Donkor et al. BMC Genomics 2012, 13:569
http://www.biomedcentral.com/1471-2164/13/569RESEARCH ARTICLE Open AccessComparative phylogenomics of Streptococcus
pneumoniae isolated from invasive disease and
nasopharyngeal carriage from West Africans
Eric S Donkor1,5, Richard A Stabler1, Jason Hinds3, Richard A Adegbola4, Martin Antonio2 and Brendan W Wren1*Abstract
Background: We applied comparative phylogenomics (whole genome comparisons of microbes using DNA
microarrays combined with Bayesian-based phylogenies) to investigate S. pneumoniae isolates from West Africa,
with the aim of providing insights into the pathogenicity and other features related to the biology of the organism.
The strains investigated comprised a well defined collection of 58 invasive and carriage isolates that were
sequenced typed and included eight different S. pneumoniae serotypes (1, 3, 5, 6A, 11, 14, 19 F and 23 F) of varying
invasive disease potential.
Results: The core genome of the isolates was estimated to be 38% and was mainly represented by gene functional
categories associated with housekeeping functions. Comparison of the gene content of invasive and carriage
isolates identified at least eleven potential genes that may be important in virulence including surface proteins,
transport proteins, transcription factors and hypothetical proteins. Thirteen accessory regions (ARs) were also
identified and did not show any loci association with the eleven virulence genes. Intraclonal diversity (isolates of
the same serotype and MLST but expressing different patterns of ARs) was observed among some clones including
ST 1233 (serotype 5), ST 3404 (serotype 5) and ST 3321 (serotype 14). A constructed phylogenetic tree of the
isolates showed a high level of heterogeneity consistent with the frequent S. pneumoniae recombination. Despite
this, a homogeneous clustering of all the serotype 1 strains was observed.
Conclusions: Comparative phylogenomics of invasive and carriage S. pneumoniae isolates identified a number of
putative virulence determinants that may be important in the progression of S. pneumoniae from the carriage
phase to invasive disease. Virulence determinants that contribute to S. pneumoniae pathogenicity are likely to be
distributed randomly throughout its genome rather than being clustered in dedicated loci or islands. Compared to
other S. pneumoniae serotypes, serotype 1 appears most genetically uniform.Background
Streptococcus pneumoniae is part of the normal bacterial
flora of the upper respiratory tract, but is also associated
with severe invasive diseases, including meningitis,
pneumonia and septicaemia as well as non-invasive dis-
eases such as otitis media [1]. Transmission of S. pneu-
moniae occurs through respiratory droplets and is more
commonly associated with healthy individuals who carry
the organism in the upper respiratory tract [2,3]. World-
wide, the annual incidence of invasive pneumococcal* Correspondence: Brendan.Wren@lshtm.ac.uk
1Department of Pathogen Molecular Biology, London School of Hygiene and
Tropical Medicine, London WC1E 7HT, UK
Full list of author information is available at the end of the article
© 2012 Donkor et al.; licensee BioMed Central
Commons Attribution License (http://creativec
reproduction in any medium, provided the ordisease (IPD) is about one million and though a global
problem, the public health impact of IPD is higher in
the developing world, where children less than 5 years of
age are most affected [4,5].
The capsule is considered the main virulence deter-
minant of S. pneumoniae, and only a few capsular types
tend to be associated with invasive disease which is
partly due to differential ability of the variant capsular
types to resist phagocytosis [6,7]. Epidemiological evi-
dence indicates that while some capsular types are often
associated with invasive disease, some may be associated
with carriage, while others are associated with both inva-
sive disease and carriage [8-12]. In addition to the cap-
sule, it is known that other pathogenic factors areLtd. This is an Open Access article distributed under the terms of the Creative
ommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
iginal work is properly cited.
Donkor et al. BMC Genomics 2012, 13:569 Page 2 of 11
http://www.biomedcentral.com/1471-2164/13/569required by S. pneumoniae for virulence [13], but the
genetic factors that explain the pathogenesis and viru-
lence of the organism is not fully understood.
Comparative whole genome analysis using DNA micro-
arrays has been utilised to investigate several bacterial
pathogens. The approach involves assessing the absence
or presence of genes from strains based on reference gen-
ome(s) fixed to microarray, followed by robust statistical
algorithms to infer the evolutionary relationships between
test strains that is usually represented as a phylogenetic
tree [14-18]. This allows interrogation of the genome con-
tent of bacterial strains from a variety of sources, environ-
ments and disease states, and the identification of genetic
markers that may explain how different strains are
adapted to their respective niches or disease capability.
