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Journal of Bacteriology, January 2006, p. 169-178, Vol. 188, No. 1
0021-9193/06/$08.00+0     doi:10.1128/JB.188.1.169-178.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evidence for Clonal Evolution among Highly Polymorphic Genes in Methicillin-Resistant Staphylococcus aureus

G. Kuhn,^* P. Francioli, and D. S. Blanc

Hospital Preventive Medicine, University Hospital of Lausanne (CHUV), Lausanne, Switzerland

Received 29 July 2005/ Accepted 7 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The evolution of Staphylococcus aureus has been described as predominantly clonal, based on evidence from seven housekeeping genes. We aimed to test if this was also true for more polymorphic genes. In a collection of 60 isolates including major European epidemic methicillin-resistant S. aureus (MRSA) and sporadic MRSA strains, we compared the partial gene sequences of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL), six core adhesion genes (present in all strains) (clfA, clfB, fnbA, map, sdrC, and spa), and four accessory adhesion genes (not present in all strains) (ebpS, fnbB, sdrD, and sdrE). Nucleotide diversity of adhesion genes was 2- to 10-fold higher than genes used for multilocus sequence typing. All genes showed evidence for purifying selection with a weakly reduced level among accessory adhesion genes. Among these highly variable genes, there was no evidence for a difference in molecular evolution between epidemic and sporadic strains. Gene trees constructed from concatenated sequences of housekeeping, core adhesion, and accessory adhesion genes were highly congruent, indicating clonality, despite some evidence for homologous exchange. Further evidence for clonality was found with an overall positive correlation of allelic and nucleotidic divergence for both seven housekeeping genes and six core adhesion genes. However, for small allelic differences that fit the demarcations of clonal complexes (CCs) there was no such correlation, suggesting that recombination occurred. Therefore, despite an overall clonal population structure, recombination between related isolates within CCs might have contributed to S. aureus evolution.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. The health conditions caused by MRSA can range from carriage without symptoms to severe life-threatening infections. Epidemiological studies have revealed that a high number of these infections were caused by single MRSA clones that spread rapidly in hospital settings (epidemic MRSA), while other clones that were present at the same time did not spread (sporadic MRSA) (6, 7, 8, 30). Genetic variation causes phenotypic differences, and population genetic approaches can be used to identify how this variation is created and maintained.

Multilocus sequence typing (MLST) has been developed to analyze and compare genetic variation in worldwide collections of bacterial isolates (27). Using the MLST sequence data of S. aureus, it was shown that genetic differences between a single-locus variant and its ancestral strains were created 15 times more frequently by a mutation than by a recombination event (14). Clearly, this indicated a predominantly clonal evolution.

MLST has been based on seven slowly evolving genes that encode proteins of central metabolic functions (housekeeping) (11), and it was used to analyze the evolutionary history of MRSA (12). A more polymorphic set of genes, the S. aureus cell surface genes (sas) that are mostly scattered around the origin of replication, have been analyzed as an extension of this typing scheme and resolved a more-detailed picture of MRSA evolution (32). Both sets showed a high congruence in their phylogenetic resolution of groups, which again suggested clonal evolution.

Recently, bacterial genomes were suggested to be viewed as two compartments of genes, one comprising "core" genes that are ubiquitously present in all clones of a given species and the other comprising "accessory" genes that are not necessarily present in all clones and that have the possibility to be transferred between strains (24). MLST is based on seven housekeeping genes that represent exclusively core genome genes; because of their definition, these genes can only evolve through mutation and gene replacement. Accessory genes also have potential to get lost and to be acquired during evolution (they are therefore considered "dispensable"), and thus the resulting population structure given by these genes might be different from that of core genome genes (13).

Comparative genome hybridization experiments were performed to address genetic variation due to the presence and absence of genes in a collection of highly diverse S. aureus strains. This study showed that up to 22% of all genes in the genome could be considered as accessory (16). Thus, there is a considerable proportion of the genome that seemed not to be essential and that had the potential to contribute to genetic differences between strains.

