The molecular epidemiology of non-penicillin-susceptible multidrug-resistant pneumococci in Poland was investigated. Thirty-seven patients who suffered mainly from upper and lower respiratory tract infections (n = 20 and 13, respectively) were enrolled in this study. In addition, three patients suffered from otitis media, a brain abscess, and conjunctivitis, respectively. The clinical diagnosis of one patient was unknown. The medical centers to which the patients were referred were located in 16 Polish towns across the country (Fig. ). The clinical and demographic parameters are listed in Table .
The 37 non-penicillin-susceptible pneumococcal strains were characterized by RFEL analysis and BOX PCR typing. RFEL analysis divided the 37 pneumococcal strains into 16 distinct types (Fig. ; Table ). Eight distinct BOX PCR types were observed, representing 14 subtypes (Fig. ; Table ). When RFEL and BOX PCR analyses were combined, four genetically distinct clusters of strains (designated I, II, III, and IV) were identified. Within these clusters, genetic relatedness of the strains was demonstrated by both RFEL analysis and BOX PCR (sub)typing. Clusters I, II, III, and IV consisted of 12, 3, 11, and 7 strains, respectively. Each cluster was represented by strains that originated from different centers (Table ). The remaining four strains did not fulfill the cluster criteria.
FIG. 2 Genetic relatedness of 37 Polish non-penicillin-susceptible pneumococcal strains based on the RFEL banding patterns of the isolates. The RFEL fingerprints, their relatedness (dendrogram), strain numbers, and genetic cluster codes (Roman numerals) are (more ...)
FIG. 3 DNA fingerprint patterns of the 14 distinct BOX PCR (sub)types represented by the 37 Polish non-penicillin-susceptible pneumococcal strains. Lane numbers indicate BOX PCR type codes. Numbers at the left indicate the sizes of standard DNA fragments in (more ...)
Comparison of the Polish RFEL types with 133 genotypes present in the international RFEL data library representing 15 other countries (13
) revealed that the genetic clusters II and IV matched the Spanish pandemic clone 23F (RFEL type 15) and the Spanish-French international clone 9V (RFEL type 23), respectively (data not shown). The remaining 14 Polish RFEL types did not match any of the 133 non-Polish types present in the international library.
The 37 non-penicillin-susceptible pneumococci from Poland represented seven distinct serotypes (Table ). The most predominant serotypes were 23F (n = 15), 6B (n = 12), and 9V (n = 6). Serotypes 6A, 14, and 19A were all observed once in the Polish collection. Strain 31 displayed a rough phenotype and could not be serotyped. Serotypes 23F, 6B, and 9V included 6, 5, and 2 RFEL types, respectively, whereas the remaining three serotypes (6A, 14, and 19A) were restricted to single RFEL types. The clusters I, II (pandemic clone 23F), and III displayed the serotypes 23F, 23F, and 6B, respectively. Genetic cluster IV (pandemic clone 9V) harbored both serotypes 9V (n = 6) and 14 (n = 1), indicating horizontal transfer of capsular genes within this cluster.
The penicillin MICs of the pneumococcal strains varied from 0.12 to 8.0 μg/ml. Within the genetic clusters, various resistance profiles were observed, ranging from lack of susceptibility to penicillin and doxycycline only to multidrug resistance against penicillins, cefotaxime, co-trimoxazole, doxycycline, chloramphenicol, erythromycin, and lincomycin. Genetic cluster I had the highest penicillin MICs (up to 8 μg/ml), as well as the greatest multidrug resistance (Table ). The lowest penicillin MICs were observed for isolates of genetic cluster III. The resistance patterns of these strains demonstrated their relative susceptibilities to the antimicrobial agents tested. Nevertheless, all members of cluster III were multidrug resistant, i.e., resistant to three or more antibiotics. None of the isolates were resistant to vancomycin, rifampin, and fluoroquinolones.
The penicillin resistance profiles of the pneumococcal strains were investigated in further detail by PBP genotyping. To this end, the penicillin resistance genes pbp1a
, and pbp2x
were subjected to RFLP analysis (Fig. ). The genetic clusters II (pandemic clone 23F) and IV (pandemic clone 9V) included two and three distinct PBP genotypes, respectively (Table ). The polymorphism was restricted to pbp2b
. Similar results were observed for cluster III: there were two distinct PBP genotypes that differed only in the pbp2b
gene. For cluster I, seven distinct PBP genotypes were observed; in contrast to the case for the other three genetic clusters, genetic polymorphism was demonstrated in pbp1a
(two types), pbp2b
(five types), and pbp2x
(four types). Except for clusters II and IV, representing pandemic clones 23F and 9V, respectively, no overlap was observed between the PBP genotypes of the four clusters. Similarly, the four genotypes that were observed only once in the Polish collection also displayed unique PBP genotypes. The polymorphism of the PBP-encoding genes within clusters II and IV was remarkable, as the majority of the pandemic 23F and 9V isolates that are now present in the international data library (51 23F strains from 10 countries [96%] and 31 9V strains from 5 countries [97%]) display PBP genotype 1-1-1 (13
FIG. 4 DNA fingerprint patterns of the pbp1a (n = 5), pbp2b (n = 7), and pbp2x (n = 8) genotypes represented by the 37 Polish non-penicillin-susceptible pneumococcal strains. Lane numbers indicate PBP genotype codes. Numbers at the left indicate the sizes of (more ...)
Comparison of the PBP genotypes of the individual genes pbp1a
, and pbp2x
between the Polish non-penicillin-susceptible strains and 185 Dutch penicillin-susceptible pneumococcal meningitis isolates (15
) revealed the exclusive presence of penicillin-susceptible pbp1a
genotypes among the Polish clusters I, III, and IV. Two strains of cluster IV (pandemic clone 9V; strains 37 and 38 with penicillin MICs of 0.5 and 1.0 μg/ml, respectively) displayed the predominant penicillin-susceptible pbp2b
genotype 2 (Fig. ; Table ). In addition, the majority of the strains belonging to cluster III displayed the predominant penicillin-susceptible pbp1a-pbp2b
genotype 2-2 (penicillin MIC of 0.12 μg/ml). Finally, the majority of cluster I displayed the penicillin-susceptible pbp1a
genotype 6 (penicillin MICs ranging from 2.0 to 8.0 μg/ml); this genotype was observed only once in the collection of 185 Dutch penicillin-susceptible pneumococci.
Among the pbp1a
, and pbp2x
genotypes that are restricted to non-penicillin-susceptible pneumococci (13
), the presence of pbp1a
genotype 1 was observed among clusters II (clone 23F) and IV (clone 9V) (Table ). In addition, the pbp2b
genotypes 1 and 18 and the pbp2x
genotype 1 were observed in three genetic clusters. These data indicate horizontal exchange of penicillin resistance genes between pneumococcal strains of the different Polish clusters.