Characterization of the AmpC β-Lactamase from Burkholderia multivorans

Abstract

is a member of the complex, a group of>20 related species of nosocomial pathogens that commonly infect individuals suffering from cystic fibrosis. β-Lactam antibiotics are recommended as therapy for infections due to , which possesses two β-lactamase genes, and PenA is a carbapenemase with a substrate profile similar to that of the carbapenemase (KPC); in addition, expression of PenA is inducible by β-lactams in Here, we characterize AmpC from ATCC 17616. AmpC possesses only 38 to 46% protein identity with non- AmpC proteins (e.g., PDC-1 and CMY-2). Among 49 clinical isolates of , we identified 27 different AmpC variants. Some variants possessed single amino acid substitutions within critical active-site motifs (Ω loop and R2 loop). Purified AmpC1 demonstrated minimal measurable catalytic activity toward β-lactams (i.e., nitrocefin and cephalothin). Moreover, avibactam was a poor inhibitor of AmpC1 ( > 600 μM), 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 ATCC 17616 after growth in subinhibitory concentrations of imipenem (1 μg/ml). AmpC is a unique inducible class C cephalosporinase that may play an ancillary role in compared to PenA, which is the dominant β-lactamase in ATCC 17616.

Resurrecting Old β-Lactams: Potent Inhibitory Activity of Temocillin against Multidrug-ResistantBurkholderiaSpecies Isolates from the United States

“…Strain genotyping was performed using a repetitive element PCR assay as previously described (48). The construction of pBC SK(ϩ) bla penA1 and bla ampC1 , expressed in E. coli DH10B, and pGEX-6p2 bla penA1 and bla ampC1 , expressed in E. coli Origami 2 DE3, was previously described; the genes were cloned from B. multivorans ATCC 17616 (34,36). Antibiotic susceptibility.…”

“…Purification of PenA1 and AmpC1 and steady-state kinetics. PenA1 and AmpC1 were purified as previously described (34,36), and theoretical molecular weight values were determined using the ProtParam tool available through the ExPASy bioinformatics resource portal (51). Steady-state kinetic parameters were determined using an Agilent 8453 diode array spectrophotometer (Santa Clara, CA), as previously described (50,52).…”

“…PenA1 is a carbapenemase with a substrate profile similar to that of KPC-2 (34). Conversely, the AmpC1 of B. multivorans possesses limited ␤-lactamase activity; thus, data to date suggest that this AmpC1 may play a role in peptidoglycan metabolism (36). Expression of these bla genes is regulated by PenR A through a system analogous to the AmpC/AmpR regulatory pathways present in Enterobacteriaceae (33,37).…”

“Switching Partners”: Piperacillin-Avibactam Is a Highly Potent Combination against Multidrug-Resistant Burkholderia cepacia Complex and Burkholderia gladioli Cystic Fibrosis Isolates

“…In Bcc and B. gladioli, the expression of the genes encoding the Pen-like and AmpC ␤-lactamases, bla pen and bla ampC , respectively , is regulated by a LysR-type transcriptional regulator, PenR through a pathway similar to the AmpC/AmpR regulon found in Enterobacteriaceae (13,15). However, comprehensive characterization of these ␤-lactamases, as well as the PenR regulatory pathway, is otherwise lacking, with only PenA1, AmpC1, and PenR A from Burkholderia multivorans ATCC 17616 being well studied to date (13,(16)(17)(18). Adding to the complexity, the bla penA and bla ampC genes are highly heterogeneric among B. multivorans strains with PenA1 to PenA38 and AmpC1 to AmpC28 annotated thus far (whole-genome sequencing was conducted on 50 B. multivorans strains; of these 50 isolates, we identified 38 different PenAs and 28 different AmpCs by primary amino acid sequence comparisons) (16,17).…”

“…However, comprehensive characterization of these ␤-lactamases, as well as the PenR regulatory pathway, is otherwise lacking, with only PenA1, AmpC1, and PenR A from Burkholderia multivorans ATCC 17616 being well studied to date (13,(16)(17)(18). Adding to the complexity, the bla penA and bla ampC genes are highly heterogeneric among B. multivorans strains with PenA1 to PenA38 and AmpC1 to AmpC28 annotated thus far (whole-genome sequencing was conducted on 50 B. multivorans strains; of these 50 isolates, we identified 38 different PenAs and 28 different AmpCs by primary amino acid sequence comparisons) (16,17). The spectrum of activity of PenA2-38 and AmpC2-28 variants is under investigation.…”