The study of accessory genes is of utmost importance since they include resistance to methicillin (mecA) (12) and virulence factors (sea, tst, and eta) (3, 26, 28, 41). To date, only the molecular evolution of locus (RD13), comprising an exotoxin-like family of genes, was analyzed. This locus likely evolved clonally through multiple gene deletions, and genes were subject to purifying selection (15).

The goal of the present study was to compare the evolution of "conserved" genes and polymorphic genes among MRSA isolates. We selected the seven housekeeping genes used for MLST analysis as representatives of conserved genes (11). For polymorphic genes, we selected cell surface genes; within these, we aimed to differentiate core cell surface genes and accessory cell surface genes. We selected cell surface genes because in contrast to the slowly evolving genomic core genes this class of genes has been shown to undergo rapid evolutionary changes, which already rendered them useful for short-term epidemiological typing (18, 21, 36). In addition, adhesion to host tissues and factors therein is important for successful host colonization; thus, these genes allow us to test the hypothesis of whether a specific genetic signature or molecular evolutionary pattern is linked to epidemic potential.

By sequencing the DNA of 17 different genes from a collection of 60 isolates, we aimed to study S. aureus genome evolution. In particular, we aimed to study molecular evolutionary patterns of 10 adhesion genes (clfA, clfB, ebpS, fnbA, fnbB, map, sdrC, sdrD, sdrE, and spa) and compare these to seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial isolates. A total of 60 isolates was used in this study (Table 1). Fourteen of these isolates were chosen because they represent members of the six MRSA clusters present in Europe that were previously described (4). The other strains were selected to study the evolution of epidemicity in MRSA. Our study aimed to confront two different groups of isolates, those that were shown to disseminate rapidly in a setting and those that were never reported to disseminate in a setting. Twenty-three MRSA isolates belonged to 12 different epidemic clones. Each of these isolates was collected at the onset of an epidemic, because we aimed to exclude isolates that might have undergone genetic changes during the epidemic, which could have affected their epidemic potential. For six of these clones (EMRSA-15, Norway, Iberian, Brazilian, The Netherlands, and Gm-S) (Table 1), we had either two or three isolates. They had the same genotype (see the references cited in Table 1) but were sampled at different locations. For two clones (B and D), we had two additional isolates that were obtained from the same patient after a 4-year period of time (isolates 2 and 4). Previous genotyping analysis using pulsed-field gel electrophoresis showed that the first and the second isolates from each patient were identical. Fourteen isolates were considered sporadic, because they were never found to be transmitted between patients, because they were never isolated from another patient in the same setting, and because they differed in pulsed-field gel electrophoresis patterns from epidemic clones. In addition, sporadic CHUV isolates were present on the patients for at least 3 weeks during hospitalization and thus had the possibility of disseminating. The sequences from the seven publicly available genome sequences were also included.

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TABLE 1. Details of typing analyses of 60 S. aureus isolates from different European countries, grouped according to MLST housekeeping typing data (CC and ST)

Cultivation of bacteria, lysis, and DNA extraction. S. aureus isolates were grown in 1 ml brain heart infusion liquid medium. Pelleted cells were lysed in 200 µl lysis buffer (1x Tris-EDTA buffer, 0.35 M NaCl, and 0.05-mg/ml lysostaphin). DNA was extracted from 100 µl lysate as described previously (10).

Design of 10 adhesion gene primers. Gene sequences of published genes clfA (Z18852), clfB (AJ224764), ebpS (U48826), fnbA (J04151), fnbB (X62992), map (AJ245439), sdrC (AJ005645), sdrD (AJ005646), sdrE (AJ005647), and spa (M18264) were taken for a BLAST analysis with the seven publicly available S. aureus genome sequences to obtain their homologous gene sequences (COL, NCTC8325, MW2, methicillin-susceptible Staphylococcus aureus 476 [MSSA476], MRSA252, Mu50, and N315). Sequences of each gene were aligned, and primers were targeted against regions with no genetic variation.

The genes sdrC, sdrD, and sdrE shared regions with high sequence similarity among each other, leading to cross-hybridization in first PCR trials. The primers were redesigned to match conserved regions that were specific for each of the three genes. Table 2 summarizes the names, the supposed or known function of the gene, the size of the amplicons, and both primer names and sequences.

