Two major Spanish clones of penicillin-resistant Streptococcus pneumoniae in Portuguese isolates of clinical origin

Manuela Caniça*, Ricardo Dias, M. Vitória Vaz-Pato and Catarina Carvalho

Antibiotic Resistance Unit, Centre of Bacteriology, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal

Received 31 July 2002; returned 18 September 2002; revised 25 September 2002; accepted 7 November 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
We studied the genetic relatedness of 47 Portuguese penicillin-resistant 9V and 23F Streptococcus pneumoniae of clinical origin, using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism analysis. PFGE fingerprinting showed that 24 isolates of serotype 9V and 23 isolates of serotype 23F were variants of the Spain9V-3 and Spain23F-1 clones, respectively. Fingerprinting of pbp1a, pbp2b and pbp2x genes showed that all the penicillin-resistant clones gave similar HinfI restriction patterns. In this study, serotypes 9V and 23F have different clonal origins and identical PBP genotypes, suggesting a horizontal transfer of resistance. Visual and computer-assisted analysis of PFGE patterns correlated well (r = 0.983).

Keywords: Streptococcus pneumoniae, Portugal, drug-resistant clones, genetic relatedness


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The resistance to penicillin as well as antibiotic multiresistance of Streptococcus pneumoniae is now widespread and is increasing all over the world. The resistance of S. pneumoniae to penicillin is due to altered penicillin-binding proteins (PBPs) encoded by mosaic pbp genes. Resistant isolates carry allelic variants, which differ greatly from pbp genes in penicillin-susceptible strains, which are acquired by horizontal gene transfer from related species.1

Penicillin resistance among S. pneumoniae has also rapidly emerged in Portugal during the last decade to become a major public health problem.2,3 The proportion of clinical isolates that were penicillin resistant (MIC >= 0.1 mg/L) was 22.4% in 1995, 23.9% in 1996 and 25.8% in 1997.3 Multiresistance is also increasing among penicillin-resistant strains: 38.8% in 1995 to 51.6% in 1997.3 Many of these clinical strains are serotypes 23F and 9V.2,3

We used PFGE and PBP genotyping to study the genetic relatedness of penicillin-resistant isolates of S. pneumoniae serotypes 9V and 23F of clinical isolates. Visual comparison and computer-assisted analysis of PFGE fingerprints were also used.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Clinical isolates

We studied 47 penicillin-resistant pneumococci with reduced susceptibility to at least one other structurally unrelated antibiotic, randomly selected from serotype 9V and 23F strains in the Antibiotic Resistance Unit collection at the National Institute of Health in Lisbon. One non-typeable (NT) penicillin-multiresistant strain and five susceptible strains, from the same collection, were also included. The isolates were obtained from Portuguese clinical specimens, from 10 hospitals (Table 1). In total, 45 strains were isolated from the respiratory tract (23 from sputum, 12 from the upper respiratory tract and 10 from the lower respiratory tract), four from middle ear, two from cerebrospinal fluid and two had unknown origin.


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Table 1.  Properties of penicillin-resistant and -susceptible S. pneumoniae of serotypes 9V and 23F, and one NT isolate
 
As previously reported,2 serotyping was carried out by latex agglutination with type-specific antiserum (Statens-Serum Institut, Copenhagen, Denmark). MICs of penicillin (Wyeth Lederle Portugal-Farma, Algés, Portugal), ceftriaxone (Roche Farmacêutica Química, Amadora, Portugal), cefotaxime, chloramphenicol (Hoechst Marion Roussel, Mem Martins, Portugal), tetracycline (Laboratórios Atral, Castanheira do Ribatejo, Portugal), ofloxacin (Farmoz, Prior Velho, Portugal), erythromycin (Abbot Laboratórios, Amadora, Portugal) and clindamycin (Upjohn Farmoquímica, Lisbon, Portugal) were determined by the agar dilution method and susceptibility to trimethoprim–sulfamethoxazole (Oxoid, Hampshire, UK) by disc test.

