JAC Advance Access originally published online on March 13, 2006
Journal of Antimicrobial Chemotherapy 2006 57(5):983-986; doi:10.1093/jac/dkl083
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Combinations of PBPs and MurM protein variants in early and contemporary high-level penicillin-resistant Streptococcus pneumoniae isolates in Spain
1 Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Ctra. Colmenar Km 9.1, Madrid 28034, Spain; 2 Departamento de Microbiología, Facultad de Medicina, Universidad Complutense, Avda Complutense s/n, Madrid 28040, Spain; 3 Departamento de Microbiología, Instituto Nacional de Salud Carlos III, Ctra Majadahonda-Pozuelo Km 2, Majadahonda 28220, Spain; 4 Servicio de Microbiología, Hospital Universitario de Bellvitge, Feixa Llarga s/n L'Hospitalet de Llobregat, Barcelona 08097, Spain
* Corresponding author. Tel: +34-91-3368542; Fax: +34-91-3368809; E-mail: rosacampo{at}yahoo.com
Received 29 December 2005; returned 12 January 2006; revised 10 February 2006; accepted 22 February 2006
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Abstract |
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Objectives: High-level penicillin resistance in Streptococcus pneumoniae requires extensive re-modulation of the penicillin-binding proteins (PBPs), and murM gene function is also required for the expression of resistance. In this work, we determined whether specific changes in PBPs were associated with specific MurM variants.
Methods: Two collections of highly penicillin-resistant (MIC 2–8 mg/L) isolates, including 10 early (1997–1998) and 23 contemporary (2002–2004) isolates, were studied.
Results: Most of the isolates belonged to clones Spain6B-2 (13 strains), Spain23F-1 (10 isolates) and Spain14-5 (20 isolates). Different protein variants of MurM (MA, MB5, MB6, MB9 and MB10), PBP1A (A–C), PBP2B (A–D) and PBP2X (A–C) were recognized, including two murM alleles not previously described. Particular [MurM-PBP1A-2B-2X] allelic combinations were predominant among the different clones, including [MA-B-B-B] for old (MIC 2 mg/L) and [MB10-C-A-B] for recent (MIC 4–8 mg/L) Spain6B-2 isolates, [MA-A-C-A] for Spain23F-1 and [MB5-A-A-A] in Spain14-5 isolates.
Conclusions: Although S. pneumoniae has a basic recombinational population structure, our results indicate remarkable conservation of PBPs and MurM protein types within each clone. This suggests that particular PBPs–MurM combinations tend to be preserved and may have an independent evolutionary history in particular clones.
Keywords: resistance , clones , mutations , penicillin-binding proteins
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Introduction |
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High-level penicillin resistance constitutes a major threat to the therapy of pneumococcal infections; in Spain, 8.4% of the current invasive isolates are highly resistant to penicillin (MIC
2 mg/L).1 The acquisition of amino acid alterations in penicillin-binding protein (PBP) sequences constitutes the classical mechanism of penicillin resistance in Streptococcus pneumoniae, which is concomitantly reflected in changes in the cell wall peptidoglycan structure. The penicillin resistance phenotype depends on particular amino acid changes in PBP2X and PBP2B; further changes in PBP1A are required to reach high-level penicillin resistance.2,3 The operon murMN encodes enzymes involved in the synthesis of branched structured muropeptide components in pneumococcal peptidoglycan.4 The preservation of a functional murMN operon seems to be critical for the expression of penicillin resistance, because inactivation of the murM gene completely restores penicillin susceptibility.5,6 Eight different protein variants (MurMB1-8) were noted in penicillin-resistant strains, resulting from considerable polymorphisms in their murM genes.4 This suggests that low-affinity PBPs that evolved to high-level penicillin resistance might eventually recover full efficiency in cell wall construction when particular MurM protein variants evolve or are acquired by the cell.7 The aim of this work was to study whether PBPs and MurM protein variant combinations changed in time from early to recent high-level penicillin-resistant S. pneumoniae isolates, and how specific these combinations are for particular clones.
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Materials and methods |
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We studied two different collections of S. pneumoniae isolates showing high-level penicillin resistance (MIC
2 mg/L), including the first Spanish isolates from 1987–1988 (n = 10), and also the most recent isolates from 2001–2004 (n = 23). Characteristics of the isolates are listed in Table 1. Genetic variability was studied by PFGE, and relatedness was analysed visually and interpreted using PhoretixTM 5.0 software (Nonlinear Dynamics Ltd, UK). Total DNA was extracted using the QIAamp® DNA kit (Qiagen, Germany). The primers and conditions used for the amplification of pbp1A,7 pbp2X,8 pbp2B9 and murM4 genes were as previously published. The nucleotide sequence of each PCR fragment was determined using the ABI PRISm BigDye Terminator Cycle Sequencing Ready Reaction kit and analysed with an ABI PRISM 377 automated sequencer (PE Applied Biosystems, Foster City, CA, USA). Novel murM sequences were deposited in the EMBL database under accession numbers DQ067557
[GenBank]
(murMB9) and DQ100160
[GenBank]
(murMB10).
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Results |
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Serotypes and clones of the studied isolates are shown in Table 1. Three major clones were detected among both early and contemporary high-level penicillin-resistant S. pneumoniae isolates: Spain23F-1 (40 and 13%, respectively), Spain14-5 (0 and 47.8%) and Spain6B-2 (50 and 34.7%). The highest penicillin MIC value detected was 8 mg/L (5 isolates), with 4 mg/L the modal MIC (18 isolates).
