Characterization of the AmpC β-lactamase from .

Burkholderia multivorans is a member of the Burkholderia cepacia complex, a group of >20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. beta-Lactam antibiotics are recommended as therapy for infections due to B. multivorans, which possesses two beta-lactamase genes, bla(penA) and bla(AmpC). PenA is a carbapenemase with a substrate profile similar to that of the Klebsiella pneumoniae carbapenemase (KPC); in addition, expression of PenA is inducible by beta-lactams in B. multivorans. Here, we characterize AmpC from B. multivorans ATCC 17616. AmpC possesses only 38 to 46% protein identity with non-Burkholderia AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of B. multivorans, we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Omega loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward beta-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 (K-i (app) > 600 mu M), and acyl-enzyme complex formation with AmpC1 was slow, likely due to lack of productive interactions with active-site residues. Interestingly, immunoblotting using a polyclonal anti-AmpC antibody revealed that protein expression of AmpC1 was inducible in B. multivorans ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 mu g/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in B. multivorans compared to PenA, which is the dominant beta-lactamase in B. multivorans ATCC 17616.