DISCUSSION The global increase in highly penicillin-resistant and multidrug-resistant pneumococci appears, in large part, to result from the spread of individual highly resistant pneumococcal clones. This phenomenon has been particularly highlighted by the worldwide spread of the serotype 23F pneumococcal clone (Spain 23F-1), first identified in Spain in the early 1980s ( 37) and resistant to penicillin, chloramphenicol, and tetracycline (and sometimes erythromycin). This successful clone has now been found in the United States ( 26, 37), South Africa ( 23), the United Kingdom ( 7), South America ( 5, 6, 12, 43), several other countries in Europe ( 29, 30, 42, 46, 48, 50, 58, 61), and the Far East ( 34, 47, 59). Serotype 19F ( 7), 14 ( 4), 19A ( 9), 9N ( 40), and 3 ( 40) and serogroup 6 ( 54) variants of this clone have also been reported in various regions of the world. Of these serotype variants of the Spain 23F-1 clone, those expressing the serotype 19F capsule are the most commonly encountered. A recent study conducted in the United States showed that close to 40% of all highly penicillin-resistant pneumococci belong to this Spain 23F-1 clone ( 10). The penicillin-resistant serotype 9V clone (Spain 9V-3), first identified in Spain ( 7) and France ( 29), has become widely disseminated throughout the world and has been identified in Italy ( 30), Sweden ( 35), Germany ( 42), the United States ( 10), the United Kingdom ( 7), South America ( 6, 12), and the Far East ( 34, 47). Serotype 14 ( 11, 36) and 9A ( 46) and serogroup 19 ( 10, 43) variants of this clone have been reported in numerous countries. The serotype 14 variants of the Spain 9V-3 clone are the most commonly encountered. The overwhelming majority of S. pneumoniae isolates for which MICs were about 1.0 μg/ml or higher (107 of 119 isolates, or 89.9%, collected during surveillance from 1996 through 1998) and colonizing the nasopharynges of children attending day care centers in Lisbon, Portugal, belonged to serotype variants of the Spain 23F-1 and Spain 9V-3 clones ( 44, 45). The multidrug-resistant serotype 6B clone (Spain 6B-2), recovered in Spain for over a decade, has become the dominant pneumococcus isolated from children in Iceland ( 52), where it accounts for approximately 75% of multidrug-resistant pneumococci ( 25). It has also been identified in other countries in Europe ( 29, 42) and elsewhere, including the United Kingdom ( 52), the United States ( 62), and Taiwan ( 47). Interestingly, Vilhelmsson et al. ( 63) recently described the emergence of antibiotic-resistant variants of this 6B clone in Iceland which have lost one or more of the antibiotic resistance phenotypes and/or resistance genes, singly or in combination. Studies from other countries that have a high incidence of antibiotic-resistant pneumococci have identified additional distinctive clones that have presumably emerged within these countries. An example is the serotype 23F pneumococci resistant to extended-spectrum cephalosporins and originally isolated in Tennessee in the United States ( 33). This clone (Tennessee 23F-4), which probably evolved locally and is unrelated to the Spain 23F-1 clone, is characterized by a mutation at position 550 in the pbp2X gene which confers high-level cephalosporin resistance. Pneumococci belonging to a multidrug-resistant serotype 14 clone (Spain 14-5) have been identified among isolates recovered from six Spanish hospitals between 1990 and 1994 ( 8). A clone of multidrug-resistant serotype 19A strains first identified in Hungary (Hungary 19A-6) ( 31) has been described in the Czech Republic and Slovakia ( 16). A second, highly penicillin-resistant clone of serotype 19A (CSR 19A-10) and a serotype 14 clone (CSR 14-11) have also been described in Slovakia and the Czech