Journal of Bacteriology, March 2004, p . 1518-1530, Vol . 186, No . 5

eBURST: Inferring Patterns of Evolutionary Descent among Clusters of Related Bacterial Genotypes from Multilocus Sequence Typing Data

Edward J . Feil,^1* Bao C . Li,² David M . Aanensen,² William P . Hanage,² and Brian G . Spratt²

Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY,¹ Department of Infectious Disease Epidemiology, Imperial College London, St . Mary's Hospital Campus, London W2 1PG, United Kingdom²

Received 29 August 2003/ Accepted 14 November 2003

 
The introduction of multilocus sequence typing [MLST] for theprecise characterization of isolates of bacterial pathogenshas had a marked impact on both routine epidemiological surveillanceand microbial population biology . In both fields, a key prerequisitefor exploiting this resource is the ability to discern the relatednessand patterns of evolutionary descent among isolates with similargenotypes . Traditional clustering techniques, such as dendrograms,provide a very poor representation of recent evolutionary events,as they attempt to reconstruct relationships in the absenceof a realistic model of the way in which bacterial clones emergeand diversify to form clonal complexes . An increasingly popularapproach, called BURST, has been used as an alternative, butpresent implementations are unable to cope with very large datasets and offer crude graphical outputs . Here we present a newimplementation of this algorithm, eBURST, which divides an MLSTdata set of any size into groups of related isolates and clonalcomplexes, predicts the founding [ancestral] genotype of eachclonal complex, and computes the bootstrap support for the assignment.The most parsimonious patterns of descent of all isolates ineach clonal complex from the predicted founder[s] are then displayed.The advantages of eBURST for exploring patterns of evolutionarydescent are demonstrated with a number of examples, includingthe simple Spain^23F-1 clonal complex of Streptococcus pneumoniae,"population snapshots" of the entire S . pneumoniae and Staphylococcusaureus MLST databases, and the more complicated clonal complexesobserved for Campylobacter jejuni and Neisseria meningitidis.

 
The ability to accurately determine the genetic relatednessof isolates of bacterial pathogens [or other disease agents]is fundamental to molecular epidemiological and evolutionarystudies . In recent years, the use of nucleotide sequence variationat multiple housekeeping loci has become increasingly popularfor strain characterization, as it has advantages for inferringlevels of relatedness between strains and the reconstructionof evolutionary events [1, 2, 6-14, 18-23, 25, 28, 29].

In many bacterial species, genetic variation at housekeepingloci accumulates as frequently or more frequently by homologous recombination [replacement of small chromosomal segments withthose from related isolates] as by point mutation [15] . Over the long term, recombination may prevent the true relationships between distantly related isolates of a species from being discerned. Epidemiological studies, however, are typically concerned with disease outbreaks or the spread of antibiotic-resistant or virulent strains between countries . Over these very short evolutionary timescales, of weeks to a few hundred years, recombination is unlikely to prevent the recognition of clones and clonal complexes within most bacterial populations . Thus, although the phylogenetic complexities introduced by homologous recombination may be problematic over long periods of evolutionary time [14, 15], given an appropriatemodel of bacterial evolution, it should be possible to accuratelyreconstruct evolutionary events that occur over short timescales,even if rates of recombination are high.

Characterization of isolates of bacterial pathogens on the basis of sequence variation is carried out by multilocus sequencetyping [MLST], which generates approximately 450 bp of nucleotidesequence for internal fragments of seven housekeeping loci foreach isolate [23, 33] . The different sequences at each locusare assigned different allele numbers, and each strain is definedby the alleles at the seven loci [the allelic profile] . Eachunique allelic profile [or genotype] is assigned a sequencetype [ST], which is a convenient and unambiguous descriptorfor the strain [or clone] . Analyses of isolates of several bacterialspecies by MLST [7-10, 23, 25] support the view obtained from earlier studies [3, 26, 32] that a considerable proportion ofa population belongs to a limited number of clusters of closelyrelated genotypes, here referred to as clonal complexes . Clonalcomplexes are typically composed of a single predominant genotypewith a number of much less common close relatives of this genotype[15].

The simplest model for the emergence of clonal complexes isthat a founding genotype increases in frequency in the population,as a consequence either of a fitness advantage or of randomgenetic drift, to become a predominant clone [15] . As it increases in frequency in the population, the founding genotype gradually diversifies, to result in a clonal complex . In terms of MLST, descendants of the founder will initially remain unchanged inallelic profile, but over time variants in which one of theseven alleles has changed [by point mutation or recombination]will arise . These genotypes, which have allelic profiles thatdiffer from that of the founder at only one of the seven MLSTloci, are called single-locus variants [SLVs] . Eventually, SLVswill diversify further, to produce variants that differ at twoof the seven loci [double-locus variants [DLVs]], at three ofthe loci [triple-locus variants [TLVs]], and so on.

