Ambler class C-type β-lactamases in Enterobacter spp. and Klebsiella aerogenes in the Netherlands, 2012-2023

We investigated the genomic epidemiology of Ambler class C (AmpC-type) {beta}-lactamases in Enterobacter spp. 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 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. Considerable proportions of Enterobacter spp. (32%) and K. aerogenes (52%) isolates produced carbapenemase, without detectable major carbapenemase genes (IMP, KPC, NDM, OXA-48-like, VIM), a phenotype termed CIM+Carba-. These isolates were mostly (82%) 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, correlating with chromosome-encoded AmpC-type {beta}-lactamases like blaACT-28, blaACT-52, blaMIR-3, blaMIR-11 or ampC of which the majority (63%) yielded a positive CarbaNP. These CIM+Carba-isolates carried only few plasmids, and there was limited nosocomial spread. CIM+Carba-E. kobei carrying blaACT-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 {beta}-lactamases with putative carbapenemase activity and represent a low-risk for public health. ImportanceCPE represents an important healthcare problem worldwide. This study highlights the diverse genetic Enterobacter spp. population in the Dutch CPE surveillance with E. hormaechei subsp. steigerwaltii as the most common carbapenemase-producing (CIM+Carba+) species. However, a significant proportion of E. kobei, E. roggenkampii, E. ludwigii, and K. aerogenes obtained in the Netherlands carry chromosomal AmpC-type {beta}-lactamases with putative carbapenemase activity in the absence of major carbapenemases (CIM+Carba-), were mostly susceptible for meropenem, and showed limited nosocomial spread. We recommend whole-genome sequencing for accurate Enterobacter/K. aerogenes species classification, and AmpC-type {beta}-lactamase gene identification. Despite limited carbapenem resistance and dissemination, proper infection control measures are necessary. The work outlined here underscores the importance of distinguishing E. kobei, E. roggenkampii, E. ludwigii, and K. aerogenes isolates and its AmpC-type {beta}-lactamases from true CPE by whole-genome sequencing to avoid misclassification and unnecessary infection prevention and public health interventions.