Republic ( 16). An analysis of a wide geographic diversity of resistant pneumococcal isolates in South Africa has identified two clones of serogroup 19A, one intermediately resistant to penicillin (South Africa 19A-7) and the other multiply resistant (South Africa 19A-13) ( 51). In addition, a unique penicillin-resistant serotype 6B clone (South Africa 6B-8) has emerged locally in South Africa ( 51). The widespread use of erythromycin may have led to the emergence of a serotype 14 clone (England 14-9) resistant only to erythromycin in various parts of the United Kingdom ( 17). In Finland, pneumococcal strains resistant to multiple antibiotics have been characterized as belonging to a serogroup 6B clone (Finland 6B-12) ( 48). Shi et al. ( 47) have recently used MLST to describe two antibiotic-resistant clones that have emerged in Taiwan, a serotype 19F clone (Taiwan 19F-14) and a serotype 23F clone (Taiwan 23F-15). The former clone has been recovered from several countries in the Far East and elsewhere (J. Zhou and B. G. Spratt, unpublished results). Penicillin resistance among pneumococcal isolates has rapidly emerged in Poland during the last decade. Although the pandemic Spain 23F-1 and France 9V-3 clones are present in that country, a novel serotype 23F clone (Poland 23F-16) has clearly contributed to the increase in the prevalence of penicillin-resistant pneumococci nationwide ( 41). A search of the literature indicates that the initial characterization of clones within a population of resistant pneumococci can be conveniently carried out using several molecular typing methods, including BOX-PCR ( 60), repetitive extragenic palindromic-PCR ( 62), multilocus enzyme electrophoresis ( 37), and PFGE ( 52). Most studies have analyzed penicillin-resistant isolates, and each of the above techniques can successfully identify clusters of closely related isolates within a population. However, determination of whether clones are novel or related to previously described clones is more problematic. Some of the clones are relatively old (e.g., 20 years), and substantial variations may have accumulated in the PFGE or BOX profiles of the descendents of the ancestral member of a clone. It can therefore be difficult to decide whether a resistant strain is sufficiently similar by PFGE or BOX-PCR analysis to be considered a member of a known clone or should be considered a distinct and previously unrecognized clone. MLST provides a less ambiguous and highly portable approach to global epidemiological studies by defining the genotypes of antibiotic-resistant isolates based on the alleles at each of seven housekeeping loci ( 13, 47, 64). MLST studies have shown that isolates of each of the Spain 23F-1, Spain 6B-2, Spain 9V-3, and Spain 14-5 clones typically have identical allelic profiles or differ from the typical profile at only one of the seven loci ( 64). MLST therefore assigns an isolate as a member of one of the resistant clones if it has the allelic profile of the reference isolate of that resistant clone or differs from it at a single locus. Isolates whose allelic profiles differ at three or more of the seven loci used in MLST can be regarded as distinct clones ( 13, 47, 64). Clearly, PFGE, BOX-PCR, and MLST results do correlate well, although the DNA fragment patterns used in PFGE and BOX-PCR are believed to diversify more rapidly than the allelic profiles used in MLST. Thus, PFGE provides increased levels of discrimination which may be useful for local epidemiological studies, whereas MLST provides a more rigorous way of assigning isolates to one of the major clones. It should be remembered that similarity of genotype does not necessarily imply that two resistant isolates are members of the same clone. Penicillin resistance has emerged predominantly