J Med Microbiol 57 (2008), 992-999; DOI: 10.1099/jmm.0.2008/000505-0
© 2008 Society for General Microbiology
ISSN 1473-5644
A new serotype 14 variant of the pneumococcal Spain9V-3 international clone detected in the central region of Argentina
Andrea G. Albarracín Orio1,
Paulo R. Cortes1,2,3,
Miguel Tregnaghi2,
German E. Piñas1,
Argentinean Network Pneumococcus Study Group
and
José R. Echenique1
1 Departamento de Bioquimica Clinica-CIBICI (CONICET), Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Cordoba, Argentina
2 Centro de Desarrollo de Proyectos Avanzados en Pediatria (CEDEPAP), Cordoba, Argentina
3 Hospital Pediatrico del Niño Jesus, Cordoba, Argentina
Correspondence
José R. Echenique
jeche{at}fcq.unc.edu.ar
Received 17 January 2008
Accepted 7 April 2008
The penicillin-resistant Spain9V-3 clone of Streptococcus pneumoniae is widespread and presents different serotype variants originating from recombination of the capsular genes. In this work, the genetic relatedness of 29 invasive pneumococci isolated from the central region of Argentina (Cordoba, Buenos Aires, Santa Fe and La Pampa provinces) was assessed by multilocus sequence typing (MLST). All of the penicillin-non-susceptible isolates studied (21/29) belonged to a serotype 14 variant of the Spain9V-3 clone. This clone was predominant, suggesting that it was responsible for the penicillin resistance spread in this region. Interestingly, this serotype 14 variant (named Cordoba S14V) could be differentiated from the European one by its pbp1a gene, suggesting a different recombinational replacement of the capsular genes. The putative recombination sites were analysed, resulting in the proximal crossover point being clearly localized in the spr0309 gene, with the distal site restricted to the recU gene, confirming a different recombination event. Analysis of the dexB, cpsB, aliA and pbp1a genes from these strains showed a high similarity with the corresponding genes of the Spain14-5 clone, suggesting that the capsular genes were provided by this international clone. Analysis of the genetic polymorphisms of the pbp1a (nt 1473–1922) and spr0309 (nt 1–790) genes is proposed as an epidemiological tool to help recognize the Cordoba S14V of the Spain9V-3 clone. On the other hand, BOX-repeat-based PCR and MLST analyses of serotype 14 strains revealed a divergent epidemiology of the Cordoba S14V, suggesting a non-recent dissemination in the paediatric population. It is suggested that this molecular epidemiology work will be a reference for monitoring the evolution of S14Vs of Spain9V-3, the emergence of new clones and the impact of pneumococcal vaccination programmes in Argentina.
Abbreviations: BOX-PCR, BOX-repeat-based PCR; MLST, multilocus sequence typing; ST, sequence type.
The Argentinean Network Pneumococcus Study Group comprises the following people and institutions: Silvia Yudowski, Lydia Carvajal, Catalina Culasso, Carmen Nobile and Myriam Figueroa (Centro de Desarrollo de Proyectos Avanzados en Pediatria, CEDEPAP, Córdoba, Argentina); Horacio Lopardo and Claudia Hernández (Hospital de Pediatria ‘Professor J. P. Garrahan’, Buenos Aires, Argentina); and Mabel Regueira (Servicio de Bacteriología Clínica, INEI-ANLIS', ‘Dr Carlos Malbrán’, Buenos Aires, Argentina). 
