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Journal of Clinical Microbiology, August 2008, p. 2745-2750, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00189-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Use of Phenotypic and Molecular Serotype Identification Methods To Characterize Previously Nonserotypeable Group B Streptococci

Fanrong Kong,¹ Lotte Munch Lambertsen,² Hans-Christian Slotved,² Danny Ko,¹ Hui Wang,^1,3 and Gwendolyn L. Gilbert^1*

Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia,¹ Neisseria & Streptococci Reference Laboratory, Department of Bacteriology, Mycology & Parasitology, Division of Microbiology & Diagnostics, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen S, Denmark,² Research Laboratory for Infectious Skin Diseases, Department of Dermatology, Wuhan First Hospital, Wuhan, Hubei Province, People's Republic of China³

Received 30 January 2008/ Returned for modification 11 April 2008/ Accepted 10 June 2008

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Among 1,762 isolates of Streptococcus agalactiae (group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning cpsH to cpsM). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsG region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the cpsE-cpsF-cspG region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516; P < 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.

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Conjugate vaccines containing the common polysaccharide capsule antigens (24) of Streptococcus agalactiae (group B streptococcus [GBS]) are under development (20). Monitoring of serotype distribution is therefore important for epidemiological and future vaccine-related studies (24). We recently described a new GBS serotype, IX (30), which brings the number to at least 10 (Ia, Ib, and II to IX) (18, 30). Serotype distribution varies with geographical region and ethnic origin (8). In the United States, Europe, and Australasia, serotypes Ia, II, III, and V account for 80 to 90% of clinical isolates, while serotypes IV, VI, VII, VIII, and IX are rarely found (10, 30, 34, 37). However, in Japan, serotypes VI and VIII are relatively common (18). In recent years, 7 to 32% of S. agalactiae isolates have been reported to be nonserotypeable (12) and the proportions are similar between invasive (8, 26-28, 30) and noninvasive (1, 9, 23) isolates. However, a much higher proportion of GBS isolates from animals are nonserotypeable, presumably because typing antisera were initially developed for human isolates (8, 36).

For many years, serotyping of GBS isolates has been based on phenotypic methods such as that described by Lancefield (29, 31). The proportion that are nonserotypeable has decreased recently with the improvement in methods, including better growth medium to improve capsule production (2) and more sensitive conventional serotyping (CS) (31) and latex agglutination assays (29). Molecular serotyping methods (15) such as multiplex PCR-based reverse line blot (mPCR/RLB) assays (16) have made it possible to assign molecular serotypes (MSs) to most nonserotypeable isolates (16). However, the MS cannot be used directly (36) to explain why some isolates are nontypeable with antisera (21).

In this study, we used improved phenotypic and molecular serotyping methods to characterize a collection of human and bovine GBS isolates that were initially nonserotypeable (36) and sequenced a variable region of the cps gene cluster of those that still could not be serotyped to determine a genetic basis for this finding.

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S. agalactiae isolates. The 1,762 GBS clinical isolates (1,623 from humans and 139 from cows) in our collection were obtained from colleagues in eight countries (see Table 1 and Acknowledgments). Of the isolates from humans, 784 were from normally sterile sites (blood or cerebrospinal fluid), 150 were from nonsterile sites (colonizing or superficial), and 689 were from unknown sites. They included eight serotype IX isolates identified and described in a previous study (30), which were also used to validate a new serotype IX-specific PCR. Nine S. agalactiae serotype reference strains (Ia, Ib, and II to VIII) were kindly provided by Lawrence Paoletti, Channing Laboratory, Boston, MA (15).

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TABLE 1. GBS isolates tested in this study

Initial CS. Initial CS was performed and reported to us by the donor laboratory, and the methods used for isolation, identification, and CS are described in the references cited in Table 1. Isolates from Australia and New Zealand were serotyped by the Streptococcus Reference Laboratory, Institute of Environmental Science & Research Limited, Kenepuru Science Centre, Porirua, New Zealand (15), with antisera against serotypes Ia, Ib, and II to V for New Zealand isolates and against serotypes Ia, Ib, and II to VIII for Australian isolates. About 50 of these isolates were subsequently tested with antisera against all nine previously recognized serotypes (Ia, Ib, and II to VIII) at the World Health Organization Collaborating Centre for Diphtheria and Streptococcal Infections, Health Protection Agency, Colindale, London, United Kingdom (15, 36).