MLST data are typically represented by a dendrogram [e.g., the unweighted pair-group method with arithmetic averages [UPGMA]]on the basis of a matrix of pairwise differences in the allelicprofiles of the isolates . This dendrogram provides a convenientmeans of identifying isolates that are identical or closelyrelated in genotype and that can be assigned to the same cloneor clonal complex . However, the topology of such dendrogramscan be somewhat arbitrary, and they provide essentially no informationon the patterns of evolutionary descent of the isolates withina clonal complex or the identity of the founder.

Here we describe a new implementation of the BURST algorithm, called eBURST . This approach subdivides large MLST data setsinto nonoverlapping groups of related STs or clonal complexesand then discerns the most parsimonious patterns of descentof isolates within each clonal complex from the predicted founder.As this approach is dependent upon the correct assignments offounding genotypes, a bootstrapping procedure is introducedto gauge the level of confidence in these assignments . Througha very simple set of rules, eBURST can be used to explore howbacterial clones diversify and can provide evidence concerningthe emergence of clones of particular clinical relevance . Wedemonstrate the utility of this approach by using MLST datafrom antibiotic-resistant Streptococcus pneumoniae [8, 35] andfrom Staphylococcus aureus [9, 11, 16], Campylobacter jejuni[7, 31, 34], and Neisseria meningitidis [20, 23] . The rapidrate of clonal diversification in the latter two species [13, 31] provides a particularly challenging test of procedures thataim to untangle the short-term evolutionary history of a bacterialspecies.

 
eBURST algorithm. The eBURST algorithm is implemented as a Java applet at http://eburst.mlst.net, and detailed guidance in its use is available at this website. A description of the algorithm is given below.

Subdivision of input data into groups. The first step of the eBURST algorithm is to subdivide STs intogroups . Every ST within an eBURST group has a user-defined minimumnumber of identical alleles in common with at least one otherST in the group . eBURST groups therefore are mutually exclusive;no ST can belong to more than one group . The default settingin eBURST is the most exclusive group definition, in which STsare included within the same group only if they share identicalalleles at six or seven of the seven MLST loci with at leastone other ST in the group . Thus defined, each group equatesto a single clonal complex [see below].

The above procedure provides a list of the STs assigned to each group along with their observed frequencies in the data set.STs that cannot be assigned to any group are called singletons.For example, with the default group definition, singletons aredefined as STs differing at two or more alleles from every otherST in the sample . eBURST also allows all of the input data tobe treated as a single group by selecting a group definitionof zero of seven shared alleles . This procedure allows the clusteringpatterns among all isolates within a complete MLST databaseto be visualized as a single eBURST diagram ["population snapshot"].

eBURST groups and clonal complexes. We draw an important distinction between an eBURST group anda clonal complex . Whereas an eBURST group is simply a collectionof STs that are placed together according to the selected groupdefinition, a clonal complex refers to a biologically meaningfulcluster of STs that have diversified very recently from a commonfounder . The STs within an eBURST group obtained with the moststringent [exclusive] group definition are closely related andare considered to belong to a single clonal complex . Groupsobtained with a less stringent group definition should not beequated with clonal complexes . eBURST displays only the mostlikely patterns of evolutionary descent within each clonal complexand does not attempt to reconstruct pathways between clonal complexes, even if they are closely related . Clonal complexes therefore are defined conservatively as a cluster of STs inan eBURST diagram in which all STs are linked as SLVs to atleast one other ST . These may be represented individually whenthe default group definition is used or as separate clustersof linked STs when a less stringent group definition is used.

Assignment of primary founders. For each clonal complex, eBURST identifies the ST that is mostlikely to represent the founding genotype [the primary founder].eBURST also attempts to identify the most likely founder ofa group when a more relaxed group definition is used, althoughoften in such situations the assigned founder is unlikely torepresent the original genotype of the entire group . The primaryfounder is predicted on the basis of parsimony as the ST thathas the largest number of SLVs in the group or clonal complex.This method of assigning the founder takes into account the way in which clones emerge and diversify; most of the initial diversification of a clone results in variants of the founderthat differ at only one of the seven alleles [i.e., SLVs ofthe founder] . If two STs in a group have the same number ofSLVs, then the one with the larger number of DLVs is chosen.In such situations, the confidence in the assignment is low,as reflected in the bootstrap values . In some groups, typicallythose composed of a very limited number of STs, it may not bepossible to assign a primary founder . The frequency of a givenST in the input data is not used in the procedure to assignfounders; however, founders often correspond to the most predominantSTs, a fact that adds independent support to the assignments.

