i.e. E. faecium, E. hirae, E. durans, E. villorum, E. mundtii and E. ratti. The 16S rRNA genes of these species show similarities of 98·899·7 % (Devriese et al., 2002
), but the highest gene sequence similarities observed for rpoA, pheS and atpA were 97, 86 and 89·9 %, respectively. This also demonstrates the advantage of using several housekeeping genes for species identification studies (Stackebrandt et al., 2002
). Consequently, all currently known Enterococcus species were clearly differentiated on the basis of rpoA and pheS gene sequences (Figs 1 and 2
).
Both genes provide efficient screening methods for the detection of novel species. At the interspecies level, the simultaneous analysis of rpoA and pheS gene sequences offers an alternative to DNADNA hybridization to differentiate closely related Enterococcus species. To evaluate the intraspecies rpoA and pheS gene sequence similarities, multiple strains of each species and, in particular, 16 well characterized strains of E. faecium were included. The results showed that rpoA and pheS genes had a high degree of homogeneity among strains of the same species. Consequently, this indicated the low discriminatory power of these genes for intraspecies differentiation. Therefore, we could conclude that strains of the same enterococcal species will have at least 99 % rpoA and 97 % pheS gene sequence similarity, respectively. In comparison, strains of a single Enterococcus species have at least 96·3 % atpA gene sequence similarity.
Rapid and robust classification using MLSA may be used with a universal set of protein-coding genes that are widely distributed among bacterial genomes and present in single-copy, that show levels of variation below saturation for the group being analysed and that are not unusually prone to recombination (Zeigler, 2003). The fact that different chronometers provide different closest neighbours of a given strain does not hamper their use to unambiguously circumscribe bacterial species. Several factors account for the different topologies determined for different housekeeping genes, i.e. the level of the information content, the different rates of evolution due to different selection forces on various genes and the length of partial sequences compared (Christensen et al., 2004
). The use of several housekeeping genes in bacterial taxonomy is best suited for analysis at the species and genus levels as it integrates the information of different molecular clocks around the bacterial chromosome (Lerat et al., 2003
; Palys et al., 1997
; Stackebrandt et al., 2002
; Ventura et al., 2004
; Zeigler, 2003
). This type of data may aid the development of better species definition for Enterococcus.
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ACKNOWLEDGEMENTS |
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Received 19 December 2004;
revised 20 April 2005;
accepted 20 April 2005.
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