Improved CS. The improved CS, which consisted of (i) the use of antigens extracted with 0.1 N HCl (as well as the standard 0.2 N HCl extracts) (31) and (ii) a latex agglutination assay, as described previously (31), was performed at the Neisseria and Streptococci Reference laboratory, Statens Serum Institut (SSI), Copenhagen, Denmark. All nonserotypeable isolates were also tested with the serotype IX antiserum, raised at SSI, as described previously (30). For serotype IX, both latex-coupled and regular serotype IX antisera (Lancefield reaction) were used (30).

Genotyping and sequencing. Genotyping and sequencing were performed at the Centre for Infectious Diseases and Microbiology, Sydney, Australia. The MS identification of nine serotypes (Ia, Ib, and II to VIII) was done by mPCR/RLB targeting serotype-specific sequences in various genes, i.e., cpsH for MS Ia, Ib, III, IV, V, and VI; cpsK for MS II; cpsM for MS VII; and cpsJ for MS VIII (16). Partial cpsE-cpsF-cpsG sequencing (800 bp) was performed by nested PCR as previously described (36). The structures of the cps cluster and target regions for sequencing and serotype-specific PCR (including mPCR/RLB) are shown schematically in Fig. 1.

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FIG. 1. Schematic diagram of GBS cps gene cluster showing (labels at the bottom) conserved (bold) and variable (italic) regions and sites of sequencing and serotype-specific PCR (and mPCR/RLB) for serotype identification. (Modified from reference 6 with permission.)

To more easily identify serotype IX (30), we designed three new PCR primers, based on a portion of cpsH (the primer locations are shown according to GenBank sequence EF157290), i.e., IXS1 (₄GCT CAT TTA CAA CTT GTA GAC GGC₂₇), IXS2 (₃₀AAC TCT TTT TGG AAA TAG TTT TAA GGA G₅₇), and IXA (₃₅₀GCC ATA TCA GAG CAA ATA TGT CAT ATA TC₃₂₂), which were used in two combinations, IXS1/IXA and IXS2/IXA. These were evaluated with nine serotype reference strains (Ia, Ib, and II to VIII) as negative controls and previously identified serotype IX isolates as positive controls (20).

Statistical analysis. Proportions were compared by the chi-square or Fisher exact test, where appropriate.

Nucleotide sequence accession numbers. New sequence data (i.e., sequences containing mutations) generated for partial cps gene clusters (800 bp in the cpsE-cpsF-cpsG region) have been deposited in GenBank (see Table 3 for accession numbers).

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TABLE 3. Results for 34 isolates in which mutations were identified in partial cpsE-cpsF-cpsG sequence

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Serotype identification by initial CS. Of the 1,762 GBS isolates in our collection, 140 (8.6%) of 1,623 human isolates and 67 (48.2%) of 139 bovine isolates (36) were reported to be nonserotypeable by the donor laboratories (Table 1). Four isolates gave apparent cross-reactions and/or equivocal results on initial CS and the initial CS and MS results were discrepant for 9 isolates (all 13 isolates were from humans).

Comparison of improved CS and MS identification by mPCR/RLB. After testing by improved CS, serotypes were assigned to 53 of 140 previously nonserotypeable isolates from humans, including 3 that were identified as serotype IX (Table 2). Eighteen of the 67 previously nonserotypeable bovine isolates were identified as serotype III, and the rest remained nonserotypeable. The 87 isolates from humans that remained nonserotypeable included 38 (4.8%) of 784 from sterile sites, 9 (6.0%) of 150 from superficial sites, and 40 (5.8%) of 689 from unknown sites (no significant differences).

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TABLE 2. Comparison of improved CSa results and MS identification by mPCR/RLB, partial sequencing, and/or serotype-specific PCR for the 140 human isolates that were initially nonserotypeable

Of nine human isolates with initially discrepant CS and MS results (Table 1), five (initially identified by CS/MS, respectively, as IV/Ia, VI/Ia, VII/V, V/Ib, and III/V) were found to be nonserotypeable by improved CS. The CS and MS results were consistent for one (there had been a typographical error in the original documentation), two were serotype IX (previously identified as serotypes Ib and II), and the results remained discrepant, after repeated testing of single colony subcultures, for one isolate (98-055-2166; initially serotype Ib by CS, serotype III by improved CS, and serotype II by MS).

Three of four isolates with initially mixed or indeterminate CS results had MSs (and protein gene profiles; data not shown) by mPCR/RLB that were consistent with their being mixtures of two serotypes rather than single strains with copies of two different cps clusters and protein antigen genes. These results were confirmed by retesting subcultures of several individual colonies of each, separately, by mPCR/RLB (for MS and PGP), serotype-specific PCR, partial cps sequencing, and improved CS. The fourth isolate initially reacted with both serotype III and Ib antisera; the MS identified by mPCR/RLB was Ib, and improved CS confirmed this result.

