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Use of phenotypic and molecular serotype identification methods to characterise previously 1
non-serotypeable group B streptococci (GBS). 2
Running title: Characterization of nontypeable GBS isolates 3
Fanrong Kong,1 Lotte Munch Lambertsen,
2 Hans-Christian Slotved,
2 Danny Ko,
1 Hui Wang,
1,3 4
Gwendolyn L. Gilbert1*
5
1. Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and 6
Medical Research (ICPMR), Westmead, New South Wales, Australia 7
2. Neisseria & Streptococci Reference Laboratory, Department of Bacteriology, Mycology & 8
Parasitology, Division of Microbiology & Diagnostics, Statens Serum Institut, Artillerivej 5, 9
DK-2300 Copenhagen S, Denmark 10
3. Research Laboratory for Infectious Skin diseases, Department of Dermatology, Wuhan First 11
Hospital, Wuhan, Hubei Province, P. R. China 12
*Author for correspondence: 13
Professor Gwendolyn L. Gilbert, 14
Centre for Infectious Diseases and Microbiology (CIDM), 15
Institute of Clinical Pathology and Medical Research (ICPMR), 16
Westmead Hospital, Darcy Road, Westmead, 17
New South Wales, 2145 AUSTRALIA 18
Phone: (612) 9845 6255 19
Fax: (612) 9893 8659 20
Email: [email protected] 21
Keywords: Streptococcus agalactiae; group B streptococcus; nontypeable; multiplex PCR-based 22
reverse line blot hybridization assay (mPCR/RLB); latex assay; Lancefield test 23
Subject category: Typing 24
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00189-08 JCM Accepts, published online ahead of print on 18 June 2008
2
ABSTRACT 1
Among 1762 isolates of Streptococcus agalactiae (GBS; group B streptococcus), 207 (12%) 2
initially non-serotypeable isolates were tested by improved conventional serotyping methods 3
(Lancefield antigen extraction using 0.1 and 0.2N HCl, latex agglutination assays and use of 4
antisera against all known serotypes (Ia, Ib and II-IX) and a molecular serotype identification 5
system (multiplex PCR-based reverse line blot [mPCR/RLB] assays, targeting serotype-specific 6
sites in the region cpsH-cpsM). Serotypes were assigned to 71 of 207 isolates (34%) using antisera 7
and 204 (98.5%) using mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsG region, of 141 8
persistently nonserotypeable isolates and one with discrepant conventional and molecular 9
serotyping results, was attempted. Major mutations were identified in 34 isolates (24%), including 10
11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared 11
with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By 12
contrast, mutations were identified in only 12 of 516 (0.02%) serotypeable isolates, for which this 13
region has been sequenced previously. In summary: an improved serotyping scheme allowed 14
serotype identification of more than one third of previously nonserotypeable GBS isolates. 15
Molecular serotypes were assigned by mPCR/RLB to almost all isolates, using mPCR/RLB. 16
Significant mutations (no amplicons or with associated amino acid changes) were found in the 17
cpsE-cpsF-cspG region of a higher proportion of non-serotypeable than of serotypeable isolates 18
(32/141 vs 8/516; p<0.001) but further investigation is needed to determine the genetic basis for 19
most non-serotypeable GBS isolates. 20
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INTRODUCTION 1
Conjugate vaccines containing the common polysaccharide capsule antigens (24) of Streptococcus 2
agalactiae (group B streptococcus, GBS) are under development (20). Monitoring of serotype 3
distribution is therefore important for epidemiological and future vaccine-related studies (24). We 4
recently described a new GBS serotype IX (30), which brings the number to at least ten (Ia, Ib, II-5
IX) (18;30). Serotype distribution varies with geographical region and ethnic origin (8). In the US, 6
Europe and Australasia, serotypes Ia, II, III, and V account for 80-90% of clinical isolates, while 7
serotypes IV, VI, VII, VIII and IX are rarely found (10;30;34;37). However, in Japan, serotypes VI 8
and VIII are relatively common (18). In recent years, 7%-32% of S. agalactiae isolates have been 9
reported to be non-serotypeable (12) and the proportions are similar between (8;26-28;30) invasive 10
