JAC Advance Access originally published online on February 16, 2006
Journal of Antimicrobial Chemotherapy 2006 57(4):654-660; doi:10.1093/jac/dkl028
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Molecular epidemiology and variants of the multidrug-resistant Streptococcus pneumoniae Spain14-5 international clone among Spanish clinical isolates
1 Servicio de Microbiología, Hospital Donostia Paseo Dr Beguiristain s/n 20014 San Sebastián, Spain; 2 Departamento de Medicina Preventiva y Salud Pública, Universidad del País Vasco, Paseo Dr Beguiristain s/n 20014 San Sebastián, Spain; 3 Centro Nacional de Microbiología, Majadahonda, Madrid, Spain
* Corresponding author. Tel: +34-94-300-7046; Fax: +34-94-300-7063; E-mail: mikrobiol{at}terra.es
Received 23 November 2005; returned 5 January 2006; revised 17 January 2006; accepted 19 January 2006
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Abstract |
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Objective: To analyse the molecular structure of several antimicrobial resistance determinants in isolates of the Spain14-5 clone to better understand its emergence and spread.
Methods: The distinct genes and mechanism of resistance to penicillin, erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim were studied in an apparently homogeneous group of 117 isolates of the multidrug-resistant Spain14-5 major clone isolated in Spain between 1981 and 2004.
Results: Several genotyping techniques such as PFGE, BOX-PCR and multilocus sequence typing (MLST) revealed a high degree of homogeneity among these isolates over time. Nevertheless, distinct variants of the clone could be established according to the restriction fragment length polymorphism (RFLP) patterns of the penicillin-binding protein (pbp) genes and the sequences of the dihydrofolate reductase (dhfr) gene. In addition, an association between the pbp2b RFLP patterns, the ddl alleles identified by MLST and the dhfr alleles was found. The emergence of variants of the Spain14-5 clone, which had lost macrolide and tetracycline resistance, while harbouring the ins and xis genes of the Tn916–Tn1545 family of conjugative transposons, was documented. Two different tet(M) alleles were detected in isolates of the clone, one of them with a mosaic structure.
Conclusions: The finding of different patterns or alleles of the genes responsible for antibiotic resistance among isolates of the Spain14-5 clone from different Spanish cities indicates different evolutionary events within isolates of a unique Streptococcus pneumoniae clone.
Keywords: Multilocus sequence typing (MLST) , resistance genes , major clones
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Streptococcus pneumoniae with resistance to penicillin and other antibiotics are highly prevalent throughout the world. Such resistance is achieved by two different genetic mechanisms. The first involves an alteration of the genes encoding antibiotic targets, which may involve inter- or intra-specific recombinational exchanges as in the case of penicillin-binding protein (pbp) and topoisomerase genes, that lead to ß-lactam and fluoroquinolone resistance, respectively.1–4 In other cases, these gene alterations involve point mutations as is the case in the dihydrofolate reductase (dhfr) gene, genes encoding the beta-subunit of the RNA polymerase (rpoB) and the genes encoding the subunits of DNA topoisomerase IV (parC and parE) and DNA gyrase (gyrA and gyrB), which are responsible for trimethoprim, rifampicin and fluoroquinolone resistance, respectively.5–7
The second mechanism of resistance is usually due to the incorporation of new genetic material, which in S. pneumoniae is usually mediated by transposons that carry distinct genes of resistance.8 This is the case for the chloramphenicol acetyltransferase gene (cat), the tet(M) and the erythromycin methylase (erm) genes, responsible for resistance to chloramphenicol, tetracyclines and macrolides, respectively. In S. pneumoniae, cat has been associated with the 65.5 kb composite conjugative transposon Tn5253, which consists of two independent conjugative elements: Tn5251, which resembles the Tn916 class of elements and carries the tet determinant of tetracycline resistance, and Tn5252, which carries the cat gene.9,10
