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Antimicrob Agents Chemother. Mar 2008; 52(3): 1021–1027.
Published online Dec 26, 2007. doi:  10.1128/AAC.01082-07
PMCID: PMC2258499

Highly Variable Penicillin Resistance Determinants PBP 2x, PBP 2b, and PBP 1a in Isolates of Two Streptococcus pneumoniae Clonal Groups, Poland23F-16 and Poland6B-20[down-pointing small open triangle]

Abstract

Penicillin-binding proteins (PBPs) in representatives of two Streptococcus pneumoniae clonal groups that are prevalent in Poland, Poland23F-16 and Poland6B-20, were investigated by PBP profile analysis, antibody reactivity pattern analysis, and DNA sequence analysis of the transpeptidase (TP) domain-encoding regions of the pbp2x, pbp2b, and pbp1a genes. The isolates differed in their MICs of β-lactam antibiotics. The majority of the 6B isolates were intermediately susceptible to penicillin (penicillin MICs, 0.12 to 0.5 μg/ml), whereas all 23F isolates were penicillin resistant (MICs, ≥2 μg/ml). The 6B isolates investigated had the same sequence type (ST), determined by multilocus sequence typing, as the Poland6B-20 reference strain (ST315), but in the 23F group, isolates with three distinct single-locus variants (SLVs) in the ddl gene (ST173, ST272, and ST1506) were included. None of the isolates showed an identical PBP profile after labeling with Bocillin FL and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only one pair of 6B isolates and one pair of 23F isolates (ST173 and ST272) each contained an identical combination of PBP 2x, PBP 2b, and PBP 1a TP domains. Some 23F isolates contained PBP 3 with an apparently higher electrophoretic mobility, and this feature also did not correlate with their STs. The data document a highly variable pool of PBP genes as a result of multiple gene transfer and recombination events within and between different clonal groups.

Streptococcus pneumoniae (the pneumococcus) is a common cause of pneumonia, bacteremia, meningitis, otitis media, and sinusitis; and β-lactam antibiotics, especially penicillins, are the most important drugs in the treatment of these infections. Therefore, the emergence and rapid spread of penicillin-nonsusceptible S. pneumoniae (PNSP) represent a serious public health problem. Resistance to β-lactams in S. pneumoniae arises from alterations in penicillin-binding proteins (PBPs), which are enzymes involved in bacterial cell wall biosynthesis (16). Of the six PBPs, modifications of PBP 2x, PBP 2b, and PBP 1a which result in their decreased affinity for β-lactams are mainly responsible for penicillin resistance in clinical isolates (3). PBP 2x and PBP 2b are primary resistance determinants, and their rearrangements confer low-level resistance (19). The presence of a low-affinity PBP 1a is essential for high-level resistance but requires a modified PBP 2b and/or PBP 2x (33, 38). Since the expanded-spectrum cephalosporins do not interact with PBP 2b (22), only PBP 2x and PBP 1a play a role in resistance to these compounds (33).

The PBP-mediated resistance in S. pneumoniae is the result of intra- and interspecies gene transfer events involving related commensal species, such as Streptococcus mitis and Streptococcus oralis (10, 11, 27, 28). This is apparent by the mosaic structure of the PBP genes in resistant isolates, which contain sequence blocks that differ from those in susceptible isolates by approximately 20%, corresponding to about 10% different amino acids (11, 27, 28). Most of these amino acid changes are due to the fact that they originate from homologous PBPs, and the identification of mutations relevant for resistance represents a major challenge. All mutations whose role in resistance has been confirmed by mutagenesis experiments are located within the penicillin-binding/transpeptidase (TP) domains (2, 4, 19, 30, 31, 36, 37, 42, 43). These domains contain the active centers of these enzymes, as defined by three amino acid motifs: SXXK with the active-site serine, SXN, and KT/SG (15).

The initial study of β-lactam resistance in the pneumococci circulating in Poland indicated a high degree of variability of PBP 2x, PBP 2b, and PBP 1a in PNSP isolates (40), including those of two major clonal groups, identified by the Pneumococcal Molecular Epidemiology Network as Poland23F-16 and Poland6B-20 (29, 35). This observation was based on the results of restriction fragment length polymorphism (RFLP) analysis of the PCR amplicons of the pbp2x, pbp2b, and pbp1a genes (PCR-RFLP). The objective of this study was to characterize the PBPs of representative isolates of these two clonal groups in detail.

MATERIALS AND METHODS

Bacterial strains.

