Molecular Evolution of {szligbeta}-Lactam-Resistant Haemophilus influenzae: 9-Year Surveillance of Penicillin-Binding Protein 3 Mutations in Isolates from Japan

A total of 621 clinical isolates of Haemophilus influenzae collected in Japan between 1995 and 2003 were studied for their susceptibilities to several antimicrobial agents, {szligbeta}-lactamase production, and amino acid substitutions in penicillin-binding protein 3 (PBP 3). Over the four study periods (first period, 1995 to 1996; second period, 1997 to 1998; third period, 2000 to 2001; fourth period, 2002 to 2003), the susceptibilities to {szligbeta}-lactam agents decreased and the incidence of isolates with substitutions at positions 377, 385, 389, 517, and/or 526 in PBP 3 increased from 28.8% to 52.0%. Five hundred seventy-one {szligbeta}-lactamase-nonproducing isolates were grouped into 18 classes, based on the pattern of the five mutations in PBP 3. The Asp526Lys substitution led to 6.0-, 4.3-, 2.4-, and 5.4-fold increases in amoxicillin-clavulanic acid, cefdinir, cefditoren, and faropenem resistance, respectively. PBP 3 with multiple substitutions (Met377Ile, Ser385Thr, and/or Leu389Phe) together with Asp526Lys resulted in increased resistance compared to that for PBP 3 with the Asp526Lys substitution alone. These results indicate that mutations at these five positions increased resistance to most {szligbeta}-lactams. Although a significant change in the prevalence of {szligbeta}-lactamase-producing strains was not observed, the proportions of those possessing both PBP 3 alterations and {szligbeta}-lactamase production have slightly increased (from 1.4% to 5.0%). The ROB-1 {szligbeta}-lactamase was rare, but this is the first report of this {szligbeta}-lactamase in Japan.