Analysis of penicillin-binding protein lb and 2a genes from Streptococcus pneumoniae.
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.
Du Plessis
M
M
Department of Clinical Microbiology and Infectious Diseases, South African Institute for Medical Research, Johannesburg. mignondp@hotmail.com
Smith
A M
AM
Klugman
K P
KP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016172:DNA Fingerprinting; D005798:Genes, Bacterial; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010403:Penicillin Resistance / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000494:pharmacology; D010453:Peptide Synthases; D010458:Peptidyl Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011008:Pneumococcal Infections / Q000382:microbiology*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D046915:Penicillin-Binding Proteins; D010406:Penicillins; C478699:penicillin-binding protein 2a, Streptococcus; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D010453:Peptide Synthases