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

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 Spain(23F)-1, Spain(6B)-2, Spain(9V)-3 and Spain(14)-5 international clones; however, 8 of the 14 strains belonged to the Spain(9V)-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 >= 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.