The study isolates were a part of the collection used in an earlier study on the nonsusceptibility of Polish pneumococci to β-lactams; and they presented with various PCR-RFLP patterns for the pbp2x, pbp2b, and pbp1a genes (40). The isolates included reference strains of Poland23F-16 and Poland6B-20 (strains ATCC BAA-343 and ATCC BAA-612, respectively) (35), as well as 11 other PNSP isolates (Table (Table1).1). Of these, seven isolates of serotype 23F represented Poland23F-16, sequence type (ST) 173, or its single-locus variants (SLVs), ST272 and ST1506. Four isolates of serotype 6B represented Poland6B-20, ST315. The serotypes, STs, β-lactam MICs, and PCR-RFLP patterns of the pbp genes of the study isolates were reported previously (40).

TABLE 1.
Selected clinical data, serotypes, STs, and antimicrobial susceptibilities of the S. pneumoniae study isolatesa

Visualization of PBPs with Bocillin FL.

The bacteria were grown at 37°C without aeration in C medium supplemented with 0.2% yeast extract (25). The growth was monitored by nephelometry. Cell pellets, harvested from 6-ml aliquots of cultures of 80 to 100 nephelometry units, were resuspended in 120 to 150 μl of 20 mM sodium phosphate buffer (pH 7.2) with 0.2% Triton X-100. Ten-microliter aliquots were used for labeling with Bocillin FL (Molecular Probes, Eugene, OR) (45). After incubation at 37°C for 30 min with 1.1 μg/ml Bocillin FL, the proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with the buffers and under the conditions reported previously (21, 26) with 5% stacking gels and 7.5% resolving gels. The gels were analyzed with a FluorImager 595 apparatus (Molecular Dynamics, Sunnyvale, CA). For determination of binding affinity, the assay was performed with decreasing amounts of Bocillin FL at the concentrations described in the legend to Fig. Fig.22.

FIG. 2.
SDS-PAGE with the Bocillin FL-binding affinity assay with strain R6 and two selected study isolates. Lanes 1, 1.1 μg/ml of Bocillin FL; lanes 2, 0.36 μg/ml of Bocillin FL; lanes 3, 0.11 μg/ml of Bocillin FL; lanes 4, 0.036 μg/ml ...

Western blots.

The transfer of proteins onto a nylon membrane and immunostaining with mouse monoclonal antibodies (MAbs) anti-PBP 2b (MAbs I6, 410, and 517) and anti-PBP 1a (MAbs 221 and 301), as well as with anti-PBP 1a rabbit antiserum 1220, were performed as described previously (21).

Amplification and sequencing of PBP genes.

Bacterial DNAs were prepared by using a Genomic mini kit (A&A Biotechnology, Gdynia, Poland). The PBP genes were amplified with primers specific for pbp2x, pbp2b, and pbp1a (7, 12, 32). Direct sequencing of the PCR products was performed with consecutive primers and an ABI Prism 3100 Avant genetic analyzer (Applied Biosystems, Foster City, CA). The nucleotide and deduced amino acid sequences were aligned by using Lasergene (version 6.1) software (DNASTAR, Madison, WI) and were compared to the corresponding DNA and amino acid sequences from penicillin-susceptible S. pneumoniae strain R6 (ATCC 27336) and all other sequences deposited in databases.

EMBL nucleotide sequence accession numbers.

The nucleotide sequences of the following genes obtained in this study appear in the EMBL database under the indicated accession numbers: pbp2x, AM748666 to AM748675 for reference strains Poland23F-16 and Poland6B-20 and isolates 2907, k-421, 4134, 187, 40, 2904, k-585, and 909, respectively; pbp2b, AM748676 to AM748680 for reference strains Poland23F-16 and Poland6B-20 and isolates k-421, 187, and k-585, respectively; and pbp1a, AM748681 to AM748684 for reference strains Poland23F-16 and Poland6B-20 and isolates 2907 and 4134, respectively.

RESULTS

PBP profiles and affinity of PBPs for Bocillin FL.

The PBP-Bocillin FL complexes of 11 isolates of the 23F and 6B clonal groups (except for the two reference strains) were visualized by fluorometry (Fig. (Fig.1).1). It should be noted that alterations in the apparent molecular weights of PBPs in resistant isolates are not due to changes in the actual lengths of the proteins but rather to their different amino acid compositions. All isolates contained a PBP pattern distinct from that of susceptible reference strain R6 and from each other. The 23F isolates had highly variable PBP 2x, 2b, and 2a patterns, while they had almost identical PBP 1a and 1b patterns. On the other hand, a high degree of variability for PBP 1a was found in the 6B isolates, and in some of these, including penicillin-intermediate isolates 657 and 2907, the PBP 1a band appeared much farther below the PBP 1b band. Five 23F isolates contained a PBP 3 with a lower electrophoretic mobility, similar to that observed in several other isolates of various serotypes (24, 34, 39).

