1 Microbiology Service, Hospital Donostia, Paseo Dr. Beguiristain s/n, 20014 San Sebastián (Gipuzkoa); 2 Department of Preventive Medicine and Public Health, Faculty of Medicine, University of the Basque Country, San Sebastián, Spain
Received 4 November 2003; returned 23 November 2003; revised 10 December 2003; accepted 11 December 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: From 1981 to 2002, the presence of the multidrug-resistant Spain14-5 clone was studied among the 4201 S. pneumoniae isolated in Gipuzkoa (northern Spain).
Results: Overall, 93 isolates belonging to the Spain14-5 clone were identified. The first isolate of this clone was detected in 1981 and was already resistant to ß-lactams, erythromycin, clindamycin and chloramphenicol. The reference strain from the international collection for this clone was susceptible to macrolides and lincosamides whereas most of the isolates studied, including the first isolate detected in 1981, were resistant to macrolides and had the erm(B) gene encoding macrolide resistance.
Conclusions: The clone was genetically stable through time, was multiresistant since its inception and has recently become highly resistant to fluoroquinolones. The characteristic antibiotic resistance pattern of this clone should include erythromycin resistance.
Keywords: multiresistance, PFGE, MLST, fluoroquinolone resistance, macrolide resistance
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Resistant pneumococci probably emerged in Spain earlier than in other parts of the world, with penicillin-resistant strains appearing in the late 1970s and early 1980s.7,8 Multidrug-resistant pneumococci appeared in Gipuzkoa at about the same time.9
Reports of multidrug-resistant pneumococci have increased in the last few years.1013 Although much is known about the prevalence of antibiotic resistance, the mechanisms and determinants of these resistances, and the international spread of the major antibiotic-resistant clones, few studies have analysed the evolution of these clones over long periods. In this study we describe the evolution of one of the major multidrug-resistant international clonesthe Spain14-5 cloneover more than 20 years in a region of the north of Spain. At the same time, the phenotypic and genotypic characterization of this international clone initially described by Coffey et al.14 in 1996 is completed.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
From January 1981 to December 2002, all clinical isolates of S. pneumoniae in Gipuzkoa, Basque Country, northern Spain, were collected and stored at 80°C. All were identified to the species level by their colony morphology, optochin sensitivity and/or bile solubility tests. Isolates were also serogrouped or serotyped by the Quellung reaction (Quellung antisera, Staten Seruminstitut, Copenhagen, Denmark).
Susceptibility testing
Susceptibility to antibiotics was initially undertaken using the disc diffusion method on MuellerHinton agar or Iso-Sensitest agar supplemented with 5% horse or sheep blood following the recommendations of the NCCLS in force at the time.15 The antibiotic MICs for all invasive isolates (blood, CSF, pleural fluid, etc.) and all penicillin non-susceptible isolates (1 µg oxacillin disc inhibition zone < 20 mm) isolated between 1981 and 1997, as well as the antibiotic MICs for all S. pneumoniae isolated between January 1998 and December 2002, were determined by the broth microdilution method using microtitre trays (Sensititre; Trek Diagnostics Systems, West Sussex, UK) and MuellerHinton broth supplemented with 3% lysed horse blood and interpreted according to the NCCLS criteria.16 S. pneumoniae ATCC 49619 was used as control for the disc diffusion and broth microdilution methods.
Molecular typing methods
For the molecular typing methods, the previously characterized6 isolate of the Spain14-5 international multiresistant clone, strain MS22 ATCC 700902, was used as a control.
PFGE, multilocus sequence typing (MLST) and BOX-PCR were carried out as previously described.17 The PFGE patterns were analysed with the Diversity Database fingerprinting software version 2 (Bio-Rad, Hercules, CA, USA) and a dendrogram was constructed by the unweighted pair group method with arithmetic averages, the Dice coefficient and a position tolerance of 1%. Isolates with a genetic relatedness of >85% were considered to represent the same PFGE type.
Materials and methods common for all protocols using PCR
Bacterial DNA was extracted after lysozyme and proteinase K incubations using Qiagen columns (Valencia, CA, USA), according to the manufacturers instructions. All the primers were synthesized by Amersham Pharmacia (Buckinghamshire, UK). PCR products were automatically sequenced with the ABI 300 Genetic Analyzer (Perkin-Elmer Applied Biosystems, Foster City, CA, USA).