in a small number of serotypes, and although isolates of the same serotype are often only distantly related in terms of overall genotype ( 13), most isolates of a few serotypes appear to have very similar genotypes ( 13, 57). For example, almost all isolates of serotype 9V appear to be similar in genotype, and two resistant clones of this serotype that emerged independently will inevitably appear to be closely related ( 64). Rigorous assignment of penicillin-resistant isolates to the same clone should therefore also involve an analysis of their PBP1A, PBP2B, and PBP2X genes. PBP analysis of the 16 clones showed that the Spain 23F-1 and Spain 9V-3 clones harbored identical PBP1A, PBP2B, and PBP2X genes (1-1-1), as previously described by Coffey et al. ( 7). The presence of identical PBP genes (8-6-7) in the chromosomes of the two unrelated clones from the Czech Republic and Slovakia (CSR 14-10 and CSR 19A-11) suggests the horizontal transfer of these PBP genes. Nine of the 16 clones were resistant to erythromycin, and 6 clones carried the ermB resistance determinant, which codes for target modification by 23S rRNA methylase ( 27). Three isolates showed the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible) and carried the mefA gene, which codes for an efflux mechanism ( 56). In conclusion, the PMEN includes the molecular characterization and nomenclature of 16 important national and international antibiotic-resistant clones that have contributed to the rapid increase in resistance in pneumococci worldwide. The biological basis of the successful spread of these particular clones remains to be established. |
REFERENCES 1. Allen, K D. Penicillin-resistant pneumococci. J Hosp Infect. 1991;17:3–13. [PubMed]2. Appelbaum, P C. Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clin Infect Dis. 1992;15:77–83. [PubMed]3. Ausubel, F M; Brent, R; Kingston, R E; Moore, D D; Seidman, J G; Smith, J A; Struhl, K. Short protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1998. pp. 61–63. 4. Barnes, D M; Wittier, S; Gilligan, P H; Soares, S; Tomasz, A; Henderson, F W. Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo. J Infect Dis. 1995;171:890–896. [PubMed]5. Camou, T; Hortal, M; Tomasz, A. The apparent importation of penicillin-resistant capsular type 14 Spanish/French clone of Streptococcus pneumoniae into Uruguay in the early 1990s. Microb Drug Resist. 1998;4:219–224. [PubMed]6. Castañeda, E; Tomasz, A; Vela, M C T. Penicillin-resistant Streptococcus pneumoniae in Colombia: presence of international epidemic clones. Microb Drug Resist. 1998;4:233–239. [PubMed]7. Coffey, T J; Dowson, C G; Daniels, M; Zhou, J; Martin, C; Spratt, B G; Musser, J M. Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae. Mol Microbiol. 1991;5:2255–2260. [PubMed]8. Coffey, T J; Berrón, S; Daniels, M; Garcia-Leoni, E; Cercenado, E; Bouza, E; Fenoll, A; Spratt, B G. Multiply antibiotic-resistant Streptococcus pneumoniae recovered from Spanish hospitals (1988–1994): novel major clones of serotypes 14, 19F and 15F. Microbiology. 1996;142:2747–2757. [PubMed]9. Coffey, T J; Enright, M C; Daniels, M; Wilkinson, P; Berrón, S; Fenoll, A; Spratt, B G. Serotype 19A variants of the Spanish serotype 23F multiresistant clone of Streptococcus pneumoniae. Microb Drug Resist. 1998;4:51–55. [PubMed]10. Corso, A; Severina, E P; Petruk, V F; Mauriz, Y R; Tomasz, A. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates causing respiratory disease in the United States. Microb Drug Resist. 