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TABLE 2. Details of the 10 adhesion genes and the regions amplified and sequenced in this study

PCR and sequencing. PCR amplifications for each gene were carried out in 25-µl reaction volumes containing 40 ng of bacterial DNA, 0.5 µM of each primer, 1 U of Taq DNA polymerase (Invitrogen), 1x PCR buffer, 1.5 mM MgCl₂ (supplied with the Taq polymerase), and 0.1 mM deoxynucleotide triphosphates (Invitrogen). Cycling conditions were 3 min of initial denaturation at 94°C; 32 cycles consisting of 30 s at 94°C, and 30 s at primer-annealing temperature (Table 2), 1 min (2 min, if amplicons were <1,000 bp) at 72°C; and a final extension for 5 min at 72°C.

Standard gel electrophoresis was performed, and stained gels were evaluated for success and specificity, i.e., one clear band visible per PCR. All reactions with no amplification were repeated to exclude the possibility of technical errors. In addition, some of these reactions were repeated with newly extracted DNA and/or lowered annealing temperatures. However, none of the findings was altered by a second reaction. PCRs were then purified with the Montage PCR u96 kit (Millipore) according to the manufacturer's instructions.

Sequencing reactions were carried out with the Big Dye Terminator kit, version1.1 (Applied Biosystems), according to the manufacturer's instructions. Sequencing reaction cleanup was performed with the Montage Seq96 Sequencing Reaction Cleanup kit (Millipore), according to the manufacturer's instructions. Purified samples were analyzed with the ABI Prism 3100 Avant sequencer (Applied Biosystems), according to standard protocols.

Preparation of sequence data sets. First, alignments of all genes were created using ClustalW 1.81. Then, gaps introduced by ClustalW were, if needed, shifted to either side by one or two bases to produce sequences that are in frame. These alignments were taken for analysis of the genetic relationships between isolates. In a second procedure, indel sites were removed from alignments to produce data sets without gaps and that were in frame. Finally, for each gene a data set was compiled that comprised one copy of each allele. These were the data sets used to calculate nucleotide diversity and strength of purifying selection and to compare allelic and nucleotidic divergence.

Determination of sequence types. MLST was performed as previously described (11) using the S. aureus MLST database at http://www.mlst.net/ (Table 1). Sequences of unknown alleles were confirmed by repeating the procedure from a new DNA extraction of the isolate in question and were submitted to the MLST database. Furthermore, the obtained sequence types (STs) were assigned to previously identified clonal complexes (CCs) (CC1, CC5, CC8, CC22, CC30, and CC45) (12, 14).

spa typing. The spa region C contains at its 3' end the beginning of the first 24-bp spa repeat (39). Beginning with this repeat, Ridom spa allele numbers were determined at http://www.ridom.de/spaserver/. The concatenated allele numbers were taken to determine the Ridom spa type.

Molecular evolution. We calculated allelic diversity and nucleotide diversity (mean number of nucleotide differences between alleles) by using DNASP, version 4.00 (33). The proportion of synonymously (ds) and nonsynonymously (dn) mutated sites was determined using the implemented function in the software Start (37). dn and ds values were plotted against each other to produce data points in a graph. By measuring the strength of purifying selection (dn/ds ratio), the evolutionary forces of a gene are usually classified into three categories. Values of <1 indicate purifying selection, values approximately equal to 1 indicate balanced selection or neutral evolution, and values of >1 indicate diversifying selection or accelerated evolution.

Genetic relationship between isolates. For a reference and starting point, we calculated the gene tree of all MLST housekeeping genes. Gene sequences of all seven loci were concatenated to produce an in-frame sequence. To this, we compared gene trees of each gene. For representation of the other genomic compartments, we equally concatenated sequences of six core adhesion genes and four accessory adhesion genes. Maximum parsimony trees were calculated by using MEGA, version 3.0 (22). The statistical significance of branches was inferred by performing a bootstrap analysis with 1,000 repetitions.