Assigning levels of confidence in founding genotypes. A measure of statistical confidence in each of the assignedprimary founders is made by a bootstrap resampling procedure.eBURST is used to divide the input population into groups accordingto the selected group definition; for each group, one exampleof each ST is extracted, and a user-defined [default, 1,000]number of random data sets of the same size as the extractedST set is produced by resampling with replacement . eBURST isrun on the resampled data sets from each group, and the ST thatis assigned as the primary founder in each resampling is determined.Conditional bootstrap values for each ST in the group are generatedaccording to the percentage of times that the ST is assignedas the founder; resamplings in which the ST cannot be assignedas the founder due to its absence from the resampled data setare omitted . An ST that is assigned as the founder in each ofthe resamplings in which it is present therefore has a bootstrapvalue of 100% . The computation of bootstrap values is restrictedto gauging the confidence of the assignment of primary foundersfor individual clonal complexes by using the default [most stringent]group definition.

Assignment of subgroups and subgroup founders. Large clonal complexes typically contain subgroups and thereforehave both primary and subgroup founders . For example, an SLVof the primary founder may have increased in frequency and diversifiedto generate a number of its own SLVs, thus becoming a subgroupfounder . The promotion of an ST to a subgroup founder dependsupon the number of previously unassigned SLVs that it defines[see below] . This definition can be user defined, but the defaultsetting is at least two previously unassigned STs [i.e., atleast three links to other STs, including the link to its assumedprogenitor].

A single ST may be an SLV of more than one founder . When anST is an SLV of both primary and subgroup founders, the ST is preferentially assigned to the primary founder . When an ST isan SLV of two or more subgroup founders, eBURST initially assignsSLVs on the basis of the distance from the primary founder,but a local optimization procedure then reassigns SLVs preferentiallyto the largest subgroup . In this way, the same model of clonalexpansion is used for both primary and subgroup founders whilelinks between subgroups are retained [see the readme file at http://eburst.mlst.net for more details] . Although any givenST may be an SLV of more than one founder, this procedure allowsan ST to be assigned to only one founder . This means that thenumber of SLVs of each subgroup founder shown in the eBURST diagram often is smaller than the total number shown in the initial eBURST output table, as some SLVs will have been preferentially assigned to the primary founder or to other subgroup founders.

Text and graphical output from eBURST. eBURST has a variety of input options [including direct inputfrom MLST databases; http://www.mlst.net] and produces an output table defining the STs in each group, the number of isolatesof each ST, and the number of SLVs, DLVs, and TLVs of each ST.The predicted primary founder is identified [where possible]along with the percent recovery of each ST as the primary founderof the clonal complex in the bootstrap resamplings.

The eBURST diagrams display the patterns of descent of all STs within each clonal complex from the primary founder . The earlier version of the algorithm [BURST] positioned SLVs and DLVs ofthe primary founder within concentric rings [11, 15], whereaseBURST shows a radial link from the primary founder to eachof its SLVs by a solid line . A second difference is that only links to SLVs are shown; DLVs of the primary founder are linked only when the intermediate SLV on the path from the founderto the DLV is present in the input data . With the default groupdefinition [six of seven shared alleles], all STs must be SLVsof at least one ST in the group, and the eBURST diagram willshow a single cluster [a clonal complex] in which all STs arelinked . With the less stringent group definition [five of sevenshared alleles], more than one cluster of linked STs [each ofwhich is a clonal complex] may be displayed along with a numberof individual unlinked STs . The lack of linking between twoclusters within a single group implies that no ST in one clusteris an SLV of any ST in the other cluster . Similarly, individualunlinked STs are not SLVs of any ST in the group . Thus, eBURSTis very conservative and only shows links between STs that havediverged very recently, that differ at only a single locus, and that are considered to belong to the same clonal complex.

Each ST is represented as a circle; the number beside the circle is the ST [except in Fig . 1, the ST numbers have been removed for increased clarity] . The frequency of each ST [i.e., the number of isolates of the ST in the input data] is indicatedby the area of the circle . The primary founder is given in blue,while subgroup founders are given in yellow . The initial eBURSTdiagrams were edited, as required, to produce the final figures;details of the editing functions within eBURST are providedat http://eburst.mlst.net . Editing only changes the positionsof the STs to improve the clarity of the diagram and does notchange any of the links between the STs.

