Structure of ADC-68, a novel carbapenem-hydrolyzing class C extended-spectrum β-lactamase isolated fromAcinetobacter baumannii

Abstract

Outbreaks of multidrug-resistant bacterial infections have become more frequent worldwide owing to the emergence of several different classes of β-lactamases. In this study, the molecular, biochemical and structural characteristics of an Acinetobacter-derived cephalosporinase (ADC)-type class C β-lactamase, ADC-68, isolated from the carbapenem-resistant A. baumannii D015 were investigated. The blaADC-68 gene which encodes ADC-68 was confirmed to exist on the chromosome via Southern blot analysis and draft genome sequencing. The catalytic kinetics of β-lactams and their MICs (minimum inhibitory concentrations) for A. baumannii D015 and purified ADC-68 (a carbapenemase obtained from this strain) were assessed: the strain was resistant to penicillins, narrow-spectrum and extended-spectrum cephalosporins, and carbapenems, which were hydrolyzed by ADC-68. The crystal structure of ADC-68 was determined at a resolution of 1.8 Å. The structure of ADC-68 was compared with that of ADC-1 (a non-carbapenemase); differences were found in the central part of the Ω-loop and the C-loop constituting the edge of the R1 and R2 subsites and are close to the catalytic serine residue Ser66. The ADC-68 C-loop was stabilized in the open conformation of the upper R2 subsite and could better accommodate carbapenems with larger R2 side chains. Furthermore, a wide-open conformation of the R2-loop allowed ADC-68 to bind to and hydrolyze extended-spectrum cephalosporins. Therefore, ADC-68 had enhanced catalytic efficiency against these clinically important β-lactams (extended-spectrum cephalosporins and carbapenems). ADC-68 is the first reported enzyme among the chromosomal class C β-lactamases to possess class C extended-spectrum β-lactamase and carbapenemase activities.

Structural Basis for Carbapenem-Hydrolyzing Mechanisms of Carbapenemases Conferring Antibiotic Resistance

“…Compared with chromosomal enzymes, plasmid-encoded class C β-lactamases are more problematic because they are transmissible to other bacterial species and are often expressed in large amounts [79]. Recently, five class C carbapenemases (namely ACT-1, DHA-1, CMY-2, CMY-10, and ADC-68) have been reported [9,11,12,66]. ACT-1, DHA-1, CMY-2, and CMY-10 are plasmid-encoded class C β-lactamases that exhibit catalytic activity for imipenem [8,80].…”

“…ADCs can hydrolyze cephalosporins such as cefotaxime and ceftazidime but not cefepime or carbapenems. ADC-68 from A. baumannii D015 was the first reported enzyme among chromosomal class C β-lactamases to possess class C extended-spectrum β-lactamase and carbapenemase activities [12]. The catalytic efficiencies ( K cat / K m ) of ACT-1 [8], CMY-2 [8], CMY-10 [80], and ADC-68 [12] for imipenem were 0.007, 0.04, 0.14, and 0.17 µM −1 ·S −1 , respectively.…”

“…In the next deacylation step, the acyl-enzyme adduct is attacked by a water molecule, releasing hydrolyzed antibiotics [86,87]. Crystal structures of class C carbapenemases, CMY-10 and ADC-68, have been determined [12,80]. …”

“…They are plasmid-encoded class C β-lactamases that exhibit the catalytic activity for imipenem [8]. Recently, ADC-68 was identified from carbapenem-resistant A. baumannii D015 [12]. Class D carbapenemases belong to the OXA (oxacillinase) family and were identified in Acinetobacter clinical isolates [13]; they hydrolyze carbapenems weakly and are poorly inhibited by clavulanate [6].…”

Resistência de bactérias isoladas em equipamentos em unidade de terapia intensiva

“…(VRE) and, more recently, extended-spectrum beta-lactamases (ESBL) and carbapenem-resistant Acinetobacter baumannii. (6)(7)(8)(9) Bacterial resistance is natural and unavoidable, (3) but the frequent and undistinguished use of antimicrobials (mainly broad-spectrum drugs) are crucial factors for the development and acceleration of this process. (3,10) Given these facts, the purpose of this study was to isolate and determine the drug resistance profile of bacteria isolated from ICU equipment in a hospital in Caruaru-PE.…”