INTRODUCTION

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Streptococcus pneumoniae is a common cause of invasive diseases, such as meningitis and bacteremia, and of respiratory tract infections (10, 15). S. pneumoniae isolates that are resistant to penicillin and/or to non--lactam agents have been reported with increasing frequency worldwide (15). This global increase in antibiotic-resistant and especially of multidrug-resistant (MDR) pneumococci appears, in part, to result from the spread of individual highly resistant pneumococcal clones (1, 4, 5, 12-14). A penicillin-resistant MDR clone of serotype 6B was initially isolated from Spain (14) and then spread to Iceland (20). This clone has been designated the Spain^6B-2 clone (11). Additional clones of antibiotic-resistant serotype 6B pneumococci have been found in Europe, North America, and South Africa (2, 4, 6, 7, 17, 19); it is not clear whether they share a common ancestor or emerged independently.

Recently, penicillin-susceptible MDR serotype 6B pneumococci, resistant to chloramphenicol, tetracycline, erythromycin, clindamycin, and trimethoprim-sulfamethoxazole (SXT) were isolated from young carriers in Greece. Specifically, this notable serotype 6B resistance pattern was described for the first time in the report of a study that was performed between December 1995 and February 1996 in the city of Patras, southwestern Greece (21). Following the initial isolation of pneumococci with this phenotype, between the years 1996 and 1999, isolates with the same characteristics were recovered from young carriers living in various Greek areas (21, 22). In addition, similar serotype 6B penicillin-susceptible MDR pneumococci were isolated from young carriers in Italy and Israel in 1997 to 1998 (23).

The present study was undertaken to investigate, by using restriction fragment end labeling (RFEL) analysis and penicillin-binding protein (PBP) genotyping, the molecular characteristics of the Greek serotype 6B penicillin-susceptible MDR pneumococci and their genetic relatedness with other serotype 6B pneumococcal isolates, both penicillin-susceptible and penicillin-resistant strains, isolated in various parts of the world.

	MATERIALS AND METHODS

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Study population. Penicillin-susceptible MDR pneumococci were isolated from nasopharyngeal cultures obtained from children during two independent studies in Greece (21, 22). The first study was performed with 338 children attending seven day-care centers in the city of Patras, southwestern Greece, during the 2-month period from 19 December 1995 to 15 February 1996. In this study, 20 penicillin-susceptible MDR pneumococcal isolates were recovered from 132 carriers. The second study, the Hellenic Antibiotic-Resistant Respiratory Pathogens' (HARP) Study, was conducted from 10 February 1997 to 10 February 1999. Nasopharyngeal cultures for S. pneumoniae from 2,448 children younger than 24 months old living in central and southern Greece were performed. Ninety-five (3.9%) of the 2,448 children attended day-care centers. In the HARP Study, screening of the children revealed 46 penicillin-susceptible MDR pneumococci.

Bacteriological procedures. Isolation, identification, and susceptibility testing of the Greek S. pneumoniae isolates were performed at the Laboratory of the Division of Pediatric Infectious Disease of the University of Patras as described previously (22). Penicillin and erythromycin MICs for the Greek isolates were determined by the Etest method (AB Biodisk, Solna, Sweden). Susceptibility to clindamycin, chloramphenicol, tetracycline, and SXT was determined by the disk diffusion method (16). Multidrug resistance was defined as resistance to at least three classes of antimicrobial agents. Pneumococci were serotyped at the Centre National de Référence du Pneumocoque, Créteil, France, by latex agglutination using specific antisera prepared by the investigators at the Centre or at the Laboratory of the Division of Pediatric Infectious Disease of the University of Patras by the capsular swelling method using commercially available antisera (Statens Seruminstitut, Copenhagen, Denmark).