 
Input data. The complete current sets of isolates [as of July 2003], withtheir STs and allelic profiles, were extracted from public MLSTdatabases at the following websites: S . pneumoniae [http://spneumoniae.mlst.net; 1,638 isolates, 893 STs]; S . aureus [http://saureus.mlst.net; 1,072 isolates, 191 STs]; C . jejuni [http://campylobacter.mlst.net; 2,001 isolates, 796 STs]; and N . meningitidis [http://neisseria.mlst.net; 3,730 isolates, 2,609 STs] . eBURST was applied to the entire database for each species with the group definitions specifiedto identify the groups, and eBURST diagrams were generated.

Construction of trees. UPGMA dendrograms were constructed from the matrix of pairwisedifferences in the allelic profiles of the isolates by usingthe Statistica package [Statsoft Inc., Tulsa, Okla.].

 
A multiply antibiotic-resistant clone of S . pneumoniae. As discussed above, eBURST is primarily an epidemiological tool designed for examining clonal diversification over short evolutionary timescales . Antibiotic-resistant strains therefore provide asimple test case, as these are unlikely to predate the introductioninto medicine of the antibiotics to which they show resistanceand should have diversified little from their primary founderwithin this very short period of time.

Strains of S . pneumoniae that are resistant to multiple classes of antibiotics were first reported from South Africa and Spain in the late 1980s; one of the first of these to be characterizedis the Spanish multiply antibiotic-resistant serotype 23F clone[Spain^23F-1] [24] . The majority of isolates assigned as Spain^23F-1 by molecular typing methods have been shown by MLST to havethe same allelic profile [ST81] [35] . All isolates with an allelicprofile similar to that of ST81, sharing four or more of the seven MLST alleles, were extracted from the pneumococcal MLST database . In order to compare the results from eBURST with thosefrom more traditional techniques, a UPGMA dendrogram was constructedfrom the matrix of pairwise differences in the allelic profilesof the extracted isolates . Figure 1 shows multiple isolates of ST81, a cluster of STs very closely related to ST81 [all SLVs of ST81], and one slightly more distantly related ST [aDLV of ST81]; all of these isolates were multiply antibioticresistant . None of the isolates on the dendrogram that weremore distantly related to ST81 [linkage distance of greaterthan 0.4] were multiply antibiotic resistant, and they werevery unlikely to have descended from ST81.

The entire pneumococcal MLST database was entered into eBURST,and groups were defined with the stringent [default] group definition [six or more shared alleles] . The group containing ST81 wasdisplayed as an eBURST diagram [Fig . 1, inset] . Consistent with its high frequency, ST81 was assigned as the primary founderof the ST81 [Spain^23F-1] clonal complex, with bootstrap support of 100%; all of the other isolates in this eBURST group were SLVs of ST81 . The prevalence of ST81 in the input data set is reflected by the area of the circle in the eBURST diagram . Theone DLV of ST81 was not included, as the linking SLV was notpresent in the MLST database, although it was included whenthe group definition was made less stringent [five of sevenshared alleles] . The structure of this clonal complex thereforeis simple, with the founder radially linked to its 13 SLVs,reflecting the very short evolutionary timescale over whichST81 has diverged [less than 50 years].

Pneumococcal population snapshot. The ability of eBURST to provide an overview of the clonal complexeswithin an entire MLST database was demonstrated by an analysisof all 1,638 isolates in the pneumococcal MLST database, accountingfor 893 STs . All isolates were analyzed as a single group bysetting the group definition to zero of seven shared alleles;the eBURST diagram is shown in Fig. 2 . The diagram shows themajor clusters of linked STs [clonal complexes], the minor clusters,linked triplets and doublets, and individual unlinked STs . TheST81 clonal complex shown in Fig . 1 is labeled . Note that thespacing between unlinked STs and clonal complexes provides noinformation concerning the genetic distance between them.

 
Evolution of MRSA. S . aureus is an important gram-positive human pathogen and,since the early 1960s, methicillin-resistant S . aureus [MRSA]isolates have emerged . MRSA isolates are now particularly commonin hospitals, although their prevalence is also increasing inthe community . The gene conferring resistance to methicillinis transmitted horizontally through the S . aureus population,and MRSA clones are known to have emerged independently on multipleoccasions [11, 27].

An extensive MLST data set is available for global collectionsof MRSA isolates, and the BURST algorithm was previously usedto determine the origins of MRSA clones from their antibiotic-sensitive forebears [11] . The MLST database for S . aureus as of July 2003contains 1,072 isolates [191 STs] from global sources . Theseisolates are a mixture of MRSA and methicillin-susceptible S.aureus [MSSA] from disease cases and asymptomatic carriage. The eBURST diagram shown in Fig . 3 is the population snapshotof the entire S . aureus database showing the linked clustersof STs [clonal complexes], with the primary founders and subgroupfounders identified . There were 12 clusters of four or more STs for which the primary founder could be assigned; many of these clonal complexes were previously described from a studyof 334 isolates recovered from Oxfordshire, United Kingdom [9, 16] . Interspersed among these clonal complexes were minor groups,typically doublets joined by an SLV link, and individual unlinkedSTs that were not SLVs of any other STs in the database.

