JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1996, p. 2707–2712 Vol. 34, No. 110095-1137/96/$04.0010Copyright q 1996, American Society for Microbiology

Clonal Distribution of Penicillin-Resistant Streptococcuspneumoniae 23F in France

AGNES FERRONI,1 LAURENCE NGUYEN,1 PIERRE GEHANNO,2

ISABELLE BOUCOT,1 AND PATRICK BERCHE1*

Service de Microbiologie, Hopital Necker-Enfants Malades, 75743 Paris Cedex 15,1 andService d’Oto-Rhino-Laryngologie, Hopital Bichat-Claude Bernard,

75018 Paris,2 France

Received 1 April 1996/Returned for modification 14 June 1996/Accepted 8 August 1996

We studied the clonality of clinical isolates of Streptococcus pneumoniae 23F, the serotype most oftenassociated with penicillin resistance in France. Clinical isolates obtained between November 1992 and April1993 from nasopharyngeal samples from children with acute otitis media from different regions of the countrywere analyzed. The genetic polymorphism of penicillin-susceptible and -resistant 23F isolates (MIC, 2 mg/liter)was studied by pulsed-field gel electrophoresis. The resistant isolates were closely related, whereas thesusceptible isolates were genetically heterogeneous. PCR amplification and restriction of the genes encodingpenicillin-binding proteins (PBPs) 1A, 2B, and 2X also showed that the 24 resistant isolates had similarpatterns which were very different from those of the susceptible isolates. All resistant isolates gave the samePBP pattern, with low affinities of PBPs for penicillin. Our results indicate that, in contrast to penicillin-susceptible 23F isolates, the penicillin-resistant 23F isolates have a single clonal origin, suggesting the rapidclonal spread of a resistant epidemic strain throughout the country.

Penicillin-resistant Streptococcus pneumoniae isolates werefirst detected in the 1960s and have been isolated from patientsin many parts of the world, including South Africa, Australia,the United States, and Europe. There is evidence that penicil-lin resistance in pneumococci is mainly due to alterations in thestructural genes encoding the penicillin-binding proteins(PBPs), thus resulting in PBPs with decreased affinity for pen-icillin (4, 10). Nucleotide sequence analysis of the DNA re-gions encoding the penicillin-binding domains of PBP 2B andPBP 2X from resistant isolates strongly suggests that thesealterations of PBPs might be due to the acquisition of exoge-nous PBP-encoding DNA through transformation and recom-bination (5, 14). These events might have occurred indepen-dently on more than one occasion in various parts of the world.Resistant isolates may then be transported to geographicallydistant areas, where they may spread rapidly, as described inIceland (23).In France, the first penicillin-resistant isolate was found in

1978, and resistant pneumococci were rare until 1987 (7). Theproportion of isolates resistant to penicillin increased yearlythereafter to 6.6% in 1989 and 25% in 1993 (8). A nationwidemulticenter study of children with acute otitis media in 1993revealed that 37.5% of pneumococci isolated from the naso-pharyngeal flora of children were resistant (1). The geograph-ical distribution of resistant isolates was uneven, with a pre-dominance in urban areas, where the proportion was as high as50% in some cities. The majority of resistant isolates (65%)belonged to the 23F serogroup, suggesting a common clonalorigin (1). Here, we report an analysis of clonality involving thestudy of a panel of clinical penicillin-resistant and -susceptibleisolates of S. pneumoniae obtained from children (age, ,6years) in various parts of France. The penicillin-resistant 23Fisolates appear to be closely related and clearly different from

susceptible 23F isolates. Thus, the emergence of resistant 23Fisolates might have been due to rapid clonal spreading ofbacteria in the commensal nasopharyngeal flora of children.

