Abstract
PCR was used to identify penicillin resistance in 1,062 clinical isolates of Streptococcus pneumoniae. Three sets of primers were designed to amplify (i) a 240-bp fragment of the penicillin-binding protein (PBP) 2B gene (pbp2b) of penicillin-susceptible S. pneumoniae (PSSP), (ii) a 215-bp fragment of the class A mutations of the pbp2b gene present in penicillin-resistant S. pneumoniae, and (iii) a 286-bp fragment of the class B mutation. In addition, a set of primers that amplify 273 bp of the autolysin (lytA) gene was applied in combination with the above to identify S. pneumoniae. Of 621 isolates for which MICs of penicillin were < or = 0.06 mu g/ml, 614 (98.9%) were ascertained as having DNA fragments amplified by the PSSP primers. Of 441 isolates for which MICs of penicillin were > or = 0.125 mu g/ml, a class A mutation was detected in only 8 (1.8%), a class B mutation was detected in 310 (70.3%), and neither class A nor class B mutations were found in the remaining 123 (27.9%). However, when analysis was limited to isolates for which MICs of penicillin were > or = 1.0 mu g/ml, 247 isolates (89.8%) of 275 were found to possess a class B mutation. When PBPs were analyzed in 12 isolates with unclear mutations of the pbp2b gene by using [3H]benzylpenicillin, low affinity to PBP 2B was observed in them all. These findings suggest that a pbp2b mutation other than class A or class B is present in these isolates. These results also indicate that it may be possible to identify PSSP and penicillin-resistant S. pneumoniae by applying PCR using a combination of primers to detect the susceptible pbp2b gene, resistant pbp2b gene mutations, and the lytA gene.
Full Text
The Full Text of this article is available as a PDF (300.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Appelbaum P. C. Antimicrobial resistance in Streptococcus pneumoniae: an overview. Clin Infect Dis. 1992 Jul;15(1):77–83. doi: 10.1093/clinids/15.1.77. [DOI] [PubMed] [Google Scholar]
- Asensi F., Pérez-Tamarit D., Otero M. C., Gallego M., Llanes S., Abadia C., Cantó E. Imipenem-cilastatin therapy in a child with meningitis caused by a multiply resistant pneumococcus. Pediatr Infect Dis J. 1989 Dec;8(12):895–895. doi: 10.1097/00006454-198912000-00019. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Dowson C. G., Hutchison A., Spratt B. G. Nucleotide sequence of the penicillin-binding protein 2B gene of Streptococcus pneumoniae strain R6. Nucleic Acids Res. 1989 Sep 25;17(18):7518–7518. doi: 10.1093/nar/17.18.7518. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dowson C. G., Hutchison A., Woodford N., Johnson A. P., George R. C., Spratt B. G. Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5858–5862. doi: 10.1073/pnas.87.15.5858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dowson C. G., Johnson A. P., Cercenado E., George R. C. Genetics of oxacillin resistance in clinical isolates of Streptococcus pneumoniae that are oxacillin resistant and penicillin susceptible. Antimicrob Agents Chemother. 1994 Jan;38(1):49–53. doi: 10.1128/aac.38.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
- García P., García J. L., García E., López R. Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. Gene. 1986;43(3):265–272. doi: 10.1016/0378-1119(86)90215-5. [DOI] [PubMed] [Google Scholar]
- Gillespie S. H., Ullman C., Smith M. D., Emery V. Detection of Streptococcus pneumoniae in sputum samples by PCR. J Clin Microbiol. 1994 May;32(5):1308–1311. doi: 10.1128/jcm.32.5.1308-1311.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hakenbeck R., Tarpay M., Tomasz A. Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother. 1980 Mar;17(3):364–371. doi: 10.1128/aac.17.3.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jabes D., Nachman S., Tomasz A. Penicillin-binding protein families: evidence for the clonal nature of penicillin resistance in clinical isolates of pneumococci. J Infect Dis. 1989 Jan;159(1):16–25. doi: 10.1093/infdis/159.1.16. [DOI] [PubMed] [Google Scholar]
- Laible G., Hakenbeck R., Sicard M. A., Joris B., Ghuysen J. M. Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506. Mol Microbiol. 1989 Oct;3(10):1337–1348. doi: 10.1111/j.1365-2958.1989.tb00115.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Markiewicz Z., Tomasz A. Variation in penicillin-binding protein patterns of penicillin-resistant clinical isolates of pneumococci. J Clin Microbiol. 1989 Mar;27(3):405–410. doi: 10.1128/jcm.27.3.405-410.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Potgieter E., Chalkley L. J. Reciprocal transfer of penicillin resistance genes between Streptococcus pneumoniae, Streptococcus mitior and Streptococcus sanguis. J Antimicrob Chemother. 1991 Sep;28(3):463–465. doi: 10.1093/jac/28.3.463. [DOI] [PubMed] [Google Scholar]
- Rudolph K. M., Parkinson A. J., Black C. M., Mayer L. W. Evaluation of polymerase chain reaction for diagnosis of pneumococcal pneumonia. J Clin Microbiol. 1993 Oct;31(10):2661–2666. doi: 10.1128/jcm.31.10.2661-2666.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith A. M., Klugman K. P. Alterations in penicillin-binding protein 2B from penicillin-resistant wild-type strains of Streptococcus pneumoniae. Antimicrob Agents Chemother. 1995 Apr;39(4):859–867. doi: 10.1128/aac.39.4.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ubukata K., Nakagami S., Nitta A., Yamane A., Kawakami S., Sugiura M., Konno M. Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products. J Clin Microbiol. 1992 Jul;30(7):1728–1733. doi: 10.1128/jcm.30.7.1728-1733.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ubukata K., Yamashita N., Konno M. Occurrence of a beta-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci. Antimicrob Agents Chemother. 1985 May;27(5):851–857. doi: 10.1128/aac.27.5.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Viladrich P. F., Gudiol F., Liñares J., Rufi G., Ariza J., Pallares R. Characteristics and antibiotic therapy of adult meningitis due to penicillin-resistant pneumococci. Am J Med. 1988 May;84(5):839–846. doi: 10.1016/0002-9343(88)90061-7. [DOI] [PubMed] [Google Scholar]
- Virolainen A., Salo P., Jero J., Karma P., Eskola J., Leinonen M. Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear fluid of children with acute otitis media. J Clin Microbiol. 1994 Nov;32(11):2667–2670. doi: 10.1128/jcm.32.11.2667-2670.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang Y., Isaacman D. J., Wadowsky R. M., Rydquist-White J., Post J. C., Ehrlich G. D. Detection of Streptococcus pneumoniae in whole blood by PCR. J Clin Microbiol. 1995 Mar;33(3):596–601. doi: 10.1128/jcm.33.3.596-601.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]