The erm(B) and tet(M) genes are usually carried on the same 25.3 kb conjugative transposon Tn1545 that also encodes kanamycin resistance via aphA3.8 Tn1545 belongs to a larger class of conjugative transposons, typically represented by Tn916, which encodes tet(M)-mediated resistance to tetracycline but not resistance to erythromycin or kanamycin.11 The transference is mediated by the excisionase (xis) and integrase (int) genes characteristic of the Tn916–Tn1545 family of conjugative transposons.11,12 Erythromycin resistance can also result from an efflux mechanism of resistance associated with the mef genes, which in streptococci are generally carried in the mobile elements Tn1207.1 and mega.13,14
Worldwide molecular epidemiological surveillance of antibiotic resistance in S. pneumoniae led to the description of the major multidrug-resistant clones.15 These clones were phenotypically characterized by their serotype and antimicrobial susceptibilities, and genotypically by their PFGE, BOX-PCR and multilocus sequence typing (MLST) patterns. Subsequently, some of these major clones with widespread resistance to macrolides and fluoroquinolones have been described.16,17 Other molecular typing methods of S. pneumoniae include DNA fingerprinting of the pbp1a, pbp2b, pbp2x and dhfr and of the pspA (pneumococcal surface protein) genes.18–20
We recently reported the evolution of the antibiotic resistance in one of these clones (Spain14-5) over a 22 year period.21 The availability in Gipuzkoa of an S. pneumoniae collection dating from 1981, specially focussed on non-penicillin-susceptible isolates, prompted us to carry out a molecular epidemiological study of the determinants of antibiotic resistance in isolates of the Spain14-5 clone isolated between 1981 and 2004 in Gipuzkoa and in a sample of other Spain14-5 clone pneumococci isolated in other Spanish regions between 1998 and 2003.
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Materials and methods |
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Bacterial isolates and susceptibility testing
From January 1981 to December 2004, 98 S. pneumoniae clinical isolates belonging to the major multidrug-resistant clone Spain14-5 were detected by PFGE among the 379 serotype 14 penicillin non-susceptible (penicillin MIC 
0.12 mg/L) S. pneumoniae isolated in the Microbiology Department of Hospital Donostia, Gipuzkoa, Basque Country, northern Spain. Of these, 93 were isolated between 1981 and 2002,21 and of the remaining five, four were detected in 2003 and 1 in 2004. A sample of 19 clinical isolates of the Spain14-5 major multiresistant clone isolated in other Spanish regions was also included in the study. Thirteen were isolated between 1998 and 1999 in seven different Spanish cities and the remaining six were isolated in 2003 in Madrid.
Antibiotic susceptibility testing was performed by broth microdilution according to the CLSI (former NCCLS) guidelines using the isolate S. pneumoniae ATCC 49619 as control.21 For molecular typing methods, isolates MS22 ATCC 700902, the representative isolate of the Spain14-5 major multiresistant clone and the penicillin-susceptible S. pneumoniae isolate R6 were used as controls.
Molecular typing methods
PFGE, BOX-PCR and MLST were performed as described previously.16 Restriction fragment length polymorphism (RFLP) of the genes encoding the penicillin-binding proteins Pbp1a, 2b and 2x were obtained after 2.4, 1.5 and 2.0 kb fragments of each gene, respectively, were amplified using the primers and conditions described previously.22 The unpurified amplification products of the three pbp gene fragments were digested by restriction endonucleases AluI and HinfI (pbp1a, pbp2b and pbp2x) and HaeIII and RsaI (pbp1a) and were separated by electrophoresis in 3% agarose gels.
PBP gene sequencing
A 1 kb region of the penicillin-binding domain (PBD) of pbp1a, pbp2b and pbp2x from representative isolates of the Spain14-5 clone was sequenced in both directions using primers and conditions as described previously.23
Definition of variants and sub-variants of the clone
In this study, pbp2b, pbp2x and dhfr alleles were arbitrarily named according to their different RFLP profiles or sequences found. On the basis of the different pbp2b, pbp2x and dhfr types defined, isolates of the Spain14-5 clone were divided into variants, if they had the same pbp2b and dhfr types and further into sub-variants according to the pbp2x–RFLP types found. This division into sub-variants was only used to select the isolates whose determinants of resistance were finally studied in depth.
