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. 1996 Apr;40(4):829–834. doi: 10.1128/aac.40.4.829

Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics.

T Grebe 1, R Hakenbeck 1
PMCID: PMC163214  PMID: 8849235

Abstract

High-level resistance to beta-lactam antibiotics in Streptococcus pneumoniae is mediated by successive alterations in essential penicillin-binding proteins (PBPs). In the present work, single amino acid changes in S. pneumoniae PBP 2x and PBP 2b that result in reduced affinity for the antibiotic and that confer first-level beta-lactam resistance are defined. Point mutations in the PBP genes were generated by PCR-derived mutagenesis. Those conferring maximal resistance to either cefotaxime (pbp2x) or piperacillin (pbp2b) were obtained after transformation of the susceptible laboratory strain R6 with the PCR-amplified PBP genes and selection on agar with various concentrations of the antibiotic. In the case of PBP 2x, transformants for which the cefotaxime MIC was 0.16 microgram/ml contained the substitution of a Thr for an Ala at position 550 (Thr550-->Ala), close to the PBP homology box Lys547SerGly, a mutation frequently observed in laboratory mutants and in a high-level cefotaxime-resistant clinical isolate as well. After further selection, transformants resisting 0.3 microgram of cefotaxime per ml were obtained; they contained the substitution Gly550 as the result of two mutations in the same codon. In PBP 2b, Thr446-->Ala, adjacent to another homology box Ser443SerAsn, was the mutation selected with piperacillin. This substitution has been described in all clinical isolates with a low-affinity PBP 2b but was distinct from point mutations found in laboratory mutants. Both pbp2b with the single mutation and a mosaic pbp2b of a clinical isolate conferred a twofold increase in piperacillin resistance. Attempts to select PBP 2b variants at higher piperacillin concentrations were unsuccessful. The mutated PBP 2b also markedly reduced the lytic response to piperacillin, suggesting that such a mutation is an important step in resistance development in clinical isolates.

