Genetic Approach To Study the Relationship between Penicillin-Binding Protein 3 Mutations and Haemophilus influenzae β-Lactam Resistance by Using Site-Directed Mutagenesis and Gene Recombinants

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Abstract
To clarify the relationship between mutations commonly found for penicillin-binding protein 3 (PBP 3) of β-lactamase-nonproducing ampicillin-resistant (BLNAR) Haemophilus influenzae isolates and β-lactam resistance, single and multiple amino acid mutations at positions 377, 385, 389, 517, and 526 were introduced into PBP 3 of a β-lactam-susceptible Rd strain by site-directed mutagenesis. Twelve isogenic recombinant strains were challenged with nine β-lactam antibiotics. Replacement of the asparagine at position 526 with lysine (N526K) increased the resistance to imipenem eightfold and increased the resistance to various cephalosporins two- to eightfold. Substitution of threonine for serine at position 385 (S385T) and/or substitution of phenylalanine for leucine at position 389 (L389F), in addition to the N526K mutation, led to two- to fourfold additional increases in cephalosporin resistance. An isoleucine-to-methionine substitution at position 377 did not change the antibiotic sensitivity of any of the recombinant strains also carrying other PBP 3 mutations tested. Thirty-six clinical isolates carrying a PBP 3 gene ( ftsI ) with the S385T, L389F, R517H, and/or N526K mutation were chosen from among 279 clinical isolates collected in Japan, and the isolates were grouped into six classes on the basis of the patterns of the four mutations in PBP 3. Rd recombinants were made with each of the ftsI genes. The levels of resistance to β-lactams varied between recombinants of different classes but were comparable for those of the same class. The levels of resistance to cephalosporins of these recombinants were similar to those of the parent clinical isolates, while those to ampicillin and carbapenems were lower. These results indicate that resistance to β-lactams, at least to cephalosporins, depends in large part on the PBP 3 mutations R517H, N526K, S385T, and L389F.

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