Detection of the Tn916–Tn1545 transposons and sequence variability of the tet(M), erm(B) and cat genes
The excisionase (xis) and integrase (int) genes, which mediate transference of the Tn916–Tn1545 family of conjugative transposons were amplified using the primers and conditions described elsewhere.13,24 Amplification products from five isolates were sequenced using the same primers to confirm the specificity of the amplicons obtained and were compared with the sequence of the xis and int genes available at GenBank (accession number X61025 [GenBank] ).
Detection of the tet(M), erm(B), mef(A) and cat genes, responsible for tetracycline, erythromycin and chloramphenicol resistance, was performed using the primers and conditions described previously.24–26
On arbitrarily selected isolates representative of all the sub-variants found, erm(B) and cat genes were sequenced using the same primers employed for amplification.
With tetracycline-resistant isolates and isolates with reduced susceptibilty to tetracycline but which were formally susceptible according to CLSI criteria (MIC 2 mg/L), the forward primers 5'-TTA TCA ACG GTT TAT CAG G-3' (positions 409–427) and reverse, 5'-GAA GCC CAG AAA GGA TTC GGT-3' (positions 1311–1331) were used for sequencing an internal fragment of 923 bp of the tet(M) gene according to the S. pneumoniae tet(M) sequence available at GenBank (accession number X90939 [GenBank] ).
Analysis of dhfr gene
The sequence of the dhfr gene was studied on the single trimethoprim-susceptible isolate and on other arbitrarily selected resistant isolates that were representative of the different pbp2b and pbp2x profiles observed (Table 1) as described previously.5
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Results |
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The 117 Spanish isolates of the Spain14-5 clone studied showed decreased susceptibility or resistance to penicillin (MIC range 1–4 mg/L) and cefotaxime (MIC range 1–4 mg/L) and resistance to chloramphenicol (MIC
16 mg/L). All isolates except one were co-trimoxazole-resistant (MIC 2/38 mg/L). All isolates were tetracycline-resistant (MIC 16 mg/L) except two that showed reduced susceptibility to tetracycline (MIC 2 mg/L).
Seven isolates were erythromycin- and clindamycin-susceptible (MICs 
0.25 mg/L) and the remainder were resistant to both antibiotics (erythromycin and clindamycin MICs 128 mg/L).
Part of the antimicrobial susceptibility results of 93 of the 117 isolates included in this study has been reported previously.21
PFGE and BOX-PCR
The BOX-PCR patterns of the 117 isolates were identical to the pattern of the reference isolate of the Spain14-5 clone (isolate ATCC 700902). By PFGE, 113 isolates had an identical pattern to that of the reference isolate of the Spain14-5 clone. The homology of the PFGE patterns of the remainder four isolates with that of the reference isolate was >90%.
MLST
All 117 isolates belonged to two sequence types (ST) that differ only in the allele number of the ddl (D-alanine-D-alanine ligase) gene: 109 belonged to ST17 (ddl allelic number 47) and eight isolates, all from Gipuzkoa, belonged to ST18 (ddl allelic number 16), the same ST as the reference isolate ATCC 700902. The ddl gene was studied in all isolates of the clone by PCR–RFLP,21 while MLST was performed on the eight isolates belonging to ST18 and on 52 of the 109 isolates belonging to ST17.
RFLP patterns and sequences of the pbp genes
The 117 isolates of the Spain14-5 clone had pbp1a, pbp2b and pbp2x RFLP patterns that were different from those of the penicillin-susceptible isolate R6.
PCR–RFLP analysis of pbp1a with the four restriction enzymes AluI, HinfI, RsaI and HaeIII of 35 randomly selected isolates of the Spain14-5 clone, which included isolates of all the distinct variants (Table 1) and sub-variants, gave a single RFLP pattern for each enzyme. Therefore, the pbp1a gene was considered similar in all isolates of this clone and was not further studied in the remaining isolates.