 
Most of the clonal complexes of S . aureus are simple, with a primary founder surrounded by SLVs and, in some cases, DLVs.The ST30 and ST239 clonal complexes are more complicated [Fig.3] . The major ST30 clonal complex contains both MRSA and MSSAisolates [9, 11] . ST30 is the predicted primary founder [99% bootstrap support] of this clonal complex . All of the MRSA isolates within this complex belong to ST36, with the exception of theone SLV of ST36 that has probably descended from it . ST36 isa well-characterized epidemic MRSA clone [EMRSA16] that appearsto have emerged following the acquisition of methicillin resistanceby an SLV of ST30 [11] . One of the SLVs of ST30 appears to have diversified further to produce a DLV [ST39] that has become successful and that has formed a subgroup with its own SLVs.

The ST239 clonal complex includes the earliest known MRSA clone [ST250] [6, 11] and three other STs that represent MRSA clonescommonly encountered within hospitals [ST8, ST239, and ST247][6, 11] . All three of these major STs have diversified to producetheir own SLVs, but as ST239 has the largest number of SLVs,it has been assigned as the primary founder of the clonal complex.This assignment is based on a single SLV; ST239 has eight SLVs,whereas ST8 has seven [one of these SLVs is preferentially assignedto the primary founder, as discussed in Materials and Methods].However, additional evidence, discussed by Enright et al . [11],suggests that ST8 rather than ST239 is the true founder of thisclonal complex . The ambiguity in the assignment of the primaryfounder is reflected in the bootstrap support values obtainedwhen this clonal complex is analyzed separately by eBURST withthe default group definition of six of seven shared alleles.The bootstrap support values for ST239 and ST8 are 70% and 66%,respectively, thus alerting the user to the fact that the assignmentof ST239 as the primary founder is not robust.

C . jejuni ST21 clonal complex. C . jejuni is a gram-negative bacterial pathogen that causesgastroenteritis in humans and that is commonly isolated fromchicken and cattle . An MLST scheme for this species was presentedby Dingle et al . [7] . The C . jejuni MLST database contains 2,001isolates [796 STs] . Recombination, which is believed to be frequentin this species [31], may lead to clones that diversify rapidlyto produce complicated clonal complexes . The ST21 clonal complexis the largest within the C . jejuni database [7] . The likelyprimary founder of this complex was identified by Dingle etal . [7] by using a combination of BURST and splits decompositionanalysis . However, these authors did not use BURST to attemptto reconstruct the evolutionary pathways within this complexand instead used splits decomposition analysis for this purpose[7] . Although this analysis confirmed ST21 as the most likelyprimary founder, the relationships between the STs were characterizedby an extensive network, and recent patterns of descent couldnot be inferred.

Figure 4 shows a UPGMA clustering dendrogram containing one example of each ST that shares three or more alleles in common with ST21 . This figure illustrates the size and complexity ofthe ST21 complex and the difficulties in inferring the mostlikely evolutionary pathways . The 2,001 isolates in the publicC . jejuni MLST database were entered into eBURST and, with thestringent [default] group definition, the group including ST21was identified . Figure 5 shows the eBURST diagram for the STs assigned to this clonal complex [688 isolates; 180 STs]; manyof these STs have been added to the public MLST database subsequentto the analysis by Dingle et al . [7] . The analysis is consistent with that reported by Dingle et al . [7], in that ST21 remainsthe most likely primary founder [with 99% bootstrap support]. This ST has 37 SLVs, whereas the two next most prevalent STs each have 24 SLVs . Several SLVs [and two TLVs] of ST21 [shownin yellow in Fig . 5] have emerged as successful subgroup founders,each with its own cluster of linked SLVs.

 
The primary and subgroup founders correspond to the STs thatare the most prevalent within the ST21 complex . For example,ST21 is the most common ST within the group [123 isolates].Subgroup founders also are relatively common and, for the ninemost common STs, there is a close relationship between the frequencyof the ST and the number of SLVs of that genotype [data notshown].

