Novel mechanisms of resistance to β-lactam antibiotics in Haemophilus parainfluenzae: β-lactamase-negative ampicillin resistance and inhibitor-resistant TEM β-lactamases

To determine the mechanisms of resistance to -lactam antibiotics in clinical isolates of Haemophilus parainfluenzae. Twenty clinical isolates of H. parainfluenzae with decreased susceptibility to aminopenicillins were examined and compared with a control group of 20 fully susceptible isolates. In this collection, the presence of amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), -lactamase production and the surrounding genetic regions of bla(TEM) genes in selected isolates were analysed. Of the 20 non-susceptible isolates, 8 produced TEM -lactamase (gBLPAR), 7 had mutations in the transpeptidase domain of the ftsI gene related to decreased susceptibility to -lactams (gBLNAR) and 5 had both resistance mechanisms (gBLPACR). No resistance mechanisms were identified in the susceptible control group (gBLNAS). gBLNAR isolates had MIC90 values 4- to 16-fold higher than gBLNAS isolates for ampicillin, amoxicillin/clavulanic acid, cefuroxime, cefotaxime and cefixime, and the most common PBP3 mutation was Asn526Ser. The additional Ser385Thr substitution (III-like group) may confer decreased susceptibility to cefotaxime, cefixime and aztreonam, as in Haemophilus influenzae. In two -lactamase-positive isolates without PBP3 mutations, the inhibitor-resistant TEM (IRT) -lactamases TEM-34 and the novel TEM-182 were detected and carried by a TnA transposon of the Tn2 type; both isolates had an amoxicillin/clavulanic acid MIC of 8 mg/L. The TnA transposons of two -lactamase-positive isolates (TEM-1 and TEM-182) were inserted between the tfc20 and tfc21 genes, typically associated with integrative and conjugative elements in Haemophilus spp.; the TEM-34 IRT -lactamase was harboured in a 5.5 kb plasmid. Clinical isolates of H. parainfluenzae express a variety of aminopenicillin resistance mechanisms, either alone or in combination, including PBP3 modifications, bla(TEM-1) and IRT -lactamase production.