and non-invasive isolates (1;9;23). However, a much higher proportion of GBS isolates from 11
animals are non-serotypeable, presumably because typing antisera were initially developed for 12
human isolates (8;36). 13
For many years, serotyping of GBS isolates has been based on phenotypic methods such as 14
that described by Lancefield (29;31). The proportion that are non-serotypeable has decreased 15
recently, with improvement in methods, including better growth medium to improve capsule 16
production (2), more sensitive conventional serotyping (CS) (31) and latex agglutination assays (29). 17
Molecular serotyping methods (15), such as multiplex PCR-based reverse line blot (mPCR/RLB) 18
assays (16) have made it possible to assign a molecular serotype to most non-serotypeable isolates 19
(16). However, the molecular serotype (MS) cannot be used directly to (36) explain why some 20
isolates are nontypeable using antisera (21). 21
In this study we used improved phenotypic and molecular serotyping methods to 22
characterize a collection of human and bovine GBS isolates that were initially non-serotypeable (36) 23
and sequenced a variable region of the cps gene cluster of those that still could not be serotyped to 24
determine a genetic basis for this finding. 25
26
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MATERIALS AND METHODS 1
Streptococcus agalactiae isolates. 2
The 1762 GBS clinical isolates (1623 from humans and 139 from cows) in our collection were 3
obtained from colleagues in eight countries (See Table 1 and Acknowledgements). Of the isolates 4
from humans, 784 were from normally sterile (blood or cerebrospinal fluid), 150 from nonsterile 5
(colonising or superficial) and 689 from unknown sites. They included eight serotype IX isolates 6
identified and described in a previous study (30), which were also used to validate a new serotype 7
IX-specific PCR. Nine S. agalactiae serotype reference strains (Ia, Ib, II-VIII) were kindly provided 8
by Lawrence Paoletti, Channing Laboratory, Boston, Mass. (15). 9
10
Initial conventional serotyping. 11
Initial conventional serotyping was performed and reported to us by the donor laboratory and 12
methods for isolation, identification and conventional serotyping are described in references cited in 13
Table 1. Isolates from Australia and New Zealand were serotyped by the Streptococcus Reference 14
Laboratory, Institute of Environmental Science & Research Limited, Kenepuru Science Centre, 15
Porirua, New Zealand (15), using antisera against serotypes Ia, Ib, II-V for New Zealand isolates 16
and against serotypes Ia, Ib, II-VIII for Australian isolates. About 50 of these isolates were 17
subsequently tested using antisera against all nine previously recognised serotypes (Ia, Ib, II-VIII), 18
at the WHO Collaborating Centre for Diphtheria & Streptococcal Infections, Health Protection 19
Agency, Colindale, London U.K. (15;36). 20
21
Improved conventional serotyping. 22
The improved conventional serotyping - which consisted of a) use of antigens extracted with 0.1 N 23
HCl (as well as the standard 0.2 N HCl extracts) (31) and b) latex agglutination assay, as described 24
previously (31) - was performed at the Neisseria and Streptococci Reference laboratory, Statens 25
Serum Institut (SSI), Copenhagen, Denmark. All non-serotypeable isolates were also tested with the 26
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serotype IX antiserum, raised at SSI, as described previously (30). For serotype IX, both latex-1
coupled and regular serotype IX antisera (Lancefield reaction) were used (30). 2
3
Genotyping and sequencing. 4
Genotyping and sequencing were performed at the Centre for Infectious Diseases and Microbiology 5
(CIDM), Sydney Australia. The molecular serotype (MS) identification of nine serotypes (Ia, Ib, II-6
VIII) was done by mPCR/RLB targeting serotype-specific sequences in various genes: cpsH for MS 7
Ia, Ib, III, IV, V and VI; cpsK for MS II; cpsM for MS VII; and cpsJ for MS VIII (16). Partial cpsE-8
cpsF-cpsG sequencing (~800 bp) was performed using nested PCR as previously described (36). 9
The structure of the cps cluster and target regions for sequencing and serotype-specific PCR 10
(including mPCR/RLB) are shown schematically in the Figure 1. 11