Mechanisms of antibiotic resistance
The mechanisms of macrolide, rifampicin and fluoroquinolone resistance were investigated as previously described.18 The cat (chloramphenicol acetyltransferase) gene and the tet(M) and tet(O) genes were detected as previously described.1921 Resistance to trimethoprim was investigated in all susceptible isolates and in a sample of resistant isolates after sequencing of the dihydrofolate reductase (dhfr) gene.22
PCR-restriction fragment length polymorphism (RFLP) assay for differentiation of alleles 16 and 47 of the ddl gene
To differentiate between alleles 16 and 47 of the ddl (D-Ala-D-Ala ligase) gene, 10 µL of the unpurified amplicon of 513 bp obtained after PCR using the primers and conditions described by Enright & Spratt23 was digested for 1 h at 37°C with 10 U each of AccI and AluI. As expected, after analysing the sequences described in the GenBank, isolates displaying allele 16 (GenBank accession no. AJ232256) were not digested with AccI, and gave four fragments of 216, 161, 107 and 29 bp after digestion with AluI. In contrast, isolates displaying allele 47 (GenBank accession no. AJ387980) gave two fragments of 275 and 238 bp after digestion with AccI, and gave two fragments of 484 and 29 bp after digestion with AluI.
Analysis of the pbp2b gene
Genetic polymorphism of the penicillin-binding protein 2b (pbp2b) gene was studied by a PCR-RFLP analysis using the primers and PCR conditions previously described.24 Amplification products were digested with the restriction endonuclease HinfI and separated by electrophoresis in 2.5% agarose gels stained with ethidium bromide.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All serotype 14 isolates were studied using PFGE and 93/294 (31.6%) showed a pattern with a similarity >85% with the PFGE pattern of the S. pneumoniae reference strain of the international multiresistant Spain14-5 clone (strain MS22, ATCC 700902). Eighty-nine of these 93 isolates showed exactly the same PFGE pattern (100% similarity) and only four strains showed a pattern with less than three bands difference with the reference strain (similarity >85%). These four isolates were further characterized by MLST and gave the same ST17 as most of the isolates of this clone (see below).
These 93 isolates were from 72 patients (11 patients had multiple isolates within an interval that ranged from 8 days to more than 1.5 years). They were isolated mainly from specimens of the respiratory tract (63 isolates) and from blood (17 isolates). The remainder were isolated from ear secretions (10 isolates), eye secretions (two isolates) and CSF (one isolate).
The first multidrug-resistant S. pneumoniae belonging to the Spain14-5 clone was isolated in Gipuzkoa in April 1981 from the throat of a 1-year-old child with meningococcal meningitis. This first isolate had 100% homology with the PFGE and BOX-PCR patterns of the reference strain of this clone and a unique PCR-RFLP profile of the pbp2b gene (Figure 1, lane 1) while MLST revealed it to be ST17, the same type as other isolates of this clone. The isolate was resistant to penicillin, chloramphenicol and erythromycin but was susceptible to trimethoprimsulfamethoxazole. Until 1987, no other isolate of the Spain14-5 clone was detected. From 1989 to 1998, the number of isolates of this clone ranged from 1 to 6 per year, but in 1999, 34 isolates of this clone were obtained from 23 patients (all except two patients were aged more than 65 years old). Only one of the isolates was from blood culture, the remaining 33 being from sputum. In the last years of the study, the number of isolates of this clone decreased to 12 in 2000, four in 2001 and eight in 2002.
|
With BOX-PCR, all the 93 isolates with PFGE profiles similar to the Spain14-5 clone showed two patterns, arbitrarily named pattern I and pattern II (Table 1) that differed in only two bands. Pattern I, the same as that of the reference strain of the Spain14-5 clone was observed in 42 isolates whereas pattern II was found in 51 isolates. Of 34 isolates examined by MLST, eight belonged to ST18 (allelic profile: 1-5-4-11-9-3-16), the ST of the reference strain of the Spain14-5 clone, whereas 26 belonged to ST-17 (allelic profile: 1-5-4-11-9-3-47), which is identical except for a change in the allele number of ddl from 16 to 47.