Few comparative genomics studies have been carried out
on S. pneumoniae, and these studies were based mainly on
strains from developed countries and none from Sub-
Saharan Africa [19-23], where the organism exacts its
greatest toll. Though these studies have contributed sig-
nificantly to our understanding of S. pneumoniae, several
aspects of the organism particularly, its pathogenicity, evo-
lution and population structure in the Sub-Saharan Africa
is still inadequately understood. In view of this, we carried
out comparative phylogenomics (whole genome compari-
sons of microbes using DNA microarrays combined with
Bayesian-based phylogenies) of 58 S. pneumoniae epide-
miologically well defined isolates from West Africa with
the aim of providing insights into the pathogenicity and
other features related to the biology of the organism.
Results and discussion
Strain selection
A total of 58 isolates were used in this study, and were col-
lected from three West African countries including The
Gambia (52), Nigeria (4) and Ghana (2). All isolates were
serotyped [24] and multilocus sequence typed [25]
(Figure 1). The isolates comprised 35 invasive and 23 car-
riage isolates and were recovered from subjects of an age
range of 3 months to 58 years. The carriage isolates were
recovered from the nasopharynx of healthy human popu-
lations [9,26], while invasive isolates were recovered from
specimens of blood (87%), CSF (10%) and lung and knee
aspirates (3%) of patients with IPD [8,27,28]. Based on in-
formation from capsule serotype, the isolates were selected
to cover pneumococcal serotypes of varying invasive dis-
ease potential in West Africa [8,9,26-29]. Eight serotypes
were selected and included serotypes 1, 3, 5, 6A, 11, 14,
19 F and 23 F (Table 1). In West Africa, Serotypes 1 and 5
are common in IPD but rare in carriage and represent ser-
otypes of high invasive disease potential; serotypes 3, 11
and 19 F are common in carriage but not in invasive dis-
ease, and represent serotypes of low invasive disease po-
tential; serotypes 6A, 14, and 23 F are common in bothinvasive disease and carriage, and represent serotypes of
intermediate disease potential. Overall, the isolates studied
covered 35 different sequence types; invasive isolates cov-
ered 22 sequence types while the carriage isolates covered
16 sequence types.
Core gene set of S. pneumoniae strains
Whole genome microarray comparisons of 58 isolates of
S. pneumoniae were used to compute the minimal core
gene set. This was achieved by calculating the total num-
ber of coding sequences (CDSs) that had a GACK (Gen-
ome Analysis by Charlie Kim) score of ‘present’ in every
isolate and the control strain (TIGR4) using the
advanced filtering function available in Genespring 6.1.
The minimal core gene set for the S. pneumoniae iso-
lates was 831 CDSs, which translates to 38% of the total
genome of the isolates. Similarly, individual core gene
sets were computed for invasive and carriage isolates
and were found to be 1162 CDSs (84%) and 919 CDSs
(63%) respectively (p < 0.05). The low core genome esti-
mate of 38% observed in this study is quite similar to a
core genome of 46% reported by Hiller et al. [22] but
contrast significantly with a core genome of 73% reported
by Obert et al. [21] and 80% reported by Tettelin et al.
[20]. However, Hiller et al. [22] demonstrated that indi-
vidual strains core orthologous clusters account for
68–79% of the genome. Reported core gene sets of some
other streptococci species include 58% for S. thermophi-
lus [30], 82.5% for S. uberis [31] and 82% for S. agalac-
tiae [32]. Relatively low core gene of 28% has been
reported for some non-streptococcal organisms such as
Yersinia enterocolitica [16]. Thus core genome quantifi-
cation may vary significantly among different bacterial
strain collections and is highly dependent on the cut off
method used as well as the core genome definition. The
relatively low core genome reported in this study may
reflect the more stringent approach used to compute the
core genome (Section “Microarray data analysis and
comparative phylogenomics”).
As expected, the S. pneumoniae core gene set was
represented by many of the functional categories that
are involved in housekeeping functions such as DNA
metabolism, intermediary metabolism, protein synthesis
and cellular processes. This concurs with other S. pneu-
moniae microarray studies [20-22]. Conserved house-
keeping genes including those identified in the core
genome of the West African isolates have been shown to
be abundant in sequenced pneumococcal genomes
[33-37]. Comparison of eight nasopharyngeal S. pneumo-
niae genomes with nine published genomes (including
TIGR4 and R6) identified 1,454/3,170 (46% ) ortholo-
gous gene clusters conserved among all 17 strains [22].