To more easily identify serotype IX (30), we designed three new PCR primers, based on a 12
portion of cpsH (the primer locations are shown according to Genbank sequence EF157290): IXS1 13
(4GCT CAT TTA CAA CTT GTA GAC GGC27); IXS2 (30AAC TCT TTT TGG AAA TAG TTT 14
TAA GGA G57); and IXA (350GCC ATA TCA GAG CAA ATA TGT CAT ATA TC322), which 15
were used in two combinations: IXS1/IXA and IXS2/IXA. These were evaluated using nine 16
serotype reference strains (Ia, Ib, II-VIII) as negative controls and previously identified serotype IX 17
isolates as positive controls (20). 18
19
Statistical analysis. 20
Proportions were compared using the chi square or Fisher's exact tests, where appropriate. 21
22
Nucleotide sequence accession numbers. 23
New sequence data (i.e. sequences containing mutations) generated for partial cps gene clusters 24
(~800 bp in the region of cpsE-cpsF-cpsG) have been deposited in GenBank (see Table 3 for 25
GenBank accession numbers). 26
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1
RESULTS 2
Serotype identification by initial conventional serotyping. 3
Of the 1762 GBS isolates in our collection, 140 of 1623 (8.6%) human and 67 of 139 (48.2%) 4
bovine isolates (36) were reported to be non-serotypeable by the donor laboratory (Table 1). Four 5
isolates gave apparent cross-reactions and/or equivocal results on initial conventional serotyping 6
(CS) and the initial CS and MS results were discrepant for nine isolates (all 13 were isolates from 7
human). 8
9
Comparison of improved conventional serotyping (CS) and molecular serotype identification 10
by mPCR/RLB. 11
After testing with improved CS, serotypes were assigned to 53 of 140 previously non-serotypeable 12
isolates from humans, including three that were identified as serotype IX (Table 2). Eighteen of the 13
67 previously non-serotypeable bovine isolates were identified as serotype III and the rest remained 14
non-serotypeable. The 87 isolates from humans that remained non-serotypeable included 38 of 784 15
(4.8%) from sterile sites, nine of 150 (6.0%) from superficial sites and 40 of 689 (5.8%) from 16
unknown sites (differences not significant). 17
Of nine human isolates with initially discrepant CS and MS results (Tables 1), five (initially 18
identified as CS/MS: IV/Ia; VI/Ia; VII/V; V/Ib; III/V) were found to be non-serotypeable with 19
improved CS; CS and MS results were consistent for one (there had been a typographical error in 20
the original documentation); two were serotype IX (previously identified as serotype Ib and II) and 21
results remained discrepant, after repeated testing of single colony subcultures, for one isolate (98-22
055-2166: initial CS Ib, improved CS III, MS II ). 23
Three of four isolates with initially mixed/indeterminate CS results had MS (and protein 24
gene profiles – data not shown), by mPCR/RLB, that were consistent with their being mixtures of 25
two serotypes rather than single strains with copies of two different cps clusters and protein antigen 26
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genes. These results were confirmed by retesting subcultures of several individual colonies of each, 1
separately, by mPCR/RLB (for MS and PGP), serotype-specific PCR, partial cps sequencing and 2
improved CS. The fourth isolate initially reacted with both serotype III and Ib antisera; the MS 3
identified by mPCR/RLB was Ib and improved CS confirmed this result. 4
A molecular serotype (MS) was assigned to all but three of 1762 isolates tested of which one 5
was of bovine (NI-96-2846) and two of human (00B1198593, 00B1200564) origin. They were also 6
non-serotypeable (Table 3) but all gave positive results with the GBS-specific PCR targeting cfb. 7
8
Molecular serotype identification by partial cps sequencing. 9
We attempted to sequence a portion of the cpsE-cpsF-cpsG region of the cps gene cluster (~800bp) 10
of all 142 isolates that remained (136 - 87 human, 49 bovine) or became (5) non-serotypeable after 11
improved CS and the one (98-055-2166) for which MS and CS results remained discrepant. No 12
amplicons were produced from 11 isolates - three of 92 human (3%) and eight of 49 (16%) of 13
