False-Positive Carbapenem-Hydrolyzing Confirmatory Tests Due to ACT-28, a Chromosomally Encoded AmpC with Weak Carbapenemase Activity from Enterobacter kobei

Antimicrob Agents Chemother. 2019 Apr 25;63(5):e02388-18. doi: 10.1128/AAC.02388-18. Print 2019 May.

Abstract

In Enterobacter cloacae complex (ECC), the overproduction of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. In this study, we have characterized ACT-28, a cAmpC with weak carbapenemase activity, from a single Enterobacter kobei lineage. ECC clinical isolates were characterized by whole-genome sequencing (WGS), susceptibility testing, and MIC, and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady-state kinetic parameters were determined. Among 1,039 non-carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only 8 had a positive carbapenemase detection test (Carba NP). These eight ECC isolates were resistant to broad-spectrum cephalosporins due to AmpC derepression, showed decreased susceptibility to carbapenems, and were categorized as carbapenemase-producing Enterobacteriaceae (CPE) according to several carbapenemase detection assays. WGS identified a single clone of E. kobei ST125 expressing only its cAmpC, ACT-28. The blaACT-28 gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the blaACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing the blaACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis compared to that of ACT-1. In silico porin analysis revealed the presence of a peculiar OmpC-like protein specific to E. kobei ST125 that could impair carbapenem influx into the periplasm and thus enhance carbapenem-resistance caused by ACT-28. We described a widespread lineage of E. kobei ST125 producing ACT-28, with weak carbapenemase activity that can lead to false-positive detection by several biochemical and phenotypic diagnostic tests.

Keywords: AmpC; carbapenem hydrolysis; cephalosporinase; dissemination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Carbapenems / metabolism*
  • Carbapenems / pharmacology*
  • Cephalosporinase / genetics
  • Cephalosporinase / metabolism
  • Enterobacter / drug effects*
  • Enterobacter / enzymology
  • Hydrolysis
  • Kinetics
  • Microbial Sensitivity Tests
  • beta-Lactamases / metabolism*

Substances

  • Bacterial Proteins
  • Carbapenems
  • Cephalosporinase
  • beta-Lactamases
  • carbapenemase

Supplementary concepts

  • Enterobacter kobei