1998;4:325–337. [PubMed]11. Doit, C; Denamur, E; Picard, B; Geslin, P; Elion, J; Bingen, E. Mechanisms of the spread of penicillin resistance in Streptococcus pneumoniae strains causing meningitis in children in France. J Infect Dis. 1996;174:520–528. [PubMed]12. Echániz-Aviles, M; Carnalla-Barajas, N; Velàzquez-Meza, M E; Soto-Noguerón, A; Espinoza-de los Monteros, L E; Solórzano-Santos, F. Capsular types of Streptococcus pneumoniae causing disease in children from Mexico City. Pediatr Infect Dis J. 1995;14:907–909. [PubMed]13. Enright, M C; Spratt, B G. A multilocus sequence typing scheme for Streptococcus pneumoniae: identification of clones associated with serious invasive disease. Microbiology. 1998;144:3049–3060. [PubMed]14. Fenoll, A; Bourgon, M; Munoz, R; Vicioso, D; Casal, J. Serotype distribution and antimicrobial resistance of Streptococcus pneumoniae isolates causing systemic infections in Spain, 1979–1989. Rev Infect Dis. 1991;13:56–60. [PubMed]15. Fenoll, A; Jado, I; Vicioso, D; Pérez, A; Casal, J. Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Spain: update (1990–1996). J Clin Microbiol. 1998;36:3447–3454. [PubMed]16. Figueiredo, A M; Austrian, R; Urbaskova, P; Teixeira, L A; Tomasz, A. Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and Slovakia. Microb Drug Resist. 1995;1:71–78. [PubMed]17. Hall, L M; Whiley, R A; Duke, B; George, R C; Efstratiou, A. Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns. J Clin Microbiol. 1996;34:853–859. [PubMed]18. Hansman, D; Bullen, M M. A resistant pneumococcus. Lancet. 1967;ii:264–265. 19. Jabes, D; Nachman, S; Tomasz, A. Penicillin-binding protein families: evidence for the clonal nature of penicillin-resistance in clinical isolates of pneumococci. J Infect Dis. 1989;159:16–25. [PubMed]20. Jacobs, M R; Koornhof, H J; Robins-Browne, R M; Stevenson, C M; Vermaak, Z A; Freiman, I; Miller, G B; Witcomb, M A; Isaacson, M; Ward, J I; Austrian, R. Emergence of multiply resistant pneumococci. N Engl J Med. 1978;299:735–740. [PubMed]21. Kam, K M; Luey, K Y; Fung, S M; Yiu, P P; Harden, T J; Cheung, M M. Emergence of multiple-antibiotic-resistant Streptococcus pneumoniae in Hong Kong. Antimicrob Agents Chemother. 1995;39:2667–2670. [PubMed]22. Klugman, K P. Pneumococcal resistance to antibiotics. Clin Microbiol Rev. 1990;3:171–196. [PubMed]23. Klugman, K P; Coffey, T J; Smith, A; Wasas, A; Meyers, M; Spratt, B G. Cluster of erythromycin-resistant variant of the Spanish multiply resistant 23F clone of Streptococcus pneumoniae in South Africa. Eur J Clin Microbiol Infect Dis. 1994;13:171–174. [PubMed]24. Koeuth, T; Versalovic, J; Lupski, J R. Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria. Genome Res. 1995;5:408–418. [PubMed]25. Kristinsson, K G. Epidemiology of penicillin-resistant pneumococci in Iceland. Microb Drug Resist. 1995;1:121–125. [PubMed]26. Laible, G; Spratt, B G; Hakenbeck, R. Interspecies recombinational events during the evolution of altered PBP2X genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Mol Microbiol. 1991;5:1993–2202. [PubMed]27. Leclercq, R; Courvalin, P. Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrob Agents Chemother. 1991;35:1267–1272. [PubMed]28. Lefèvre, J C; Faucon, G; Sicard, A M; Gasc, A M. DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis. J Clin Microbiol. 1993;31:2724–2728. [PubMed]29. Lefèvre, J C; Bertrand, M A; Faucon, G. Molecular analysis by pulsed-field gel electrophoresis of penicillin-resistant Streptococcus pneumoniae from Toulouse, France. Eur J Clin Microbiol Infect Dis. 1995;14:491–497. [PubMed]30. Marchese, A; Ramirez, M; Schito, G C; Tomasz, A. Molecular epidemiology of penicillin-resistant Streptococcus pneumoniae isolates recovered in Italy from 1993 to 1996. J Clin Microbiol. 1998;36:2944–2949. [PubMed]31. Marton, A; Gulyas, M; Munoz, R; Tomasz, A. Extremely high incidence of antibiotic resistance in clinical isolates of Streptococcus pneumoniae in Hungary. J Infect Dis. 1991;163:542–548. [PubMed]32. McDougal, L K; Facklam, R; Reeves, M; Hunter, S; Swenson, J M; Hill, B C; Tenover, F C. Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States. Antimicrob Agents Chemother. 1992;36:2176–2184. [PubMed]33. McDougal, L K; Rasheed, J K; Biddle, J W; Tenover, F C. Identification of multiple clones of extended-spectrum cephalosporin-resistant Streptococcus pneumoniae isolates in the United States. Antimicrob Agents Chemother. 1995;39:2282–2288. [PubMed]34. McGee, L; Klugman, K P; Friedland, D; Lee, H J. Spread of the Spanish multi-resistant serotype 23F clone of Streptococcus pneumoniae to Seoul, Korea. Microb Drug Resist. 1997;3:253–257. [PubMed]35. Melander, E; Ekdahl, K; Hansson, H B; Kamme, C; Laurell, M; Nilsson, P; Persson, K; Söderström, M; Mölstad, S. Introduction and clonal spread of penicillin- and trimethoprim-sulphamethoxazole-resistant Streptococcus pneumoniae, serotype 9V, in southern Sweden. Microb Drug Resist. 1998;4:71–78. [PubMed]36. Moissenet, D; Valcin, M; Marchand, V; Garabédian, E-N; Geslin, P; Garbarg-Chenon, A; Vu-Thien, H. Molecular epidemiology of Streptococcus pneumoniae with decreased susceptibility to penicillin in a Paris children's hospital. J Clin Microbiol. 1997;35:298–301. [PubMed]37. Munoz, R; Coffey, T J; Daniels, M; Dowson, C G; Laible, G; Casal, J; Hakenbeck, R; Jacobs, M; Musser, J M; Spratt, B G; Tomasz, A. Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae. J Infect Dis. 1991;164:302–306. [PubMed]38. Munoz, R; Musser, J M; Crain, M; Briles, D E; Marton, A; Parkinson, A J; Sorensen, U; Tomasz, A. Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterisation by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis. Clin Infect Dis. 1992;15:112–118. [PubMed]39. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Publication M7–A5. Wayne, Pa: National Committee for Clinical Laboratory Standards; 2000. 40. Nesin, M; Ramirez, M; Tomasz, A. Capsular transformation of a multi-drug resistant Streptococcus pneumoniae in vivo. J Infect Dis. 1998;177:707–713. [PubMed]41. Overweg, K; Hermans, P W M; Trzcinski, K; Sluijter, M; de Groot, R; Hryniewicz, W. Multidrug-resistant Streptococcus pneumoniae in Poland: identification of emerging clones. J Clin Microbiol. 1999;37:1739–1745. [PubMed]42. Reichmann, P; Varon, E; Gunther, E; Reinert, R R; Luttiken, R; Marton, A; Geslin, P; Wagner, J; Hakenbeck, R. Penicillin-resistant Streptococcus pneumoniae in Germany: genetic relationship to clones from other European countries. J Med Microbiol. 1995;43:377–385. [PubMed]43. Rossi, A; Corso, A; Pace, J; Regueira, M; Tomasz, A. Penicillin-resistant Streptococcus pneumoniae in Argentina: frequent occurrence of an internationally spread serotype 14. Microb Drug Resist. 1998;4:225–231. [PubMed]44. Sá-Leão, R; Tomasz, A; Santos Sanches, I; Brito-Avô, A; Vilhelmsson, S E; Kristinsson, K G; de Lencastre, H. Carriage of internationally-spread epidemic clones of Streptococcus pneumoniae with unusual drug resistance patterns in children attending day care centers in Lisbon, Portugal. J Infect Dis. 2000;182:1153–1160. [PubMed]45. Sá-Leão, R; Tomasz, A; Santos Sanches, I; Nunes, S; Alves, C R; Brito-Avô, A; Saldanha, J; Kristinsson, K G; de Lencastre, H. Genetic diversity and clonal patterns among antibiotic-susceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units. J Clin Microbiol. 2000;38:4137–4144. [PubMed]46. Setchanova, L; Tomasz, A. Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria. J Clin Microbiol. 1999;37:638–648. [PubMed]47. Shi, Z-Y; Enright, M C; Wilkinson, P; Griffiths, D; Spratt, B G. Identification of the three major clones of multiply antibiotic-resistant Streptococcus pneumoniae in Taiwanese hospitals by multilocus sequencing typing. J Clin Microbiol. 1998;36:3514–3519. [PubMed]48. Sibold, C; Wang, J; Henrichsen, J; Hakenbeck, R. Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents. Infect Immun. 