bovine origin - including the three that were also non-genotypeable (Table 3). Sequencing results 14
were consistent with mPCR/RLB results for 102 isolates and mutations were identified in 23 - 15
seven (8%) human and 16 (33%) bovine non-serotypeable isolates. Mutations in six (6.5%) 16
serotypeable and 15 (31%) nonserotypeable isolates were associated with amino acid changes 17
(Table 3). In all, 34 of 141 non-serotypeable had major genetic changes in the cpsE-cpsF-cpsG; the 18
proportion was significant higher among bovine (24/49; 49%) than among human (10/92; 11%) 19
isolates (p <0.001). There were no mutations in the cpsE-cpsF-cpsG region of the isolate (98-055-20
2166) with discrepant MS/CS results. 21
22
Serotype IX-specific PCR. 23
The new serotype IX-specific PCRs were tested against nine serotype (Ia, Ib-VIII) reference strains 24
and the eight previously identified serotype IX strains, all of which produced expected results with 25
both combinations of primer pairs. Improved CS and serotype IX-specific PCR identified three of 26
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207 initially non-serotypeable isolates (Table 2) and two for which original CS and MS results were 1
discrepant as serotype IX (these five serotype IX isolates are included among eight previously 2
reported)(30). Four of eight serotype IX isolates were from normally sterile sites; two from blood 3
cultures of neonates, one from a blood culture of a 57 year old male diabetic and one from a lung 4
specimen. The other four isolates were from vaginal swabs (30). 5
6
DISCUSSION 7
The capsule of S. agalactiae is an important virulence factor (21) and one of the main targets for 8
vaccine development. Conventional serotyping is therefore essential for disease and vaccine related 9
surveillance. It is based on the reactions of isolates with antisera raised against capsular 10
polysaccharides of ten recognized serotypes, using the classic (Lancefield) precipitation or latex 11
agglutination tests (19, 20, 21). However, a significant proportion of S. agalactiae isolates is non-12
serotypeable (9, 11, 29, 30, 30), either because they do not express capsular polysaccharide or do 13
not react with available typing antisera, and production of new type-specific antisera is complicated 14
and expensive. 15
The development of molecular techniques that identify serotype-specific sequences in the 16
cps gene cluster of S. agalactiae, such as mPCR/RLB, has made it possible to assign a molecular 17
serotype to most non-serotypeable isolates (30). In previous studies, we have found that partial 18
sequencing of a variable ~800 bp region of partial cpsE-cpsF-cpsG can also identify molecular 19
serotypes (15;17;36), even for non-serotypeable or, rarely, non-genotypeable (by mPCR/RLB) 20
isolates and resolve discrepancies between conventional and molecular serotype identification. 21
Based on these methods, we have recently described a proposed n new serotype IX (30). 22
23
Comparison of conventional serotyping and molecular serotype identification. 24
Traditional serotyping methods, using antisera, identify the phenotype expressed at the stage of 25
testing but is limited by the quality of typing sera, the technical experience of the operator and the 26
existence of non-serotypeable isolates. Nevertheless, it remains an appropriate method for 27
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surveillance of serotype distribution. The latex assay is very practical for typing large numbers of 1
isolates (29) and is more sensitive than the conventional Lancefield test (31). In this study, we 2
showed that the latex assay results, for isolates that were initially non-serotypeable, were almost 3
always confirmed by molecular methods, indicating that the increased sensitivity is not at the 4
expense of specificity (2). Molecular serotyping methods identify genes that, if expressed, 5
determine the phenotype. They can provide a greater level of discrimination for epidemiological 6
studies and isolates are very rarely non-genotypeable; mPCR/RLB (14) is a practicable method for 7
rapid typing of relatively large numbers of isolates or, potentially, for direct application to clinical 8
specimens. 9
10
Possible new S. agalactiae serotypes. 11
