Phenotypic and Biochemical Comparison of the Carbapenem-Hydrolyzing Activities of Five Plasmid-Borne AmpC β-Lactamases

Abstract

The CMY-2, ACT-1, DHA-1, ACC-1, and FOX-1 enzymes are representative of five plasmid-mediated AmpC (pAmpC) ␤-lactamase clusters. Resistance to imipenem has been reported in Enterobacteriaceae as a result of pAmpC expression combined with decreased outer membrane permeability. The aim of this study was to determine the role of different pAmpCs in carbapenem resistance and to define the structure/activity relationship supporting carbapenemase activity. The ampC genes encoding the five pAmpCs and the chromosomal AmpC of Escherichia coli EC6, which was used as a reference cephalosporinase, were cloned and introduced into wild-type E. coli TOP10 and OmpC/OmpF porin-deficient E. coli HB4 strains. The MICs of ␤-lactams for the recombinant strains revealed that CMY-2, ACT-1, and DHA-1 ␤-lactamases conferred a high level of resistance to ceftazidime and cefotaxime once expressed in E. coli TOP10 and reduced significantly the susceptibility to imipenem once expressed in E. coli HB4. In contrast, FOX-1 and ACC-1 enzymes did not confer resistance to imipenem. Biochemical analysis showed that CMY-2 ␤-lactamase and, to a lesser extent, ACT-1 exhibited the highest catalytic efficiency toward imipenem and showed low K m values. A modeling study revealed that the large R2 binding site of these two enzymes may support the carbapenemase activity. Therefore, CMY-2-type, ACT-1-type, and DHA-1-type ␤-lactamases may promote the emergence of carbapenem resistance in porin-deficient clinical isolates.The class C (AmpC) ␤-lactamases constitute a group of enzymes widely distributed in Enterobacteriaceae. They preferentially inactivate narrow-spectrum cephalosporins and, to a lesser extent, expanded-spectrum cephalosporins (ESCs), such as ceftazidime and cefotaxime. Zwitterionic cephalosporins, such as cefepime, and carbapenems, such as imipenem, ertapenem, and meropenem, which penetrate very efficiently through the native outer membrane of Gram negatives and are poor substrates of AmpC ␤-lactamases, remain active in vitro against enterobacterial isolates that overproduce chromosome-encoded cephalosporinase (24).Plasmid-mediated AmpC (pAmpC) ␤-lactamases, which originated from chromosomal AmpC of different Gram-negative bacteria, has emerged since the 1980s (24). They can be divided into five clusters: the Citrobacter freundii cluster, represented by CMY-2, the Enterobacter cluster with MIR-1 and ACT-1, the Morganella morganii group with DHA-1, the Hafnia alvei cluster represented by ACC-1, and the Aeromonas cluster with MOX-1 (also called CMY-1) and FOX-1 enzymes, which constitute two distinct subgroups (24). pAmpC ␤-lactamases, whose constitutive expression often is triggered by strong promoters (29), confer a phenotype of resistance similar to that displayed by chromosomal AmpC-overproducing strains.Modifications of the membrane permeability can markedly change the susceptibility profile of pAmpC-producing isolates. By reducing the antibiotic concentration inside the periplasm, porin change may amplify the ␤-lactamase effects toward wea...

Characterization of KPC, NDM and VIM Type Carbapenem Resistance <i>Enterobacteriaceae</i> from North Eastern, Nigeria

“…The carbapenems, namely: imipenem, meropenem, ertapenem and doripenem became the antimicrobials of last resort used in treating infections due to these highly drug resistant bacteria [2]. These antimicrobial agents became crucial in the management of life threatening healthcare associated and community acquired infections.…”

Multi-locus sequence typing of Escherichia coli isolates with acquired ampC genes and ampC promoter mutations

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Detección de serinocarbapenemasas de clase A y otros mecanismos de resistencia enzimática a β-lactámicos en cepas de enterobacterias con susceptibilidad disminuida a carbapenémicos, aisladas de pacientes de un hospital universitario de Santiago, Chile

“…Estos resultados son consistentes con otros estudios 9,33 . La resistencia en K. pneumoniae, se asoció a BLEE; sin embargo, la ausencia de AmpC, podría estar subestimada, ya que en este estudio no se buscaron AmpC plasmidiales, siendo la CMY-2 en particular, prevalente a nivel mundial, tanto en cepas de pacientes hospitalizados como en ambulatorios 34,35 . Por otra parte, E. cloacae se asocia principalmente a sobrexpresión de AmpC, aunque es posible encontrar cepas con BLEE y AmpC simultáneamente.…”