Ambler class C-type β-lactamases and porin alterations in Enterobacter cloacae complex and Klebsiella aerogenes in the Netherlands, 2012–2023

We investigated the genomic epidemiology of Ambler class C (AmpC-type) β-lactamases in Enterobacter cloacae complex and Klebsiella aerogenes in the national carbapenemase-producing Enterobacterales (CPE) surveillance of the Netherlands between 2012 and 2023. A total of 399 E. cloacae complex and K. aerogenes isolates from 399 patients were analyzed using whole-genome sequencing to assess genetic relatedness, resistance genes, porin, and AmpC-regulatory gene profiles, plasmid replicons, and the genomic location of AmpC-genes, respectively. Of the 399 patients, 217 were male (54%), and the median age was 67 years. Carbapenemase production was assessed using the carbapenem inactivation method (CIM) and CarbaNP-test. A considerable proportion of E. cloacae complex (32%) and K. aerogenes (52%) isolates were CIM-positive in the absence of detectable carbapenemase genes (IMP, KPC, NDM, OXA-48-like, VIM) by the CarbaPCR, a phenotype termed CIM + Carba-. These isolates were mostly (81.9%) susceptible (EUCAST ≤ 2mg/L) to meropenem. The majority of CIM + Carba + isolates with major carbapenemase genes were gained from pre-emptive screening, while CIM + Carba- isolates were mainly taken for diagnostic purposes. Genomic analysis identified 18 genogroups, with E. kobei , E. roggenkampii , E. ludwigii, and K. aerogenes showing the CIM + Carba- phenotype, were mostly lacking porins and AmpC regulators, and correlated with chromosome-encoded AmpC-type β-lactamases like bla ACT-28 , bla ACT-52, bla MIR-3 , bla MIR-11 or ampC of which the majority (63%) yielded a positive CarbaNP-test. These CIM + Carba- isolates carried only few plasmids, and there was limited evidence of nosocomial spread. CIM + Carba- E. kobei carrying bla ACT-28 overproduced ACT-28 protein in the CIM. Overall, the Enterobacter and K. aerogenes population in the Netherlands is genetically diverse, with most isolates carrying species-specific AmpC-type β-lactamases. Isolates with low MIC for meropenem that lack porins and major carbapenemases represent a low-risk for public health.