RESUMO
OBJECTIVE: To observe the effect of amino acid substitution in conserved sequence of penicillin-binding protein (PBP) 1A, 2B, 2X on antimicrobial activity of beta-lactams against Streptococcus pneumoniae (SP). METHOD: Minimal inhibitory concentration (MIC) of 6 beta-lactams was determined by the E-test in 59 SP strains. The penicillin-binding protein genes pbp1a, 2b, 2x in every SP strain were amplified by nested-polymerase chain reaction (nPCR), then the PCR products were sequenced using automatic genetic analyzer directly. To analyze the amino acid substitutions, the DNA sequences were converted to protein sequences and aligned by Clustalx software. According to amino acid substitution in conserved sequence of PBP2B, 3 phenotypes were observed, including: PBP2B phenotype I (no amino acid substitution); PBP2B phenotype II (Glutamine 432-->Leucine and/or Threonine 445/451-->Alanine/Serine, Glutamic 481-->Glycine, 1 strain had proline insertion between residues 431/432); PBP2B phenotype III (Alanine 624-->Glycine with the addition of phenotype II). According to amino acid substitution in conserved sequence of PBP1A, 3 phenotypes were observed, including: PBP1A phenotype I (no amino acid substitution); PBP1A phenotype II (Threonine 574-->Asparagine, Serine 575-->Threonine, Glutamine 576-->Glycine, Phenylalanine 577-->Tyrosine, 574TSQF-->NTGY); PBP1A III (Threonine 371-->Alanine/Serine, Proline 432-->Threonine with the addition of 574TSQF-->NTGY). According to amino acid substitution in conserved sequence of PBP2X, 4 phenotypes were observed, including: PBP2X phenotype I (no amino acid substitution); PBP2X phenotype II (Histidine 394-->Leucine or Threonine 338-->Alanine); PBP2X phenotype III (Threonine 338-->Alanine, Isoleucine 371-->Threonine, Arginine 384-->Glycine and Leucine 546-->Valine); PBP2X phenotype IV (Methionine 339-->Phenylalanine, Methionine 400-->Threonine with the addition of PBP2X phenotype III). RESULT: Among 59 SP strains antibacterial activities distribution (sensitive strains, intermediate strains and resistant strains) of 6 beta-lactams were penicillin (12, 29, 18); amoxicillin(49, 9, 1); cefuroxime (16, 16, 27); ceftriaxone (47, 1, 11); cefotaxime (47, 3, 9); imipenem (49, 10, 0). beta-lactam antibiotics insensitive strains (intermediate + resistant strain) in PBP2B phenotype III, PBP1A phenotype III, PBP2X phenotype III and IV were significantly increased, the MIC(50) of these strains were significantly higher than that of the others. CONCLUSION: The amino acid substitutions in or vicinal conserved sequence of PBP of SP increase MIC for beta-lactam antibiotics.