Analysis of pbp genes

PBP genotyping was carried out with primers to amplify the genes pbp1a, pbp2b and pbp2x by PCR as previously described.4 Amplified DNA was digested with HinfI (Boehringer Mannheim, Carnaxide, Portugal) and analysed for restriction fragment length polymorphism (RFLP). A number was given to each restriction pattern for each of the three pbp genes analysed, so the PBP genotype has three numbers (example: 1-1-1).

Pulsed-field gel electrophoresis

PFGE was carried out as described previously.5 SmaI-restricted DNA from the penicillin-susceptible unencapsulated strain R6, and from the major Spanish multiresistant serotype 23F clone, Clev1 (both kindly donated by Professor A. Tomasz, The Rockefeller University, New York, NY, USA), and the Lambda ladder (Biolabs, Beverly, MA, USA) were used as markers for intra-gel normalization and inter-gel comparison. Gels were stained with ethidium bromide and photographed under UV light with a Polaroid system. Photographs were scanned, normalized and saved as TIFF files for computer analysis.

Data analysis

PFGE DNA fingerprints were analysed by visual comparison (method A).6 Major patterns (capital letters) differed by more than three fragments, and one to three fragment variations represent subtypes (capital letters with numbers). PFGE patterns were also subjected to computer-assisted analysis (method B) using Bionumerics software (version 2.5) (Applied Maths, Kortrijk, Belgium). Clustering was carried out using the Dice band-based similarity coefficient (SD), with a band position tolerance of 2% and an optimization of 1%. A dendrogram was constructed by the unweighted pair-group method using average linkages (UPGMA), and a cut-off value of 80% similarity was determined by the cluster cut-off method according to the Bionumerics program (Applied Maths). Isolates with an SD value >80% were considered to belong to the same cluster.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
PFGE fingerprint analysis showed that serotypes 9V and 23F do not group together but show similarities (Table 1; Figure 1). Computer-assisted analysis distinguished subtypes I-1 to I-6 and II-1 to II-6. Visual comparison showed two major patterns (G and A), with subtypes G1, G2, A1, A3, A5, A8/9, A12, A14, A16 and A17. Penicillin-resistant 9V isolates from subtypes I-1 to I-3 were closely related and belonged to serotype variants of the Spain9V-3 clone.7 Penicillin-resistant 9V isolates from subtypes I-4 to I-6 were possibly related, forming cluster I together with subtypes I-1 to I-3. Penicillin-resistant 23F isolates were all closely related and belonged to serotype variants of the Spain23F-1 clone,7 forming cluster II, but were genetically unrelated to susceptible 23F isolates, which formed clusters III, IV and VI. Clusters I and II were 72% related with a cophenetic correlation of 93% (Figure 1). Computer-assisted analysis gave more discriminating subtype patterns than visual comparison, for G2, M and A1. However, subtype II-6 was closely related to subtype II-5, as demonstrated by the A14 pattern obtained by visual comparison. Visual comparison gave more discriminating subtype patterns for II-1, II-3, II-4 and II-5 (Table 1). The correlation between visual comparison and computer-assisted analysis of PFGE DNA fingerprints was good (R = 0.983).



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Figure 1. Dendrogram (genetic relatedness) and PFGE fingerprint patterns of 48 resistant and five susceptible pneumococcal isolates. Strain code number, capsular serotype and MIC of penicillin are given (on the right). Clusters I and II, containing isolates of >80% similarity, are indicated by vertical bands. Codes I-1 to I-6 and II-1 to II-6 refer to PFGE patterns of S. pneumoniae showing up to 98% homology. Cophenetic correlations are shown in each branch of the dendrogram.