MATERIALS AND METHODS

Bacterial strains and culture media. We used clinical isolates of S. pneu-moniae, obtained between November 1992 andMarch 1993 from nasopharyngealsamples from children (age, ,6 years) from throughout France with acute otitismedia. They included 24 isolates of serotype 23F susceptible to penicillin G forwhich MICs ranged from#0.0075 to 0.03 mg/liter and 24 isolates of serotype 23Fresistant to penicillin G (MIC, 2 mg/liter), with most isolates being multidrugresistant (resistant to erythromycin, tetracycline, chloramphenicol, and co-tri-moxazole). Twenty-three penicillin-susceptible isolates (MICs, 0.0075 to 0.03mg/liter) belonging to various serotypes were used as controls. The isolates(listed in the legends to Fig. 1 and 4) were selected according to the MICs for theisolates and their serotypes and geographic distributions from among a collectionof 632 isolates obtained during a national survey of 1,200 children with acuteotitis media (1). We also used penicillin-susceptible S. pneumoniae ATCC 33400(serotype 1; MIC, #0.0075 mg/liter; isolated in the United States in 1981),penicillin-resistant S. pneumoniae 456 (MIC, 1 to 2 mg/liter; isolated in Spain in1984) (9), penicillin-susceptible unencapsulated strain R6 (MIC, 0.008 mg/liter;isolated in the United States in 1930), and a penicillin-resistant 23F strain, strainBM4200 (MIC, 0.5 mg/liter; isolated in France in 1978) (2). Pneumococci weregrown on Columbia agar plates containing 5% blood or in brain heart infusionmedium (Diagnostics Pasteur, Marnes-la-Coquette, France). Serotyping was car-ried out by the quellung reaction with specific antisera provided by the Statens-Serum Institute (Copenhagen, Denmark). The MIC of penicillin G was deter-mined on 5% sheep blood Mueller-Hinton agar (Diagnostics Pasteur).PFGE. Pulsed-field gel electrophoresis (PFGE) was performed as described

previously (15). DNA was digested overnight with 20 U of SmaI or ApaI (NewEngland Biolabs Inc., Beverly, Mass.), and fragments were separated on a 1%agarose gel by PFGE (CHEF mapper DRII; Bio-Rad Laboratories, Richmond,Calif.) for 24 h at 148C at 7 V/cm, with pulse times of 10 to 30 s and an angle of1208. Gels were visualized with ethidium bromide under UV light.Computer-assisted analysis of the DNA fingerprints. Coefficients of similarity

(CSs) (CS is the number of shared bands3 23 100/total number of bands in thetwo samples) or the Dice index were determined for each isolate by using thecomputer program Bio-Profil (Vilber Lourmat, Marne-la-Vallee, France), and adendrogram was calculated by the unweighted pair-group method. Strains with aCS value of more than 80% were considered to belong to the same lineage.DNA polymorphism of the PBP genes. The genes encoding PBP 1A (pbp1A),

PBP 2X (pbp2X), and PBP 2B (pbp2B) of S. pneumoniae were amplified fromchromosomal DNA by PCR with previously described oligonuclotide primers (3,6, 19). These primers amplified DNA fragments of 2,409 bp (pbp1A), 2,056 bp(pbp2X), and 1,505 bp (pbp2B). Amplification was performed with a Perkin-Elmer (Norwalk, Conn.) 9600 DNA thermal cycler with an initial denaturation

* Corresponding author. Mailing address: Service de Microbiologie,Hopital Necker-Enfants Malades, 149, rue de Sevres, 75743 ParisCedex 15, France. Phone: 44 49 49 61. Fax: 44 49 49 60. Electronic mailaddress: berche@citi2.fr.