MSs were assigned to all but 3 of the 1,762 isolates tested, of which 1 was of bovine origin (NI-96-2846) and 2 were of human origin (00B1198593 and 00B1200564). They were also nonserotypeable (Table 3), but all gave positive results with the GBS-specific PCR targeting cfb.

MS identification by partial cps sequencing. We attempted to sequence a portion of the cpsE-cpsF-cpsG region of the cps gene cluster (800 bp) of all 141 isolates that remained (n = 136 [87 human and 49 bovine]) or became (n = 5) nonserotypeable after improved CS and the 1 (98-055-2166) for which the MS and CS results remained discrepant. No amplicons were produced from 11 isolates—3 (3%) of 92 of human and 8 (16%) of 49 of bovine origin—including the 3 that were also nongenotypeable (Table 3). Sequencing results were consistent with mPCR/RLB results for 102 isolates, and mutations were identified in 23, i.e., 7 (8%) human and 16 (33%) bovine, nonserotypeable isolates. Mutations in 6 (6.5%) serotypeable and 15 (31%) nonserotypeable isolates were associated with amino acid changes (Table 3). In all, 34 of 141 nonserotypeable isolates had major genetic changes in the cpsE-cpsF-cpsG region; the proportion was significantly higher among bovine (24/49; 49%) than among human (10/92; 11%) isolates (P < 0.001). There were no mutations in the cpsE-cpsF-cpsG region of the isolate (98-055-2166) with discrepant MS and CS results.

Serotype IX-specific PCR. The new serotype IX-specific PCRs were tested against nine serotype (Ia and Ib to VIII) reference strains and the eight previously identified serotype IX strains, all of which produced the expected results with both combinations of primer pairs. Improved CS and serotype IX-specific PCR identified 3 of 207 initially nonserotypeable isolates (Table 2) and 2 for which the original CS and MS results were discrepant as serotype IX (these 5 serotype IX isolates are included among 8 previously reported) (30). Four of eight serotype IX isolates were from normally sterile sites; two were from blood cultures of neonates, one was from a blood culture of a 57-year-old male diabetic, and one was from a lung specimen. The other four isolates were from vaginal swabs (30).

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The capsule of S. agalactiae is an important virulence factor (21) and one of the main targets of vaccine development. CS is therefore essential for disease- and vaccine-related surveillance. It is based on the reactions of isolates with antisera raised against capsular polysaccharides of 10 recognized serotypes in the classic (Lancefield) precipitation and latex agglutination tests (19, 20, 21). However, a significant proportion of S. agalactiae isolates is nonserotypeable (9, 11, 29, 30, 36), either because they do not express capsular polysaccharide or because they do not react with available typing antisera, and production of new type-specific antisera is complicated and expensive.

The development of molecular techniques that identify serotype-specific sequences in the cps gene cluster of S. agalactiae, such as mPCR/RLB, has made it possible to assign an MS to most nonserotypeable isolates (30). In previous studies, we have found that partial sequencing of a variable 800-bp region of the partial cpsE-cpsF-cpsG region can also identify MSs (15, 17, 36), even for nonserotypeable or, rarely, nongenotypeable (by mPCR/RLB) isolates and resolve discrepancies between conventional and MS identification methods. Based on these methods, we have recently described a proposed new serotype, IX (30).

Comparison of CS and MS identification methods. Traditional serotyping with antisera identifies the phenotype expressed at the stage of testing but is limited by the quality of the typing sera used, the technical experience of the operator, and the existence of nonserotypeable isolates. Nevertheless, it remains an appropriate method for the surveillance of serotype distribution. The latex assay is very practical for typing large numbers of isolates (29) and is more sensitive than the conventional Lancefield test (31). In this study, we showed that the latex assay results, for isolates that were initially nonserotypeable, were almost always confirmed by molecular methods, indicating that the increased sensitivity does not come at the expense of specificity (2). Molecular serotyping methods identify genes that, if expressed, determine the phenotype. They can provide a greater level of discrimination for epidemiological studies, and isolates are very rarely nongenotypeable; mPCR/RLB (14) is a practicable method for the rapid typing of relatively large numbers of isolates or, potentially, for direct application to clinical specimens.

