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									Journal of Medical Microbiology (2005), 54, 351–356                                                                   DOI 10.1099/jmm.0.45924-0




                                        A molecular-capsular-type prediction system for 90
                                        Streptococcus pneumoniae serotypes using partial
                                        cpsA–cpsB sequencing and wzy- or wzx-specific
                                        PCR
                                        Fanrong Kong,1,5,6 Weizhen Wang,2 Jiang Tao,3 Lei Wang,3,5 Quan Wang,3
                                        Archcna Sabananthan4 and Gwendolyn L. Gilbert1,6,7
                                        1
 Correspondence                             Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical
 Gwendolyn L. Gilbert                       Research (ICPMR), Westmead, NSW 2145, Australia
 lyng@icpmr.wsahs.nsw.gov.au            2
                                            Wuhan First Hospital, Hubei Province, Wuhan 430022, PR China
                                        3
                                            TEDA School of Biological Sciences and Biotechnology and Tianjin State Laboratory of Microbial
                                            Functional Genomics, Nankai University, TEDA College, Tianjin 300457, PR China
                                        4
                                            School of Biotechnology and Biomolecular Sciences, The University of New South Wales,
                                            NSW 2052, Australia
                                        5
                                            Tianjin Biochip Technology Corporation, TEDA, Tianjin 300457, PR China
                                        6
                                            Department of Medicine, The University of Sydney, NSW 2066, Australia
                                        7
                                            National Centre for Immunisation Research and Surveillance of Vaccine Preventable Diseases,
                                            Children’s Hospital at Westmead, NSW 2145, Australia


                                        In a previous study, a molecular capsular type (MCT) prediction system for 51 Streptococcus
                                        pneumoniae serotypes was developed based on a combination of partial cpsA–cpsB sequencing
                                        and serotype(s)/group(s)-specific PCR. In this study, another 169 S. pneumoniae isolates were
                                        added to the existing database of 427 isolates, including representatives of all 39 serotypes not
                                        previously studied. In addition to the authors’ own limited sequence data for all 90 serotypes, cpsA–
                                        cpsB sequence data published by the S. pneumoniae capsular loci-sequencing group (http://
                                        www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) at the Sanger Institute or available from
                                        GenBank were incorporated into the database. All serotypes, except 25A, were represented by at
                                        least two isolates. The number of sequence types identified was 138, of which 110 corresponded to
                                        single conventional serotypes (CSs); of these, 57 were represented by two or more isolates. Twenty-
                                        six sequence types were shared by between two and four CSs. To resolve these shared cpsA–cpsB
                                        sequence types and increase the discriminatory power of our system, the genes encoding the
                                        capsular polysaccharide flippase (wzx) and polymerase (wzy) were annotated and 24 new
                                        serotype(s)/group(s)-specific PCRs targeting wzy and two targeting wzx were designed. Using
                                        both cpsA–cpsB sequencing and wzx/wzy PCR, MCT correctly predicted the CSs of 516 (73 %)
                                        and the serogroup of an additional 155 (22 %) of the 708 isolates evaluated. For 5 % of isolates,
                                        MCT could not distinguish between members of five serotype pairs (37 isolates) containing
                                        members of different serogroups. Although further study of the relationship between MCT and CS is
 Received 13 October 2004               needed, this system now allows serotype or serogroup identification of 95 % of S. pneumoniae
 Accepted 1 December 2004               isolates.




Abbreviations: CS, conventional serotype/typing; MCT, molecular capsular type/typing.
The GenBank/EMBL/DDBJ accession numbers for the new partial cpsA (wzg)– cpsB (wzh) genes are AY508586–AY508641, AY621659, AY621660
and AY661448–AY661457.
Three phylogenetic trees, a schematic representation of related wzx genes and five tables of data are available as supplementary material in JMM Online.