The core genes consisted mainly of housekeeping genes
but also contained 462 hypothetical proteins with no
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Isolate ID Ser ST Source Accessory Regions
1 2 3 4 5 6 7* 8* 9 10 11* 12* 13
PNC8719 1 ND NPS
PNC9310 1 ND NPS
PNI0197 1 612 KA
PNI0028 1 618 BLD
PNI0225 1 618 BLD
PNI0509 1 618 BLD
PNI0213 1 3579 BLD
PNI0276 1 3960 BLD
PNC223 5 3955 NPS
PNC429 5 4014 NPS
PNC492 5 1233 NPS
PNC5036 5 1233 NPS
PNC5226 5 3957 NPS
PNI121 5 289 BLD
PN1I50 5 289 BLD
PNI156 5 289 BLD
PNI0004 5 3966 BLD
PNI018 5 ND BLD
PNI0223 5 3964 BLD
PNI0144 5 3404 BLD
**PNI0163 5 3404 CSF
PNI0369 5 3404 BLD
PNC3182 3 925 NPS
PNI0145 3 3962 LA
PNI0151 3 458 BLD
PNI0243 3 3961 BLD
PNI0324 3 3961 BLD
PNC11458 11 3951 NPS
PNC2953 11 3949 NPS
PNC6612 11 3948 NPS
PNI0491 11 2158 BLD
PNC7696 19F 925 NPS
PNC882 6A 913 NPS
PNC9001 6A 913 NPS
PNC9304 6A ND NPS
PNI0046 6A 2983 BLD
PNI0320 6A 1348 BLD
PNI0170 6A 3958 BLD
**PNI0474 6A 4012 CSF
PNI097 6A 3959 BLD
NPS
PNC4436 23F 1336 
NPS
PNC4501 23F 2174 
NPS
PNC7921 23F 2174 
NPS
PNC36 23F 3969 
PNI0183 23F 4012 BLD 
PNI0229 23F 4012 CSF 
BLD
PNI0230 23F 4012 
BLD
**PNI0482 23F 4012 
BLD
PNI0424 23F 63 
BLD
PNI18 23F ND 
NPS
PNC6147 14 3310 
NPS
PNC7128 14 3968 
NPS
PNC9088 14 3321 
NPS
PNC6741 14 3321 
PNI0048 14 3321 BLD 
PNI0520 14 3321 CSF 
PNI0175 14 3963 CSF 
PNI0579 14 2108 BLD 
Green colour- AR is completely present  
Red colour- AR is completely absent 
Yellow- most genes of AR (>50%) are present 
Blue- most genes of AR (>50%) are absent 
Source: NPS-nasopharyngeal swab; BLD-blood; CSF-cerebrospinal fluid; LA-lung aspirate; KA-knee aspirate 
ND- ST not determined 
* contain virulence genes identified by signature tagged mutagenesis [41]. 
** associated with case fatality
Figure 1 Distribution of accessory regions among S. pneumoniae isolates of different serotypes and sequence types.
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Table 1 Serotype distribution of invasive and carriage S. Table 2 Genes that showed significant differences
pneumoniae isolates used for comparative between invasive and carriage isolates
phylogenomics analysis Gene Function Frequency
Serotype No. of invasive isolates No. of carriage isolates Total Invasive Carriage
1 6 2 8 (N = 35) (N = 23)
3 4 1 5 SP0071 immunoglobulin A1 protease 35 2
5 9 5 14 SP0091 ABC transporter, permease protein 18 5
6A 5 3 8 SP0161 hypothetical protein 25 7
11 1 3 4 SP0238 hypothetical protein 25 6
14 4 4 8 SP0491 hypothetical protein 26 9
19 F 0 1 1 SP0514 hypothetical protein 25 9
23 F 6 4 10 SP0743 transcriptional regulator 30 14
Total 35 23 58 SP0955 competence protein 27 9
SP1032 iron-compound ABC transporter 25 5
SP1612 hypothetical protein 30 12
SP1800 transcriptional activator 31 14
N indicates total number of invasive or carriage isolates.known function [22]. More than 70% of the West Afri-
can S. pneumoniae core genes were present in the core
gene set of Hiller et al. [22]. Virulence determinant
CDSs including transport proteins and various enzymes
such as hyaluronidase, neuraminidase A, phosphogluco-
mutase and triosephosphate isomerase were identified in
the S. pneumoniae core gene set in this study. By com-
parison, hyaluronidase and neuraminidase A were also
demonstrated to be conserved within the 17 genomes
analysed by Hiller et al. [22]. The presence of virulence
determinants in all the invasive as well as carriage iso-
lates in the current study probably indicates that these
virulence determinants are necessary, but not adequate,
to determine the ability of an isolate to cause invasive
disease. Also, analysis of the core gene set of the isolates
showed that a wide range of mobile and extrachromo-
somal elements were conserved, which agrees generally,
with information obtained from pneumococcal genomes
that have been fully sequenced [20,33-37]. Within the
Hiller et al. core gene list are twelve transposases listed
[22], which were also present in the core gene list of our
study.
