Identification of Amino Acids Conferring High-Level Resistance to Expanded-Spectrum Cephalosporins in the penA Gene from Neisseria gonorrhoeae Strain H041

The recent identification of a high-level-ceftriaxone-resistant (MIC = 2 to 4 mu g/ml) isolate of Neisseria gonorrhoeae from Japan (H041) portends the loss of ceftriaxone as an effective treatment for gonococcal infections. This is of grave concern because ceftriaxone is the last remaining option for first-line empirical antimicrobial monotherapy. The penA gene from H041 (penA41) is a mosaic penA allele similar to mosaic alleles conferring intermediate-level cephalosporin resistance (Ceph(i)) worldwide but has 13 additional mutations compared to the mosaic penA gene from the previously studied Ceph(i) strain 35/02 (penA35). When transformed into the wild-type strain FA19, the penA41 allele confers 300- and 570-fold increases in the MICs for ceftriaxone and cefixime, respectively. In order to understand the mechanisms involved in high-level ceftriaxone resistance and to improve surveillance and epidemiology during the potential emergence of ceftriaxone resistance, we sought to identify the minimum number of amino acid alterations above those in penA35 that confer high-level resistance to ceftriaxone. Using restriction fragment exchange and site-directed mutagenesis, we identified three mutations, A311V, T316P, and T483S, that, when incorporated into the mosaic penA35 allele, confer essentially all of the increased resistance of penA41. A311V and T316P are close to the active-site nucleophile Ser310 that forms the acyl-enzyme complex, while Thr483 is predicted to interact with the carboxylate of the beta-lactam antibiotic. These three mutations have thus far been described only for penA41, but dissemination of these mutations in other mosaic alleles would spell the end of ceftriaxone as an effective treatment for gonococcal infections.