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step (958C, 5 min) that was followed by 38 cycles of denaturation (988C, 20 s),annealing (558C, 20 s), DNA extension (728C, 45 s), and a final extension step(728C, 10 min). The amplified DNA was digested for 18 h at 378C with 5 U ofAluI or HinfI (New England Biolabs), which give large numbers of DNA frag-ments according to the restriction map of the nucleotide sequences (6, 13, 17).The fragments were then electrophoretically separated on a 3% MetaPhor aga-rose gel (Tebu, Le Perray, France) and visualized with ethidium bromide underUV light.[3H]penicillin labeling of PBPs. We used a previously described procedure

(25) to label the PBPs with [3H]penicillin. Briefly, bacteria (108/ml) were incu-bated for 10 min at 378C with [3H]benzylpenicillin (final concentration, 10 mg/ml)kindly provided by D. Bouanchaud (Rhone-Poulenc, Romainville, France).Binding was stopped by the addition of 5 ml of unlabeled benzylpenicillin (200mg/ml), and the cells were lysed in 0.13 M lysis buffer (pH 7) with 1% Sarkosyl.The samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) in a Mini-PROTEAN II dual slab cell (Bio-Rad Lab-oratories) at a constant voltage of 200 V. The gels were stained with Coomassiebrilliant blue and exposed to Kodak X-Omat XR3 films for 4 days at 2808C.

RESULTS

Genetic polymorphism of S. pneumoniae 23F isolates byPFGE. The clonality of S. pneumoniae 23F, the serotype whichis most often associated with penicillin resistance in France,was studied by PFGE. Forty-eight 23F isolates from nasopha-ryngeal samples from children from various regions of Francewith acute otitis media were analyzed. We compared the DNApolymorphism of 24 penicillin-susceptible 23F isolates withthat of 24 penicillin-resistant 23F isolates (MIC, 2 mg/liter).Chromosomal DNA was digested with SmaI or ApaI, generat-ing about 14 or 15 fragments in each case, as illustrated forSmaI in Fig. 1. The results obtained with ApaI were consistentwith those obtained with SmaI (data not shown). The restric-tion profiles of resistant 23F isolates were all similar and weredifferent from those of susceptible 23F isolates (Fig. 1A andB), which were more heterogeneous. A dendrogram was con-structed by computer analysis of the DNA fingerprints (Fig. 2).The isolates segregated into two clusters corresponding to thesusceptible and the resistant 23F isolates. By using a cutoffvalue of 80% similarity, at least 14 different lineages werefound among the susceptible 23F isolates, suggesting clonaldiversity in this serotype. In contrast, only one lineage wasidentified for the group of resistant 23F isolates. In this lin-eage, there were three clusters of identical isolates (four, four,and five isolates, respectively), which were isolated from vari-ous and in some cases distant parts of the country (Fig. 2). Thepenicillin-resistant serotype 23F strain 456, isolated in Spain in1984, was related to the lineage of resistant isolates, (CS, 70%)and to one penicillin-susceptible isolate (isolate 1752). In ad-dition, the first penicillin-resistant strain, strain BM4200 (MIC,0.5 mg/liter), isolated in France in 1978, had a restriction pat-tern different from that of the multidrug-resistant isolates(data not shown). The genetic polymorphism of 14 penicillin-susceptible isolates of 14 different serotypes (including the 23Fserotype) was analyzed (Fig. 1C). Each strain produced a dif-ferent banding pattern, demonstrating the high discriminatorypower of the PFGE analysis. These results suggest that peni-cillin-resistant 23F isolates from all over the country have acommon clonal origin, whereas the penicillin-susceptible 23Fisolates do not.Genetic polymorphisms of pbp1A, pbp2B, and pbp2X genes in

S. pneumoniae 23F. We then studied the genetic polymor-phisms of the genes encoding the PBPs implicated in the pen-icillin resistance of S. pneumoniae. Intragenic fragments of thepbp1A (2,409 bp), pbp2X (2,056 bp), and pbp2B (1,505 bp)genes from the 48 23F isolates were amplified by PCR anddigested with AluI or HinfI; the results for representative iso-lates are illustrated in Fig. 3. The AluI and HinfI restrictionpatterns of the three amplified pbp fragments were similar for

the 24 French penicillin-resistant isolates and Spanish strain456, but differed from those of the penicillin-susceptible iso-lates. Most of the patterns obtained for these penicillin-sus-ceptible isolates were also identical; pbp2X, however, gave at

