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
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Abstract |
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Keywords: Streptococcus pneumoniae, Portugal, drug-resistant clones, genetic relatedness
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Introduction |
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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.
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Materials and methods |
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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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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.
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Results and discussion |
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S. pneumoniae from cluster I were mostly resistant to penicillin plus trimethoprimsulfamethoxazole plus cefotaxime plus ceftriaxone, and S. pneumoniae from cluster II were mostly resistant to penicillin plus tetracycline plus chloramphenicol plus trimethoprimsulfamethoxazole 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 trimethoprimsulfamethoxazole plus cefotaxime plus ceftriaxone (isolate 1654), and the single clone penicillin plus tetracycline plus trimethoprimsulfamethoxazole 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.
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Acknowledgements |
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Footnotes |
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References |
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