45924 & 2005 SGM Printed in Great Britain                                                                                                         351
F. Kong and others


INTRODUCTION                                                               and wzy were generated by the neighbour-joining method using
                                                                           program MEGA (Kumar et al., 1994).
The importance of infections with Streptococcus pneumoniae
is being increasingly recognized (Gillespie, 1999). The use of             MCT prediction
molecular tools to speed the process of detection, culture                 Oligonucleotide primers. In addition to our previous MCT primer
identification and typing of S. pneumoniae has been widely                  pairs (Kong & Gilbert, 2003), 24 new serotype(s)/group(s)-specific
accepted (Gillespie, 1999).                                                oligonucleotide primer pairs targeting wzy and two targeting wzx were
                                                                           designed for this study. The specificities, sequences, numbered base
S. pneumoniae comprises at least 90 serotypes (Henrichsen,                 positions and melting temperatures (Tm ) are shown in Table S1
1995) distinguished by capsular polysaccharide antigens                    (available as supplementary data in JMM Online). Expected amplicon
(Garcia et al., 2000). Capsule production in S. pneumoniae                 lengths of different primer pairs were calculated from the 59-end
is largely controlled by the capsular polysaccharide synthesis             positions of the corresponding primers. Since these PCRs were designed
                                                                           to resolve serotypes with shared sequence types, all available isolates of
(cps) gene cluster (Garcia et al., 2000). While serotyping and             the relevant serotypes were tested. Finally, the specificities of all 24 new
antibiotic-susceptibility testing remain the primary methods               primer pairs were checked against a reference set of one strain of each of
for characterizing pneumococci, molecular typing can add                   the 90 serotypes.
greater discrimination and complementary information
(Hall, 1998). A molecular capsular typing (MCT) system                     DNA preparation, PCR, sequencing and sequence analysis. These
for S. pneumoniae will be of greatest value when it can fully              steps were performed as previously described (Kong & Gilbert, 2003).
replace serotyping and allow monitoring of capsule evolu-                  The only difference was that for the new PCRs 55–60 8C was used as the
tion (Lawrence et al., 2000, 2003; Enright & Spratt, 1998). In a           annealing temperature because of the low Tm values of the new primers.
previous study, we developed a MCT prediction system for
                                                                           Nucleotide sequence accession numbers. Fifty-six new sequences
51 S. pneumoniae serotypes based on sequencing and
                                                                           generated in this study for partial cpsA (wzg)–cpsB (wzh) genes were
serotype(s)/group(s)-specific PCR, targeting the cps gene                   deposited in GenBank with the following accession numbers:
cluster (Kong & Gilbert, 2003). In this study, we aimed to                 AY508586–AY508641, AY621659, AY621660 and AY661448–
extend the system to allow prediction of all 90 S. pneumoniae              AY661457 (see Table S2, available as supplementary data in JMM
serotypes.                                                                 Online).