Putative virulence determinants and accessory regions
Overall, comparison of the gene content of invasive and
carriage isolates identified at least eleven CDSs that were
significantly associated with invasive isolates compared
to carriage isolates (Table 2). IgA protease showed the
largest difference between invasive and carriage isolates.
This surface protein degrades IgA and thus helps S.
pneumoniae to evade host immune system and provide
an opportunity for more effective invasion [38,39]. Sev-
eral transport proteins of the ABC type, were also sig-
nificantly associated with invasive isolates and may be
based on the fact that these transport proteins are
involved in the transport of metal ions or nutrients
which are required by pathogenic bacteria for growth
and metabolic activities [40]. Several transcriptional
genes were associated with invasive isolates/diseasewhich has been previously reported [41]. Several other
proteins were also associated with invasive isolates, but
were mainly hypothetical proteins and therefore require
further investigation.
An accessory region was defined as three or more con-
tiguous genes not conserved in all the isolates. Thirteen
accessory regions (ARs) were identified in this study
(Table 3) and the distribution of such regions among the
study isolates is presented in Figure 1. By comparison
previous studies have reported 13–38 ARs [21,42,43].
Nine ARs identified in the current study have been pre-
viously reported and include AR2, AR3, AR4, AR5, AR6,
AR7, AR8, AR9 and AR13 [21,42,43]. Four ARs includ-
ing AR1, AR10, AR11 and AR12 identified in this study
have not been previously reported and represent novel
ARs. In the case of AR1 and AR10, none of the genes in
these regions have been associated with virulence and
thus their functional role in virulence is not clear. Two
of the novel ARs namely, AR11 and AR12 contained
genes identified by Signature Tagged Mutagenesis (STM)
as required for virulence in mice, but none of these ARs
was associated with invasive disease [41]. The poor cor-
relation of invasive isolates or serotypes of high invasive
disease potential with ARs that contain virulence genes
has also been reported by Bloomberg et al. [42]. In the
study of Bloomberg et al. [42], though 24 ARs contain-
ing virulence genes were identified, only two of such
ARs were preferentially found in invasive isolates or ser-
otypes of high invasive disease potential.
Despite the poor correlation between invasive disease
and ARs that contain virulence genes, differences in
virulence between different clones of the same serotype
could be explained by the distribution of such ARs in
some cases (evidence provided below). This indicates
that the role of ARs in pneumococcal virulence may be
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Table 3 Accessory regions identified among S. pneumoniae isolates
Accessory Region TIGR4 locus Gene annotation
AR1 SP0482 hypothetical protein
SP0483 ABC transporter, ATP-binding protein
SP0484 hypothetical protein
AR2 SP1050 transcriptional regulator, putative
SP1051 hypothetical protein
SP1052 phosphoesterase, putative
AR3 SP1062 ABC transporter, ATP-binding protein
SP1063 ABC-2 transporter, permease protein, putative
SP1064 transposase, IS200 family
AR4 SP1129 integrase/recombinase, phage integrase family
SP1130 transcriptional regulator
SP1131 transcriptional regulator, putative
AR5 SP1134 hypothetical protein
SP1135 hypothetical protein
SP1136 hypothetical protein
SP1137 GTP-binding protein, putative
AR6 SP1315 V-type ATP synthase subunit D
SP1316 V-type ATP synthase subunit B
SP1317 V-type ATP synthase subunit A
SP1318 V-type ATP synthase subunit F
SP1319 V-type sodium ATP synthase, subunit C
SP1320 V-type sodium ATP synthase, subunit E
SP1321 V-type ATP synthase subunit K
SP1322 V-type ATP synthase subunit I
SP1323 hypothetical protein
SP1324 ROK family protein
SP1325 oxidoreductase, Gfo/Idh/MocA family