Comparison of allelic and nucleotidic divergence. The underlying assumption of this analysis is that by comparing allelic and nucleotidic divergence it can be inferred whether clones in a population diversify predominantly by mutation which would result in a positive correlation between the two parameters or predominantly by recombination, which would result in a different relationship (14). Two data sets, based on either MLST housekeeping genes or core adhesion genes, were created. Pairwise comparisons of all STs were performed, and all pairs were sorted according to differences at one, two, three, four, five, six, and seven loci (up to six for core adhesion genes). For each pairing within a group, the number of nucleotide differences was then calculated per locus. The average number of differences was afterwards determined across loci. In this manner, a graph was produced where each group (x axis) was plotted against the average number of nucleotide differences across loci (y axis). The calculations were performed by using the BLUND program that was kindly provided by D. A. Robinson.

Epidemic and sporadic isolates. The rational of this comparison was to find out by means of molecular evolutionary analysis whether there was a difference related to either of the two groups of isolates. We aimed to look for each gene separately, whether or not there was a difference in nucleotide diversity and/or purifying selection between the two groups. A difference between the two groups could be imagined, due to the accumulation of mutations that occurred among sporadic isolates, which would be indicated by increased nucleotide diversity and/or reduced strength of purifying selection. We also aimed to examine whether epidemic isolates were grouped together in any of the obtained gene trees, thereby indicating a specific allele or pattern of alleles for epidemicity.

Nucleotide sequence accession numbers. Sequences were submitted to the EMBL database (accession numbers AM075836 to AM076337).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence analysis. Analysis of the 17 gene loci sequenced showed that, among the 58 MRSA isolates that cover the major clones found in Europe and the two MSSA isolates, a total of 24 strains did lack at least 1 gene locus. One strain lacked ebpS, 11 strains lacked fnbB, 1 strain lacked sdrD only, 6 strains lacked sdrE only, and 6 strains lacked both sdrD and sdrE. Therefore, these four genes were considered "accessory" for further analysis. In contrast, clfA, clfB, fnbA, map, sdrC, and spa were present in all strains and were classified as "core" adhesion genes (Table 1).

Indel mutations were observed among alleles of genes clfB, ebpS, fnbA, fnbB, map, sdrC, and spa. The great majority of these did not lead to a frameshift and produced a continuous open reading frame. Four alleles (arcC, isolate 55; clfB, isolates 25 and 40; and spa, isolate 31) comprised one or two base insertions. This led to appearance of early stop codons in the open reading frame. Thus, frameshift mutations occurred among one MLST housekeeping gene and two core adhesion genes. In addition, we found three new alleles (aroE, isolate 45; glpF, isolate 62; and gmk, isolate 33) that have not yet been submitted to the MLST database.

Our collection comprised isolates of the same clone that were sampled either at different locations or at different moments in time. For five of the clones, isolates showed the same genotype with all markers used in this study. For the remaining four clones, differences were observed (Table 1). For example, two isolates of EMRSA-15 from Portugal differed at locus gmk (one comprised allele 5, the other allele 33). This indicated either that two different strains of EMRSA-15 were introduced to Portugal or that one strain accumulated a mutation during its dissemination.

Determination of sequence types. Based on the seven MLST housekeeping genes, the complete data set with all 60 MRSA isolates contained 24 STs. BURST analysis of all 60 isolates showed that there were six distinct groups. Nineteen STs were within the five major MRSA lineages that were previously described (CC5, CC8, CC22, CC30, and CC45). One additional group was formed by the identical ST of MSSA476, community-acquired MRSA MW2, and one new ST. This group corresponded to the previously described CC1. The remaining two STs, isolates 44 and 90, were singletons and were new STs that are not yet described in the MLST database.

Molecular evolution. Variable numbers of alleles were observed for all genes (Table 3). In general, there were more adhesion gene alleles than housekeeping gene alleles, which can be expected due to the greater length of adhesion gene alleles. Calculation of nucleotide diversity between alleles takes into account the different sizes of the products and thus allows the comparison of values between genes and classes of genes. According to the classification of genes, we found that values for core adhesion genes (mean = 0.077 ± 0.046) or accessory adhesion genes (mean = 0.088 ± 0.063) were significantly greater than those for MLST housekeeping genes (mean = 0.009 ± 0.002) (P = 0.0023 and P = 0.007, respectively), meaning that there was more nucleotide diversity between adhesion genes than between housekeeping genes (Table 3). The amount of nucleotide diversity ranged from double for spa to >10 times as much for fnbA and fnbB.