1992;60:4119–4126. [PubMed]49. Sloas, M M; Barrett, F F; Chesney, P J; English, B K; Hill, B C; Tenover, F C; Leggiadro, R J. Cephalosporin treatment failure in penicillin- and cephalosporin-resistant Streptococcus pneumoniae meningitis. Pediatr Infect Dis. 1992;11:662–666. 50. Sluijter, M; Faden, H; de Groot, R; Lemmens, N; Goessens, W H F; van Belkum, A; Hermans, P W M. Molecular characterization of pneumococcal nasopharynx isolates collected from children during their first 2 years of life. J Clin Microbiol. 1998;36:2248–2253. [PubMed]51. Smith, A M; Klugman, K P. Three predominant clones identified within penicillin-resistant South African isolates of Streptococcus pneumoniae. Microb Drug Resist. 1997;3:385–389. [PubMed]52. Soares, S; Kristinsson, K G; Musser, J M; Tomasz, A. Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. J Infect Dis. 1993;168:158–163. [PubMed]53. Song, J-H; Lee, N Y; Ichiyama, S; Yoshida, R; Hirakata, Y; Fu, W; Chongthaleong, A; Aswapokee, N; Chiu, C-H; Lalitha, M K; Thomas, K; Perera, J; Yee, T T; Jamal, F; Warsa, U C; Vinh, B X; Jacobs, M R; Appelbaum, P C; Pai, C H. the ANSORP Study Group. Spread of drug-resistant Streptococcus pneumoniae in Asian countries: Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Clin Infect Dis. 1999;28:1206–1211. [PubMed]54. Song, J-H; Yang, J-W; Jin, J H; Kim, S W; Kim, C K; Lee, H; Peck, K R; Kim, S; Lee, N Y; Jacobs, M R; Appelbaum, P C. the Asian Network for Surveillance of Resistant Pathogens (ANSORP) Study Group. Molecular characterization of multidrug-resistant Streptococcus pneumoniae isolates in Korea. J Clin Microbiol. 2000;38:1641–1644. [PubMed]55. Sutcliffe, J; Tait-Kamradt, A; Wondrack, L. Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrob Agents Chemother. 1996;40:1817–1824. [PubMed]56. Tait-Kamradt, A; Clancy, J; Cronan, M; Dib-Hajj, F; Wondrack, L; Yuan, W; Sutcliffe, J. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob Agents Chemother. 1997;41:2251–2255. [PubMed]57. Takala, A K; Vuopio-Varkila, J; Tarkka, E; Leinonen, M; Musser, J M. Subtyping of common pediatric pneumococcal serotypes from invasive disease and pharyngeal carriage in Finland. J Infect Dis. 1996;173:128–135. [PubMed]58. Tarasi, A; Sterk-Kuzmanovic, N; Sieradzki, K; Schoenwald, S; Austrian, R; Tomasz, A. Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a paediatric hospital in Zagreb, Croatia. Microb Drug Resist. 1995;1:169–176. [PubMed]59. Tarasi, A; Chong, Y; Lee, K; Tomasz, A. Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea. Microb Drug Resist. 1997;3:105–109. [PubMed]60. Van Belkum, A; Sluijter, M; de Groot, R; Verbrugh, H; Hermans, P W M. Novel BOX repeat PCR assay for high-resolution typing of Streptococcus pneumoniae strains. J Clin Microbiol. 1996;34:1176–1179. [PubMed]61. Vaz Pato, M V; de Carvalho, C B; Tomasz, A. the Multicenter Study Group. Antibiotic susceptibility of Streptococcus pneumoniae isolates in Portugal. A multicenter study between 1989 and 1993. Microb Drug Resist. 1995;1:59–69. [PubMed]62. Versalovic, J; Kapur, V; Mason, E O; Shah, U; Koeuth, T; Lupski, J R; Musser, J M. Penicillin-resistant Streptococcus pneumoniae strains recovered in Houston: identification and molecular characterization of multiple clones. J Infect Dis. 1993;167:850–856. [PubMed]63. Vilhelmsson, S E; Tomasz, A; Kristinsson, K G. Molecular evolution in a multidrug-resistant lineage of Streptococcus pneumoniae: emergence of strains belonging to the serotype 6B Icelandic clone that lost antibiotic resistance traits. J Clin Microbiol. 2000;38:1375–1381. [PubMed]64. Zhou, J; Enright, M C; Spratt, B G. Identification of the major Spanish clones of penicillin-resistant pneumococci via the Internet using multilocus sequence typing. J Clin Microbiol. 2000;38:977–986. [PubMed] |