Amino acid substitutions in mosaic penicillin-binding protein 2 associated with reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae

Abstract

The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

FIG. 1.

Genetic relationship among the 58 clinical isolates. The dendrogram was constructed from the amino acid sequence of porin using the neighbor-joining method. Classification of the strains based on the sequences of PBP1 and PBP2 are shown in parentheses. Strains that were indistinguishable based on the sequences of porin, PBP1, and PBP2 and also on their susceptibilities to antibiotics are boxed. Bar, 0.1 genetic distance.

FIG. 2.

Amino acid sequences of mosaic PBP2 from clinical isolates. The amino acid sequences of the mosaic PBP2 (mosaic-1 to mosaic-4) are aligned with those of penicillin-susceptible strains, LM306 and FA1090. Dashes indicate amino acid residues identical to those of LM306 (GenBank accession no. M32091).