FIG. 1. PFGE restriction profiles of clinical isolates of S. pneumoniae be-longing to serotype 23F and other serotypes. Chromosomal DNA was digestedwith SmaI. (A) 23F resistant strains. Lanes: 1, bacteriophage l DNA ladder; 2,strain ATCC 33400; 3, Spanish strain 456; 4, 1470; 5, 1485; 6, 1531; 7, 1679; 8,1674; 9, 1002; 10, 1672; 11, 1284; 12, 1101; 13, 1506; 14, 1181; 15, 1668; 16, 1808;17, 1062; 18, 1292; 19, 1031; 20, 1032; 21, 1826; 22, 1678; 23, 1353; 24, 1487; 25,1502; 26, 1388; 27, 1457. (B) 23F susceptible strains. Lanes: 1, bacteriophage lDNA ladder; 2, strain ATCC 33400; 3, Spanish strain 456; 4, 591; 5, 1752; 6, 1118;7, 1722; 8, 1089; 9, 1877; 10, 1516; 11, 1273; 12, 1870; 13, 1004; 14, 1659; 15, 1129;16, 1261; 17, 1370; 18, 1246; 19, 1114; 20, 1427; 21, 1496; 22, 1623; 23, 1254; 24,1727; 25, 1621; 26, 1835; 27, 1056. (C) Other susceptible serotypes strains. Lanes:1, bacteriophage lDNA ladder; 2, 1519, serotype 33; 3, 1194, serotype 9; 4, 1394,serotype 35; 5, 1497, serotype 1; 6, 1260, serotype 16; 7, 1175, serotype 17; 8,1250, serotype 3; 9, 1192, serotype 4; 10, 1064, serotype 19; 11, 1113, serotype 6;12, 1534, serotype 23; 13, 1169, serotype 11; 14, 1187, serotype 18; 15, 1093,serotype 14; 16, strain ATCC 33400. Molecular markers (bars) are 48.5, 97,145.5, 194, and 242.5 kb, from bottom to top, respectively.

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least three different profiles. Among the 24 susceptible 23Fisolates, there was one exception (isolate 1835; MIC, #0.0075mg/liter), for which pbp1A AluI and HinfI restriction profileswere identical to those for the resistant isolates (data notshown). Twenty-three penicillin-susceptible non-23F isolatesbelonging to 13 different serotypes were also analyzed, as il-lustrated in Fig. 4 for representative isolates. The pbp1A andpbp2B restriction patterns were similar to those for susceptible23F isolates, whereas at least three different restriction pat-

terns were found for pbp2X (Fig. 4). Thus, the genes encodingPBP 1A, PBP 2B, and PBP 2X in penicillin-resistant isolates(MIC, 2 mg/liter) are closely related or identical and are dis-tantly related to those found in penicillin-susceptible isolates.Penicillin affinities of PBPs in S. pneumoniae 23F.We tested

the penicillin affinity of S. pneumoniae PBPs for the 48 23Fisolates. Bacterial proteins were labeled with [3H]penicillin andwere separated by SDS-PAGE. The results for representativeisolates are illustrated in Fig. 5. As expected, the penicillin

FIG. 2. Dendrogram and geographic distribution of S. pneumoniae 23F isolates. As indicated on the maps, the distributions of penicillin-susceptible and -resistantisolates (24 in each group) were comparable. The dendrogram was calculated by the unweighted pair-group method. All resistant isolates belonging to the same lineagehave a similarity value (Dice index) of greater than 80%. The isolates are listed by their reference numbers. Hollow symbols (E) indicate penicillin-susceptible isolates.Solid symbols (F) indicate resistant isolates except for undistinguishable isolates, which constitute three groups (å, ■, and }), each with 4 to 5 strains. It is shown thatidentical resistant strains were isolated from distant areas. The Spanish reference strain 456 is indicated by an asterisk.