|
|
Antibiotic resistance
The penicillin MIC for all isolates of the Spain14-5 clone ranged from 1 to 4 mg/L. The amoxicillin MIC for all isolates was always equal to or greater than the penicillin MIC (Table 2). Thirty-seven isolates showed a high level of resistance to amoxicillin (816 mg/L). The MICs (mg/L) for the first isolate of the Spain14-5 clone from Gipuzkoa (April 1981) were: penicillin 2, amoxicillin 2, cefotaxime 1, erythromycin >128, chloramphenicol 16, tetracycline 32, and trimethoprimsulfamethoxazole 0.5/9.5.
|
All isolates of this clone were chloramphenicol-resistant and 91 of 93 were tetracycline non-susceptible (MIC 4 mg/L); the presence of the cat and the tet(M) genes was detected in all 93 isolates. The two tetracycline-susceptible isolates had a tetracycline MIC of 2 mg/L and both had the tet(M) gene. The tet(O) gene was not detected in any of the isolates.
Only the first isolate of this clone was trimethoprimsulfamethoxazole-susceptible (MIC = 0.5/9.5 mg/L). The dihydrofolate reductase genes of the reference strain, of the trimethoprimsulfamethoxazole-susceptible isolate and of five trimethoprimsulfamethoxazole-resistant isolates were amplified, and sequenced and the amino acid sequences were deduced and compared with the sequence of S. pneumoniae ATCC 49619.22 The five resistant clinical isolates (trimethoprimsulfamethoxazole MICs 2/38 mg/L) and the ATCC strain 700902 (the reference strain of the clone, trimethoprimsulfamethoxazole MIC 2/38 mg/L) shared five amino acid substitutions (Glu-20
Asp, Lys-60
Gln, Asp-92
Ala, Ile-100
Leu, and Leu-135
Phe). The only amino acid substitution found in the susceptible isolate was Asp-92
Ala.
Of the 93 isolates of the Spain14-5 clone, 61 (65.6%) were levofloxacin-susceptible (levofloxacin MIC 2 mg/L; ciprofloxacin MIC
2 mg/L), one was levofloxacin-intermediate (levofloxacin MIC = 4 mg/L, ciprofloxacin MIC = 4 mg/L) and 31 (33.3%) were levofloxacin-resistant (levofloxacin and ciprofloxacin MIC
8 mg/L). Until 1999, only two levofloxacin-resistant isolates of the Spain14-5 clone (2/35; 5.4%) were isolated, both in 1993. From 1999 to 2002, 29/58 (50%) isolates of this clone were resistant to levofloxacin. All levofloxacin- and ciprofloxacin-resistant isolates had a point mutation at Ser-79 in parC and at Ser-81 in the gyrA gene. The levofloxacin-intermediate isolate showed no point mutation in parC and one point mutation at Ser-81 in the gyrA gene.
The two levofloxacin-resistant isolates of the Spain14-5 clone isolated in 1993 were isolated from the sputum of two different HIV-infected patients. These two isolates that have previously been described,18 were also erythromycin- and clindamycin-resistant and were the only two rifampicin-resistant isolates of this clone (rifampicin MIC > 4 mg/L). Both pertained to ST17 and had the same mutation at the rpoB gene.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In our region, the incidence of this clone was negligible until 1989 when its detection was limited to one to six isolates yearly for 10 years. Most of the Spain14-5 isolates found were respiratory tract isolates, a source from which more resistant pneumococci are usually isolated.32 The low incidence of this clone confirmed its circulation among the population and, as occurred in 1999, it can cause outbreaks of reactivation of chronic obstructive pulmonary disease among the elderly. This finding revealed the ability of this clone to disseminate in a small area. Since that year, its annual incidence returned to previous levels.
A frequent event among pneumococci is that the expression of certain capsular types can be altered as a consequence of the horizontal transfer of capsular genes.33 For other purposes, we used PFGE to study more than 150 penicillin non-susceptible S. pneumoniae isolated in the same period that belonged to more than 10 different serotypes (data not shown) and found no relation with the Spain14-5 clone. Although genes specifying the capsular type can be replaced by genetic transformation,34 we did not find any isolates expressing other capsular types that showed a PFGE pattern similar to that of the Spain14-5 clone. Another genetic characteristic found in the isolates of this clone was their low degree of genetic diversity through time, as PFGE revealed that most of the isolates had a similarity of >85% and that most had exactly the same PFGE pattern. Although PFGE is a typing technique that reflects small variations in the overall bacterial genome,35 only slight differences were found in four of the 93 isolates of this clone.