METHODS                                                                    RESULTS AND DISCUSSION
Pneumococcal clinical isolates. This study was based on 169 well-          CS results
characterized S. pneumoniae isolates that represented 63 serotypes,
including all 39 that were not included in our previous study (which       CS was repeated on 23 isolates, at the Department of
involved 427 isolates belonging to 51 serotypes) (Kong & Gilbert, 2003).   Microbiology, Children’s Hospital at Westmead, because of
Seventy-two isolates were obtained from the National Institute for the     apparent inconsistencies between or sharing of sequence
Control of Pharmaceutical and Biological Products, Beijing, PR China       types and serotypes. After careful repetition by two different
(Zhang et al., 1990), three from the Microbiological Diagnostic Unit,
Public Health Laboratory, Department of Microbiology and Immunol-
                                                                           operators, isolates previously identified as serotypes 42 and
ogy, University of Melbourne, Victoria and 10 from the Statens Serum       41F (Kong & Gilbert, 2003) were reassigned to serotypes 31
Institut, Denmark. Conventional serotyping (CS) was performed by           and 41A, respectively; serotypes of three additional isolates
donor laboratories and the serotypes were known at the time of receipt.    were also corrected. The serotypes of 15 isolates (including all
A subset of well-characterized isolates, the serotypes of which were       those belonging to serotypes 27, 28F and 16A, and one each of
unknown at the time of receipt, was used to evaluate our genotyping        serotypes 6A, 38 and 25F) were confirmed as previously
system. It included 35 isolates provided by the Royal College of           defined. The final results are shown in Table S2 (available as
Pathologists of Australasia, Quality Assurance Program Pty Limited,        supplementary data in JMM Online).
New South Wales, Australia and 49 by the Department of Microbiology,
Children’s Hospital at Westmead.
                                                                           Extension of partial cpsA–cpsB sequence
Isolates were retrieved from storage by subculture on blood agar plates    database
(Columbia II agar base supplemented with 5 % horse blood) and
incubated overnight at 37 8C in 5 % CO2 .                                  Partial cpsA–cpsB sequencing primers. The sequencing
                                                                           primers cpsS1–cpsA3 produced amplicons from all isolates
Annotation and analysis of wzx and wzy. Analysis of homology and
protein hydrophobicity was performed to annotate the wzx and wzy
                                                                           studied in this and our previous study, except for three
genes in S. pneumoniae cps gene clusters. BLAST and PSI-BLAST (Altschul    belonging to rare serotypes, 25F, 25A and 38, and five that
et al., 1997) were used to search GenBank and Pfam protein motif           were non-serotypable (Kong & Gilbert, 2003). Two addi-
databases (Bateman et al., 2002) for possible gene functions. The          tional primer pairs, cpsS1–cpsA1 and cpsS3–cpsA2, formed
TMHMM v2.0 analysis program (http://www.cbs.dtu.dk/services/               amplicons from isolates belonging to serotypes 25F, 25A and
TMHMM-2.0/) was used to identify potential transmembrane segments          38 and two non-serotypable isolates (the other three non-
from amino acid sequences (Chen et al., 2003). Sequence alignment and
                                                                           serotypable isolates were not studied further).
comparison were done using the program CLUSTAL_W (Thompson et
al., 1994).
                                                                           Sequence type nomenclature. Sequence types were gen-
Phylogenetic trees. The phylogenetic trees for partial cpsA–cpsB, wzx      erally named according to the corresponding serotype, with a

352                                                                                                            Journal of Medical Microbiology 54
                                                                                    Partial cps genes of 90 S. pneumoniae serotypes


suffix representing the source of the isolate in which they        Online). cpsA–cpsB sequencing alone correctly characterized
were first identified. The name given where a sequence type         the serotype of 41 isolates and the serogroup of 13. Six isolates
was shared by multiple serotypes includes all (two to four) of    belonged to one of four new sequence types, which have been
the serotypes in ascending numerical order (e.g. 15B-15C-         added to our database (Table S2, available as supplementary
22F-22A) (Henrichsen, 1995). One or two representative            data in JMM Online). Serotype(s)/group(s)-specific PCR
sequences of each sequence type were deposited into Gen-          allowed correct serotype (18) or serogroup (seven) identifi-
Bank (see Tables S2 and S3, available as supplementary data       cation of another 25 isolates (including those belonging to
in JMM Online, for sequence type nomenclature and                 new sequence types). The remaining five isolates belonged to
corresponding GenBank accession numbers).                         one of three pairs of serotypes, individual members of which
                                                                  can be rapidly distinguished using serotype-specific antisera
Phylogenetic tree based on partial cpsA–cpsB genes. A             (see Table S4, available as supplementary data in JMM
tree based on partial cpsA–cpsB sequences of all isolates         Online).
studied is shown in Fig. S1 (available as supplementary data
in JMM Online). The tree shows similarities between               Other potential uses of the cpsA–cpsB database. It has
sequence types – analogous to a multilocus sequence type          been recently reported that serotype 14 variants of the France
tree (Enright & Spratt, 1998) – rather than true phylogenetic     9VÀ3 clone from Baltimore, Maryland, can be differentiated
relationships. Adding the sequence of an isolate of unknown       by cpsB gene sequence variation (McEllistrem et al., 2004). In
serotype to the cpsA–cpsB tree, which contains all the            addition, sequence variation in cpsB is related to S. pneumo-
sequences in our database, will allow us to infer its most        niae strain virulence (Morona et al., 2004). Incorporation of
likely serotype; the predictive accuracy will increase as the     these sequence variants and related epidemiological and
number of sequences increases (see Table S2, available as         virulence data into the cpsA–cpsB database would allow
supplementary data in JMM Online).                                them to be easily recognized by other researchers.