The possibility that there are previously unrecognised S. agalactiae serotypes, which are potentially 12
virulent, has been demonstrated by our recent identification of serotype IX (30), 50% of which were 13
from normally sterile sites. The proportions of isolates that were non-serotypeable did not differ 14
significantly between those from normally sterile and those from mucous membranes or superficial 15
sites. In the present study we identified three isolates to which a serotype could not be assigned after 16
extensive testing using both improved conventional and molecular methods. Further investigation is 17
required to determine whether they represent new serotypes. Similarly, further investigation will be 18
required to determine whether the high proportion of non-serotypable bovine isolates, which were 19
identified as atypical MS III actually represent a novel serotype (Table 3) (25). Finally, results of 20
CS and MS, for one isolate (98-055-2166) remained discrepant. No mutations were identified in the 21
cpsE-cpsF-cpsG region and mPCR/RLB was positive for MS II (targeting cpsK) but negative for 22
MS III (targeting cpsH). In general, our results suggest that new serotypes among S. agalactiae 23
isolates from humans occur with low frequency. 24
25
26
27
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Comparison of human and bovine isolates. 1
A significant proportion of initially non-serotypeable isolates from humans were serotypeable when 2
retested with improved methods (29;31). However, these improved serotyping methods still failed 3
to identify a high proportion of bovine isolates. The proportions of the remaining nonserotypeable 4
isolates, which failed to produce amplicons or had cpsE-cpsF-cpsG mutations, was much higher 5
among bovine than human isolates (P<0.001) (Table 3). The high proportion of bovine isolates that 6
are non-serotypeable probably reflects the fact that the antisera were specifically developed and 7
optimized for the identification of isolates from humans, not bovine isolates. Several studies have 8
shown that bovine and human isolates belong to different genetic lineages and that cross-infection is 9
rare (32). Therefore, it is also not surprising that a high proportion of bovine isolates showed cpsE-10
cpsF-cpsG sequence differences compared with sequenced human strains. 11
12
Sequencing results. 13
Nearly a quarter (32 of 141; 23%) of non-serotypeable isolates had significant mutations associated 14
with amino acid changes in the cspE-cpsF-cpsG region, including nine (10%) human and 23 (47%) 15
bovine non-serotypeable isolates; P<0.001). By contrast, significant mutations in this region are rare 16
among serotypeable isolates. Previously, we have sequenced this region of 516 (426 from humans, 17
90 bovine) serotypeable isolates and identified mutations in only 12 – 10 (2%) human and 2 (2%) 18
bovine isolates – with amino acid changes in eight (1.6%; one produced no amplicon) – data not 19
shown. The difference in mutation rates between non-serotypeable and serotypeable isolates is 20
highly significant for both human (11/92 vs 8/516; p<0.001) and bovine isolates (23/49 vs 2/90; 21
P<0.001). However, the relationship between these mutations and the failure of these isolates to 22
produce polysaccharide antigens for which they carry corresponding genes is uncertain. Further 23
investigation is needed to identify the genetic basis for non-serotypeability of the significant 24
majority of these isolates – particularly those from humans – which have no mutations at this site. 25
26
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Conclusion. 1
In this study we have evaluated both phenotypic and molecular methods for serotype identification 2
of a large number of non-serotypeable GBS isolates and confirmed that improved CS methods can 3
significantly reduce the proportion that are non-serotypeable. Nevertheless, genotypic 4
characterization of these isolates does not assist in the development of vaccines based common 5
capsular polysaccharides. Fortunately they represent fewer than 5% of invasive isolates. 6
Presumably, protection against all invasive GBS strains would require a vaccine based on a 7