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INTRODUCTION
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Worldwide, Streptococcus pneumoniae is one of the main human pathogens, being the causal agent of infections such as otitis and sinusitis, as well as severe infections such as pneumonia, bacteraemia and meningitis. In developing countries, more than 1 million children under 5 years of age are estimated to die annually from pneumococcal pneumonia (Greenwood, 1999). The major epidemiological concerns about pneumococcal infection are determining the origin of emergence and the dissemination of drug-resistant strains. Penicillin-resistant isolates were first detected in the late 1960s, and their prevalence increased rapidly during the 1980s. Since 1989, the incidence of penicillin-resistant pneumococci has remained fairly constant at about 20–30 % of all isolates (Lynch & Zhanel, 2005). Molecular characterization of penicillin-resistant strains from around the world has highlighted an important diversity among isolates, but has also identified a number of successful pneumococcal clones with a high level of penicillin resistance, some of which have spread globally (Crook & Spratt, 1998). Countries such as Spain, South Africa and Hungary reported high frequencies of penicillin-resistant pneumococcal infections in the mid-1970s (Fenoll et al., 1998), followed later by France, Iceland and the USA in the mid-1980s to early 1990s (Breiman et al., 1994; Lefevre et al., 1995). The increasing number of penicillin-resistant clones, the diversity of the genotypes and the serotype exchange make it difficult to assign isolates to these clones. It is now recommended that new genotype assignments should be made only after the strain has undergone careful PFGE, BOX-repeat-based PCR (BOX-PCR) or multilocus sequence typing (MLST) analysis (McGee et al., 2001). One of the best-characterized clones is Spain9V-3, which appears to have emerged in Spain during the 1980s (Fenoll et al., 1991). This clone has been prevalent in France for the last 15 years and has been found in many other countries including Germany, the Netherlands, Poland, Denmark, Uruguay, Argentina, Brazil, Taiwan and South Africa (Castanheira et al., 2004; Coffey et al., 1999; Corso et al., 1998; Hermans et al., 1997a, b; Shi et al., 1998).
Strains that are highly related by MLST may express different serotypes. This phenomenon is caused by a recombinational replacement occurring in neighbouring regions of the capsular (csp) genes. The Spain9V-3 clone has been isolated as serotype 9A, 9V, 14 and 19 variants (McGee et al., 2001). For the European serotype 14 variant (S14V) of this clone (isolated in Uruguay, Denmark and Spain), the proximal recombination site was detected in the cspA gene, whereas the distal site was localized in the pbp1a gene (Coffey et al., 1999).
The main purpose of this work was to assess the genetic relatedness of invasive pneumococcal strains isolated from children in Cordoba, Argentina, using genotypic methods such as MLST, BOX-PCR and RFLP analysis of the penicillin-binding protein (pbp) genes. Our results showed that all penicillin-non-susceptible strains belonged to a new S14V of the Spain9V-3 clone; therefore, we decided to investigate whether this variant was similar to others described previously by analysing the recombinational replacement of the capsular genes and their flanking regions. We also examined whether this S14V was disseminated among other provinces in Argentina.
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METHODS
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Bacterial strains and antimicrobial susceptibility testing.
All of the pneumococcal strains were obtained from invasive infections of paediatric patients and were collected from public children's hospitals of different provinces (Cordoba, Buenos Aires, La Pampa, Santa Fe and Buenos Aires City) during the period 2003–2005. The original Spain9V-3 clone (ATCC 700671) and the Spain14-5 clone (ATCC 700902) were utilized as references. Serotyping was performed using the Quellung reaction with sera produced by the Statens Serum Institut, according to the manufacturer's instructions. For antimicrobial susceptibility testing, strains were grown at 37 °C in an atmosphere of 5 % CO2 on Mueller–Hinton agar with 5 % defibrinated sheep blood. Penicillin MICs were determined by agar dilution following a CLSI protocol (CLSI, 2007).
RFLP analysis of pbp genes.
pbp genes were amplified by PCR using the following primers: F1a/R1a for pbp1a, F2b/R2b for pbp2b and F2x/R2x for pbp2x (Table 1
). The reaction mixtures contained 2 mM MgCl2, 0.5 µM each dNTP and 2 U Taq DNA polymerase (Invitrogen). PCR was performed using a Bio-Rad Gene Cycler for 30 cycles of 95 °C for 1 min, 53 °C for 30 s and 72 °C for 45 s. Purified PCR product (1 µg) was digested with MseI/DdeI (3 U each enzyme) or 2 U HinfI for 2 h at 37 °C and analysed by 7 % non-denaturing PAGE.
PCR conditions.
For amplification of the pbp1a DNA fragments, the primer pairs F1a/R1a2, F1aS/R1aS, F1aM/R1aM and F1aF/R1a were utilized. An internal fragment of the cpsB gene was amplified using the primers cpsB-down (Coffey et al., 1998) and cpsS3 (Kong & Gilbert, 2003). These PCR products were all amplified using the following PCR parameters: initial denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s and elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. A 639 bp internal fragment from the dexB gene and an 842 bp internal fragment from the aliA gene were amplified using the primer pairs Fdex/Rdex and Fali/Rali, respectively. The PCR parameters were initial denaturation at 94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 52 °C for 45 s and 72 °C for 1 min. The spr0309 and recU genes were amplified using the primer pairs Fspr/Rspr and FrecU/RrecU, respectively. All primer sequences are shown in Table 1
. Chromosomal DNA was isolated using a Wizard Genomic DNA purification kit (Promega) following the manufacturer's instructions. The PCR products were amplified from chromosomal DNA and sequenced in both directions using the primers used for amplification. The cpsB sequences from the Baltimore strains were kindly provided by Dr Lee Harrison (McEllistrem et al., 2004).