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TABLE 3. Nucleotide sequence variation and purifying selection among alleles of 7 MLST housekeeping and 10 adhesion genes

The strength of purifying selection was then calculated as dn/ds, and showed that variation was high between genes and classes of genes. When tested for significance, there was no significant difference in strength of purifying selection between the MLST housekeeping genes (mean dn/ds = 0.176 ± 0.054) and core adhesion genes (mean dn/ds = 0.18 ± 0.041) (P = 0.879). However, when each of these two classes was compared to the class of accessory adhesion genes (mean dn/ds = 0.347 ± 0.165), there was less purifying selection in the latter class of genes, with weak significance (P = 0.0287 and P = 0.0408, respectively).

Values of dn and ds of all genes were plotted in a graph (Fig. 1). All genes were subject to purifying selection and were therefore found in the bottom part of the plot. A regression line with a good correlation (R² = 0.9151) could be calculated on the basis of all values. MLST housekeeping genes had very low values of dn and ds, due to their low nucleotide diversity. They grouped together at the lower-left-hand part of the plot. Synonymous and nonsynonymous substitution rates of adhesion genes were more variable between genes, as was previously indicated by the variation of nucleotide diversity. The weaker purifying selection in accessory adhesion genes was indicated in the graph by the fact that these genes were all plotted above the regression line (Fig. 1).

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FIG. 1. Purifying selection. The aligned sequence data of each gene were taken to calculate the proportion of synonymous (ds) and nonsynonymous (dn) substitutions. The plotted values of each gene are shown. The ratio of dn/ds indicates purifying selection (positive Darwinian selection) if values are <1, diversifying selection if values are >1, and balancing selection or neutral evolution if values are close to 1. Three classes of genes are represented: housekeeping genes arcC, aroE, glpF, gmk, pta, tpi, and yqiL (circles); core adhesion genes (triangles); and accessory adhesion genes (diamonds). Gene names of the latter two classes are shown in the graph. Data points of all genes are located in the area of purifying selection. The dotted line shows the regression line calculated on the basis of all data points (R2 = 0.9151).

Genetic relationship between isolates. The concatenated set of MLST housekeeping genes (Fig. 2A) produced a gene tree with weakly supported groups of isolates (low bootstrap values). However, all five groups of isolates known as major MRSA CCs were identified.

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FIG. 2. Genetic relationship between isolates. The sequences obtained from each isolate for each gene were taken to produce alignments without gaps. We then concatenated separately the gene sequences to produce data sets for seven MLST housekeeping genes (A), six core adhesion genes (B), and four accessory adhesion genes (C). Maximum parsimony trees were constructed for all three data sets with MEGA 3.0. For the data shown in panels A and B, the branch lengths were also calculated and their scales are indicated at the lower left of each panel. Bootstrap values were calculated for 1,000 repetitions and are indicated at branches. In addition, STs were determined for all isolates based on housekeeping genes using the MLST database, and these were used to assign each ST to published CCs. CCs are indicated in all three panels. We observed that all groups of CCs were resolved in each concatenated gene tree.

The concatenated set of core adhesion genes produced a gene tree with very well-supported groups of isolates (Fig. 2B). High bootstrap values were found at nodes that separated not only the major groups of clonal complexes but also groups previously separated by multiprimer randomly amplified polymorphic DNA (RAPD) analysis. In CC8, all isolates of ST239 were well grouped together. This group was previously identified as 2a by multiprimer RAPD (4). Within CC45, two isolates (71 and 92) were distinctly separated and corresponded to RAPD group 6 (4).

The reconstruction of the accessory adhesion gene tree differed from the other gene trees in that calculation of branch lengths was impossible because there were no sites in common in the concatenated data set. Therefore, only the stepwise differences between isolates based on maximum parsimony were shown (Fig. 2C). Still, all major MRSA complexes were found to be concordant in the grouping of the tree.

Visibly, there was high congruence between the gene trees of the different classes of concatenated data sets, indicating that recombination had little effect on the population structure. When individual gene trees were constructed and compared, we also observed a high congruence between trees (data not shown), while the best congruence was observed with clfA and the least was observed with clfB.