N . meningitidis clonal complexes. High rates of recombination also are a feature of the meningococcalpopulation, and clones diversify rapidly [13] . The entire public meningococcal MLST database [3,730 isolates; 2609 STs] was analyzed by eBURST with the stringent [default] group definition of sixof seven shared alleles . Groups corresponding to the ET37 [ST11],A4 [ST8], and ET5 [ST32] clonal complexes [3, 4, 20, 23] weredisplayed as eBURST diagrams . Figure 6 shows the eBURST diagram for the ST32 clonal complex [4], which has a primary founder [ST32; 100% bootstrap support] surrounded by a ring of SLVs, one of which [ST33] has diversified to become a large subgroup founder . In addition, there are numerous DLVs and TLVs of theprimary founder and the major subgroup founder.

 
In both the ST8 and the ST11 clonal complexes, there was alsoa single strongly supported primary founder [100% bootstrapvalues] and a simple pattern of diversification from the founderto produce a large number of linked SLVs and DLVs . On a UPGMAdendrogram, isolates of the ST8 and ST11 complexes [4] appearedto be related [Fig . 7], and this finding was explored by relaxing the eBURST group definition to five of seven shared alleles. Under these conditions, isolates of both clonal complexes wereplaced within a single group, although they formed two separateclusters, since no ST within the ST8 complex was an SLV of anyST in the ST11 complex [Fig . 8].

 
A dendrogram separates STs assigned to lineage 3 of N . meningitidis [4, 23, 30] into two major clusters of lineages representingthe ST41 and ST44 clonal complexes [Fig . 9] . Isolates of bothST41 and ST44 complexes are assigned as a single clonal complexby eBURST [six of seven shared alleles], and this clonal complexis the largest so far observed by MLST for any species [411isolates; 304 STs] . ST41 was assigned as the primary founderof the lineage 3 clonal complex, with 69 SLVs [79% bootstrapsupport], and ST44 was identified as a large subgroup founder,with 64 SLVs [57%] . The eBURST diagram [Fig. 10] confirms thatthe lineage 3 complex is divided into two major subgroups, thefounders of which [ST44 and ST41] are connected through ST303.The ST41 subgroup is the largest, consistent with the statusof ST41 as the primary founder . Curiously, ST303 is observedonly three times in the database and yet has a total of 35 SLVs[25 of which are not apparent in Fig . 10, as they have beenpreferentially assigned to either ST44 or ST41, as these arelarger subgroups; see Materials and Methods] . ST41 and ST44 are both SLVs of ST303, and it is possible that ST303 is the real primary founder of this complicated [and presumably relatively old] clonal complex but now is rarely encountered among contemporary isolates.

 
The two major subgroups of the ST41-ST44 [lineage 3] clonalcomplex contain a number of subgroups, and the complexity ofthe diagram in Fig . 10 reflects the presumably rapid diversification of this highly successful complex . This example also further illustrates that eBURST is able to reveal possible evolutionary pathways even for the largest and most complicated clonal complexes.

 
The relationships among isolates of bacterial species typicallyare displayed with a clustering algorithm, which identifiesclosely related genotypes but, in the absence of a realisticmodel of clonal expansion, provides no information about thefounding genotypes or the likely patterns of evolutionary descentwithin the clusters . We address this important problem by usinga new implementation of an algorithm that extracts this informationfrom MLST data [or, in principle, other multilocus data] . Afull description of the features of eBURST is available in thedocumentation provided at http://eburst.mlst.net . The BURST algorithm was also recently incorporated as a set of "priority rules" into the minimum-spanning-tree method within the latest BioNumerics cluster analysis module [Applied Maths, Sint-Martens-Latem, Belgium].

The S . pneumoniae example is a very simple one, because the selected clonal complex is less than 50 years old and all isolates [except for a single DLV] are SLVs of the phylogenetically central ST81, which is likely the founder [35] . The example is also simple because no antibiotic-susceptible isolates with genotypes similar to ST81 have been identified, and resistance appearsto have occurred within a rare genotype that subsequently hasincreased greatly in frequency under strong selection.

S . aureus clones diversify mainly by point mutation [16], andin most cases, the clonal complexes also have a simple structure, with a single founder and a number of linked SLVs [Fig . 3]; the ST239 complex is more complicated and is discussed further below.

However, the clonal complexes containing the major MRSA clonesare more complex than the pneumococcal example, because resistanceto methicillin has emerged in successful MSSA clones withinpreexisting and presumably relatively old methicillin-susceptibleclonal complexes [11] . In contrast, the pneumococcal Spain^23F-1 antibiotic-resistant ST81 clone has an allelic profile thathas not been observed among antibiotic-susceptible isolates.