The S. pneumoniae Spain14-5 clone was first detected in our area in 1981 and is, to the best of our knowledge, the oldest strain belonging to this clone described anywhere. It was an ST17 isolate, had a unique pbp2b-RFLP pattern and was already multiresistant. It was the only trimethoprimsulfamethoxazole-susceptible isolate of this clone but was erythromycin-resistant. The reference strain of this clone is trimethoprimsulfamethoxazole-resistant and erythromycin-susceptible. All these findings indicate that this first isolate of the Spain14-5 clone was an intermediate strain in the evolution of this particular clone.
An association between penicillin-resistance and particular ddl alleles has been described for S. pneumoniae isolates as the recombinational exchanges at the pbp2b gene have extended in many cases into or through the ddl gene.36 We also found a relationship between pbp2b profiles and alleles at the ddl locus in isolates of the Spain14-5 clone, as almost all the isolates with the ddl allele 47 had the same pbp2b profile. The finding of isolates with identical PFGE profiles but different pbp genes is relatively frequent in other penicillin non-susceptible S. pneumoniae isolates.37
Throughout the study period, the isolates of this clone have not only maintained their spectrum of multidrug resistance but have incorporated a high level of resistance to fluoroquinolones. Most of the Spain14-5 S. pneumoniae isolates in our area showed resistance to erythromycin owing to the presence of the erm(B) gene. All ST17 (ddl allele 47) variants of the Spain14-5 clone were resistant to erythromycin probably as a result of a more active spread of this variant of the Spain14-5 clone, which included strains with the MLSB phenotype of macrolide resistance. Possibly, isolates of this clone would be better represented if the reference strain were a ST17 erythromycin-resistant isolate. Among the S. pneumoniae isolated in hospitals located in different regions of Spain between 1990 and 1996, a parallel increase in both serotype 14 and erythromycin-resistant isolates was observed.38 Isolates belonging to the most common clones usually share identical susceptibility patterns, but regional selective pressure in different parts of the world can add resistance to erythromycin and extended-spectrum cephalosporins.26
The finding that the two tetracycline-susceptible isolates harboured the tet(M) gene encoding tetracycline resistance but did not express it was surprising. The tetracycline MICs for these two isolates were 2 mg/L, which is on the breakpoint recommended by the NCCLS.16 Only the first isolate of this clone was trimethoprimsulfamethoxazole-susceptible and it had only a single amino acid substitution at dhfr, which was not associated with resistance to trimethoprim.21 The remaining five trimethoprimsulfamethoxazole clinical isolates studied, as well as the reference strain, all of which were resistant to trimethoprimsulfamethoxazole, had several mutations in the dhfr gene, known to confer resistance to this antibiotic.
From a molecular point of view, the main genetic characteristics of this clone over the 22 year period were the lack of horizontal transfer of capsular genes, the overall homogeneity found by the three genotyping techniques employed (PFGE, BOX-PCR and MLST) and the less successful intercontinental spread to date compared with the three other Spanish major clones, Spain23F-1, Spain6B-2 and Spain9V-3.2628 Study of antibiotic resistance revealed an increase in the spectrum of antibiotic resistance in isolates of this clone over time. Nearly all the isolates studied harboured the well known determinants of antibiotic resistance [cat, tet(M), erm(B) genes] or had the mutations known to confer antibiotic resistance in the genes encoding the corresponding antibiotic targets (point mutations in the dhfr, rpoB and parC and gyrA genes). Since 1999, nearly half of the isolates were fluoroquinolone-resistant and all but one isolate were ST17 erythromycin-resistant.
![]() |
Acknowledgements |
---|
The Spanish Pneumococcal Infection Study Network (G03/103). General Coordination: Roman Pallares (rpallares{at}bell.ub.es). Participants and Centres: Ernesto García (Centro 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 (Hospital Ramón y Cajal, Madrid); Francisco Soriano, José Prieto (Fundación Jiménez Díaz y Facultad de Medicina de la Universidad Complutense, Madrid); Roman Pallares, Josefina Liñares (Hospital Universitari de Bellvitge, Barcelona); Javier Garau, Javier Martínez Lacasa (Hospital Mutua de Terrassa, Barcelona); Cristina Latorre (Hospital Sant Joan de Deu, Barcelona); Emilio Pérez-Trallero, María Ercibengoa (Hospital Donostia, San Sebastián); Juan García de Lomas (Hospital Clínico, Valencia); Ana Fleites (Hospital Central de Asturias).