SP1326 neuraminidase, putative
SP1327 hypothetical protein
SP1328 sodium: solute symporter family protein
SP1329 N-acetylneuraminate lyase
SP1330 N-acetylmannosamine-6-phosphate 2-epimerase
SP1331 phosphosugar-binding transcriptional regulator, putative
AR7 SP1341 ABC transporter, ATP-binding protein
SP1342 toxin secretion ABC transporter, ATP-binding/permease protein
SP1343* prolyl oligopeptidase family protein
SP1344* hypothetical protein
AR8 SP1433 transcriptional regulator, AraC family
SP1434* ABC transporter, ATP-binding/permease protein
SP1435 ABC transporter, ATP-binding protein
SP1436 hypothetical protein
AR8 SP1437 hypothetical protein
SP1438 ABC transporter, ATP-binding protein
AR9 SP1616 allulose-6-phosphate 3-epimerase
SP1617 PTS system, IIC component
SP1618 PTS system, IIB component
SP1619 PTS system, IIA component
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Table 3 Accessory regions identified among S. pneumoniae isolates (Continued)
SP1620 PTS system, nitrogen regulatory component IIA
SP1621 putative transcription anti terminator BglG family protein
SP1622 transposase, IS200 family
AR10 SP1738 guanylate kinase
SP1739 hypothetical protein
SP1740 hypothetical protein
AR11 SP1856* transcriptional regulator, MerR family
SP1857 cation efflux system protein
SP1858 transcriptional regulator, TetR family
SP1859* transporter, putative
SP1860 choline transporter
AR12 SP1896* sugar ABC transporter, permease protein
SP1897 sugar ABC transporter, sugar-binding protein
SP1898* alpha-galactosidase
AR13 SP2159 fucolectin-related protein
SP2160 hypothetical protein
SP2161 PTS system, IID component
SP2162 PTS system, IIC component
SP2163 PTS system, IIB component
* contain virulence genes identified by signature tagged mutagenesis [41].serotype dependent which has also been reported
[21,42]. Included in this study, were four invasive iso-
lates of the serotype 5 virulent PMEN clone ST 289, and
also two serotype 5 carriage isolates of ST 1233 which is
considered less virulent. The pattern of AR distribution
of the ST 289 isolates was the same and carried all the
ARs associated with virulence in this study (ARs 7, 8, 11
and 12). However, the ST 1233 isolates were deficient in
three of the four ARs associated with virulence including
AR7, AR8 and AR11. Thus these differences in ARs of
the two serotype 5 clones may explain the enhanced
virulence of ST 289. A similar observation has been
reported for two serotype 19 F clones namely, ST 162
which is a virulent clone and ST 425, a non-virulent
clone [42]. These observations also highlight the varia-
tions in virulence of clones of the same serotype and are
important in pneumococcal vaccination, where virulent
clones of a serotype rather than non-virulent clones of
that serotype, undergo capsular switching and emerge
with non-vaccine serotypes [44,45]. For an invasive sero-
type like serotype 5, it also shows that the ability of an
isolate to cause invasive disease is not only dependent
on the capsule type but also the genetic background of
the strain.
Though ARs may have some relevance in pathogen-
icity, the extent to which ARs contribute to pneumococ-
cal virulence is still not very clear. In this study, the 13
ARs identified did not show loci association with any of
the eleven potential virulence genes identified. Analysis
of the distribution of virulence genes identified by Havaand Camilli in TIGR4 indicates that the virulence genes
did not cluster [41]. These observations suggest that
virulence determinants that contribute to S. pneumoniae
pathogenicity are likely to be distributed randomly
throughout its genome rather than being clustered in
dedicated loci or islands. This agrees with the findings of
Obert et al. [21] which showed that ARs are more likely
to adapt S. pneumoniae to carriage rather than invasive
disease. Thus ARs may not play a highly prominent in
pathogenicity as observed in pathogens such as uro-
pathogenic Escherichia coli [46].