International collection of serotype 6B S. pneumoniae strains. Twenty-five serotype 6B penicillin-susceptible or -resistant pneumococci, isolated from various parts of the world, were compared to 41 randomly selected Greek penicillin-susceptible MDR isolates of serotype 6B (Table 1). The international collection of serotype 6B pneumococci included strains from Thailand (n = 6), the United States (n = 5), The Netherlands (n = 4), Iceland (n = 3), Poland (n = 3), Spain (n = 1), South Africa (n = 1), Finland (n = 1), and Cuba (n = 1) (7).

RFEL typing. Pneumococcal strain typing by RFEL was done as described by van Steenbergen et al. (24) and adapted by Hermans et al. (8, 9). Briefly, purified pneumococcal DNA was digested by the restriction enzyme EcoRI. The DNA restriction fragments were end labeled at 72°C with [-³²P]dATP using DNA polymerase (Goldstar; Eurogentec, Seraing, Belgium). After the radiolabeled fragments were denatured and separated electrophoretically on a 6% polyacrylamide sequencing gel containing 8 M urea, the gel was transferred onto filter paper, vacuum dried (HBI, Saddlebrook, N.Y.), and exposed for various times at room temperature to ECL hyperfilm (Amersham Laboratories, Amersham, United Kingdom).

PBP genotyping. Genetic polymorphism of the penicillin resistance genes pbp1a, pbp2b, and pbp2x of the penicillin-resistant isolates was investigated by restriction fragment length polymorphism (RFLP) analysis as described previously (9). The different PBP genotypes received a three-number code (i.e., 6-12-34), referring to the RFLP patterns of the genes pbp1a (6), pbp2b (12), and pbp2x (34), respectively (18).

Computer-assisted analysis of DNA band patterns. RFEL autoradiographs were converted to images (Image Master DTS; Pharmacia Biotech, Uppsala, Sweden) and analyzed by computer (Windows version Gelcompar software, version 4; Applied Mathematics, Kortrijk, Belgium). DNA fragments were analyzed as described previously (9). For evaluation of the genetic relatedness of the isolates, we used the following definitions: (i) strains of a particular RFEL type are 100% identical by RFEL analysis; (ii) an RFEL cluster represents a group of RFEL types that differ in only one band (>95% [approximate] genetic relatedness); and (iii) an RFEL lineage represents a group of RFEL types that differ in fewer than four bands (>85% [approximate] genetic relatedness).

	RESULTS

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Epidemiology of the Greek penicillin-susceptible MDR pneumococci. Of the 66 penicillin-susceptible MDR pneumococci isolated during the two studies in Greece, 79% displayed capsular type 6B. These serotype 6B isolates were invariably susceptible to penicillin and resistant to erythromycin and clindamycin, while 98, 94, and 92% of them were resistant to tetracycline, chloramphenicol, and SXT, respectively.

RFEL analysis. DNA fingerprinting of 41 penicillin-susceptible MDR 6B pneumococci from Greece and an international group of 25 serotype 6B pneumococcal strains revealed 34 distinct types (Fig. 1 and Table 1). The 41 randomly selected Greek isolates represented a total of nine RFEL types. Forty (98%) of the 41 isolates were strongly related, representing a single lineage (genetic relatedness, 91%). RFEL analysis showed six clusters of strains. Only one strain had a unique type that was genetically unrelated to those of the other isolates (genetic relatedness, <80%). There was no correlation between the slightly different resistance patterns and the distinct genotypes (Table 1).

View larger version (61K): [in this window] [in a new window]   FIG. 1.   Dendrogram depicting genetic distances and overall relatedness of 6B pneumococcal isolates on the basis of RFLP data. Numbers of base pairs of the single-stranded DNA are indicated above the banding patterns. See Table 1 for the characteristics of the isolates.

PBP genotyping. All Greek serotype 6B isolates harbored PBP types which were clearly distinct from those of the penicillin-resistant isolates. They invariably displayed a penicillin-susceptible PBP 1A-2B-2X genotype, namely, 2-2-71 (Table 1).