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affinities of PBP 1A, PBP 2B, and PBP 2X were reduced forthe 24 resistant isolates and for Spanish strain 456. All resistantisolates displayed similar PBP patterns (Fig. 5).

DISCUSSION

We have shown in the present work that penicillin-resistantisolates of S. pneumoniae 23F are genetically closely related,suggesting a common clonal origin. The PFGE restriction pat-terns of some of the resistant 23F isolates from diverse regionsof France were identical (Fig. 1), indicating that clones havespread throughout the country in the commensal nasopharyn-geal flora of children. In contrast, penicillin-susceptible 23Fisolates were genetically diverse: at least 14 distinct lineageswere distinguished by PFGE (Fig. 1). None of these lineagesgave restriction patterns resembling those of the penicillin-resistant 23F isolates or those of the penicillin-susceptible iso-lates of unrelated serotypes (Fig. 1). Resistant 23F isolates aretherefore not closely related to susceptible 23F or non-23Fisolates. Our results suggest that the emergence of penicillin-resistant 23F isolates in France is due to the rapid spread of arecently introduced 23F clone. Reichmann et al. (20), usingmultilocus enzyme electrophoresis, discriminated six lineagesamong resistant 23F isolates displaying intermediate or high-level penicillin resistance. One lineage corresponds to the mul-tiresistant Spanish 23F clone and to highly resistant isolatesfrom France, Germany, and Hungary (20). This also agreeswith several reports indicating that resistant 23F clones canspread to distant areas (18, 19). A resistant 23F clone has beenshown to have spread from Spain to the United States (Ohio)(19), where it was rapidly disseminated (18). Similarly, a ge-netic relationship has been observed between resistant 23Fclones from Spain and South Africa (12, 21). The dissemina-tion of resistant clones of S. pneumoniae has also been de-scribed for the penicillin-resistant clones of the 6B serotype,which has been propagated from Spain to Iceland, where it isnow prevalent (23). This clone is also related to resistant 6Bclones isolated in Alaska and Texas (24). Interestingly, thePFGE restriction patterns of our resistant 23F isolates resem-ble that of an epidemic 23F strain isolated in Barcelona, Spain,in 1984 (9), suggesting that these clones might have a commonorigin.The results obtained by PFGE were confirmed by studying

the restriction patterns of three PBP genes (pbp1A, pbp2B, andpbp2X). These restriction patterns were identical in all resis-tant 23F clones, including the Spanish strain 456, confirmingthe close relatedness of these isolates. Multiple transformationevents producing resistant clones are expected to generategenetic heterogeneity of the PBP genes (5, 9, 10, 16), but therewas no evidence for this in our study. The susceptible 23F andnon-23F isolates also displayed relatively similar PBP generestriction patterns, consistent with previous reports (5, 11, 16,25). However, a low level of genetic polymorphism was ob-served for pbp2X in these susceptible isolates. Such geneticpolymorphism has been described for highly susceptible strainsbelonging to serotypes 6 and 19 (22). In addition, we found ahighly susceptible 23F isolate exhibiting a pbp1A restrictionpattern identical to that found in resistant 23F isolates, sug-gesting horizontal gene transfer in this strain without detect-able alteration of the MIC of penicillin. The PBPs of resistant23F isolates, including the Spanish strain 456, displayed lowaffinities for penicillin. The migration profiles of PBPs fromresistant 23F isolates were similar, further eliminating the pos-sibility that these resistant isolates had been generated inde-pendently of each other by multiple genetic events. In conclu-sion, our results demonstrate that the emergence of penicillin-