From Figure 1, it can be observed that some isolates of
the same serotype and ST were found to express different
patterns of ARs, which can be seen for ST 1233 (serotype
5), ST 3404 (serotype 5) and ST 3321 (serotype 14). This
phenomenon of intraclonal diversity has also been
observed in studies carried out by Silva et al. [43] and
Bloomberg et al. [42], and shows that strains of the same
serotype and ST may exhibit genetic and phenotypic dif-
ferences. In the study by Silva et al. [43] different patterns
of ARs was observed among pneumococcal isolates of ST
124 (serotype 14), while Bloomberg et al. [42] observed
different AR patterns among isolates of ST 176 (serotype
6B), ST 124 (serotype 14) and ST 156 (serotypes 14 and
19 F). Thus the current study provides evidence of the
phenomenon of intraclonal diversity beyond clones and
serotypes that have been previously reported. Bloomberg
et al. [42] pointed out that intraclonal diversity was rare
among serotypes of high invasive disease potential, as it
was not observed among clones of serotypes 1, 4 and 7 F
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http://www.biomedcentral.com/1471-2164/13/569included in their study. This finding contrasts with the
current study, where intraclonal diversity was consistently
exhibited by clones (ST 3404 and ST 1233) of serotype 5,
a serotype of high invasive disease potential. Nevertheless,
it can be observed that while intraclonal diversity occurred
among several serotype 5 clones, it did not occur among
the more virulent ST 289 (serotype 5) PMEN clone, indi-
cating that intraclonal diversity may be relatively rare
among more virulent clones. This is also the case for the
virulent ST 618 (serotype 1) clone and also the ST 4012
(serotype 23 F) clone, which is a novel clone and inferred
to be virulent, as it was the most frequent cause of mortal-
ity (Figure 1). This suggests some association of these
virulent clones with stability (uniform genetic content).
Dagerhamn et al. [47] have demonstrated that some
pneumococcal accessory regions may predict genetic re-
latedness similar to that predicted by MLST, which they
attributed to the influence of recombination on variations
in housekeeping genes (used for MLST) and as well as
accessory regions. Data on intraclonal diversity from the
current study further suggests that in some cases
accessory regions may also provide better resolution than
MLST, as highly genetically similar isolates of the same
serotype and MLST can be distinguished by their
accessory regions patterns. This shows the potential as a
pneumococcal typing scheme based on accessory regions
which would provide similar results to MLST but of better
resolution. However, it should be noted that typing by
analysis of ARs could be especially susceptible to being
confounded by horizontal gene transfer.
Comparative phylogenomics
The data obtained from microarray analysis was used to
generate a phylogenetic tree which is shown in Figure 2.
Three of the isolates (PNI676, PNI0108 and PNC12026)
subjected to phylogenomic analysis showed a distant as-
sociation with all the other isolates. These three isolates
were subjected to molecular serotyping using another
type of microarray [48] to confirm their identity as S.
pneumoniae. This showed that the three isolates were
not S. pneumoniae isolates but likely to be a closely
related Streptococcus species such as S. mitis or S. oralis,
and hence their separation from S. pneumoniae isolates
in the phylogenetic tree, which confirms the credibility
of the phylogenetic relationship among the isolates.
MLST of the three isolates also showed that the sequences
were divergent from those of known MLST alleles. The
three non-pneumococcal isolates were excluded in analysis
of the core genome (Section “Core gene set of S. pneumo-
niae strains”) as well as analysis of putative virulence deter-
minants and accessory regions (Section “Putative virulence
determinants and accessory regions”).
Phylogenetic analysis of the S. pneumoniae isolates
showed two major clades, with each clade comprising amixture of invasive and carriage isolates of varied sero-
types (Figure 2). Despite the heterogeneous clustering
of serotypes, all of the eight serotype 1 isolates (six in-
vasive and two carriage isolates) formed a subclade
(Figure 2A). Recently, Donati et al. [23] constructed a
phylogenetic tree based on 44 sequenced pneumococcal
genomes covering 19 different serotypes and 24 MLST
clonal clusters. By comparison, in this study, the poor
correlation observed between a serotype of an isolate
and its position in the tree except for serotype 1, agrees
well with the study by Donati et al. [23]. Similarly, the
poor correlation observed between an isolate from an
invasive or carriage source and its position in the tree
also agrees with the study by Donati et al. [23]. The
high level of heterogeneity among isolates in the phylo-
genetic tree of this study is probably due to recombin-
ation which occurs frequently among pneumococci. A
recent study by Croucher et al. [49] found more than
700 recombination events in 240 strains of the PMEN1
(Spain23F-1) multidrug-resistant lineage. According to
Feil et al. [50], evolution of the pneumococcal popula-
tion is dominated by recombination, and can abolish
any deep-rooted phylogenetic signal resulting in a pat-
tern of heterogeneity as observed in this study. The
homogeneous clustering observed among the serotype
1 isolates agrees with the uniform distribution of ARs
observed among the serotype 1 isolates, and reflects the
fact that, because this serotype is rarely carried, it is less
likely to undergo recombination. Within the phylogen-
etic tree, clustering of isolates of the same MLST was
observed (Figure 2B), which has also been reported by
Donati et al. [23] and Dagerhamn et al. [47], and pro-
vides evidence of the agreement between microarray
and MLST. This implies that the frequent pneumococ-
cal recombination did not eliminate phylogenetic sig-
nals related to a common ancestor though it may have
weakened such signals.
