title: Alterations of the penicillin-binding proteins and murM alleles of clinical Streptococcus pneumoniae isolates with high-level resistance to amoxicillin in Spain.
abstract: OBJECTIVE
  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.
authors.affiliation: Departamento de Microbiología, Facultad de Medicina, Universidad Complutense de Madrid, Avda Complutense s/n, 28040 Madrid, Spain.
authors.forename: Fabio
authors.initials: F
authors.lastname: Cafini
authors.forename: Rosa
authors.initials: R
authors.lastname: del Campo
authors.forename: Luis
authors.initials: L
authors.lastname: Alou
authors.forename: David
authors.initials: D
authors.lastname: Sevillano
authors.forename: María Isabel
authors.initials: MI
authors.lastname: Morosini
authors.forename: Fernando
authors.initials: F
authors.lastname: Baquero
authors.forename: José
authors.initials: J
authors.lastname: Prieto
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics; D000658:Amoxicillin / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010403:Penicillin Resistance / Q000235:genetics*; D046915:Penicillin-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D010406:Penicillins / Q000494:pharmacology*; D010453:Peptide Synthases / Q000235:genetics*; D011008:Pneumococcal Infections / Q000382:microbiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013030:Spain / Q000453:epidemiology; D013296:Streptococcus pneumoniae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D010406:Penicillins; C478699:penicillin-binding protein 2a, Streptococcus; C072223:PBP 2x protein, Streptococcus; D000658:Amoxicillin; D019881:Aminoacyltransferases; C478701:penicillin-binding protein 2b, Streptococcus; C408066:MurM protein, Streptococcus pneumoniae; D010453:Peptide Synthases