False-positive carbapenem-hydrolyzing confirmatory tests due to ACT-28, a chromosomally-encoded AmpC with weak carbapenemase activity from Enterobacter kobei [Mechanisms of Resistance]

In Enterobacter cloacae complex (ECC), the over-production of the chromosome-encoded cephalosporinase (cAmpC) associated with decreased outer membrane permeability may result in carbapenem resistance. Here, 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, minimal inhibitory concentration and carbapenemase activity was monitored using diverse carbapenem hydrolysis methods. ACT-28 steady state kinetic parameters were determined.

Among 1039 non carbapenemase-producing ECC isolates with decreased susceptibility to carbapenems received in 2016-2017 at the French National Reference Center for antibiotic resistance, only eight displayed 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 a 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. Bla_ACT-28 gene was expressed in a wild type and in a porin-deficient E. coli background and compared to bla_ACT-1 gene. Detection of carbapenemase activity was positive only for E. coli expressing bla_ACT-28 gene. Kinetic parameters of purified ACT-28 revealed a slightly increased imipenem hydrolysis as compared to 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.

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