An attempt was made to use MacClade 4 to identify
CDSs which were associated with relevant clades and sub-
clades in the S. pneumoniae phylogenetic tree (Figure 2).
The two major clades formed, were associated with pres-
ence/absence of AR6, AR8, AR9 and AR13 (Table 3). These
ARs have been reported to have some importance in
pneumococcal pathogenicity [21,51,52]. The fact that each
clade comprised a mixture of invasive and carriage isolates
probably support the earlier claim in this study that ARs
may have little relevance in pneumococcal pathogenicity.
The formation of the serotype 1 cluster of isolates
(Figure 2A) was associated with 10 CDSs, all of which were
highly divergent or absent from these isolates.
Conclusions
The current study is unique in that it is based on a rela-
tively large number (58) of S. pneumoniae isolates from
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A
SUBCLADE 
A 
SUBCLADE 
B 
             _______________ 
0.1
B
SUBCLADE 
A 
SUBCLADE 
B 
             _______________           
0.1
Figure 2 (See legend on next page.)
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(See figure on previous page.)
Figure 2 A: Phylogeny of S. pneumoniae isolates (serotype analysis). Isolate names are shown in brackets while the corresponding serotypes
are indicated outside brackets; Non-pneumococcal isolates are shown by dotted lines. Invasive isolates are shown in red while carriage isolates
are shown in green; + indicates p = 1.0 while × indicates p > 0.9. B: Phylogeny of S. pneumoniae isolates (sequence type analysis). Isolate
names are shown in brackets while the corresponding MLST are indicated outside brackets; Non-pneumococcal isolates are shown by dotted lines.
Invasive isolates are shown in red while carriage isolates are shown in green; STND indicates MLST of the isolate was not determined; + indicates
p = 1.0 while × indicates p > 0.9.the developing world (West Africa), while other studies
were based mainly on isolates from developed countries.
Comparative phylogenomics of invasive and carriage S.
pneumoniae isolates identified a number of putative
virulence determinants that may be important in the
progression of S. pneumoniae from the carriage phase to
invasive disease. These putative virulence determinants
are currently being investigated by mutagenesis to con-
firm their role in pneumococcal pathogenicity. Virulence
determinants that contribute to S. pneumoniae patho-
genicity are likely to be distributed randomly throughout
its genome rather than being clustered in dedicated loci
or islands. Compared to other S. pneumoniae serotypes,
serotype 1 maintains a more uniform genetic content
which implies that serotype 1 strains are more likely to
be clonally related than strains of other serotypes.
Limitations
There are a number of limitations of the study. Firstly, the
microarray used was based on only two sequenced gen-
omes including TIGR4 and R6 strains, which are reference
strains from developed countries rather than the developing
world where the study isolates were collected. This means
that genes that are absent in the reference strains but
present in the study isolates may not be detected. Secondly,
it is not known if the genes detected are expressed in vivo
or not and if expressed under what conditions. The second
limitation is partly addressed by the fact that expres-
sions of some of the virulence genes identified (SP0071,
SP0743 and SP1032) have been demonstrated by other
investigators [53,54].
Methods
Identification of S. pneumoniae isolates and extraction of
DNA
The study isolates were confirmed to be S. pneumoniae by
the optochin test [55]. The isolates were purified on
5% blood agar plates and bacterial chromosomal DNA was
prepared using the Wizard gDNA purification kit (Pro-
mega). The concentration and purity of extracted DNA
was determined by means of a NanoDropW ND-1000
spectrophotometer (NanoDrop, Wilmington, USA).
Microarray analysis
S. pneumoniae genomic DNA extracted from the study
isolates and reference strain were analysed using theBμG@S SPv1.1.0 microarray as described previously
[43]. This microarray consisted of duplicate spotted PCR
products, representing all annotated genes in S. pneumo-
niae strains TIGR4 and R6. Briefly, 1 μg of DNA was la-
belled by random priming with Klenow polymerase to
incorporate either Cy3 or Cy5 dCTP (GE Healthcare)
for the reference strain or the test strain, respectively.