The murM gene was entirely sequenced from 33 isolates, yielding five different DNA alleles. The deduced amino acid sequences represented three previously published MurM variants: MurMA (42.4%), MurMB5 (21.2%) and MurMB6 (3%). We also found two variants, named MurMB9 (3%) and MurMB10 (30.3%) (Table 1).
In the case of the pbp1A gene, five different DNA alleles encoded three different protein variants (A–C) with a divergence of 95–87% compared with the allele of strain R6. Identical alleles have been published under the numbers AF467822 [GenBank] (allele A), AF467821 [GenBank] (allele B) and AF387163 [GenBank] (allele C).
Sequence analysis of the pbp2B gene yielded four different alleles, equivalent to GenBank AJ243054 [GenBank] (allele A), DQ056793 [GenBank] (allele B), AY963281 [GenBank] (allele C) and AJ842018 [GenBank] (allele D). The divergence among the different DNA alleles compared with R6 sequences ranged from 92% for allele A to 82% for allele D; these encoded four different protein variants (A–D), showing a divergence from 96 to 89%.
In the case of the pbp2X gene, four different alleles were found presenting identity values from 81 to 84% with the previously described sequence of strain R6. All four alleles were included in the EMBL database under the numbers X65136 [GenBank] , AJ560762 [GenBank] , AJ238587 [GenBank] and X78215 [GenBank] .
Particular [MurM-PBP1A-PBP2B-PBP2X] protein combinations were more frequently represented in specific serotypes of highly resistant S. pneumoniae isolates (Table 1). In isolates belonging to the clone Spain6B-2, which were recovered from 1987 to 1988, the predominant combination was [MA-B-B-B], which was present in 80% of the isolates. Nevertheless, among recent Spain6B-2 isolates, the predominant combination was [MB10-C-A-B], which was found in 75% of the isolates. Interestingly, this change in combination correlated with increases in the penicillin MICs for isolates (2 mg/L in early isolates and 4–8 mg/L in recent isolates). In the Spain23F-1 clone, the most frequent combination was [MA-A-C-A], which was detected in 71% of the total sequenced strains. Finally, in the Spain14-5 clone, different combinations were observed, with [MB5-A-A-A] being the most frequently represented.
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Discussion |
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Modifications in PBPs 2X, 2B and 1A have been classically associated with high-level penicillin resistance in S. pneumoniae. Our study confirmed previous findings about PBP changes involved in penicillin resistance. Other consistent amino acid changes, some of them not previously described, have been found in the sequenced PBP fragments, whose involvement in high-level resistance remains uncertain. It is important to remember that among different clones the nucleotide sequence corresponding to every particular PBP or MurM protein remains extremely conserved, supporting the hypothesis of a possible common origin.
MurM protein changes are probably more related to the maintenance of PBP changes (e.g. by possibly lowering biological cost) than directly with the levels of penicillin resistance.10 In a previous study we found that isolates expressing high-level resistance to amoxicillin showed extensive alterations in PBP sequences and yet carried the same MurMA proteins as susceptible isolates.11 Nevertheless, in the present work, we were able to detect changes in MurM-PBP combinations between early and recent Spain6B-2 isolates; the change correlated with increases in penicillin MICs (2 to 4–8 mg/L).
The most significant result obtained in this work was that particular clones tend to collect particular combinations of PBP and MurM protein variants (Table 1). The [MurM-PBP1A-2B-2X] combinations predominant in old and recent isolates of clones Spain6B-2, Spain23F-1 and Spain14V-5 were [MA-B-B-B] and [MB10-C-A-B], [MA-A-C-A], and [MB5-A-A-A], respectively, which suggests independent evolution of highly penicillin-resistant clones. S. pneumoniae has a basic recombinational population structure. As expected, a number of isolates within each clone share some PBP or MurM variants, illustrating the possibility of inter-clonal horizontal gene transfer. This might also be due to independent access to identical sequences from other streptococcal species. Nevertheless, our results indicate that inside each highly penicillin-resistant clone there was a remarkable conservation of DNA sequence for its predominant PBPs and MurM protein types. This observation suggests that particular MurM-PBP combinations tend to be preserved and may have a separate evolutionary history in particular clones.
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None to declare.
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Acknowledgements |
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R. C. is a recipient of a contract from the Spanish Pneumococcal Infection Study Network (G03/103) from the Spanish Health Ministry. This work was partially supported by research grants from the Spanish Pneumococcal Infection Study Network (G03/103) and the Microbial Sciences Foundation. General coordination for the Spanish Pneumococcal Infection Study Network (G03/103) was provided by Román Pallarés. The following Spanish Pneumococcal Infection Study Network (G03/103) members and centres participated in this study: Ernesto García, Centro de Investigaciones Biológicas, Madrid; Julio Casal, Asunción Fenoll and Adela G. de la Campa, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid; Emilio Bouza, Hospital Gregorio Marañón, Madrid; F. B., Hospital Ramón y Cajal, Madrid; Francisco Soriano and J. P., Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid; Román Pallarés and J. L., Hospital Universitario de Bellvitge, Barcelona; Javier Garau and Javier Martínez Lacasa, Hospital Mutua de Terrassa, Barcelona; Cristina Latorre, Hospital Sant Joan de Déu, Barcelona; Emilio Pérez-Trallero, Hospital Donostia, San Sebastián; Juan García de Lomas, Hospital Clínico, Valencia; and Ana Fleites, Hospital Central de Asturias.
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References |
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