Sequence type vs. mutation. Based on our sequence type            Are shared sequence types plausible? In order to explain
definition, heterogeneity at one or more sites defines a new        the sharing of cpsA–cpsB sequence types by more than one
sequence type (Kong & Gilbert, 2003), which is consistent         serotype, we studied their antigenic formulae (Henrichsen,
with the widely accepted principle for definition of a multi-      1995). Among the 24 shared cps sequence types (genotypes),
locus sequence type (Enright & Spratt, 1998, 1999). This          the majority involved closely related serotypes (or pheno-
strategy does not allow us to distinguish significant evolu-       types). However, four (2-41A, 10A-17A, 10A-23F, 13-20)
tionary mutations from ‘accidental’ point mutations, but it is    were shared between apparently unrelated serotypes (no
a consistent, unambiguous basis for sequence type nomen-          antigenic cross-reactions) and three (11A-11D-18F, 15B-
clature (see Tables S2 and S3, available as supplementary data    15C-22F-22A, 17F-35B-35C-42) between both cross-react-
in JMM Online).                                                   ing and non-cross-reacting serotypes (Henrichsen, 1995)
                                                                  (see Table S3, available as supplementary data in JMM
Extension and ongoing evaluation of our sequence type             Online). The latter probably can be explained by recombina-
database. Our database has been extended to 90 serotypes,         tion events (Coffey et al., 1998, 1999).
but its development will continue as new sequence types or        S. pneumoniae is characterized by high-frequency recombi-
even serotypes are identified. In future, more isolates of each    nation within the cps gene cluster, including wzx, leading to
serotype will be examined as they become available and the        serotype ‘switching’ among isolates within genetic lineages
results added to our database. It may be necessary to modify      defined by relationships between the more conserved house-
the database when additional sequence types are identified         keeping genes (Coffey et al., 1998; Jiang et al., 2001).
and discrepancies are resolved (for example, our data differ      Although wzx sequences are usually highly variable (Samuel
from cps gene cluster sequences reported by the Sanger            & Reeves, 2003), we found that those of 24 serotypes share
Institute for serotypes 3, 25A and 28F).                          high-level (72–100 %) homology. We found three main
Progress so far demonstrates that it is possible to generate an   recombination sites within these 24 wzx sequences (base
accessible cpsA–cpsB sequence database for practical use by S.    positions 395, 775 and 1150) using PhylPro 1.0 (Weiller,
pneumoniae serotyping reference laboratories (McEllistrem         1998), which generated the diagrammatic representation of
et al., 2004). In general, the more serotypes, sequence types     polymorphic sites and hypothetical recombination events as
and isolates of each that are included in the database, the       shown in Fig. S2 (available as supplementary data in JMM
greater the accuracy of serotype prediction using sequence        Online). These regions of high-level similarity in wzx suggest
data. The rationale for making our database available at this     recent recombination.
stage is to allow others to use and contribute to further
evaluation of the effectiveness of the serotype prediction        Are wzx and wzy helpful?
system.
                                                                  In our previous study, we showed that wzx- and wzy-based
The results of our preliminary evaluation of 84 selected well-    PCRs increase the accuracy of cpsA–cpsB sequence-based
characterized isolates, representing 46 serotypes, are shown      serotype prediction (Kong & Gilbert, 2003; Rubin & Rizvi,
in Table S4 (available as supplementary data in JMM               2004). Therefore, in order to extend our serotype-prediction

http://jmm.sgmjournals.org                                                                                                    353
F. Kong and others