MLST.
MLST was performed as described previously (http://www.mlst.net). The internal fragments from the aroE, gdh, gki, recP, spi, xpt and ddl genes were amplified by a PCR utilizing primers described previously (Enright & Spratt, 1998). DNA sequencing was performed by Macrogen and DNA sequences were edited using BioEdit software (Hall, 1999). Alleles and sequence types (STs) were assigned using the database available at http://www.mlst.net.
BOX-PCR typing.
The BOX elements were amplified from genomic DNA using the primer BOXA1R (Koeuth et al., 1995). The reaction conditions were 30 cycles of denaturation at 95 °C for 1 min, annealing at 52 °C for 2 min and elongation at 72 °C for 2 min. PCR was performed using a PTC-100 MJR thermal cycler. PCR products were analysed by electrophoresis on a 2 % TAE/agarose gel.
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RESULTS
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Determination of the genetic relatedness of invasive pneumococcal strains
Initially, 66 invasive pneumococcal strains were isolated from children under 5 years of age attending public children's hospitals in Cordoba, Argentina, during the period 2003–2005, and 20 of these were non-susceptible to penicillin (Table 2). To analyse the genetic relationship of this pneumococcal population by MLST, we selected nine penicillin-non-susceptible isolates with a high penicillin MIC and eight penicillin-susceptible isolates, based on the serotype prevalence in our region. The results showed that all penicillin-non-susceptible strains belonged to the S14V of the Spain9V-3 clone. Seven strains presented an identical allelic profile to ST156 and two strains showed a single-locus variant, ST370 (Table 3). To test the clonal evolution of the Cordoba S14V strains, we used molecular markers that diverge more frequently than housekeeping genes, such as BOX-PCR and RFLP of pbp genes. Our Spain9V-3 strains showed five BOX profiles (data not shown), indicating an epidemiological divergence of this clonal complex and a non-recent dissemination in the paediatric population. All nine penicillin-non-susceptible strains isolated from Cordoba showed identical pbp RFLP patterns (pbp1a/AluI, HinfI or MseI/DdeI, pbp2b/HinfI and pbp2x/HinfI; data not shown), indicating a lower discriminatory power than BOX-PCR.
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Table 2. Phenotypic and molecular characterization of non-susceptible strains isolated from Cordoba
Cba, Cordoba; CSF, cerebrospinal fluid; NT, not typable.
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The penicillin-susceptible strains showed genetic diversity by MLST and were classified into minor international clones (Table 4). Two of the eight strains presented different serotypes to those described originally, namely a serotype 7F variant of the England14-9 clone and a S14V of the Sweden15A-25 clone. We also found two new STs, 2240 and 2241, which we registered in the MLST database.