PBD of the pbp1a gene was sequenced in eight isolates that belonged to all the distinct sub-variants of the clone found and showed a similarity >99% between them and with the sequence of the reference isolate of the Spain14-5 clone (isolate ATCC 700902). All of them had, besides a T371A and a P432T substitution, the same amino acid substitution described for other penicillin-resistant S. pneumoniae isolates.23
PCR–RFLP analysis of the pbp2b of the 117 isolates of the Spain14-5 clone with HinfI yielded four distinct patterns, arbitrarily named ‘a’ to ‘d’ (Table 1). These four patterns matched the four patterns obtained after digestion with the enzyme AluI. The eight isolates of this clone that had the MLST ST18 had a pbp2b pattern ‘b’, a pattern not seen in any of the remaining 109 isolates of this clone. Only one of these pbp2b pattern ‘b’ isolates harboured the macrolide-resistance gene erm(B).
PBD of the pbp2b gene was sequenced in 10 isolates of the Spain14-5 clone comprising isolates of the four different RFLP patterns found. Their sequences differed from that of reference isolate R6 by 69–187 nt (similarity 94–84%, respectively), which corresponded to a substitution between 13 and 42 amino acids (translated protein similarity ranging from 89 to 97%). PBD sequences of isolates with the pbp2b RFLP patterns ‘a’ and ‘d’ differed in only 11 nt and in one unique amino acid when translated. All pbp2b genes of the four RFLP patterns had the amino acid substitution T445A, a mutation frequently found in penicillin-resistant pneumococci.23
PCR–RFLP analysis of the pbp2x gene after digestion with the enzymes HinfI and AluI showed five and seven distinct RFLP patterns, which did not match with the pbp2b RFLP patterns found. The four variants defined (Table 1) could be divided into 12 different sub-variants according to the pbp2x RFLP patterns found, but this distribution into sub-variants was of limited practical value and therefore has not been detailed.
The PBD of the pbp2x was sequenced in 10 isolates of the seven different RFLP patterns found and revealed a similarity between 81.7 and 82.8% with the pbp2x gene of the penicillin-susceptible isolate R6. With independence of the pattern they belonged to, the PBD of the Ppb2b proteins deduced from the 10 isolates sequenced had two amino acid substitutions (T338A and L546V), substitutions strongly related to penicillin resistance in S. pneumoniae.23
Analysis of the dhfr gene
The deduced proteins from the dhfr sequences of the 65 randomly selected isolates, which included isolates of all the distinct sub-variants, gave four different types, arbitrarily named ‘a’, ‘b’, ‘c’ and ‘c1’. Types ‘c’ and ‘c1' differed in only one amino acid (Table 2). Considering ‘c' and ‘c1'as a single type, an association between the pbp2b profiles and the dhfr type (a-a, b-b, c-c, and d-c, respectively) was found (Table 1).
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Other resistance genes and mobile elements
The int and xis genes of the Tn916–Tn1545 family of transposons were detected in all isolates, including those that were susceptible to erythromycin (seven isolates) and/or tetracycline (two isolates). All isolates were resistant to chloramphenicol and had the cat gene. The 110 erythromycin-resistant isolates showed the MLSB phenotype of resistance and had the erm(B) gene; only one erythromycin-resistant isolate harboured both the erm(B) gene and the mef(A) [subtype mef(E)] genes. None of the seven erythromycin-susceptible isolates had the erm(B) gene. No differences were found in any of the sequences of the erm(B) and cat genes studied from 47 randomly selected isolates, which included isolates of all the different sub-variants.