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Selected References

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  1. Charlier P., Buisson G., Dideberg O., Wierenga J., Keck W., Laible G., Hakenbeck R. Crystallization of a genetically engineered water-soluble primary penicillin target enzyme. The high molecular mass PBP2x of Streptococcus pneumoniae. J Mol Biol. 1993 Aug 5;232(3):1007–1009. doi: 10.1006/jmbi.1993.1452. [DOI] [PubMed] [Google Scholar]
  2. Coffey T. J., Daniels M., McDougal L. K., Dowson C. G., Tenover F. C., Spratt B. G. Genetic analysis of clinical isolates of Streptococcus pneumoniae with high-level resistance to expanded-spectrum cephalosporins. Antimicrob Agents Chemother. 1995 Jun;39(6):1306–1313. doi: 10.1128/aac.39.6.1306. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dowson C. G., Coffey T. J., Kell C., Whiley R. A. Evolution of penicillin resistance in Streptococcus pneumoniae; the role of Streptococcus mitis in the formation of a low affinity PBP2B in S. pneumoniae. Mol Microbiol. 1993 Aug;9(3):635–643. doi: 10.1111/j.1365-2958.1993.tb01723.x. [DOI] [PubMed] [Google Scholar]
  4. Dowson C. G., Hutchison A., Brannigan J. A., George R. C., Hansman D., Liñares J., Tomasz A., Smith J. M., Spratt B. G. Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8842–8846. doi: 10.1073/pnas.86.22.8842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dowson C. G., Hutchison A., Spratt B. G. Extensive re-modelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Streptococcus pneumoniae. Mol Microbiol. 1989 Jan;3(1):95–102. doi: 10.1111/j.1365-2958.1989.tb00108.x. [DOI] [PubMed] [Google Scholar]
  6. Guenzi E., Gasc A. M., Sicard M. A., Hakenbeck R. A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae. Mol Microbiol. 1994 May;12(3):505–515. doi: 10.1111/j.1365-2958.1994.tb01038.x. [DOI] [PubMed] [Google Scholar]
  7. Hakenbeck R., Martin C., Dowson C., Grebe T. Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants. J Bacteriol. 1994 Sep;176(17):5574–5577. doi: 10.1128/jb.176.17.5574-5577.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hakenbeck R. Target-mediated resistance to beta-lactam antibiotics. Biochem Pharmacol. 1995 Oct 12;50(8):1121–1127. doi: 10.1016/0006-2952(95)00158-v. [DOI] [PubMed] [Google Scholar]
  9. Hakenbeck R., Tornette S., Adkinson N. F. Interaction of non-lytic beta-lactams with penicillin-binding proteins in Streptococcus pneumoniae. J Gen Microbiol. 1987 Mar;133(3):755–760. doi: 10.1099/00221287-133-3-755. [DOI] [PubMed] [Google Scholar]
  10. Kuzin A. P., Liu H., Kelly J. A., Knox J. R. Binding of cephalothin and cefotaxime to D-ala-D-ala-peptidase reveals a functional basis of a natural mutation in a low-affinity penicillin-binding protein and in extended-spectrum beta-lactamases. Biochemistry. 1995 Jul 25;34(29):9532–9540. doi: 10.1021/bi00029a030. [DOI] [PubMed] [Google Scholar]
  11. LACKS S., HOTCHKISS R. D. A study of the genetic material determining an enzyme in Pneumococcus. Biochim Biophys Acta. 1960 Apr 22;39:508–518. doi: 10.1016/0006-3002(60)90205-5. [DOI] [PubMed] [Google Scholar]
  12. Laible G., Hakenbeck R. Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae. J Bacteriol. 1991 Nov;173(21):6986–6990. doi: 10.1128/jb.173.21.6986-6990.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laible G., Hakenbeck R. Penicillin-binding proteins in beta-lactam-resistant laboratory mutants of Streptococcus pneumoniae. Mol Microbiol. 1987 Nov;1(3):355–363. doi: 10.1111/j.1365-2958.1987.tb01942.x. [DOI] [PubMed] [Google Scholar]
  14. Laible G., Spratt B. G., Hakenbeck R. Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Mol Microbiol. 1991 Aug;5(8):1993–2002. doi: 10.1111/j.1365-2958.1991.tb00821.x. [DOI] [PubMed] [Google Scholar]
  15. Liu H. H., Tomasz A. Penicillin tolerance in multiply drug-resistant natural isolates of Streptococcus pneumoniae. J Infect Dis. 1985 Aug;152(2):365–372. doi: 10.1093/infdis/152.2.365. [DOI] [PubMed] [Google Scholar]
  16. Martin C., Sibold C., Hakenbeck R. Relatedness of penicillin-binding protein 1a genes from different clones of penicillin-resistant Streptococcus pneumoniae isolated in South Africa and Spain. EMBO J. 1992 Nov;11(11):3831–3836. doi: 10.1002/j.1460-2075.1992.tb05475.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Moreillon P., Tomasz A. Penicillin resistance and defective lysis in clinical isolates of pneumococci: evidence for two kinds of antibiotic pressure operating in the clinical environment. J Infect Dis. 1988 Jun;157(6):1150–1157. doi: 10.1093/infdis/157.6.1150. [DOI] [PubMed] [Google Scholar]
  18. Muñoz R., Coffey T. J., Daniels M., Dowson C. G., Laible G., Casal J., Hakenbeck R., Jacobs M., Musser J. M., Spratt B. G. Intercontinental spread of a multiresistant clone of serotype 23F Streptococcus pneumoniae. J Infect Dis. 1991 Aug;164(2):302–306. doi: 10.1093/infdis/164.2.302. [DOI] [PubMed] [Google Scholar]
  19. Muñoz R., Dowson C. G., Daniels M., Coffey T. J., Martin C., Hakenbeck R., Spratt B. G. Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol Microbiol. 1992 Sep;6(17):2461–2465. doi: 10.1111/j.1365-2958.1992.tb01422.x. [DOI] [PubMed] [Google Scholar]
  20. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sibold C., Henrichsen J., König A., Martin C., Chalkley L., Hakenbeck R. Mosaic pbpX genes of major clones of penicillin-resistant Streptococcus pneumoniae have evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis. Mol Microbiol. 1994 Jun;12(6):1013–1023. doi: 10.1111/j.1365-2958.1994.tb01089.x. [DOI] [PubMed] [Google Scholar]
  22. 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 Oct;60(10):4119–4126. doi: 10.1128/iai.60.10.4119-4126.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Spratt B. G., Pardee A. B. Penicillin-binding proteins and cell shape in E. coli. Nature. 1975 Apr 10;254(5500):516–517. doi: 10.1038/254516a0. [DOI] [PubMed] [Google Scholar]
  24. Tiraby J. G., Fox M. S. Marker discrimination and mutagen-induced alterations in pneumococcal transformation. Genetics. 1974 Jul;77(3):449–458. doi: 10.1093/genetics/77.3.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tuomanen E., Pollack H., Parkinson A., Davidson M., Facklam R., Rich R., Zak O. Microbiological and clinical significance of a new property of defective lysis in clinical strains of pneumococci. J Infect Dis. 1988 Jul;158(1):36–43. doi: 10.1093/infdis/158.1.36. [DOI] [PubMed] [Google Scholar]
  26. Williamson R., Hakenbeck R., Tomasz A. In vivo interaction of beta-lactam antibiotics with the penicillin-binding proteins of Streptococcus pneumoniae. Antimicrob Agents Chemother. 1980 Oct;18(4):629–637. doi: 10.1128/aac.18.4.629. [DOI] [PMC free article] [PubMed] [Google Scholar]

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