![]() |
Footnotes |
---|
Participants in the Spanish Pneumococcal Infection Study Network are listed in the Acknowledgements.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Appelbaum, P. C. (1996). Epidemiology and in vitro susceptibility of drug-resistant Streptococcus pneumoniae. Pediatric Infectious Disease Journal 15, 9329.[CrossRef][ISI][Medline]
3 . Fenoll, A., Martín Bourgon, C., Muñoz, R. et al. (1991). Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 19791989. Review of Infectious Diseases 13, 5660.[ISI][Medline]
4 . Baquero, F. (1996). Trends in antibiotic resistance of respiratory pathogens: an analysis and commentary on a collaborative surveillance study. Journal of Antimicrobial Chemotherapy 38, Suppl. A, 11732.[Abstract]
5 . Ritcher, S. S., Heilmann, K. P., Coffman, S. L. et al. (2002). The molecular epidemiology of penicillin-resistant Streptococcus pneumoniae in the United States, 19942000. Clinical Infectious Diseases 34, 3309.[CrossRef][ISI][Medline]
6
.
McGee, L., McDougal, L., Zhou, J. et al. (2001). Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network. Journal of Clinical Microbiology 39, 256571.
7 . Baquero, F., Martínez-Beltrán, J. & Loza, E. (1991). A review of antibiotic resistance patterns of Streptococcus pneumoniae in Europe. Journal of Antimicrobial Chemotherapy 28, Suppl. C, 318.[ISI][Medline]
8 . Casal, J. (1982). Antimicrobial susceptibility of Streptococcus pneumoniae: serotype distribution of penicillin-resistant strains in Spain. Antimicrobial Agents and Chemotherapy 22, 2225.[ISI][Medline]
9 . Perez-Trallero, E., García-Arenzana, J. M., Jiménez Alfaro, J. A. et al. (1981). Penicillin-resistance and serotype distribution in pneumococci. In Abstracts of the Eighth National Congress of Microbiology, Madrid, Spain, 1981. Abstract 329, p. 183. Sociedad Española de Microbiología, Madrid, Spain.
10
.
Ip, M., Lyon, D. J., Yung, R. W. H. et al. (1999). Evidence of clonal dissemination of multidrug-resistant Streptococcus pneumoniae in Hong Kong. Journal of Clinical Microbiology 37, 28349.
11
.
Shi, Z. Y., Enright, M. C., Wilkinson, P. et al. (1998). Identification of three major clones of multiple antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals by multilocus sequence typing. Journal of Clinical Microbiology 36, 35149.
12
.
Greenberg, D., Speert, D. P., Mahenthiralingam, E. et al. (2002). Emergence of penicillin-nonsusceptible Streptococcus pneumoniae invasive clones in Canada. Journal of Clinical Microbiology 40, 6874.
13 . Vela, M. C., Fonseca, N., Di Fabio, J. L. et al. (2001). Presence of international multiresistant clones of Streptococcus pneumoniae in Colombia. Microbial Drug Resistance 7, 15364.[CrossRef][ISI][Medline]
14 . Coffey, T. J., Berron, S., Daniels, M. et al. (1996). Multiple antibiotic-resistant Streptococcus pneumoniae recovered from Spanish hospitals (19881994): novel major clones of serotypes 14, 19F and 15F. Microbiology 142, 274757.[Abstract]
15 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.
16 . National Committee for Clinical Laboratory Standards. (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFifth Edition: Approved Standard M7-A5. NCCLS, Wayne, PA, USA.
17 . Marimon, J. M., Iglesias, L., Vicente, D. et al. (2003). Molecular characterization of erythromycin-resistant clinical isolates of the four major antimicrobial-resistant Spanish clones of Streptococcus pneumoniae (Spain23F-1, Spain6B-2, Spain9V-3 and Spain14-5). Microbial Drug Resistance 9, 1337.[CrossRef][ISI][Medline]
18
.