FIG. 3. Restriction patterns of pbp1A, pbp2B, and pbp2X from penicillin-resistant and -susceptible 23F isolates of S. pneumoniae. (A) AluI restriction. (B)HinfI restriction. pbp1A. (A and B) Lanes 2 to 12, resistant strains (MICs, 2mg/liter); lanes 13 to 18, susceptible strains (MICs, #0.03 mg/liter). Lanes: 1,strain ATCC 33400; 2, Spanish strain 456; 3, 1485; 4, 1531; 5, 1679; 6, 1674; 7,1672; 8, 1284; 9, 1101; 10, 1506; 11, 1181; 12, 1668; 13, 591; 14, 1516; 15, 1114; 16,1659; 17, 1261; 18, 1370; 19, bacteriophage l DNA ladder. pbp2B. (A) Lanes 2to 12, resistant strains; lanes 13 to 20, susceptible strains. Lanes: 1, strain ATCC33400; 2, Spanish strain 456; 3, 1485; 4, 1531; 5, 1679; 6, 1674; 7, 1672; 8, 1284;9, 1101; 10, 1506; 11, 1181; 12, 1668; 13, 591; 14, 1089; 15, 1877; 16, 1516; 17,1659; 18, 1129; 19, 1261; 20, 1370; 21, bacteriophage l DNA ladder. (B) Lanes2 to 12, resistant strains; lanes 13 to 21, susceptible strains. Lanes: 1, strain ATCC33400; 2, Spanish strain 456; 3, 1485; 4, 1531; 5, 1679; 6, 1674; 7, 1672; 8, 1284;9, 1101; 10, 1506; 11, 1181; 12, 1668; 13, 591; 14, 1089; 15, 1877; 16, 1516; 17,1114; 18, 1659; 19, 1129; 20, 1261; 21, 1370; 22, bacteriophage l DNA ladder.pbp2X. (A and B) Lanes 2 to 12, resistant strains; lanes 13 to 21, susceptiblestrains. Lanes: 1, strain ATCC 33400; 2, Spanish strain 456; 3, 1485; 4, 1531; 5,1679; 6, 1674; 7, 1672; 8, 1284; 9, 1101; 10, 1506; 11, 1181; 12, 1668; 13, 591; 14,1089; 15, 1877; 16, 1516; 17, 1114; 18, 1659; 19, 1129; 20, 1261; 21, 1370; 22,bacteriophage l DNA ladder. Molecular markers (bars) are 67, 110, 147, 190,320, and 500 bp, from bottom to top, respectively.

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resistant 23F isolates accounts for the majority of penicillin-resistant S. pneumoniae in France and is the result of the clonalexpansion of isolates rapidly spreading in the nasopharyngealflora of children.

ACKNOWLEDGMENTS

We thank Patrice Courvalin for the gift of strain BM4200 andEdouard Bingen for helpful discussion and for the gift of strain R6 andthe Spanish epidemic strain.This work was supported by INSERM and the University Rene

Descartes Paris V.

REFERENCES

1. Berche, P., P. Gehanno, F. Duval, and G. Lenoir. 1994. Epidemiologiebacterienne des otites moyennes aigues de l’enfant en France en 1993. Lett.Infect. 18:11–23.

2. Buu-Hoı, A., and T. Horodniceanu. 1980. Conjugative transfer of multipleantibiotic resistance markers in Streptococcus pneumoniae. J. Bacteriol. 143:313–320.

3. Coffey, T. J., C. G. Dowson, M. Daniels, J. Zhou, C. Martin, G. Spratt, andJ. M. Musser. 1991. Horizontal transfer of multiple penicilin-binding proteingenes, and capsular biosynthetic genes, in natural populations of Streptococ-cus pneumoniae. Mol. Microbiol. 5:2255–2260.

4. Dowson, C. G., T. J. Coffey, and B. G. Spratt. 1994. Origin and molecularepidemiology of penicillin-binding-protein-mediated resistance to b-lactamantibiotics. Trends Microbiol. 2:361–366.

5. Dowson, C. G., A. Hutchison, J. A. Brannigan, R. C. George, D. Hansman,J. Linares, A. Tomasz, J. Maynard-Smith, and B. G. Spratt. 1989. Horizontaltransfer of penicillin-binding protein genes in penicillin-resistant clinicalisolates of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 86:8842–8846.