FIG. 1.
SDS-PAGE of PBPs from the study isolates bound to Bocillin FL. Black dots indicate PBP 1a.

The affinity of the PBPs for Bocillin FL was tested by using decreasing concentrations of the compound between 1.1 μg/ml and 1.1 ng/ml; Fig. Fig.22 shows the results obtained for two selected isolates. The analysis revealed a reduced affinity of PBP 2x in all the study isolates. A similar change was observed for PBP 2b, unless this protein could not be separated from PBP 2a (as shown in Fig. Fig.22 for strain k-421). In the case of PBP 1a, the only isolate which did not demonstrate a significant decrease in affinity was 6B penicillin-intermediate isolate 657 (result not shown). No changes in affinity were observed for PBP 1b, PBP 2a, or PBP 3.

Antigenic variation of PBP 1a and PBP 2b.

The variability of PBP 1a and PBP 2b was further tested by Western blotting (Table (Table2).2). Except for 6B isolate k-421, all isolates reacted with the PBP 1a-specific antibodies in a similar way, despite the variability of PBP 1a observed in the Bocillin FL staining assay. In contrast, four patterns of reactivity were found with anti-PBP 2b antibodies. Three patterns were observed among the 23F isolates, and these correlated with the three STs for all isolates except ST173 isolate k-598, which had the same pattern as the ST272 isolates. All four 6B isolates showed one reactivity pattern that was distinct from those seen in the group of 23F isolates.

TABLE 2.
Selected data for the Bocillin FL-binding and reactivity patterns of anti-PBP antibodies with PBPs and amino acid sequence variants of PBP TP domains in the study isolates

PBP sequences.

Regions corresponding to the TP domains of pbp2x, pbp2b, and pbp1a from all isolates, including the Poland23F-16 and Poland6B-20 reference strains, were sequenced. The pbp2x sequence was determined from nucleotides 796 to 1848 (amino acids 266 to 616), the pbp2b sequence was determined from nucleotides 937 to 2002 (amino acids 313 to 667), and the pbp1a sequence was determined from nucleotides 790 to 1959 (amino acids 264 to 653). Table Table22 shows variants of the deduced amino acid PBP TP domain sequences identified. The comparison of the DNA and amino acid sequences between the study isolates and strain R6 is summarized in Table Table33 and Fig. Fig.33.

FIG. 3.
Alignment of the deduced amino acid sequences of the PBP 2x, PBP 2b, and PBP 1a TP domains of the study isolates compared with the amino acid sequence of S. pneumoniae R6. Only the different amino acid residues are indicated. Vertical numbers indicate ...
TABLE 3.
Nucleotide and amino acid alterations and variants of deduced amino acid sequences of PBP 2x, PBP 2b, and PBP 1a TP domains in the study isolates

Nine PBP 2x, five PBP 2b, and four PBP 1a sequence variants were found, several of which were new. Some variants were identical to those identified earlier at both the amino acid and the nucleotide sequence levels. Variant II of PBP 2x was observed in PNSP isolates from Canada (17), whereas variant VI of PBP 2x was observed in S. pneumoniae 1100-03 (EMBL accession no. DQ056809), whose origin was not reported. In a recent study carried out with S. pneumoniae isolates from the Alexander Project, variants I, IV, VI, and IX of PBP 2x were identified in a group of Polish isolates; and variant IV of PBP 2x was identified in a single isolate from France (44). Variant II of PBP 2b was indistinguishable from those in Canadian (18) and French (5) PNSP isolates, while variant V of PBP 2b was indistinguishable from S. pneumoniae 52328 and S. oralis 5296 from South Africa and the United Kingdom, respectively (13). Variant I of PBP 1a was found before in S. pneumoniae 1100-03 (EMBL accession no. DQ056764) and was recently found in several Polish isolates (44). Variant II of PBP 1a was identical to that in Canadian S. pneumoniae strain 14759; however, a shorter pbp1a fragment of that strain had been analyzed (18).

Identical sets of PBP TP domains were detected in only two pairs of study isolates; these were the 6B reference Poland6B-20 strain and isolate 657 and two 23F isolates, isolates k-598 and 187. The intermediately susceptible Poland6B-20 reference strain and isolate 657 probably had the same set of all PBP TP domains analyzed as intermediate 6B strain 14759 from Canada (17, 18). None of the combinations of the 23F isolates was identified in other studies, including the Alexander Project, which included 11 isolates from Poland (44).