Clonal complexes of C . jejuni and N . meningitidis were selected for analysis because recombination rates are known to be high in these species [13, 31], resulting in rapid diversificationof clones and thus providing a challenging test of the utilityof the eBURST algorithm . In both species, the complex and somewhatarbitrary branching patterns among STs produced by a dendrogramwere transformed by eBURST into patterns of evolutionary descentthat are relatively easy to interpret . With the exception of lineage 3, which was more complicated, eBURST resolved the major meningococcal clonal complexes into a single primary founder surrounded by a large number of descendant SLVs and occasional subgroups [Fig . 6 and 8].

The advantages of the conservative approach used by eBURST,in which links are shown only between STs that differ at a singlelocus, are demonstrated by the analysis of the meningococcalclonal complexes . With the default group definition, eBURSTshows that the great majority of isolates of both the ST8 andthe ST11 clonal complexes are SLVs of their respective stronglysupported primary founders . Relaxing the stringency of the groupdefinition to five of seven shared alleles places both of theseclonal complexes into a single group, although the ST8 and ST11clusters themselves are not linked . A less conservative approachthat would allow links to be drawn between DLVs would connectthese two clusters, but the validity of the links would be doubtful,as links between DLVs are expected to be less robust than thosebetween SLVs . These two clonal complexes clearly are related[5, 23] and probably emerged as two subgroups of the same clonalcomplex, although the precise evolutionary events that resultedin their divergence cannot be unambiguously reconstructed.

This conservative approach will result in the exclusion of some STs that should be connected to a primary founder by virtueof descent . For example, there is a single DLV of the S . pneumoniae Spain^23F-1 clone [ST81] that is multiply antibiotic resistant and therefore almost certainly descended from ST81; however,in the absence of an intermediate SLV in the MLST database,it is not linked to the other STs in the cluster . However, thebenefits of the conservative approach, which attempts to identifyclusters of STs with the highest level of confidence in theircommon descent, are considered to outweigh the omission of theoccasional DLV from the eBURST diagram.

An eBURST diagram clearly provides far more information about founding genotypes and patterns of evolutionary descent thana dendrogram . The analysis of the major C . jejuni ST21 clonal complex shows that eBURST also sheds more light on its diversification than could be achieved with splits decomposition [7] . eBURSTconfirmed ST21 as the ancestor of the C . jejuni ST21 clonalcomplex and also revealed that several SLVs of ST21 appear to have diversified to form major subgroups . It may be illuminating to map to the eBURST diagram other information about these isolates, such as host preference, although such an analysis is outside the scope of this study.

It should be stressed that the eBURST program provides onlya hypothesis about the origins and patterns of descent withinclonal complexes . The assignments of primary founders are likelyto be correct when only a single ST in a clonal complex hasvery strong bootstrap support, but care should be used in inferringpatterns of descent when more than one ST has considerable bootstrapsupport or when no ST has strong bootstrap support [which isoften the case for very small complexes] . It must also be emphasizedthat the bootstrapping procedure is designed for use with thedefault group definition, in which all STs are part of a singleclonal complex.

The presence of two [or more] STs with good bootstrap support occurs mainly within large clonal complexes and provides analert that the assignment of the primary founder by eBURST isunlikely to be robust and that further exploration of the datais required . The ST239 complex of S . aureus was used to illustratethis type of situation . Three STs within this complex are veryprevalent in the S . aureus MLST database, and two of them havehigh and approximately equal bootstrap values . One of them [ST239]is predicted to be the group ancestor, as it has one more SLV,but consideration of the presence of the mecA gene [which confers resistance to methicillin], the structure of the mec region, and the frequencies of variant alleles within SLVs suggest thatthis prediction is incorrect and that ST8 is the most likelyprimary founder [11] . This latter reassignment is biologically plausible, as it makes the primary founder phylogenetically central; the other two major STs become the founders of major subgroups, which are derived from ST8 by a change at a singlelocus [Fig . 3].

A similar situation occurs with lineage 3 of N . meningitidis, in which two major STs, which are DLVs of each other, have substantial bootstrap support . Although one of these is assigned as the primary founder, by analogy with the S . aureus ST239 complex, it is equally possible that these two STs are the founders oflarge subgroups and that the primary founder is the phylogenetically central ST303 [Fig . 10] . On the basis of this hypothesis, ST41and ST44 are both successful SLVs of ST303 that have diversified to become the founders of large subgroups . This example demonstrates the power of this approach, as the rarity of ST303, combined with the relatively complicated structure of the lineage 3 clonal complex, makes it very unlikely that this possible pattern ofdescent would have been revealed by other clustering techniques.