6. Dowson, C. G., A. Hutchison, and B. G. Spratt. 1989. Extensive re-modellingof the transpeptidase domain of penicillin-binding protein 2b of a penicillin-resistant South African isolate of Streptococcus pneumoniae. Mol. Microbiol.3:95–102.

7. Geslin, P., A. Buu-Hoi, A. Fremaux, and J. F. Acar. 1992. Antimicrobialresistance in Streptococcus pneumoniae: an epidemiological survey in France,1970–1990. Clin. Infect. Dis. 15:95–98.

8. Geslin, P., A. Fremaux, G. Sissia, C. Spicq, and S. Aberrane. 1994. Epide-miologie de la resistance aux antibiotiques de Streptococcus pneumoniae enFrance. Reseau national de surveillance (1984–1993). Med. Mal. Infect. 24(Special):948–961.

9. Hakenbeck, R., T. Briese, L. Chalkley, H. Ellerbrok, R. Kalliokoski, C.Latorre, M. Leinonen, and C. Martin. 1991. Antigenic variation of penicillin-binding proteins from penicillin-resistant clinical strains of Streptococcuspneumoniae. J. Infect. Dis. 164:313–319.

10. Hakenbeck, R., M. Tarpay, and A. Tomasz. 1980. Multiple changes of pen-icillin-binding proteins in penicillin-resistant isolates of Streptococcus pneu-moniae. Antimicrob. Agents Chemother. 17:364–371.

11. Jabes, D., S. Nachman, and A. Tomasz. 1989. Penicillin-binding proteinfamilies: evidence for the clonal nature of penicillin resistance in clinicalisolates of pneumococci. J. Infect. Dis. 159:16–25.

12. Klugman, K. P., T. J. Coffey, A. Smith, A. Wasas, M. Meyers, and B. G.Spratt. 1994. Cluster of an erythromycin-resistant variant of the Spanishmultiply resistant 23F clone of Streptococcus pneumoniae in South Africa.Eur. J. Clin. Microbiol. Infect. Dis. 13:171–174.

13. Laible, G., R. Hakenbeck, M. A. Sicard, B. Joris, and J. M. Ghuysen. 1989.Nucleotide sequences of the pbpX genes encoding the penicillin-bindingproteins 2X from Streptococcus pneumoniae R6 and a cefotaxime-resistantmutant, C506. Mol. Microbiol. 3:1337–1348.

14. Laible, G., B. G. Spratt, and R. Hakenbeck. 1991. Interspecies recombina-tional events during the evolution of altered PBP 2X genes in penicillin-

FIG. 4. Restriction patterns of pbp1A, pbp2B, and pbp2X from penicillin-susceptible isolates of S. pneumoniae belonging to different serotypes. (A) AluIrestriction. (B) HinfI restriction. pbp1A. (A and B) Lanes: 1, strain ATCC 33400;2, 1497, serotype 1; 3, 1250, serotype 3; 4, 1113, serotype 6; 5, 1194, serotype 9;6, 1169, serotype 11; 7, 1093, serotype 14; 8, 1260, serotype 16; 9, 1175, serotype17; 10, 1064, serotype 19; 11, 1516, serotype 23; 12, bacteriophage lDNA ladder.pbp2B and pbp2X. (A and B) Lanes: 1, strain ATCC 33400; 2, 1497, serotype 1;3 and 4, 1250 and 1396 (serotype 3), respectively; 5 and 6, 1113 and 1878(serotype 6), respectively; 7 and 8, 1194 and 1540 (serotype 9), respectively; 9 and10, 1169 and 1080 (serotype 11), respectively; 11 and 12, 1093 and 1172 (serotype14), respectively; 13 and 14, 1260 and 1236 (serotype 16), respectively; 15 and 16,1175 and 1624 (serotype 17), respectively; 17 and 18, 1187 and 1348 (serotype18), respectively; 19 and 20, 1064 and 1098 (serotype 19), respectively; 21, 1877,serotype 23; 22, bacteriophage l DNA ladder. Molecular markers (bars) are 67,110, 147, 190, 320, and 500 bp, from bottom to top, respectively.