Analysis of the tet(M) gene
The fragments of nearly 300 amino acids deduced from the tet(M) sequences of the same 65 isolates studied for the dhfr gene gave six different TetM types arbitrarily named ‘a' and ‘b1’ to ‘b5’. Types ‘b1’ to ‘b5’ only differed in one to two amino acids between them and with the S. pneumoniae TetM protein sequence described at GenBank (accession number X90939 [GenBank] ) and were considered as the single type ‘b’ (Figure 1). The initial part of the sequence of both TetM protein types, ‘a’ and ‘b’ between amino acids 161 and 194, was very similar to the amino acid sequence of TetM carried by Tn916. The final part of the protein sequence of both types, between amino acids 246 and 400, was very different coinciding in type ‘a’ sequence with that of Tn916, while type ‘b’ sequence matched with the sequence of TetM carried by Tn1545. All erythromycin-susceptible isolates [erm(B) negative] had the TetM protein of type ‘a’.
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The tet(M) gene of the two tetracycline-susceptible isolates (tetracycline MIC = 2 mg/L) was entirely sequenced, and no mutations, insertions or deletions were found in their sequences.
Analysis of the variants found
According to the pbp2b gene profile and dhfr alleles found, four distinct variants were established and arbitrarily named ‘A’ to ‘D’ (Table 1). Variant ‘C’ (103 of 117 isolates, 88.0%) was the most frequently found, was first detected in 1987 and was also found in isolates from the seven different Spanish cities. Contrarily, variant ‘D’ was only detected in Gipuzkoa between 1990 and 1999.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, the 117 isolates analysed formed a highly homogenous population of S. pneumoniae within the Spain14-5 clone according to three different genotyping methods (PFGE, MLST and BOX-PCR). Nevertheless, four main variants of the clone could be established according to the RFLP patterns and sequences of the penicillin-binding proteins pbp2b and the sequences of the dhfr.
In S. pneumoniae, high-level penicillin resistance is achieved by inter- or intra-specific recombinational exchanges leading to altered forms of PBPs1,2,19,27 and, therefore, distinct patterns of the pbp1a, pbp2b and pbp2x genes could be established if different recombinational events had taken place among isolates of a determinate clone. The study of pbp2b in isolates of the Spain14-5 clone revealed four distinct profiles, which did not always match with the seven different pbp2x profiles found. Analysis of pbp2x gave the highest number of distinct patterns of the three Pbp genes studied, as found by other authors studying more heterogeneous populations of S. pneumoniae.22,28,29 This finding in isolates of the same clone confirms that the structure of pbp2x may be more predisposed to frequent recombination or point mutation events, conferring it with a highly variable nucleotide sequence as compared with either of the other Pbp genes.28 Indeed, analysis of pbp1a with the four distinct enzymes and sequencing revealed the full homogeneity of the gene encoding this protein among isolates of this clone. Sequences of the pbp1a, pbp2b, and pbp2x of all penicillin non-susceptible and resistant isolates studied revealed point amino acid substitutions associated with penicillin resistance in S. pneumoniae.23
An association between the pbp profiles and dhfr and ddl alleles within isolates of the Spain14-5 clone isolated in Spain was found in this study. The association between pbp1a–pbp2b–pbp2x restriction profiles and dhfr has been described previously in S. pneumoniae isolates from different genetic backgrounds.22 The association between pbp2b and ddl is less surprising, as other authors have found a hitchhiking effect of the ddl in transformation of pbp2b.30 The different pbp2b patterns and dhfr and ddl alleles found allowed the division of isolates of the Spain14-5 clone into four distinct variants, suggesting distinct evolutionary events within isolates of the same clone, each one having a different capacity of spreading.
The finding of variant ‘C’ isolates from 1987 to 2004 in all the cities where the clone was detected implies that this variant or subclone has a greater ability to spread than the other variants found.
