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Journal of Clinical Microbiology, March 2004, p. 1326-1329, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1326-1329.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Multiplex PCR Assay for Direct Identification of Group B Streptococcal Alpha-Protein-Like Protein Genes

Roberta Creti,* Francesca Fabretti, Graziella Orefici, and Christina von Hunolstein

Laboratorio di Batteriologia, Istituto Superiore di Sanità, 00161 Rome, Italy

Received 3 October 2003/ Returned for modification 10 November 2003/ Accepted 4 December 2003


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ABSTRACT
 
We developed a group B streptococcus multiplex PCR assay which allows, by direct analysis of the amplicon size, determination of the surface protein antigen genes of alpha-C protein, epsilon protein, Rib, Alp2, Alp3, and Alp4. The multiplex PCR assay offers a rapid and simple method of subtyping Streptococcus agalactiae based on surface protein genes.


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INTRODUCTION
 
Streptococcus agalactiae (group B streptococcus [GBS]) is the leading pathogen in newborn infants in both developing and industrialized countries throughout the world, causing sepsis and meningitis. It also causes disease in parturient women and immunocompromised adults (18).

Epidemiological studies on S. agalactiae infections are mainly based on capsule serotyping. Currently, nine different S. agalactiae serotypes have been described (serotypes Ia, Ib, and II to VIII). Serotype distribution varies with geographical region and ethnic origin, and the virulence of clinical isolates with similar capsular composition can vary widely, suggesting that other bacterial virulence factors, in addition to capsule, are involved in the pathogenesis of GBS (19).

The major surface-localized protein antigens of group B streptococci belong to a family of surface proteins named the alpha-C protein, Rib, Alp2, Alp3, Alp4, and the epsilon protein (alpha-protein-like proteins). They contain large internal tandem repeats and are potential virulence factors (2, 5, 10, 12, 15, 16, 20). All these proteins are encoded by stable mosaic genes, generated by a recombination of modules at the same chromosomal locus (4, 12). The proteins exhibit size variation between strains depending on the number of repeats in the corresponding gene. Moreover, it has been demonstrated that, in the course of infection, the number of repeats inside the alpha-C proteins can undergo internal deletions as a means for evading the host immune response (14).

Protein typing of GBS is determined by the use of C and R antisera. Indeed, antisera are not protein specific (10, 12). While the alpha-C and Rib proteins are immunologically distinct from each other, the alpha-C, Alp2 and Alp3 (Alp2/3), and the epsilon proteins cross-react during Western blotting (11).

C antiserum also recognizes beta antigen, a GBS immunoglobulin A binding protein that was not included in this study because its structure is unrelated to that of the family of alpha-protein-like proteins (7, 8).

Protocols to identify serotypes and surface protein antigen genes by molecular methods have been recently published (3, 9, 10). For the protein subtyping, parallel PCRs using different primer pairs for each gene were set up; detection of epsilon gene had not previously been considered. We developed a multiplex PCR that allows determination of the following GBS surface protein genes directly by the analysis of the amplicon size: the alpha-C protein, the epsilon protein, Rib, Alp2/3, and Alp4.

While the repeats present extensive homology between alpha-protein-like proteins, the N-terminal portion is distinctive. The analysis of a ClustalW amino acid multisequence alignment of the N-terminal portions of the alpha-C and epsilon proteins, Rib, Alp2, Alp3, and Alp4 identified distinctive strings for each protein (Fig. 1), except for the Alp2 and Alp3 proteins, which are identical over the first half of their length.



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  		FIG. 1. Amino acid multisequence alignment of the N-terminal portions of S. agalactiae alpha-C protein, Alp2, Alp3, Rib, and the epsilon protein. The nucleotide sequence corresponding to the string from positions 11 to 17, indicated in grey, was used as the forward universal primer. The reverse primers sequences, specific for each protein gene (except for Alp2/3), are also indicated in grey.

