Cefotaxime-non-susceptibility of Haemophilus influenzae induced by additional amino acid substitutions of G555E and Y557H in altered penicillin-binding protein 3

Abstract

Cefotaxime-non-susceptible Haemophilus influenzae has rarely been isolated from clinical specimens. Although several reports have shown that amino acid (AA) alteration in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, reduces activity of cefotaxime, precise mechanisms conferring the non-susceptibility have been unclear. We analyzed the ftsI gene of two clinically isolated cefotaxime-non-susceptible H. influenzae strains, 16-11 and 20-07 (minimum inhibitory concentrations [MICs]: 16 and 8 μg/mL, respectively), and found that their deduced AA sequences of PBP3 included two AA substitutions of G555E and Y557H in addition to previously described AA alterations. To clarify whether the two additional substitutions are requisite for cefotaxime non-susceptibility, we produced transformants of Rd KW20 (cefotaxime MIC: ≤0.06 μg/mL) with the ftsI gene of 16-11. Cefotaxime MICs against transformants M1 and M2, of which deduced PBP3s were altered with that of 16-11 entirely and partially (only the N-terminal side up to the AA position 519), were 8 and 0.25 μg/mL, respectively. We also produced M2-555/7 through site-directed mutagenesis inducing additional substitutions of G555E and Y557H into the PBP3 of M2, against which cefotaxime MIC was 8 μg/mL. These findings show that the additional substitutions of G555E and Y557H in PBP3 with previously described alterations cause cefotaxime non-susceptibility. An additional substitution of either G555E or Y557H alone in altered PBP3 reduced cefotaxime activity but the elevation of MICs were within the category of susceptibility. To our knowledge, this is the first study clarifying a genetic factor in the PBP3 causing cefotaxime non-susceptibility among H. influenzae strains.

Keywords: Amino acid substitution; Cefotaxime; Haemophilus influenzae; Penicillin-binding protein 3.