common protein antigen. Although the rates of mutation are significantly higher for non-8
serotypeable than serotypeable isolates, this does not provide an explanation for non-serotypeability 9
in the majority of isolates. 10
We have also demonstrated a much higher incidence of significant mutations, in cpsE-cpsF-11
cpsG regions of bovine isolates compared with those from humans (36), which supports previous 12
studies that indicate that bovine GBS represent a distinct S. agalactiae lineage from that of isolates 13
from humans (5). 14
15
ACKNOWLEDGMENTS 16
Fanrong Kong, Lotte Munch Lambertsen and Hans-Christian Slotved had similar contribution to the 17
work, so would be seen as co-first authors. We wish to thank Maryann Pincevic for their precious 18
help in sequencing, and Kirsten Burmeister, SSI, for her experience help with serotyping. 19
20
Isolates include in this study were kindly provided by: Dr Diana Martin and Julie Morgan, 21
Streptococcus Reference Laboratory, ESR, Wellington, New Zealand; Drs Lawrence Paoletti and 22
Catherine Lachenauer, Channing Laboratory, Boston USA; Professor Johan Maeland, Department 23
of Microbiology, School of Medicine, Norwegian University of Science and Technology, 24
Trondheim, Norway; Dr Nicola Jones, Nuffield Department of Clinical Laboratory Sciences, 25
Institute for Molecular Medicine, John Radcliffe Hospital, Oxford UK; Drs Dele Davies, Shannon 26
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Manning, Department of Epidemiology, University of Michigan School of Public Health, Ann 1
Arbor, USA; Reinhard Berner Department of Pediatrics, University Children’s Hospital, D-79106 2
Freiburg, Germany; Professor Yunsop Chong and Dr Kyungwon Lee, Research Institute of 3
Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea; Catherine Satzke and 4
Professor Roy Robins-Browne, Department of Microbiology and Immunology, University of 5
Melbourne, Australia; Dr Margaret Ip, Department of Microbiology, The Chinese University of 6
Hong Kong, Prince of Wales Hospital, Hong Kong; Drs Gabriela Martinez and Marcelo Gottschalk, 7
Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de médecine vétérinaire, 8
Université de Montréal, St-Hyacinthe, Québec J2S 7C6, Canada. 9
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FIGURE LEGEND. 1
Figure 1. Schematic diagram of GBS cps gene cluster showing conserved & variable regions 2
and sites of sequencing and serotype-specific PCR (and mPCR/RLB) for serotype identification. 3
(Modified from Chaffin et al. J Bacteriol 2000; 182:4466-4477) (6). 4
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Table 1. GBS isolates tested in this study 1
Country of
origina
Isolates
tested
N =
Antisera
used in
donor labb
Initially
NSTc
N =
References NST using improved
methods (NGT
d)
N =
Mixed isolates
(serotypes)e’
N =
CS/MS*e
discrepant
N =
Isolates with
cps mutations
N =
Australia 343 Ia-VIII 27 (17;35) 20 1 4 3
Hong Kong 132 Ia-VIII 22 (35) 22 (2) - 1 2+2 na
South Korea 186 Ia-VIII 5 (19;35) 5 2 1 0
New Zealand 551 Ia-Vf 63 (13;35;35) 30 - 2+1 na
Canada 76 Ia-VIII 8 (7;22) 2 - 1 0
Japan 50 Ia-VIII 1 (18) 1 - 0 0
United Kingdom 83 Ia-VIII 4 (11;33) 2 - 0 0
Germany 202 Ia-Vf 10 (3;4) 5 - 2 0
Total human 1623 140 87 - 9
Canada (bovine) 139 Ia-Vf 67 (22) 49 (1) 1 0 16+8 na
Total 1762 207 136 (3) 4 9 23+11na
Abbreviations: CS/MS conventional serotype/molecular serotype ; na = not amplified (or no PCR amplicons); NST/NGT = non-serotypeable/non-genotypeable 2
Notes. 3
a. All isolates are from humans, except those specified. 4
b. Antisera used in donor laboratories, as reported in corresponding references. 5
c. Non-serotypeable; as reported by donor laboratory. 6
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d. See text for details of improved serotyping methods. NGT using mPCR/RLB. 1
e. See text for details. 2
f. Isolates identified as MS VI-VIII were later tested with corresponding antisera to confirm result and by improved CS. 3
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Table 2. Comparison of improved conventional serotyping results and molecular serotype 1
identification by mPCR/RLB, partial sequencing and/or serotype-specific PCR for the 140 2
human isolates that were initially non-serotypable. 3
MS
Final CS