Perez-Trallero, E., Marimon, J. M., Gonzalez, A. et al. (2003). Spain14-5 international multiresistant Streptococcus pneumoniae clone resistant to fluoroquinolones and other families of antibiotics. Journal of Antimicrobial Chemotherapy 51, 7159.
19
.
Widdowson, C. A., Adrian, P. V. & Klugman, K. P. (2000). Acquisition of chloramphenicol resistance by the linearization and integration of the entire staphylococcal plasmid pC194 into the chromosome of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 3935.
20
.
Doherty, N., Trzcinski, K., Pickerill, P. et al. (2000). Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 44, 297984.
21 . Widdowson, C. A., Klugman, K. P. & Hanslo, D. (1996). Identification of the tetracycline resistance gene, tet(O), in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 28913.[Abstract]
22 . Adrian, P. V. & Klugman, K. P. (1997). Mutations in the dihydrofolate reductase gene of trimethoprim-resistant isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 41, 240613.[Abstract]
23 . Enright, M. C. & Spratt, B. G. (1998). A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive diseases. Microbiology 144, 304960.[Abstract]
24 . Dowson, C. G., Hutchison, A. & Spratt, B. G. (1989). Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae. Molecular Microbiology 3, 95102.[ISI][Medline]
25 . Liñares, J., Pallares, R., Alonso, T. et al. (1992). Trends in antimicrobial resistance of clinical isolates of Streptococcus pneumoniae in Bellvitge Hospital, Barcelona, Spain (19791990). Clinical Infectious Diseases 15, 99105.[ISI][Medline]
26 . Hermans, P. W. M., Overweg, K., Sluijter, M. et al. (2000). Penicillin-resistant Streptococcus pneumoniae: an international molecular epidemiological study. In Streptococcus pneumoniae Molecular Biology and Mechanism of Disease (Tomasz, A., Ed.), pp. 45766. Mary Ann Liebert, Inc., Larchmont, NY, USA.
27 . Soares, S., Kristinsson, K. G., Musser, J. M. et al. (1993). Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. Journal of Infectious Diseases 168, 15863.[ISI][Medline]
28 . Koornholf, H. J., Wasas, A. & Klugman, K. (1992). Antimicrobial resistance in Streptococcus pneumoniae: a South African perspective. Clinical Infectious Diseases 15, 8494.[ISI][Medline]
29 . Song, J. H., Lee, N. Y., Ichiyama, S. et al. (1999). Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Clinical Infectious Diseases 28, 120611.[ISI][Medline]
30
.
Marchese, A., Ramirez, M., Schito, G. C. et al. (1998). Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996. Journal of Clinical Microbiology 36, 29449.
31 . Henriques, N. B., Kalin, M., Ortqvist, A. et al. (2001). Dynamics of penicillin-susceptible clones in invasive pneumococcal disease. Journal of Infectious Diseases 184, 8619.[CrossRef][ISI][Medline]
32 . Ubukata, K., Asahi, Y., Okuzumi, K. et al. (1996). Incidence of penicillin-resistant Streptococcus pneumoniae in Japan, 19931995. Journal of Infection and Chemotherapy 1, 16676.
33 . Coffey, T. J., Enright, M. C., Daniels, M. et al. (1998). Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Molecular Microbiology 27, 7383.[CrossRef][ISI][Medline]
34 . Coffey, T. J., Dowson, C. G., Daniels, M. et al. (1991). Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae. Molecular Microbiology 5, 225560.[ISI][Medline]
35 . Hermans, P. W., Sluijter, M., Hoogenboezem, T. et al. (1995). Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains. Journal of Clinical Microbiology 33, 160612.[Abstract]
36
.
Enright, M. C. & Spratt, B. G. (1999). Extensive variation in the ddl gene of penicillin-resistant Streptococcus pneumoniae results from a hitchhiking effect driven by the penicillin-binding protein 2b gene. Molecular Biology and Evolution 16, 168795.
37 . Gherardi, G., Whitney, C. G., Facklam, R. R. et al. (2000). Major related sets of antibiotic-resistant pneumococci in the United States as determined by pulsed-field gel electrophoresis and pbp1a-pbp2b-pbp2x-dhf restriction profiles. Journal of Infectious Diseases 181, 21629.[CrossRef][ISI][Medline]
38
.
Fenoll, A., Jado, I., Vicioso, D. et al. (1998). Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Spain: update (19901996). Journal of Clinical Microbiology 36, 344754.