Equal amounts of the Cy3- and Cy5-labeled samples
were copurified through a Qiagen MinElute column
(Qiagen), mixed with hybridization solution (4× SSC
0.3% SDS), and denatured at 95°C for 2 min. The la-
belled sample was loaded on to a prehybridized micro-
array under one 22 mm by 22 mm Lifter Slip (Erie
Scientific), sealed in a humidified hybridization cassette
(Corning), and hybridized overnight by immersion in a
water bath at 65°C for 16 to 20 h. Slides were washed
once in 400 ml 1 × SSC, 0.06% SDS at 65°C for 2 min
and twice in 400 ml 0.06 × SSC for 2 min at room
temperature. The microarray slides were then scanned
with a GMS 418 Scanner (Genetic Microsystems) and
spot fluorescence intensities were determined with Ima-
Gene 5.5 (BioDiscovery Inc.). All the S. pneumoniae
study isolates were hybridized once against the TIGR4
reference strain and the microarray hybridization experi-
ments were repeated for isolates which gave poor
hybridization results.
Microarray data analysis and comparative phylogenomics
Analysis of the microarray data and comparative phylo-
genomics were carried out with GeneSpring v6.1 (Silicon
Genetics). Data were median normalized in GeneSpring
and normalized intensity data for each channel from
each microarray were used to run GACK (Genomotypy-
ing Analysis Charlie Kim), to determine whether genes
were present, absent, or divergent [56]. To run GACK
analysis, the raw values were divided by the control
values for each sample and then transformed into log2
ratio data. This was saved as a tab delimited file and
used as the input file for the GACK software. GACK
uses the log2 ratio data to categorize CDSs based upon
estimated probability of presence (EPP). Computation of
EPP was done by dividing the mapped normal curve
value (the expected value for a distribution in which all
spots have signal present on the hybridized microarray)
by the actual observed data distribution value for any
given ratio [56]. Two stringent cut-offs were used;
Donkor et al. BMC Genomics 2012, 13:569 Page 10 of 11
http://www.biomedcentral.com/1471-2164/13/569‘present’ is called only if a GACK EPP was ≥100% , ‘ab-
sent (or highly divergent)’ was only called in GACK EPP
was ≤0% EPP, ‘divergent’ genes were between 0 and
100% EPP. While this cut-off for absent is highly strin-
gent, the stringent hybridisations conditions equate to
divergence of greater than approximately 5% which may
result in an ‘absent’ call to a coding sequence that is
present hence ‘absent/highly divergent’. The resulting
assigned CDS from GACK analysis were re-entered into
GeneSpring 6.1 and a core genome of the isolates was
determined: core genome was defined as the set of genes
present in all the isolates investigated. Genetic differ-
ences among the isolates were also determined at a sig-
nificant level of p < 0.05 and Chi square was used to
confirm virulence genes (ie genes that were significantly
associated with invasive isolates).
The output of GACK was transformed into NEXUS
format, and the relationship of the strains was deter-
mined based on Bayesian method-based algorithms
implemented through Mr Bayes v3.0 software [57]. The
resulting phylogenetic trees were viewed using TREE-
VIEW (http://taxonomy.zoology.gla.ac.uk/rod/treeview.
html). Coding sequences (genes) associated with the
phylogenomic relationships of isolates and also the for-
mation of clades and subclades were evaluated using
MacClade 4 [58].Ethical considerations
The study was approved by the ethics committee of the
Medical Research Council (The Gambia). The isolates
used were gathered from various laboratories and
human subjects were not enrolled in the study.
Competing interests
The authors declare that they have no competing interests.Authors’ contributions
The study was conceived by BWW, RAS, MA and RAA. Microarray
experiments were performed by ESD. Bioinformatics analyses were done by
RAS, ESD and JH. The manuscript was drafted and revised by ESD, BWW,
RAS, MA, RAA and JH.Acknowledgements
We acknowledge the Wellcome trust for funding BμG@S (Bacterial
Microarray Group at St. George’s, University of London) where microarrays
used in the study were obtained. We also acknowledge the Medical
Research Council in The Gambia for providing isolates for the study.
Author details
1Department of Pathogen Molecular Biology, London School of Hygiene and
Tropical Medicine, London WC1E 7HT, UK. 2Vaccinology Theme, Medical
Research Council Unit, The Gambia. 3Bacterial Microarray Group, St. George’s
University of London, London SW17 0RE, UK. 4GlaxoSmithKline Vaccines,
Wavre, Belgium. 5Department of Microbiology, University of Ghana Medical
School, Accra, Ghana.
Received: 30 March 2012 Accepted: 18 October 2012
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doi:10.1186/1471-2164-13-569
Cite this article as: Donkor et al.: Comparative phylogenomics of
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