Analysis of the capsular gene region to detect recombinational replacements
It has been reported that a S14V of the Spain9V-3 clone isolated in Baltimore (MD, USA) could be differentiated from the original Spain9V-3 clone (serotype 9V) by cpsB gene polymorphisms (in the 72–548 region) and from the Uruguayan/European variant by the pbp1a gene (in the 1498–1710 region) (McEllistrem et al., 2004). To determine whether our S14V was derived from the other S14Vs that have been described, the cpsB (72–548 region) and pbp1a (1–2160 region) genes were amplified and sequenced. All nine Cordoba S14V strains showed identical cpsB sequences to the Baltimore S14V and presented one polymorphism at position 446 compared with the European S14V. However, there was a 17 % divergence (82 out of 476 bp) from the original Spain9V-3 clone (Fig. 1). For the pbp1a gene, from position 1473 to 1922, the Cordoba S14V was identical to the Baltimore S14V (McEllistrem et al., 2004) and to the Spain14-5 clone, but had 60 bp differences compared with the original Spain9V-3 clone and the European S14V (Coffey et al., 1999) (Fig. 2). From position 1473 to 1872, our strains were also different to the Polish S14V (Coffey et al., 1999) with 48 polymorphisms, but were identical to the Polish and Baltimore S14Vs from position 1873 to 1922. A single polymorphism at position 2045 was present only in the Cordoba variant and the Spain14-5 clone, although the DNA sequence of this pbp1a region was not available for the Baltimore S14V (Fig. 2). All of these results suggested that the Baltimore S14V and our S14Vs had similar clonal origins, and that both had a different clonal origin to the European S14V, also suggesting a different capsular replacement. It has been proposed that serotype variants of penicillin-non-susceptible clones have arisen by recombinational exchanges at the capsular biosynthetic locus, cps. Molecular evidence in support of this hypothesis was reported for the European S14V, which arose by large recombinational replacements in the cpsA–pbp1a region (Coffey et al., 1999). The cps locus contains a series of alphabetically named capsular genes, which are flanked by the conserved genes dexB and aliA (Fig. 3). In the European S14V, the crossover point of the proximal end was localized to the cpsA gene (Coffey et al., 1999). To identify this point in our isolates, the DNA region upstream from the cpsB gene was analysed. We found that partial dexB sequences of the Cordoba S14V strains were different from that of the original Spain9V-3, indicating that the crossover occurred upstream from dexB (Fig. 3). Consequently, the spr0309 gene (as named in the R6 strain, or SP0341 in the TIGR4 strain) of three strains (Cba-6, -10 and -28) was amplified and sequenced. The recognition of a divergent block at position 198 of spr0309 revealed the proximal crossover point of the recombinational exchange that introduced the serotype 14 cps locus into our S14Vs (Fig. 1). The distal crossover point of the European S14V was localized in pbp1a (Coffey et al., 1999). We could not identify the distal crossover point along the pbp1a sequences from Cordoba isolates. Based on the identical sequence homology found in the 5' region of the pbp1a gene and for the complete recU gene among our strains and the Spain14-5 and Spain9V-3 clones, we suspected that the distal crossover point could be along this region (Fig. 2). To study the continuity of the extensive recombinational replacement in the spr0309–recU region, aliA, a gene located approximately in the centre of the replaced DNA region (Fig. 3), was also analysed. Its DNA sequence showed differences from that of the original Spain9V-3, but complete sequence similarity with the same gene described in the Spain14-5 clone (data not shown). This result was in agreement with the other gene analyses and suggested no interruption of the DNA replacement, which apparently was provided by the Spain14-5 clone.
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Fig. 1. Comparison of polymorphic sites within the spr0309–cpsB region. The DNA sequences shown correspond to the spr0309–cpsB region from different serotype variants of the Spain9V-3 clone: Sp9V-3, original Spain9V-3 clone (ATCC 700671); URU, S14V strains of the Spain9V-3 clone isolated in Uruguay (Coffey et al., 1999); BALT, S14V strains of the Spain9V-3 clone isolated in Baltimore (strain I) (McEllistrem et al., 2004); Cba, S14V strains of the Spain9V-3 clone isolated in Cordoba. The Sp9V-3 sequence is shown as a reference and identical sites in other strains are represented by dots, with only polymorphisms being indicated. The nucleotide numbers of the polymorphic sites are indicated vertically and are positioned according to the transcription sense indicated by arrows. The symbol ‘/’ represents unsequenced DNA regions. The solid arrow indicates the proximal crossover point of the recombinational replacement. This site is located at position 198 of the spr0309 gene from the Cordoba S14V of the Spain9V-3 clone. Position 446 of the cpsB gene is underlined. The DNA sequences from the Buenos Aires, Santa Fe and La Pampa strains were identical to that of the Cordoba strains; therefore we used Cba-6 as a representative strain.
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Fig. 2. Polymorphic sites within the pbp1a gene from different strains. See Fig. 1 legend for details of the different serotype variants; Sp14-5 indicates the Spain14-5 clone (ATCC 700902). From position 1711 to 1874 and from position 2000 to the end of the sequence, our strains were identical to the Spain9V-3 clone, except for a single polymorphism at position 2045, which was found only in the Spain14-5 clone. The DNA sequences from the Buenos Aires, Santa Fe and La Pampa strains were identical to that of the Cordoba strains; therefore, we used Cba-6 as a representative strain. The Uruguayan (URU) and Polish (POL) pbp1a sequences were obtained from Coffey et al. (1999).