Ia Ib II III IV V VI VII VIII NT Total
CS
Ia 3 3
Ib 19 19
II 7 7
III 4 4
IV 3 3
V 7 7
VI 3 3
VII 2 2
VIII 2 2
IX 3a 3
NT 17 3 4 24 2 28 5 1 1 2 87
Total MS 20 22 11 28 5 38* 8 3 3 2 140
Notes. 4
CS=conventional serotyping by latex assay; MS=molecular serotype identification by mPCR/RLB (16). 5
a. The three serotype IX isolates were initially non-serotypeable with antisera and were incorrectly identified as MS V. 6
using mPCR/RLB (16). They were subsequently identified as serotype IX using antisera and partial cps sequencing (30) and 7
confirmed using serotype IX-specific PCR.. 8
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Table 3. Results for 34 isolates in which mutations were identified in partial cpsE-cpsF-cpsG 1
sequence. 2
Strain
number
ICS MS cpsa GenBank
accession
no.a
4060 NT Ia Ia/III-1478-1623 145bp deletion (significant aa
changes) &1751 t-c (no aa changes)
EF524093
02B0541114 NT Ia Ia/III-1751 t-c (no aa changes) EF524086
96B0748375 NT Ia Ia/III-2022 c-t (CpsG 5 T-I) EF524088
4032 NT V V-1523-1672 150bp deletion (significant aa changes) EF524092
04-324-2173 NT V V- 2042 a insertion (significant aa changes)
EF524090
05-118-4243 NT V V-1785-1807 23bp deletion (significant aa changes) EF524091
99-294-1551 NT VI VI-1594 a-g (CpsF 12 H to R) EF524089
SH-97-1504b NT II II-1717 c-t (CpsF 53 P-L) AY257681
SF-96-6545b NT II II-1720 c-t (CpsF 54 T-I) AY257680
ASS-96-3633b NT II II-1968 a-g (CpsF 137 E-K) AY257679
SF-96-5508b NT II II-2034 a-t (CpsG 9 H-L) AY257682
AL-96-1853b NT III Ia/III-3-1525 g-a (CpsE 14 G-S) AY257678
SF-96-4036 b
NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SH-96-4072b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SH-96-4136b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SF-96-4707b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SF-96-5169b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SF-97-1330b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
ASS-97-079b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
SF-96-4961b NT III Ia/III-3-1526 g-a (CpsE 17 G-D) =AY257684
RF-96-1993b NT III Ia/III-3-1553 g-a (no aa changes) AY257677
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AL-96-1683b NT III Ia/III-3-1553 g-a (no aa changes)
&1828 c-t (CpsF 90 P-L)
AY257676
AL-96-1802b NT III Ia/III-3-1553 g-a &1828 c-t (CpsF 90 P-L) =AY257676
AL-96-1828b NT II No amplicon
c -
NI-96-2521b NT II No amplicon
c -
NI-96-2846b NT NT No amplicon
c -
SF-96-5660 b
NT III No ampliconc -
SH-96-2851b NT II No amplicon
c -
SH-96-4281b NT II No amplicon
c -
SH-96-5171b NT II No amplicon
c -
SH-96-5345b NT II No amplicon
c -
4071 NT V No ampliconc -
00B1198593 NT NT No ampliconc -
00B1200564 NT NT No ampliconc -
Abbreviations: ICS = improved conventional serotyping; MS=molecular serotype identification by mPCR/RLB (16); cps 1
indicates serotype identified by partial sequencing of capsular polysaccharide cluster genes - cpsE-cpsF-cpsG 2
Notes. 3
a. Partial cpsE-cpsF-cpsG partial sequencing - mutation site shown; nucleotide position and base change (in lower case) 4
refer to GenBank accession number AF332908 and amino acid changes (in upper case) refer to the first amino acid of 5
the relevant gene; large deletions caused amino acid changes. Please also refer to our GenBank submissions for more 6
detailed sequence annotation. “=” means the sequences were identical with the relevant GenBank submissions 7
sequences. 8
b. Bovine isolates; the rest are from humans. 9
c. No amplicons were obtained with cps PCR & sequencing primers, which probably contain high heterogeneity or large 10
mutation (insertion or deletion). 11
12
13
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Figure legend:
Schematic diagram of GBS cps gene cluster showing conserved & variable regions
and sites of sequencing and serotype-specific PCR (and mPCR/RLB) for serotype
identification. (Modified from Chaffin et al. J Bacteriol 2000; 182:4466-4477).
regulation
& transport
subunit assembly
& polymerization
sialyl
transferase
transport
sialic acid synthesis
Serotype/subtype:
~800 bp sequence ST-specific PCR: cpsH Ia, Ib, III-VI;
cpsJ VIII; cpsK II; cpsM VII
cpsA cpsBcpsCcpsD cpsE cpsFcpsGcpsH cpsI/M cpsJ cpsK cpsL
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