strategy to all 90 serotypes, we examined all known wzx and        differs from our partial cpsA–cpsB sequence for the same
wzy sequences (see Table S3, available as supplementary data       serotype 25A strain (supplied to us and the Sanger Institute
in JMM Online). This was facilitated by the timely publica-        by Statens Serum Institut). Our partial sequence results for
tion, by the Sanger Institute (http://www.sanger.ac.uk/Pro-        serotypes 38, 25F and 25A also show that serotype 25A cps is
jects/S_pneumoniae/CPS/), of the complete sequences of the         not identical to 29.
cps gene clusters of 90 serotypes. We independently anno-
                                                                   Most (110) sequence types correspond to a single serotype
tated all 89 available wzx and wzy sequences (not including
                                                                   and, of these, 57 are represented by two or more isolates. For
serotype 3, which lacks these genes), using 17 available
                                                                   516 of 708 (73 %) isolates and published sequences, CS and
serotype cps gene cluster sequences from GenBank as
                                                                   MCT are identical. MCT of another 155 (22 %) isolates
reference (see Table S5, available as supplementary data in
                                                                   identified the correct serogroup. For the remaining 37 (5 %)
JMM Online) (Kong & Gilbert, 2003). Our sequence data
                                                                   isolates, MCT could not distinguish between members of five
showed significant discrepancies when compared with the
                                                                   pairs of CSs which shared the same sequence type (7B/40,
Sanger Institute sequences for serotypes 3, 25A and 28F,
                                                                   12A/46, 25F/38, 35F/47F, 35C/42; see Table S2, available as
which have not been resolved despite repetition of sequen-
                                                                   supplementary data in JMM Online). Two antisera would be
cing.
                                                                   required to identify individual members of these groups.
Based on previous studies, both wzx and wzy should be
serotype-specific (Jiang et al., 2001; Kong & Gilbert, 2003),
but the present study suggests that this is not straightforward.   Relationship between the partial cpsA–cpsB, wzx
Our analysis showed that, for most serotypes, wzy is shorter       and wzy trees
but more heterogeneous than wzx (see Tables S3 and S5,             In the partial cpsA–cpsB tree (see Fig. S1, available as
available as supplementary data in JMM Online). These              supplementary data in JMM Online), and as suggested in
observations, as well as evidence of wzx recombination events      Table S2 (available as supplementary data in JMM Online),
(see above and Fig. S2, available as supplementary data in         some serotypes are clustered because they share the same or
JMM Online), suggest that wzy is a more suitable target for        very similar sequences. In the wzx and wzy trees these show
serotype(s)/group(s)-specific PCR for all 90 serotypes except       similar relationships (see Figs S3 and S4, available as
serotype 3, which lacks these genes and so will need to be         supplementary data in JMM Online), but there are differ-
identified on the basis of other serotype 3-specific cps genes       ences between the trees. For example, serotypes 17A, 34 and
(Kong & Gilbert, 2003).                                            10B, which are closely related in the cpsA–cpsB tree (see Fig.
To increase the predictive accuracy of our system, we              S1 ), are only distantly related in both the wzx and the wzy
designed 26 serotype(s)/group(s)-specific PCRs targeting            trees (see Figs S3 and S4), suggesting that they have different
wzy and two targeting wzx, in addition to those developed          evolutionary histories. This illustrates the potential risk of
in our previous study. The sensitivities and specificities of the   using a single gene or even one gene cluster to infer
26 new PCR primer pairs were assessed, initially, for the          phylogenetic relationships (Trzcinski et al., 2003). It also
corresponding shared sequence types and then with a                implies that, for a final accurate MCT prediction system, the
reference set of all 90 serotypes (see Table S1, available as      combination of different cps genes may increase the pre-
supplementary data in JMM Online for primer pair specifi-           dictive accuracy. Based on our study, we recommend the
city). All primer pairs amplified isolates belonging to             combined use of both cpsA–cpsB and wzy, at least.
corresponding serotypes (see Tables S1–S4, available as
supplementary data in JMM Online) and did not amplify
unrelated serotypes. As shown in our previous study (Kong &        PCR or microarray?
Gilbert, 2003), partial wzy and wzx sequencing can distin-         In future, microarray (genechip or equivalent)-based tech-
guish serotypes 7B and 7C from 40, 10F from 10C, 11F/11B           nology should be a practical solution for MCT prediction of
(identical) from 11C, 12A/46 (identical) from 12F/12B/44           all 90 pneumococcal serotypes (Magee et al., 2001). As a
(identical), 35A from 35C/42 (identical) and 35F from 47F.         prototype, we have developed a practical multiplex PCR and
However, they cannot resolve individual serotypes within           reverse line blot hybridization assay (van den Brule et al.,
some isolates of sequence types 25F-38 and 6A-6B.                  2002; Wang et al., 2004) to identify the 23 serotypes included
                                                                   in the polysaccharide vaccine. This assay showed very
Comprehensive MCT results. The final MCT results for 596            promising results in preliminary evaluation, using the 90-
isolates (427 previously studied and 169 new isolates) (Kong       serotype reference panel and a small number of clinical
& Gilbert, 2003) and 112 previously published cps sequences        isolates (data not shown). The results will be reported
are shown in Table S2 (available as supplementary data in          separately after systemic evaluation of a large number of
JMM Online). Our database now includes all 90 S. pneumo-           clinical isolates. We are also trying to develop a genechip
niae serotypes and 140 partial cpsA–cpsB sequence types            microarray to identify all the 90 serotypes. Meanwhile, we
(including two non-serotypable strains). We have at least two      will use the cpsA–cpsB sequencing and selected wzy/wzx PCR
isolates or sequences of 89 serotypes. We did not use the cps      strategy we previously described (Kong & Gilbert, 2003), for
sequence published by Sanger Institute for serotype 25A,           which we now have 26 additional primer sets, to resolve
which was reported to be identical to that of serotype 29 and      shared sequence types (Rubin & Rizvi, 2004).

