Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V
               The GenBank accession number for the sequence reported in this paper is AF402095.

Selm, Saskia van; Kolkman, Marc A. B.; Zeijst, Bernard A.M. van der; Zwaagstra, K A; Gaastra, Wim; Putten, Jos P. M. van

doi:10.1099/00221287-148-6-1747

Cited by 35 publications

(27 citation statements)

References 56 publications

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“…Indeed, this was true for serotype 9V, where sequetyping of our strains matched both serotypes 9A and 9V of the representative strains initially interrogated. However, additional 9V strain sequences deposited in GenBank during our study matched precisely our 9V sequence, resulting in correct identification (63). Conversely, some serotypes could not be sequetyped, contrary to the in silico predictions.…”

Section: Discussionmentioning

confidence: 48%

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

et al. 2012

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dThe introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.

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Section: Discussionmentioning

confidence: 48%

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

et al. 2012

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“…Moreover, there is the possibility of expanding the method to cover the detection of more capsular types. Based on the recent publication of the complete sequence of a 9V capsular locus (29) and the public release of partial sequences of capsular loci 6A and 6B by Griffiths and Hall (GenBank accession no. AF246898, AY078347, AY078342, AY078343, AY078344, AY078341, AY078339, AY078345, AY078340, and AY078346), we designed new primers to allow the identification of these serotypes and the expansion of our typing scheme.…”

Section: Discussionmentioning

confidence: 99%

“…The three additional capsular determinants for serotypes 5, 7F, and 9V also present in this vaccine had not been sequenced at the time this method was developed; the sequence for serotype 9V was recently published (29). Based on Southern hybridization results (14,19,22) and sequence homologies, the primers in this second class were designed to target different genes which were as follows: gene wzy for serotypes 1 (21), 4 (11), 6B (11), 14 (13), and 19F (6) (initially named cpsH and cpsI for serotypes 14 and 19F, respectively); gene capB for serotype 3 (2); genes wciY and gct for serotype 18C (11); gene cpsK for serotype 19A (19); and gene cpsG for serotype 23F (22).…”

Section: Molecular Capsular Typing (I) Oligonucleotide Primersmentioning

confidence: 99%

Serotyping Streptococcus pneumoniae by Multiplex PCR

2003

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The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.

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“…Eleven serotype 14 strains and 39 serotype 9V strains from a previous study were included (17). In addition, the cpsB genes from 15 international pneumococcal clones (18) and 23 strains listed in GenBank (3,9,[11][12][13][14][15]19,20,22,23,25,29) were analyzed ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

Serotype 14 Variants of the France 9V ⁻³ Clone from Baltimore, Maryland, Can Be Differentiated by the cpsB Gene

McEllistrem¹,

Noller

Visweswaran³

et al. 2004

J Clin Microbiol

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European serotype 14 variants of the France 9V؊3 clone, which have arisen through recombination events involving the penicillin binding protein 1a (pbp1a) gene, have cpsB sequences distinct from those of the 9V ؊3 clone. Serotype 14 variants of the 9V؊3 clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible Streptococcus pneumoniae strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), pbp1a PCR restriction profiles, and cpsB and pbp1a sequences. The cpsB genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V ؊3 clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V ؊3 clone nor to the R6 strain from positions 1498 to 1710 of the pbp1a gene. All serotype 14 strains had cpsB sequences that differed by <1% (0 to 5 of 476 bp) from each other and that were >16% (78 to 83 of 476 bp) divergent from that of the 9V ؊3 clone. Allowing for a 2-bp difference in the cpsB sequence resulted in the highest correlation between the cpsB gene and serotype. Overall, 95% (84 of 88) of the strains were classified correctly by serotype with the cpsB sequence. The distal recombination site of the Baltimore serotype 14 variants of the 9V ؊3 clone was not identical to that of the European serotype 14 variants. The cpsB gene was serotype specific regardless of whether capsular switching occurred. Although the correlation between serotype and the cpsB sequence was high, the overall diversity of the cpsB gene within a serotype likely will limit the role of this gene in a sequence-based serotyping method.Pneumococci are naturally transformable, with recombination rates 10-fold higher than mutation rates (5). Griffith et al. (8a) first described pneumococcal transformation in 1928, while Avery et al. determined that the transforming factor was DNA (1). Strains which are found to be highly related by pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST) may have different serotypes, indicating serotype capsular recombination (2,3,18,24). The capsular locus is comprised of a series of alphabetically named capsular genes, which are flanked by the conserved genes dexB and aliA; approximately 5.8 kb downstream from aliA is the penicillin binding protein 1a (pbp1a) gene (2). Since a capsular locus has been identified for a variety of serotypes, the capsular genes are attractive targets for a sequence-based serotyping method. An association between some capsular gene sequences and serotype has been noted in the literature, even for strains which have undergone capsular transformation (2, 3).The France 9V Ϫ3 pneumococcal clone is 1 of approximately 20 international clones and has been detected as serot...

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Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Cited by 35 publications

References 56 publications

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Serotyping Streptococcus pneumoniae by Multiplex PCR

Serotype 14 Variants of the France 9V ⁻³ Clone from Baltimore, Maryland, Can Be Differentiated by the cpsB Gene

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Organization and characterization of the capsule biosynthesis locus of Streptococcus pneumoniae serotype 9V The GenBank accession number for the sequence reported in this paper is AF402095.

Cited by 35 publications

References 56 publications

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Serotyping Streptococcus pneumoniae by Multiplex PCR

Serotype 14 Variants of the France 9V −3 Clone from Baltimore, Maryland, Can Be Differentiated by the cpsB Gene

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Serotype 14 Variants of the France 9V ⁻³ Clone from Baltimore, Maryland, Can Be Differentiated by the cpsB Gene