Possible new S. agalactiae serotypes. The possibility that there are previously unrecognized S. agalactiae serotypes that are potentially virulent has been demonstrated by our recent identification of serotype IX isolates (30), 50% of which were from normally sterile sites. The proportions of isolates that were nonserotypeable did not differ significantly between those from normally sterile sites and those from mucous membranes or superficial sites. In the present study, we identified three isolates to which a serotype could not be assigned after extensive testing by both improved conventional and molecular methods. Further investigation is required to determine whether they represent new serotypes. Similarly, further investigation is required to determine whether the high proportion of nonserotypeable bovine isolates which were identified by MS as atypical serotype III isolates actually represent a novel serotype (Table 3) (25). Finally, the CS and MS results of one isolate (98-055-2166) remained discrepant. No mutations were identified in the cpsE-cpsF-cpsG region, and mPCR/RLB was positive for MS II (targeting cpsK) but negative for MS III (targeting cpsH). In general, our results suggest that new serotypes among S. agalactiae isolates from humans occur with low frequency.

Comparison of human and bovine isolates. A significant proportion of the initially nonserotypeable isolates from humans were serotypeable when retested by improved methods (29, 31). However, these improved serotyping methods still failed to identify a high proportion of bovine isolates. The proportions of the remaining nonserotypeable isolates, which failed to produce amplicons or had cpsE-cpsF-cpsG mutations, was much higher among bovine than human isolates (P < 0.001) (Table 3). The high proportion of bovine isolates that are nonserotypeable probably reflects the fact that the antisera were specifically developed and optimized for the identification of human isolates, not bovine isolates. Several studies have shown that bovine and human isolates belong to different genetic lineages and that cross-infection is rare (32). Therefore, it is also not surprising that a high proportion of bovine isolates showed cpsE-cpsF-cpsG sequence differences compared with sequenced human strains.

Sequencing results. Nearly a quarter (32 [23%] of 141) of our nonserotypeable isolates had significant mutations associated with amino acid changes in the cspE-cpsF-cpsG region, including 9 (10%) human and 23 (47%) bovine nonserotypeable isolates (P < 0.001). By contrast, significant mutations in this region are rare among serotypeable isolates. Previously, we have sequenced this region of 516 (426 human and 90 bovine) serotypeable isolates and identified mutations in only 12 (10 [2%] human and 2 [2%] bovine) isolates, with amino acid changes in 8 (1.6%; 1 produced no amplicon) (data not shown). The difference in mutation rates between nonserotypeable and serotypeable isolates is highly significant for both human (11/92 versus 8/516; P < 0.001) and bovine (23/49 versus 2/90; P < 0.001) isolates. However, the relationship between these mutations and the failure of these isolates to produce polysaccharide antigens for which they carry corresponding genes is uncertain. Further investigation is needed to identify the genetic basis for the nonserotypeability of the significant majority of these isolates—particularly those from humans—which have no mutations at this site.

Conclusion. In this study, we have evaluated both phenotypic and molecular methods for the serotype identification of a large number of nonserotypeable GBS isolates and confirmed that improved CS methods can significantly reduce the proportion that are nonserotypeable. Nevertheless, genotypic characterization of these isolates does not assist in the development of vaccines based on common capsular polysaccharides. Fortunately, they represent fewer than 5% of invasive isolates. Presumably, protection against all invasive GBS strains would require a vaccine based on a common protein antigen. Although the rates of mutation are significantly higher for nonserotypeable than for serotypeable isolates, this does not provide an explanation for the nonserotypeability of the majority of isolates.

We have also demonstrated a much higher incidence of significant mutations in the cpsE-cpsF-cpsG region of bovine isolates compared with those from humans (36), which supports previous studies that indicate that bovine GBS isolates represent an S. agalactiae lineage that is distinct from that of isolates from humans (5).

 
Fanrong Kong, Lotte Munch Lambertsen, and Hans-Christian Slotved made similar contributions to this work and so should be considered co-first authors.

We thank Maryann Pincevic for help in sequencing, Kirsten Burmeister, SSI, for help with serotyping, and Ping Zhu for help with the figure. Isolates included in this study were kindly provided by Diana Martin and Julie Morgan, Streptococcus Reference Laboratory, ESR, Wellington, New Zealand; Lawrence Paoletti and Catherine Lachenauer, Channing Laboratory, Boston, MA; Johan Maeland, Department of Microbiology, School of Medicine, Norwegian University of Science and Technology, Trondheim, Norway; Nicola Jones, Nuffield Department of Clinical Laboratory Sciences, Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; Dele Davies and Shannon Manning, Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor; Reinhard Berner, Department of Pediatrics, University Children's Hospital, D-79106 Freiburg, Germany; Yunsop Chong and Kyungwon Lee, Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, Korea; Catherine Satzke and Roy Robins-Browne, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Australia; Margaret Ip, Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong; Gabriela Martinez and Marcelo Gottschalk, Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada.

 
* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Road, Westmead, New South Wales 2145, Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: l.gilbert{at}usyd.edu.au

Published ahead of print on 18 June 2008.

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Journal of Clinical Microbiology, August 2008, p. 2745-2750, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.00189-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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