PBP 2x.

Six PBP 2x variants occurred among the 23F isolates and four variants occurred among the 6B isolates, and they formed four sequence homology groups (Tables (Tables22 and and3;3; Fig. Fig.3).3). The first group included variants IV, V, and IX, whose sequences differed from each other by up to five amino acids. These variants were observed in both 23F and 6B isolates. The second group, found only among the 23F isolates, consisted of variants I, VI, VII, and VIII, whose sequences were almost identical in the central region of their mosaic blocks and differed from each other by one to nine changes. The two remaining groups were formed by single variants II and III and were present in 6B penicillin-intermediate isolates 657 and 2907, respectively. The sequence of variant II of PBP 2x contained only 10 differences compared to the sequence of strain R6, whereas the sequences of the other PBP 2x variants differed from the sequence of R6 by 21 to 44 amino acids. Variant III of PBP 2x was exceptional in that its sequence was highly similar (four amino acid differences) to that of PBP 2x of the susceptible S. mitis M3 strain (41), initially described as S. oralis but recently reidentified by molecular approaches (6, 20).

Except for variant III, all PBP 2x TP domains contained mutations within the S337TMK motif: T338A in variant II, T338P in the first homology group (variants IV, V, and IX), and T338AM339F in the second group (variants I, VI, VII, and VIII). These changes have been shown to be important in resistance by determination of the acylation efficiency of PBP 2x mutants and by structural analysis (1, 4, 31, 43). The substitutions L364F, I371T, R384G, M400T, Y595F, and N605T have also been implicated in penicillin resistance (1, 4, 31, 43); and all these occurred in the second homology group. The A369V substitution, described as a resistance determinant as well (1, 4, 31, 43), occurred in the three remaining groups, and A369T was present in variant VIII of PBP 2x. A369V was the only critical substitution among those that differentiated variant III from PBP 2x of the susceptible S. mitis M3 strain.

PBP 2b.

Of the five PBP 2b variants identified (Tables (Tables22 and and3;3; Fig. Fig.3),3), three were observed only in the group of 23F isolates (variants I, IV, and V). Their distribution correlated with the STs of these isolates except for ST173 isolate k-598, which, similarly to the ST272 isolates, carried variant IV of PBP 2b. Variant II of PBP 2b dominated among the 6B isolates, in which only isolate k-421 contained the unique variant III. Variants I and II of PBP 2b had six or seven consecutive alterations between residues 426 and 432, characteristic of the so-called class A PBP 2b (1, 4, 31, 43). They contained relatively short mosaic blocks, and their overall difference from PBP 2b of strain R6 was lower (3.6 to 5.7% at the DNA level) than the differences among the remaining sequences identified (10.5 to 17.3%). Variant IV of PBP 2b differed from variant V by 13 substitutions located inside a block of about 70 amino acids, identified before in resistant S. mitis strain B22 (EMBL accession no. AY187721). All PBP 2b variants had the T446A substitution adjacent to the S443SN motif, which has been found to be important for resistance (19, 36).

PBP 1a.

Variant I of PBP 1a was uniformly present in the group of 23F isolates, whereas three others occurred in the group of 6B isolates (variants II, III, and IV) (Tables (Tables22 and and3;3; Fig. Fig.3).3). Variant II was specific for 6B penicillin-intermediate strains, the Poland6B-20 reference strain, and isolate 657 but not for isolate 2907. Its sequence had only one change, E388D, compared to the strain R6 sequence, but E388D is probably not involved in resistance (42). The sequences of all other variants contained a large mosaic block and were similar to each other, differing mostly in their terminal regions. These TP domains contained a four-residue block, N574TGY, shown before to be involved in the decrease in the affinity of PBP 1a for penicillin (8, 42). The only change found in conserved motifs was T371S in variant III, which has been shown to contribute to resistance (42).