354                                                                                              Journal of Medical Microbiology 54
                                                                                        Partial cps genes of 90 S. pneumoniae serotypes


The relationship between cps gene clusters and                     biotic resistance and virulence markers (Magee et al., 2001).
CSs                                                                However, unresolved controversies between CS and MCT
                                                                   deserve further study to improve our understanding of CS
Because cpsA–cpsB, wzx and wzy PCR/sequencing cannot
                                                                   and the accuracy of the MCT system.
resolve all serotypes, we studied selected whole cps gene
cluster sequences, especially for serotypes in which wzx and
wzy were very similar (see Table S3, available as supplemen-       ACKNOWLEDGEMENTS
tary data in JMM Online). Nevertheless, some serotypes             We are grateful for sequence data from and constructive dialogue with
remain unresolved, either because the heterogeneity between        Dr Stephen Bentley of the S. pneumoniae Capsular Loci Sequencing
their cps sequences was minor and inconsistent (e.g. sero-         Group at the Sanger Institute (ftp://ftp.sanger.ac.uk/pub/pathogens/
types 6A and 6B) (Kong & Gilbert, 2003) or because the             spn/) and Dr Margit Staum Kaltoft, Statens Serum Institut, Denmark.
serotype-specific gene was located outside the cps gene cluster     We thank Dr Michael Watson (Children’s Hospital at Westmead),
(e.g. serotype 37) (Llull et al., 2001). We cannot rule out the    Professor Geoffrey Hogg and Jenny Davis (Microbiological Diagnostic
possibility that some rare serotypes have arisen as a result of    Unit) and Dr Margit Staum Kaltoft for providing isolates. This study
                                                                   was funded, in part, by the National Centre for Immunization Research
aberrant gene replication and expression – such as serotypes       and Surveillance of Vaccine Preventable Disease, Children’s Hospital at
15B and 15C (van Selm et al., 2003) – or as an isolated            Westmead, NSW and a Chinese government 863 Program Grant
accidental event (Waite et al., 2001, 2003), without a             (2002AA2Z2051) to Lei Wang. We thank Mitchell Brown and Marianne
consistent molecular basis, which could explain their rarity       Gail Stewart for serotyping selected isolates, and Mark Wheeler and Ilya
(Henrichsen, 1995).                                                Henner for sequencing.


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http://jmm.sgmjournals.org                                                                                                                  355
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