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

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 bla(ACT-28) gene was expressed in a wild-type and in a porin-deficient Escherichia coli background and compared to the bla(ACT-1) gene. Detection of carbapenemase activity was positive only for E. coli expressing the bla(ACT-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.