FIG. 5. Pattern of high-molecular-weight PBPs of susceptible and resistant23F isolates of S. pneumoniae. Lanes 2 to 6 and 13, resistant strains; lanes 7 to12, susceptible strains. Lanes: 1, strain ATTC 33400; 2, Spanish strain 456; 3,1485; 4, 1531; 5, 1679; 6, 1674; 7, 1089; 8, 1516; 9, 1114; 10, 1659; 11, 1261; 12,1370; 13, 1672.

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resistant clinical isolates of Streptococcus pneumoniae. Mol. Microbiol.5:1993–2002.

15. Lefevre, J. C., G. Faucon, A. M. Sicard, and A. M. Gasc. 1993. DNAfingerprinting of Streptococcus pneumoniae strains by pulsed-field gel elec-trophoresis. J. Clin. Microbiol. 31:2724–2728.

16. Markiewicz, Z., and A. Tomasz. 1989. Variation in penicillin binding proteinpatterns of penicillin-resistant clinical isolates of pneumococci. J. Clin. Mi-crobiol. 27:405–410.

17. Martin, C., C. Sibold, and R. Hakenbeck. 1992. Relatedness of penicillin-binding protein 1a genes from different clones of penicillin-resistant Strep-tococcus pneumoniae isolated in South Africa and Spain. EMBO J. 11:3831–3836.

18. McDougal, L. K., R. Facklam, M. Reeves, S. Hunter, J. M. Swenson, B. C.Hill, and F. C. Tenover. 1992. Analysis of multiply antimicrobial-resistantisolates of Streptococcus pneumoniae from the United States. Antimicrob.Agents Chemother. 36:2176–2184.

19. Munoz, R., T. J. Coffey, M. Daniels, C. G. Dowson, G. Laible, J. Casal, R.Hakenbeck, M. Jacobs, J. M. Musser, B. G. Spratt, and A. Tomasz. 1991.Intercontinental spread of a multiresistant clone of serotype 23F Streptococ-cus pneumoniae. J. Infect. Dis. 164:302–306.

20. Reichmann, P., E. Varon, E. Gunther, R. R. Reinert, R. Luttikens, A. Mar-

ton, P. Geslin, J. Wagner, and R. Hakenbeck. 1995. Penicillin-resistant Strep-tococcus pneumoniae in Germany: genetic relationship to clones from otherEuropean countries. J. Med. Microbiol. 43:377–385.

21. Sibold, C., J. Wang, J. Henrichsen, and R. Hakenbeck. 1992. Genetic rela-tionships of penicillin-susceptible and -resistant Streptococcus pneumoniaestrains isolated on different continents. Infect. Immun. 60:4119–4126.

22. Smith, A. M., K. P. Klugman, T. J. Coffey, and B. G. Spratt. 1993. Geneticdiversity of penicillin-binding protein 2B and 2X genes from Streptococcuspneumoniae in South Africa. Antimicrob. Agents Chemother. 37:1938–1944.

23. Soares, S., K. G. Kristinsson, J. M. Musser, and A. Tomasz. 1993. Evidencefor the introduction of a multiresistant clone of serotype 6B Streptococcuspneumoniae from Spain to Iceland in the late 1980s. J. Infect. Dis. 168:158–163.

24. Versalovic, J., V. Kapur, E. O. Mason, U. Shah, T. Kœuth, J. R. Lupski, andJ. M. Musser. 1993. Penicillin-resistant Streptococcus pneumoniae strainsrecovered in Houston: identification and molecular characterisation of mul-tiple clones. J. Infect. Dis. 167:850–856.

25. Zighelboim, S., and A. Tomasz. 1980. Penicillin-binding protein of multiplyantibiotic-resistant South African strains of Streptococcus pneumoniae. An-timicrob. Agents Chemother. 17:434–442.

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