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Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae
M Du Plessis1, A M Smith, K P Klugman
1Department of Clinical Microbiology and Infectious Diseases, South African Institute for Medical Research, Johannesburg. mignondp@hotmail.com
Abstract:
Fifty clinical isolates (penicillin MICs, 0.03-8 microg/mL) of Streptococcus pneumoniae were randomly selected from hospitals throughout South Africa, together with seven strains isolated in Hungary (penicillin MICs, 16-32 microg/mL). Penicillin-binding protein (pbp) 1b and 2a genes were amplified by PCR, and the purified DNA was digested with HinfI, StyI, and MseI + DdeI restriction enzymes. The fragments were radioactively end-labeled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. A collection of isolates was further selected for sequence analysis of pbp1b and 2a. DNA fingerprint analysis revealed a uniform profile amongst all isolates for both genes. All isolates revealed a maximum of only seven nucleotide substitutions in their pbp1b genes, resulting in a maximum of three amino acid substitutions in PBP 1B. In the case of the pbp2a gene, up to 13 nucleotide substitutions were observed randomly distributed amongst penicillin-susceptible and resistant isolates, revealing a maximum of five amino acid substitutions in PBP 2A. No amino acid substitutions were found to be common amongst all penicillin-resistant isolates. Transformation experiments with pbp1b and 2a genes isolated from two resistant strains (MICs, 4 and 16 microg/mL) failed to transform pneumococcal strains to increased levels of penicillin resistance. These results show that the pbp1b and 2a genes examined here do not display the typical mosaic gene patterns observed in the pbp2x, 2b, and 1a genes of penicillin-resistant pneumococci. In addition, the transformation studies suggest that PBPs 1B and 2A may not play a role in the development of penicillin resistance in some pneumococci.
Insights
Penicillin-binding protein (PBP) 1b and 2a genes in Streptococcus pneumoniae do not show typical resistance patterns. Transformation studies suggest these PBPs may not contribute to penicillin resistance in some strains.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Streptococcus pneumoniae is a major cause of bacterial infections.
- Penicillin resistance in S. pneumoniae is a growing public health concern.
- Understanding the genetic basis of penicillin resistance is crucial for effective treatment.
Purpose of the Study:
- To investigate the role of penicillin-binding protein (pbp) 1b and 2a genes in penicillin resistance of Streptococcus pneumoniae.
- To analyze the genetic diversity and substitution patterns within pbp1b and 2a genes.
- To determine if these genes contribute to increased penicillin resistance through transformation experiments.
Main Methods:
- DNA fingerprinting using PCR amplification and restriction enzyme digestion (HinfI, StyI, MseI + DdeI).
- Sequence analysis of pbp1b and pbp2a genes.
- Transformation experiments using pbp1b and 2a genes from resistant strains.
Main Results:
- DNA fingerprinting revealed uniform profiles for pbp1b and 2a genes across all isolates.
- Limited nucleotide and amino acid substitutions were observed in pbp1b and 2a genes.
- No common amino acid substitutions were found in penicillin-resistant isolates.
- Transformation experiments failed to confer increased penicillin resistance.
Conclusions:
- The pbp1b and 2a genes do not exhibit the mosaic patterns seen in other penicillin resistance genes.
- PBPs 1B and 2A may not play a significant role in the development of penicillin resistance in some pneumococcal strains.