DISCUSSION

PNSP isolates of two serotypes and clonal groups were investigated here. The 6B isolates were all representatives of Poland6B-20 (ST315). The 23F isolates encompassed ST173, typical for Poland23F-16, and its two SLVs, ST272 and ST1506. These SLVs differed from ST173 in the ddl gene, which may vary due to a hitchhiking effect driven by pbp2b in gene transfer events (14). Indeed, we observed a good correlation between the STs, the PBP 2b variants, and the PBP 2b antibody reactivity patterns in the group of 23F isolates. As suggested by the PCR-RFLP data (40), despite the close relatedness of the isolates, the PBPs in both clonal groups were highly variable, as shown by their PBP profiles, antibody reactivity patterns, and TP domain sequences. Each isolate had another PBP profile, and only one pair of 6B isolates and one pair of 23F isolates each carried an identical set of the PBP 2x, PBP 2b, and PBP 1a TP domains. This observation is further confirmed by the recent data on 216 S. pneumoniae isolates from the Alexander Project, 11 of which were from Poland (including isolates of ST173, ST272, and ST1506). Even though they shared some PBP 2x and PBP 1a variants with our isolates, none had the same combination of all PBPs analyzed (44). In earlier studies, identical PBP profiles have been used as an indication of clonality (23), and indeed, clones such as Spain23F-1 and Spain6B-2 appear to be rather stable regarding their PBPs (28, 32). However, within the Hungary19A-6 clonal group, a high degree of diversity of PBP genes has been observed (38), and this, together with all the data on Polish isolates, suggests that multilocus sequence typing alone may prove to be not discriminatory enough for clonal structure analyses of certain pneumococcal subpopulations, such as PNSP.

PBP 2x, PBP 2b, and PBP 1a TP domains identical or related to those reported before in S. pneumoniae or commensal streptococci occurred individually in particular isolates, and unique TP variants were also detected. Moreover, identical or similar variants of PBP 2x of the first homology group (variants IV, V, and IX) were observed in both the 6B and the 23F isolates. These results indicate that the variability of PBPs and their combinations in the study isolates resulted from multiple inter- and intraspecies DNA exchange events (10, 11, 27, 28). However, despite the overall heterogeneity, one aspect of PBP stability could be observed as well. The Poland6B-20 reference strain, identified in 1996, likely contained the same PBP 2x, PBP 2b, and PBP 1a sequences as a Canadian S. pneumoniae 6B isolate from 2001 (isolate 14759, which was not typed by multilocus sequence typing) (17, 18). This pattern was the only one in this study that occurred in another 6B isolate (isolate 657), also identified in 2001, and all three of these isolates from Poland and Canada were intermediate to penicillin. These data suggest that this particular “subclone” has been stable over a longer period and has spread widely.

Sequence analysis of the PBP TP domains of the study isolates identified numerous mutations that have previously been shown to contribute to β-lactam resistance (1, 4, 8, 19, 31, 36, 42, 43). The presence of these mutations was mirrored by the reductions in affinity for Bocillin FL of the PBPs analyzed, and they were all probably responsible for the resistance of the isolates. Three of the 6B isolates were intermediate to penicillin, namely, the Poland6B-20 reference strain (not analyzed in the Bocillin FL assays) and isolates 657 and 2907. The Poland6B-20 strain and isolate 657 (penicillin MIC, 0.12 μg/ml) had variant II of PBP 1a, with only one difference in their sequences compared to the sequence of sensitive strain R6; this difference has previously been described as not affecting penicillin susceptibility (42). Consistently, PBP 1a of isolate 657 did not show a significant decrease in affinity for Bocillin FL, and its phenotype was probably due only to PBP 2x and PBP 2b. In isolate 2907 (penicillin MIC, 0.5 μg/ml), PBP 1a had several resistance-related mutations (variant III of PBP 1a). However, this isolate was characterized by the unique variant III of PBP 2x, whose sequence differed from that of PBP 2x of penicillin-susceptible strain S. mitis M3 (41) by only four amino acids including only one resistance-associated mutation, A369V (43). It is possible that this was responsible for the relatively low level of resistance in this isolate.

In summary, this study identified several new mosaic structures of the PBP 2x, PBP 2b, and PBP 1a TP domains in PNSP isolates, as documented earlier for these three resistance determinants (7, 9, 11, 13, 41). The extreme diversity of the individual genes highlights that pbp gene recombination occurs even within particular genetic lineages, suggesting that clonal structure studies with PNSP isolates may require additional analyses of PBP polymorphisms.

Acknowledgments

We are very thankful to M. Nuhn for his excellent help in the bioinformatic analysis of DNA sequences.

This study was partially financed by a FEMS research fellowship (2005-2) to R.I., EU (LSHM-CT-2003-503413) and Forschungsschwerpunkt Biotechnologie grants to the University of Kaiserslautern, and Polish Ministry of Science and Higher Education grant 401 178 32/3572 to the National Medicines Institute in Warsaw.

Footnotes

[down-pointing small open triangle]Published ahead of print on 26 December 2007.

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