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Fig. 3. Graphic representation of the possible recombinational replacements from which the Cordoba S14V of the Spain9V-3 clone may have originated. The srp0309–recU region is shown as an open box for the original Spain9V-3 clone, as a filled box for the Spain14-5 clone, and is shaded for an unknown serotype 14 strain. DNA regions that were introduced by recombination from a serotype 14 donor into a serotype 9V receptor are shown as filled or shaded zones. Horizontal arrows below the Cordoba S14V represent the regions that were amplified and sequenced. A solid cross indicates the proximal crossover point identified in the srp0309 gene. The distal site of the recombinational replacement was not clearly detected. The hatched area represents the zone where the unidentified crossover event is expected to have occurred.
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Detection of the Cordoba S14V in other provinces
As this new S14V was predominant in Cordoba, we decided to investigate the putative dissemination of this subclone in different geographical regions near this province. Consequently, we requested invasive serotype 14 strains from children's hospitals located in other regions, such as Buenos Aires City and the provinces of Buenos Aires, Santa Fe and La Pampa, covering an approximate area of 800 km2. We obtained 12 penicillin-non-susceptible strains recovered from blood cultures that corresponded to serotype 14 and these were analysed by MLST (Table 5). Ten isolates were ST156; however, we identified two new sequence types, ST3119 and ST3120, which presented a single locus variant of the allelic profile of the Spain9V-3 clone. To identify whether these strains belonged to the Cordoba S14V, the genetic polymorphisms of the pbp1a (nt 1473–1922) and spr0309 (nt 1–790) genes were analysed and their sequences were found to be identical to those found in the S14V isolated in Cordoba (Figs 1 and 2). These results suggested that the Cordoba S14V is disseminated in the central provinces corresponding to the most densely populated regions of Argentina (Fig. 4).
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Table 5. Phenotypic and molecular characterization of the Cordoba S14Vs of the Spain9V-3 clone isolated from other provinces
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DISCUSSION
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In this work, we determined the genetic background of invasive pneumococcal strains circulating in Cordoba, showing that a new S14V of the Spain9V-3 clone is predominant among penicillin-non-susceptible strains and is disseminated in the central region of Argentina. Previously, a PFGE-based molecular epidemiological study indicated that a S14V of the Spain9V-3 clone was predominant among penicillin-non-susceptible isolates in Argentina (Rossi et al., 1998). Another epidemiological study conducted by the Pan American Health Organization analysed invasive pneumococcal strains isolated from children under 5 years of age in six Latin-American countries from 1993 to 1996 (Tomasz et al., 1998). This PFGE study revealed that the Spain9V-3 and Spain23F-1 clones were highly represented in these countries and that a S14V of the Spain9V-3 clone showed greater than 80 % prevalence in Argentina and Uruguay, with penicillin MICs of greater than 1 µg ml–1. The identical PFGE profile identified in Argentina and Uruguay suggested that the clonal origin was common, and that the importation of this clone in both countries was coincident in time. The majority of these isolations were detected in large cities, sustaining the hypothesis that the spread of these clones has been facilitated by international travel (Tomasz et al., 1998). However, a detailed molecular characterization of the Cordoba S14V revealed that its epidemiological history is different to that of the Uruguayan strains. Coffey et al. (1999) described several Uruguayan strains with identical ST profiles and pbp1a sequences to those of the European S14V. However, in contrast, we found clear differences between the Cordoba and European S14Vs, suggesting that a different recombinational event has occurred in Cordoba to that which occurred in Uruguayan strains. We found that the proximal crossover point in our strains was located precisely in the spr0309 gene (Fig. 1), confirming a new recombinational event. When dexB, cpsB, aliA and pbp1a sequences were analysed, they were found to be identical to the homologous genes described in the Spain14-5 clone, indicating that the origin of serotype 14 cps genes was this international clone and not other serotype 14 strains, as occurring in the European S14V. In contrast to the proximal site, the distal crossover point was not clearly identified. The 1473–1922 region of pbp1a was identical between our strains and the Spain14-5 clone, but we found 60 bp differences compared with the European S14V. Also, the pbp1a sequences (2000–2160 region) were identical for the original Spain9V-3 clone, the Spain14-5 clone and our clones, except that the Spain9V-3 clone did not have a polymorphism at position 2045. Up to this position, we consider that the replaced DNA region in our strains belongs to the Spain14-5 clone, but after this site, we could not detect a divergent block in the DNA sequences from these clones. For this reason, we assume that the distal crossover occurred in the recU region.