Independent of the dhfr allele found, all trimethoprim-resistant isolates had the same single amino acid substitution (I100L) in dihydrofolate reductase as that reported by other authors.5
In S. pneumoniae, tet(M) is usually carried by the conjugative transposon Tn916 or together with the erm(B) and the aphaA3 determinants of macrolide and kanamycin resistance, respectively, in the conjugative transposon Tn1545.8 It can also be found in composite structures like Tn5253 and Tn3872 that carry other resistance determinants.10,31 erm(B) can also be carried alone by Tn917.32 The presence of the int and xis elements of the Tn916–Tn1545 family was detected in the 117 Spanish isolates of the Spain14-5 clone, including the seven erythromycin-susceptible isolates in which the erm(B) gene was not present. In these seven erm(B)-negative isolates, the high similarity of the tet(M) sequence with that of Tn916 points to Tn916 as the genetic element carrying the determinant of tetracycline resistance. The only isolate of variant ‘A’ in which the erm(B) gene was detected (isolate C-71381) as well as the only isolate of variant ‘B’ (C-12950) found had a tet(M) sequence similar to that of Tn916, suggesting that tet(M) was carried by Tn916. To know whether in these two isolates the erm(B) gene was carried by a separate transposon such as Tn917 is under investigation.
In the 110 erythromycin-resistant isolates of the Spain14-5 clone studied both erm(B) and tet(M) were detected.
The tet(M) sequence of the rest of the isolates of the Spain14-5 clone studied, named in this study as tet(M) type ‘b’, had a mosaic-like structure with an initial DNA block similar to the tet(M) sequence of Tn916 and a final DNA block similar to the tet(M) sequence of Tn1545. This fact suggests that the origin of this tet(M) allele has probably arisen in an isolate of the Spain14-5 clone carrying Tn916 following transformation and recombination with a tet(M) gene from an isolate in which tet(M) was carried by Tn1545. Recombinational events in other genes of these isolates, such as rpoB, have been well documented.33
In isolates of variants C and D, it seems likely that erm(B) and tet(M) were carried in a Tn1545-like element, which has been shown to spread clonally.34
Two isolates carrying the Tn1545-like element became phenotypically tetracycline-susceptible (MIC = 2 mg/L) without losing the tet(M) gene. A deletion of 10 amino acids in the deduced protein from the tet(M) in tetracycline-susceptible tet(M)-positive isolates (positions 619–628) has previously been found, but the sequences of the tet(M) gene of the two tetracycline-susceptible isolates found in our study showed no deletions as compared with the tet(M) sequence of tetracycline-resistant isolates.13 Other authors have found the same loss of tetracycline resistance without loss of the genes responsible for that resistance.35
Analysis of the variants detected in Spanish isolates indicated the presence of at least four different evolutionary lineages within the Spain14-5 clone. Two lineages comprise isolates of variant ‘A’ and ‘D’; these isolates were detected only in Gipuzkoa and had little ability to spread compared with the more successful variant, variant C. All isolates from outside Gipuzkoa belonged to this variant ‘C’.
In conclusion, in Spanish isolates of the Spain14-5 clone, both clonal spreading and individual changes probably due to recombinational events and/or point mutations were responsible for the evolution of antibiotic resistance. The different evolutionary lineages that emerged over time within isolates of the Spain14-5 clone differed in their ability to spread and showed different patterns in their determinants of antibiotic resistance.
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Acknowledgements |
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This work was supported in part by a grant from the Basque Country University UPV/EHU, Spain, GIU05/54. Spanish Pneumococcal Infection Study Network (G03/103). General Coordination: Román Pallarés (rpallares{at}ub.edu). Participants and Centres: Ernesto García (Consejo de Investigaciones Biológicas, Madrid); Julio Casal, Asunción Fenoll, Adela G. de la Campa (Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid); Emilio Bouza (Hospital Gregorio Marañón, Madrid); Fernando Baquero, Rafael Cantón (Hospital Ramón y Cajal, Madrid); Francisco Soriano, José Prieto (Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid); Román Pallares, Josefina Liñares (Hospital Universitari de Bellvitge, Barcelona); Javier Garau, Javier Martínez Lacasa (Hospital Mutua de Terrassa, Barcelona); Cristina Latorre, Carmen Muñoz-Almagro (Hospital Sant Joan de Déu, Barcelona); Emilio Pérez-Trallero (Hospital Donostia, San Sebastián); Juan García de Lomas (Hospital Clínico, Valencia); Ana Fleites (Hospital Central de Asturias).
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