Primer nucleotide sequences corresponding to the distinctive strings were used in a multiplex assay as the reverse primers, while a nucleotide string, common to all the surface protein genes, was used as the forward primer (Table 1). Briefly, total DNA preparations were prepared from GBS cultures, according to the instructions provided with a DNeasy tissue kit (QIAGEN), and 50 ng was used as a template in a final volume of 25 µl of PCR mixture containing the following: 1x PCR buffer; 2 mM MgCl2; 200 µM concentrations of dATP, dCTP, dGTP, and dTTP; 400 nM concentrations of each of the six primers; and 0.25 U of TaqDNA polymerase (Life Technologies).


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  		TABLE 1. Nucleotide primer sequences and amplicon size expected for each S. agalactiae surface protein gene considered in this study

The samples were amplified on a DNA thermal cycler (MJ Research, Inc.) by heating for 3 min at 96°C, followed by 30 cycles of 95°C for 60 s, 58°C for 45 s, and 72°C for 45 s and concluding with a cycle of 72°C for 10 min.

The PCR products were analyzed by electrophoresis in a 2% (wt/vol) agarose-1000 gel (Life Technologies). UV transillumination of the bands on the agarose gel showed different sizes of amplicons, which allowed direct identification of the surface protein gene possessed by each GBS strain tested (Fig. 2).



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  		FIG. 2. Gel electrophoresis of multiplex PCR amplification products. Direct analysis of amplicon size allowed the determination of the genes encoding the surface protein of each GBS strain as follows: Rib (lanes 1, 2, and 8), alpha-C protein (lanes 3 and 10), Alp2/3 (lanes 4, 6, 9, and 12), Alp4 (lane 7), and the epsilon protein (lanes 5 and 1l). Lane 13 is the negative control. M, 2-log DNA ladder (0.1 to 10.0 kb; New England Biolabs, Inc.).

A serologically typed collection of GBS reference strains (10 strains), selected clinical isolates (55 strains), and bovine strains (9 strains) was analyzed by multiplex PCR, and results derived from the direct analysis of amplicon size were correlated with the gene sequence of the corresponding amplified product for each individual strain (Table 2). As noted above, C and R antisera cross-reacted with different protein genes. Moreover, as previously reported (4, 10), GBS strains can possess an alpha-protein-like protein gene even if the protein is not expressed on the surface. In two cases (reference strain Prague 10/84 and a bovine strain), no surface protein gene was detected. The absence of a surface protein gene in GBS isolates, although rare, has previously been reported (10).


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  		TABLE 2. Characteristics of GBS strains used in this study

Relationships between serotypes and surface protein genes were noted but to a lesser degree than previously reported (10, 12). The association of serotypes Ia, Ib, and II with the alpha protein, of serotype III with Rib, and of serotypes V and VIII with Alp3 was found, but these associations were not absolute. In particular, the alpha protein was present in most serotypes, Rib was detected in serotypes II, V, and VIII as well as in nontypeable strains, and Alp3 was also present in serotype VII. The Alp2 protein, first detected in serotype V (12) and subsequently detected in serotypes Ia and III (10), was also associated with type VIII in our study. No epidemiological studies surveyed the distribution of the epsilon protein that, in our study, was evenly distributed among GBS serotypes and prevalent in bovine strains.

The possibility of looking at the protein gene profile increases the potential of GBS subtyping by limiting the use of C and R antisera to the most appropriate strains after PCR has been used to ascertain protein expression.

A glycoconjugate vaccine to prevent GBS disease is being tested in clinical trials (1, 17). Promising data on using GBS surface proteins as protein carriers instead of tetanus toxoid have been published (6, 13). In this context, it is extremely important to have a rapid and reliable molecular test capable of determining the GBS surface protein genes by direct evaluation of amplicon size, which would permit extensive epidemiological studies on the profiles of protein subtypes of circulating GBS strains without consuming additional resources.


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ACKNOWLEDGMENTS
 
We thank M. Pataracchia for serotyping GBS isolates.

This work was supported by the Italian Ministry of Health Project 1%, grant 0AD-F (C.V.H.), and by ISS grant 1024/RI (C.V.H.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratorio di Batteriologia, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39 06 49902036. Fax: 39 06 49387112. E-mail: roberta.creti{at}iss.it. Back


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Journal of Clinical Microbiology, March 2004, p. 1326-1329, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1326-1329.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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