Evidence for homologous exchange. Gene transfer was suspected when the same adhesion gene allele was found in isolates from distant phylogenetic lineages (CCs) (Table 1). This way, four events of homologous exchange were identified. For the gene clfB, allele 1 was found in some isolates of CC8 and in all isolates of CC5; allele 6 was found in the remaining isolates of CC8 and in all isolates of CC30. For the gene fnbA, allele 13 was found in isolates of both CC1 and CC22. For the gene spa, allele 8 was found in some isolates of CC8 and one isolate of CC30. We emphasize that the spa gene tree must be taken with care; due to many indel mutations throughout the whole sequence, only very few repeats and domains were taken to construct an alignment. Thus, there was potential for error. Nucleotide diversification could have occurred after an event of gene transfer. In this case, if highly similar alleles were found in different phylogenetic lineages, four other events of transfer could be suspected (data not shown).

Comparison of allelic and nucleotidic divergence. The goal of this analysis was to identify whether MLST housekeeping and core adhesion genes diversified the same way. All 24 STs of MLST housekeeping genes and all 34 STs of the core adhesion genes were taken for analysis. The nucleotidic divergence was >10 times greater among adhesion genes than among MLST housekeeping genes, which was expected due to the differences in nucleotide diversity (Fig. 3). To be able to observe differences in the shape of the curve, we plotted each data set independently. Both plots clearly showed a positive correlation, indicating accumulation of de novo mutations. However, a small difference was observed between the two curves, in that the slope was null for up to two allelic differences for MLST housekeeping genes, whereas it was null for up to three allelic differences for core adhesion genes.

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FIG. 3. Sequence diversity versus allelic diversity. Two data sets were analyzed separately: MLST housekeeping genes (A) and core adhesion genes (B). They comprised 24 and 34 STs, respectively. Pairwise comparisons of all STs were performed. They were grouped according to differences at one, two, three, four, five, six, and seven loci (up to six for core adhesion genes). Within each group, the number of nucleotide differences was then calculated for each locus separately; the average was afterwards determined across loci. In this way, a graph was produced where each group (x axis) was plotted against the average number of nucleotide differences across loci (y axis). The calculations were performed using the BLUND program that was kindly provided by D. A. Robinson. We observed a positive trend for MLST housekeeping genes, indicating that diversification was mainly due to accumulation of mutations. There was also a positive trend from three to six different loci among core adhesion genes. However, there was no positive correlation for up to three different loci among core adhesion genes; this could be explained by recombination.

Epidemic and sporadic isolates. Nucleotide diversity and purifying selection were similar between epidemic and sporadic isolates (data not shown). Also, neither any of the single-gene trees nor a concatenated gene tree showed a specific grouping of epidemic or sporadic isolates (Fig. 2). Thus, there was no specific allele (genetic element) or pattern of alleles (genetic signature) of epidemic or sporadic isolates.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sequence data of 17 genes from a diverse collection of 58 MRSA and 2 MSSA isolates were used to study the evolution of the three well-defined groups of genes that represent conceptually different parts of the S. aureus genome. Based on their continuous or discontinuous presence in our collection of strains, we classified the 10 adhesion genes into two groups, core adhesion genes and accessory adhesion genes, next to the housekeeping MLST genes. These genes represented a cross section of the S. aureus genome from the slowly evolving genomic core (MLST housekeeping genes) to the highly polymorphic accessory part of the genome (accessory adhesion genes).

Molecular evolution. High degrees of polymorphism were found among the alleles of adhesion genes. The great number of nucleotide differences was expected, because cell surface genes are generally considered to be highly polymorphic and because a similar finding was previously reported for sas genes, a group of genes that carried the LPXTG protein motif for exportation to the cell surface (32). The studied sas genes were located in one region of the S. aureus chromosome, which (at least for strains of CC8) was subject to a particular evolution comprising homologous exchange (31). The 10 adhesion genes were distributed on seven different places over the entire chromosome; thus, we are confident that the great number of nucleotide differences is a general characteristic of cell surface genes in S. aureus.