Here, we have described a new capsular replacement in the predominant penicillin-non-susceptible Spain9V-3 clone, with this phenomenon also being observed in penicillin-susceptible isolates. We found that the Cba-32 strain was serotype 14 and ST782, belonging to the Sweden15A-25 clone (Table 4). In addition, the Cba-24 strain was serotype 7F and ST9, corresponding to the England14-9 clone. In this case, this capsular replacement has relevance in the application of vaccination programmes, as serotype 7F is currently not included in the vaccine formulation, and this is therefore a risk to be taken into account when considering the pneumococci circulating in our paediatric population.
Due to the geographical proximity with Uruguay, and also the active tourist exchange with our country, we expected to find the same S14V as that observed in Uruguay. However, the Uruguayan S14V presented the same genetic profile as the European S14V (Coffey et al., 1999). From an MLST analysis of our 21 strains belonging to the Spain9V-3 clone, 17 strains were ST156, two were ST370 and two were new STs, ST3119 and ST3120, which were single variants of ST156. By coincidence, a search in the MLST database (http://spneumoniae.mlst.net) showed three STs associated with S14V strains isolated in Uruguay: the original ST156 and two single variants in the xpt allele, which were represented by ST164 and ST168. These data revealed that the ST divergence was different in Argentina. Analysis of the BOX regions, an evolutionary marker that diverges more frequently than housekeeping genes, suggested that the Spain9V-3 clone was not disseminated recently in Argentina.
All of our Spain9V-3 strains presented reduced penicillin (Tables 2 and 4) and cefotaxime (data not shown) susceptibility. Castanheira et al. (2003) analysed a cefotaxime-resistant population isolated from six Latin-American countries. A major clone, which presented serotype 14 and the same ribotype as Spain9V-3, was present in Argentina, Brazil, Chile and Uruguay. However, the Argentinean and Uruguayan strains showed small differences in the PFGE analysis. Therefore, we may assume that our S14V is the source of the cefotaxime resistance in Cordoba and also in other parts of Argentina, but not in Uruguay.
So far, characterization of the Cordoba S14V has shown the same genetic profile as that of the Baltimore S14V. There are no data about the putative crossover points of the serotype 14 cps genes of the Baltimore strains, but when we analysed the published cpsB and pbp1a sequences (McEllistrem et al., 2004), we also found homology with the Spain14-5 clone, suggesting that the cps cassette had the same origin. Here, we have performed a relevant characterization of this new S14V of the Spain9V-3 clone that should be considered for further epidemiological studies to determine whether these S14V strains are disseminated in other countries and also where this clone originated. For this, we propose that the pbp1a (nt 1473–1922 region) and spr0309 (nt 1–790 region) genes should be utilized to help recognize the Cordoba S14V. We suggest that the results of this study will provide a reference for monitoring the evolution of the variants of Spain9V-3, the emergence of new clones and the impact of pneumococcal vaccination programmes in Argentina.
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ACKNOWLEDGEMENTS
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We thank Lee Harrison for providing the cpsB sequences obtained from the S14V of the Spain9V-3 strains isolated in Baltimore, MD, USA. We also thank Luis Patrito, Jose Bocco and Alex Saka for their critical review of this article and Dr Paul Hobson for revising this manuscript. This work was principally supported by a grant and a PhD fellowship to A. G. A. O. from the National Agency of Scientific and Technological Promotion (ANPCYT, PICT 05-10894 BID 1201 OC-AR). Grants from the National Council of Scientific and Technological Research (CONICET) and the Scientific and Technological Secretary of the National University of Cordoba (SECYT-UNC) are also acknowledged. We thank GlaxoSmithKline for funds granted to M. T. (CEDEPAP) to support microbiology laboratory personnel in the provision of pneumococcal isolates. We acknowledge the use of the S. pneumoniae MLST database, which is located at Imperial College London and is funded by the Wellcome Trust. A. G. A. O. and G. E. P. are PhD fellows of CONICET and J. R. E. is a member of the Research Career of CONICET.
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INTRODUCTION
METHODS
