Alterations of the penicillin-binding proteins and murM alleles of clinical Streptococcus pneumoniae isolates with high-level resistance to amoxicillin in Spain

J Antimicrob Chemother. 2006 Feb;57(2):224-9. doi: 10.1093/jac/dki442. Epub 2005 Dec 20.


Aims: The aim of this study was to analyse the nucleotide sequences of regions encoding the penicillin-binding domains of pbp1A, pbp2B and pbp2X genes and murM alleles from 14 selected amoxicillin-resistant Streptococcus pneumoniae isolates (MICs 8-16 mg/L) obtained in Spain.

Methods: PFGE and dideoxynucleotide chain termination sequencing were used.

Results: Analysis of PFGE profiles showed that the amoxicillin-resistant S. pneumoniae strains belonged to six different PFGE patterns including the Spain23F-1, Spain6B-2, Spain9V-3 and Spain(14)-5 international clones; however, 8 of the 14 strains belonged to the Spain9V-3 clone. These strains showed the typical changes in penicillin-binding proteins (PBPs) 1A and 2X and had 10 unique changes in the 590-641 region of PBP2B as described previously. Transformation experiments tried to incorporate the transpeptidase domain of PBP2B including the 590-641 region associated with amoxicillin-resistant pneumococci. Sequencing of the pbp2B genes revealed that part of the 3' region of the pbp2B sequence encoding a region of the domain (around amino acid 514-538 to the C terminus of PBP2B) did not recombine with the R6 pbp2B gene. The murM sequence analysis showed that 6, 6 and 2 amoxicillin-resistant S. pneumoniae strains had murMA, murMB5 and murMB6 alleles, respectively. However, strains with murMB5 or murMB6 alleles showed a single mutation (N537D) in the 537-581 region of PBP2B, while strains with the murMA allele had 12 unique changes.

Conclusions: Ten unique changes in the 590-641 region of PBP2B and no specific murM alleles were found in S. pneumoniae strains isolated in Spain with an amoxicillin MIC>or=8 mg/L (MICs from 6 to 12 mg/L by 1 mg/L step dilution). In addition, the presence of specific mutations in PBP2B seems to play a key role in the presence of different murM alleles in these amoxicillin-resistant pneumococcal strains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Aminoacyltransferases / genetics
  • Amoxicillin / pharmacology*
  • Bacterial Proteins / genetics*
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Microbial Sensitivity Tests
  • Molecular Sequence Data
  • Penicillin Resistance / genetics*
  • Penicillin-Binding Proteins / genetics*
  • Penicillin-Binding Proteins / metabolism*
  • Penicillins / pharmacology*
  • Peptide Synthases / genetics*
  • Pneumococcal Infections / microbiology*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Analysis, DNA
  • Spain / epidemiology
  • Streptococcus pneumoniae / drug effects
  • Streptococcus pneumoniae / genetics*
  • Streptococcus pneumoniae / metabolism*
  • Transformation, Bacterial


  • Bacterial Proteins
  • DNA, Bacterial
  • Penicillin-Binding Proteins
  • Penicillins
  • penicillin-binding protein 2a, Streptococcus
  • PBP 2x protein, Streptococcus
  • Amoxicillin
  • Aminoacyltransferases
  • penicillin-binding protein 2b, Streptococcus
  • MurM protein, Streptococcus pneumoniae
  • Peptide Synthases