There was reason to suspect that one of the mechanisms to create nucleotide differences among adhesion genes would be diversifying selection. However, in our data set, genes of all classes showed similar levels of purifying selection. Only among accessory adhesion genes, purifying selection was a little less strong than in the other classes. This indicates that functionality of adhesion genes is very important and that despite the great divergence between alleles there is no or little accumulation of deleterious mutations. In fact, the dn/ds plot of all genes followed a remarkable linear regression. If no indication of diversifying selection is observed in cell surface genes, which are likely the most polymorphic, one could suspect this is true for all genes in the S. aureus genome.

Genetic relationship. The three concatenated gene trees reconstructed from core or accessory genome genes revealed a remarkably high congruence to previously published data. They were highly congruent to CCs that were found in another study using BURST on MLST housekeeping genes (12), and they were also in agreement with a multiprimer RAPD approach where 14 of the 60 S. aureus isolates were already analyzed (4). This means that adhesion genes followed the clonal evolution that was previously described for MLST housekeeping genes.

One of the problems using MLST housekeeping genes in phylogenetic approaches is the small number of nucleotide differences between alleles. Therefore, only analyses that were based on the concatenated sequences of all seven genes led to resolution of groups that are congruent with CCs. Increased divergence between adhesion gene alleles offered the possibility to construct groupings with higher bootstrap values. These results support the hypothesis that CCs are significant genetic entities within the S. aureus population.

In this study, it was possible to correctly classify all isolates in previously defined CCs with only one marker, clfA. It was even possible to separate the two groups of strains within CC8 which were the result of gene transfer (31), thereby showing that their separation-specific mutations have already occurred in each lineage. This renders it a good candidate for future typing approaches.

We would like to emphasize that clfB, which is located at a different genomic position than clfA, was the least congruent of all studied genes. This was in contrast to an earlier study of adhesion genes, where clfB was proposed as potentially useful for phylogenetic analyses because it showed highest congruence to the expected groups of isolates (18). We think that this difference could be because the previous study was based on another part of clfB that contained various numbers of repeats. Such a region is difficult to analyze phylogenetically, and its evolution might differ from the rest of the gene.

Evidence for homologous exchange. As had been shown in previous studies, we observed the same alleles for clfB and spa in isolates of CC30 and of ST239 (CC8), indicating gene transfer between both lineages. This transfer has already been described previously with the passage of >200 kb of DNA (which includes the genes arcC, clfB, and spa). In addition, we observed an as-yet-unreported transfer where isolates of CC8 were characterized by a clfB allele which was shared by all isolates of CC5, suggesting also the passage of this allele from CC5 to CC8. Taken together, all alleles of CC8 for clfB were acquired from other CCs, while the ancestral allele could not be found in our collection of isolates. Further studies will need to be performed to know whether this is true for the whole population and whether the ancestral allele was irreversibly lost.

To our surprise, the gene arcC, which on all sequenced S. aureus genomes is located directly next to clfB, was not shown to be part of this transfer. It was previously argued that the nonclonal evolutionary history of arcC might be a "hitchhiking effect," due to the physical proximity to clfB, which was predicted to undergo a different evolution (14). This homologous exchange is at odds with the hitchhiking suggestion, but it might be an exception. Further studies are needed to know whether or not hitchhiking has greatly contributed to the evolution of arcC.

The overall relatively small amount of evidence for homologous exchange is in contrast to a previous study that analyzed a set of accessory virulence genes and suspected frequent events of transfer (28). There, evidence for gene transfer was considered, due to the observed presence and absence of accessory virulence genes within the same phylogenetic lineage. We think that such a pattern could still be explained with a predominantly clonal evolution, where genes were subsequently lost from certain isolates within lineages; such a pattern does not in itself account as evidence for homologous exchange. Such a model was already proposed for the molecular evolution of an accessory pathogenicity island (RD13), where the consecutive loss of genes was the most parsimonious model of evolution, indicating a predominantly clonal evolution (15).

Because we included the most variable class of genes, because these were distributed over the whole chromosome, and because we analyzed a highly diverse collection of isolates, we have maximized the chances to find evidence for homologous exchange. Therefore, we think that the high congruence between gene trees as presented in this study completed a panel of evidence for a predominantly clonal evolution of S. aureus (14, 15, 32).

