title: Novel mechanisms of resistance to β-lactam antibiotics in Haemophilus parainfluenzae: β-lactamase-negative ampicillin resistance and inhibitor-resistant TEM β-lactamases.
abstract: OBJECTIVE
  To determine the mechanisms of resistance to β-lactam antibiotics in clinical isolates of Haemophilus parainfluenzae.
  
  
  METHODS
  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), β-lactamase production and the surrounding genetic regions of blaTEM genes in selected isolates were analysed.
  
  
  RESULTS
  Of the 20 non-susceptible isolates, 8 produced TEM β-lactamase (gBLPAR), 7 had mutations in the transpeptidase domain of the ftsI gene related to decreased susceptibility to β-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 β-lactamase-positive isolates without PBP3 mutations, the inhibitor-resistant TEM (IRT) β-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 β-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 β-lactamase was harboured in a ∼5.5 kb plasmid.
  
  
  CONCLUSIONS
  Clinical isolates of H. parainfluenzae express a variety of aminopenicillin resistance mechanisms, either alone or in combination, including PBP3 modifications, blaTEM-1 and IRT β-lactamase production.
authors: Antibiotic Laboratory, Bacteriology Service, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors: S
authors: S
authors: García-Cobos
authors: M
authors: M
authors: Arroyo
authors: J
authors: J
authors: Campos
authors: M
authors: M
authors: Pérez-Vázquez
authors: B
authors: B
authors: Aracil
authors: E
authors: E
authors: Cercenado
authors: B
authors: B
authors: Orden
authors: N
authors: N
authors: Lara
authors: J
authors: J
authors: Oteo
mesh_terms: D000667:Ampicillin / Q000494:pharmacology*; D000668:Ampicillin Resistance; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005838:Genotype; D006192:Haemophilus Infections / Q000382:microbiology; D006193:Haemophilus influenzae / Q000187:drug effects* / Q000201:enzymology* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests; D046915:Penicillin-Binding Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; D000667:Ampicillin; D001618:beta-Lactamases