Even in situations in which the primary founder of a large group cannot be assigned unambiguously, the relationships betweenSTs are still likely to approximate the true patterns of descent,and it is only the direction of descent between the differentsubgroups [i.e., the assignment of primary as opposed to subgroupfounders] that tends to be uncertain . The problem of assigninga clear primary founder in some groups may result from a shiftin ST frequencies over time, so that for old clonal complexes,there may be few examples of the primary founder [and its SLVs]relative to subgroup founders in contemporary samples of thepopulation . This problem also may be exacerbated by samplingbias . For the ST239 clonal complex, sampling bias could havearisen from an overrepresentation of antibiotic-resistant [MRSA]strains within the data set, as these strains are of particularclinical relevance, and many strains within the S . aureus MLSTdatabase originate from hospital collections [9, 11].

Alternatively, natural selection may impose a bias within the population owing to the emergence of strains with a strong adaptive advantage, such as antibiotic resistance . For example, in Spainabout 40% of pneumococci from carriage and disease cases areantibiotic resistant [17] . A well-sampled contemporary collection of isolates from this country will be very different from that obtained 50 years ago, due to the strong selective advantageof genotypes that have become resistant, and such a major shiftin ST frequencies could have an impact on the assignment ofprimary founders . The selective advantage of resistant strainsmay have led to the increase in the frequency of MRSA cloneST239 in the population, with subsequent diversification resultingin a larger number of SLVs of ST239 than of its immediate ancestor,ST8 . It is not clear how to solve these problems within theconfines of an algorithm, although the bootstrapping procedurecan help to identify cases in which assignments of foundersare not secure . When bootstrapping indicates that there maybe more than one candidate primary founder, sampling bias withinthe data set should be considered, and any additional phenotypic,genotypic, or epidemiological data that are available shouldbe used to examine the relative plausibility of the alternativefounders and patterns of descent.

A general feature of bacterial clonal complexes is that the primary founder predicted by eBURST usually corresponds to a prevalent ST . STs that become founders of major clonal complexes[or subgroups] must predate their descendants and will haveincreased in frequency in the population . Thus, in the absenceof strong selection and with a reasonably unbiased samplingframe, they are likely to outnumber their descendants . Examinationof the eBURST diagrams shows that the primary and subgroup founderstypically are prevalent STs . The number of isolates of eachST is not used by eBURST for the assignment of founders, butthe predominance of STs assigned as predicted founders providesadditional independent support for the assignments.

Although the assignments of primary founders, the computationof the confidence of these assignments, and the patterns ofdescent are all designed for use with individual clonal complexes,eBURST also can be used to produce an overall view of a bacterialpopulation [the population snapshot] . Figures 2 and 3 show examplesof this type of display, which allows the overall structure of a bacterial population to be visualized . eBURST also can help to describe the clonal structures of populations in a quantitative way . For example, the number of clonal complexes observed within a population and the numbers of founders and subgroup founders [i.e., the number of nodes within a complex] provide a meansof describing and comparing the structures of different populationson a purely quantitative level . However, any comparisons between populations require similar sampling frames to produce meaningful results . Finally, the identification of well-supported founding genotypes and their respective SLVs allows an estimate of the relative contributions of recombination and point mutation toward clonal diversification, as discussed elsewhere [13, 15].

 
This work depended on the availability of the public MLST databases, which are kept in the laboratory of Brian Spratt at ImperialCollege London [S . aureus and S . pneumoniae, curated by Mark Enright and Angela Brueggemann] and the laboratory of MartinMaiden at the University of Oxford [C . jejuni and N . meningitidis, curated by Kate Dingle and Keith Jolley] . We acknowledge all those who submit strains to these databases . We also thank Christophe Fraser for helpful discussions.

This research was supported by the Wellcome Trust . B.G.S . isa Wellcome Trust principal research fellow . E.J.F . is supportedby an MRC career development award.

 
* Corresponding author . Mailing address: Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom . Phone: 44 1225 383021 . Fax: 44 1225 386779 . E-mail: e.feil@bath.ac.uk .

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What Is Staphylococcus Aureus?, What Is Genetics?, What Is Biotechnology?, What Is Protein?, What Is Salmonella?, o, Microbe, i, Microorganism, n, Microorganisms, s, Bacteria, s, Microbiology, i, Shigella, a, Antibiotic resistance, n, Escherichia coli, r, Candida albicans, r, Yeasts, r, S. cerevisiae, e, Culture medium, o, Proteus, n, Haemophilus, i, Aeromonades, n, Multidrug resistant, r, Microflora, a, Microflora, o, Penicillin, e, Yeasts, a, Microbiological, o, Candida albicans, o, Vibriosis, e, Salmonella, c, Bacillus, c, Culture medium




 

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