	DISCUSSION

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The molecular and epidemiological factors that control the emergence of antibiotic resistance, increase in resistance levels, and the geographic spread of antibiotic-resistant pneumococci are not fully understood. The present study investigated the emergence and spread of antibiotic resistance in S. pneumoniae strains of serotype 6B.

In Greece, serotype 6B is currently the leading capsular type among the penicillin-susceptible, non--lactam-resistant pneumococci isolated from young carriers, representing 46% of these isolates (references 21 and 22 and our unpublished data). Seventy-three percent of these serotype 6B pneumococci were resistant to four or five classes of non--lactam agents. The penicillin-susceptible pneumococci of serotype 6B that are resistant to several non--lactams have to be considered potentially epidemic, since between the years 1996 and 1999, they were recovered from a significant number of carriers living in various areas of Greece. These pneumococci were isolated from children both attending day-care centers and cared for at home.

The relative importance of the clonal spread of strains and horizontal spread of resistance genes depends on the extent of recombination between chromosomal genes in nature. We used the combination of a method that indexes the overall genetic relatedness of isolates and one that can assess whether the isolates carry the same penicillin resistance determinants (altered PBP genes) to distinguish the clonal spread of strains from the horizontal spread of resistance genes.

RFEL fingerprinting showed that the Greek serotype 6B penicillin-susceptible MDR pneumococcal isolates indicate the spread of a single clone. Another molecular technique, BOX PCR fingerprinting, was also tested (data not shown), but it did not increase the discriminatory power. All Greek isolates displayed an identical penicillin-susceptible PBP 1A-2B-2X genotype, namely, 2-2-71. This PBP type has been previously described for penicillin-susceptible pneumococci after application of the same molecular analysis (18).

A comparison of the RFEL patterns of the Greek serotype 6B isolates with those of serotype 6B pneumococci recovered from diverse geographic locales has brought to light a notable finding: the close relatedness between the Greek and Icelandic serotype 6B isolates. Since the Greek penicillin-susceptible MDR serotype 6B isolates are closely related to the penicillin-resistant MDR serotype 6B pneumococci that spread from Spain to Iceland in the late 1980s, these results suggest that the penicillin-susceptible MDR 6B clone that was found throughout Greece represents the ancestor of one of the three most virulent, internationally disseminated, penicillin-resistant MDR clones, the Spain^6B-2 clone (11, 14).

Another molecular technique, multilocus sequence typing (3), has demonstrated that the Greek penicillin-susceptible MDR serotype 6B pneumococcal isolate GR 890, representative of the RFEL cluster of isolates that match their Spanish ancestor 6B, is genetically related to the Spain^6B-2 clone but is not identical (five of seven loci are identical). In addition, isolate GR 890 is closely related to a Finnish penicillin-resistant MDR serotype 6B strain (six of seven loci are identical). The Finnish strain is related to the Spain^6B-2 clone, with five of seven loci being identical (B. G. Spratt, personal communication).

Our findings suggest that the penicillin-susceptible MDR 6B clone that was found in Greece represents the ancestor of the penicillin-resistant MDR Spanish-Icelandic clone. An alternative hypothesis to explain these findings is that a Spanish-Icelandic strain restored penicillin susceptibility by mutations and that this genetic event gave rise to the Greek isolates. However, restoration of penicillin susceptibility through reacquisition of penicillin-susceptible PBP genes is a process that, in a natural setting, has not been observed until now.

The clinical implication of the significant spread of this penicillin-susceptible, MDR 6B clone in Greece is important, as it limits the available options for the therapy of pneumococcal infection, particularly in penicillin-allergic patients. In addition, caution should be directed to the fact that penicillin-susceptible MDR 6B strains are not identified when the susceptibility testing is performed by screening only for penicillin susceptibility status. Clinical microbiologists and infectious disease specialists have to be aware that, apart from widely spread penicillin-resistant pneumococci, other MDR, penicillin-susceptible pneumococcal strains circulate widely as well.