 
Resistance to penicillin can emerge independently in closely related clones. We analysed genetic polymorphism of the penicillin resistance genes pbp1a, pp2b and pbp2x: all the penicillin-resistant clones of serotype 23F and 9V gave similar HinfI restriction patterns for the three pbp genes (PBP genotype 1-1-1) (Table 1). The penicillin-susceptible clones, in clusters III, IV, V and VI, had the PBP genotype 2-2-2 (Table 1). All isolates (except NT strain 1258) in each cluster had the same PBP genotype and the same serotype, suggesting that penicillin resistance in different lineages of S. pneumoniae may result from horizontal transfer of pbp genes, as previously reported by Coffey et al.8

S. pneumoniae from cluster I were mostly resistant to penicillin plus trimethoprim–sulfamethoxazole plus cefotaxime plus ceftriaxone, and S. pneumoniae from cluster II were mostly resistant to penicillin plus tetracycline plus chloramphenicol plus trimethoprim–sulfamethoxazole plus cefotaxime plus or minus ceftriaxone. Cluster II-4 contained a greater diversity of phenotypes than the other clusters/subtypes, possibly a consequence of the presence of insertion sequences specific to the addition of resistance genes, leading to mosaic genes.9 Interestingly, both the single clone with phenotype penicillin plus erythromycin plus clindamycin plus chloramphenicol plus trimethoprim–sulfamethoxazole plus cefotaxime plus ceftriaxone (isolate 1654), and the single clone penicillin plus tetracycline plus trimethoprim–sulfamethoxazole plus cefotaxime plus ceftriaxone (isolate 1258), were in cluster II-4. Isolate 1258, despite having an NT capsular polysaccharide, is closely related to serotype 23F. This isolate appears to be an NT variant of the Spain23F-1 clone, which may have lost the ability to produce capsule in vivo. The loss of serotype reactivity by strain 1258 may have resulted from a recombination event, involving the horizontal transfer of capsular biosynthesis genes, as frequently occurs in vivo.8,10 As a variant of the Spain23F-1 clone, it may have lost chloramphenicol resistance but acquired cefotaxime (MIC 2 mg/L) and ceftriaxone (MIC 1 mg/L) resistance. The subcluster II-4 is the most divergent between the two analysis methods, method A classifying it as three subtypes A1, A8 and A12.

We concluded that computer-assisted analysis is easier and less time consuming than visual analysis, but the methods are complementary.

In this study, Portuguese serotype 9V- and 23F-resistant S. pneumoniae showed different genetic patterns by PFGE but identical PBP genotypes, similar to those of the major Spanish clones Spain9V-3 and Spain23F-1,7 except for five 9V isolates. These resistant serotypes appear to have a common clonal origin of resistance despite being isolated in different hospitals throughout the country. These findings, reported in Portuguese clinical S. pneumoniae isolates, suggest spread of resistance by clonal dissemination and horizontal transfer. The increasing movements of populations, which contribute to the emergence in antimicrobial resistance worldwide, emphasize the need for vaccination to interrupt the transmission of resistant clones. Further studies are needed to examine the contributions of the Portuguese clinical isolates from these and other serotypes to the emergence of penicillin-resistant and multidrug-resistant S. pneumoniae.


    Acknowledgements
 
This paper is in memory of Catarina Carvalho, who made an important contribution to the understanding of pneumococcal resistance mechanisms in Portugal. We would like to thank B. G. Spratt (St Mary’s Hospital, London, UK) for critical review of the manuscript. We thank R. Tenreiro (Faculdade de Ciências, Lisbon, Portugal) for making the Bionumerics program available for computer analysis, and both A. Fenoll (Instituto Carlos III, Madrid, Spain) and H. Konradsen (Statens-Serum Institut, Copenhagen, Denmark) for the confirmation of strain 1258 serotype. This work was supported by Comissão de Fomento da Investigação em Cuidados de Saúde grant 237/99 from Ministério da Saúde, Portugal. Part of this work was presented at the Fifth International Meeting on Bacterial Epidemiology Markers, Noordwijkerhout, The Netherlands, 2000.


    Footnotes
 
* Corresponding author. Tel/Fax: +351-21-751-9246; E-mail: manuela.canica{at}insa.min-saude.pt Back


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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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