Comparison of allelic and nucleotidic divergence. The graph produced by MLST housekeeping gene analysis showed a clear linear and positive trend between nucleotidic and allelic divergence, suggesting a predominantly clonal evolution. It confirmed the analysis of a previous study using a more extensive MLST gene data set obtained from a different collection of isolates (14) and thus confirmed the robustness of our data. The same general trend was observed with adhesion genes. The fact that a predominantly clonal evolution was observed in both conserved housekeeping genes and highly polymorphic cell surface genes suggests that the majority of S. aureus genes followed this evolution.

Surprisingly, a more detailed look at both graphs revealed that for small numbers of allelic differences (up to two in the case of MLST housekeeping genes and up to three in the case of core adhesion genes), there was no clear positive correlation with nucleotidic diversity. This was also true for the previous analysis but did not receive attention (14). No correlation is generally perceived as the result of recombination that overcomes the effects of divergence due to de novo point mutations. Our data suggest that recombination occurs between isolates differing by only few alleles. Thus, despite a predominantly clonal population structure of S. aureus, we postulate that recombination occurs more frequently between closely related strains than between distant phylogenetic lineages. Such model of a population structure was proposed by M. Tibayrenc and was called "strict homogamy" (38). This model supposed the existence of "discrete genetic entities" within a species that can be seen as isolated populations between which no gene flow occurs. Importantly, we should note that CCs generally contain pairs of strains that differ by no more than three loci, which would indicate that a clonal complex delimits an isolated population.

Biological significance. The attachment of a bacterial cell to the host is one fundamental step in the line of events for host infection and thus could contribute to epidemicity of MRSA strains. One of the reasons that we found no evidence for genetic differences between epidemic and sporadic isolates might be the limited number of adhesion genes used in this study. However, the analysis of seven other S. aureus surface genes (sas) also showed high congruence with MLST housekeeping genes, supporting the hypothesis that there are no phylogenetic differences between epidemic and sporadic isolates. Thus, it seems that there is no link or correlation between epidemic potential and a certain adhesion gene or allele. However, it could also be imagined that only localized parts of a protein are linked to epidemic behavior, resulting in a specific nucleotide signature within genes. This has not yet been addressed. Also, due to the likely homogamic population structure, it is possible that epidemic clones have independently evolved in each CC and cannot be compared between CCs. Indeed, an approach including only strains of CC8 isolated in the same country clearly showed that epidemic strains (Brazilian clone) displayed significant physiological differences to other strains of the same CC. The epidemic strains produced more biofilm and showed stronger adherence to epithelial cells (1).

Concluding remarks. S. aureus adhesion genes can be regarded as highly polymorphic and, compared to housekeeping genes, represent a highly variable genomic compartment. We showed that purifying selection was present among all genes, whereas it was less strong among accessory adhesion genes. High congruence between gene trees of different genomic compartments indicated a clonal population structure and showed that it is remarkably prevalent in S. aureus genome evolution. Therefore, the association of a specific genetic element with epidemic MRSA clones failed. However, evidence for homologous exchange was detected, and we showed a previously undescribed transfer between CC5 and CC8. We also found that closely related S. aureus isolates that fit the demarcation of CCs showed evidence of recombination. This suggests that despite the remarkably overall clonal S. aureus population structure, recombination might significantly contribute to evolution of CCs.

 
We thank H. de Lencastre, M. Aires de Sousa, K. W. Larssen, W. Wannet, M. Struelens, J. Etienne, and F. Vandenesch for providing us with epidemic and sporadic MRSA strains. We are very grateful to D. A. Robinson for kindly providing the BLUND program and for helpful online discussions.

This work was funded with grant 3200B0-101044 from the Swiss National Foundation for Research.

 
* Corresponding author. Mailing address: Hospital Preventive Medicine, University Hospital of Lausanne (CHUV), Avenue du Bugnon 48, 1011 Lausanne, Switzerland. Phone: 41 21 314 0268. Fax: 41 21 314 4060. E-mail: Gerrit.Kuhn{at}chuv.ch.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, January 2006, p. 169-178, Vol. 188, No. 1
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