Genomic Epidemiology and Resistome Dynamics of Enterobacter Species in a Portuguese Open Air Laboratory: The Emergence of the FRI-8 Carbapenemase

Pedro Teixeira¹Miguel Ramos¹Rani Rivière¹Mónica Azevedo^1,2,3Mário Ferreira¹Maria Manuela Cano⁴Patrícia Vieira¹Lígia Reis¹Rui Matias¹João Rodrigues¹Carina Menezes⁵Tânia Rosado⁵António Sequeira⁶Olga Moreira⁶Werner Ruppitsch^7,8Adriana Cabal Rosel⁷Solveig Sølverød Mo⁹Elsa Dias^2,3,5Markus Woegerbauer⁷Manuela Caniça^1,10,2,3*Vera Manageiro^1,10,3

• ¹Department of Infectious Diseases, National Institute of Health Dr Ricardo Jorge, Lisbon, Portugal
• ²Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisboa, Lisboa, Portugal
• ³AL4AnimalS, Associate Laboratory for Animal and Veterinary Sciences, Portugal, Lisbon, Portugal
• ⁴Air Quality Laboratory, Department of Health Environmental Health, INSA, Portugal, Lisbon, Portugal
• ⁵Laboratory of Biology and Ecotoxicology, Department of Environmental Health, INSA, Portugal., Lisbon, Portugal
• ⁶Strategic Research Unit for Animal Production and Health, National Institute of Agrarian and Veterinary Research, Lisbon, Portugal
• ⁷Austrian Agency for Health and Food Safety (AGES), Vienna, Vienna, Austria
• ⁸Faculty for Food Technology, Food Safety and Ecology, University of Donja Gorica, Podgorica, Montenegro
• ⁹Department of Animal Health, Welfare and Food Safety, Norwegian Veterinary Institute, Ås, Norway., Ås., Norway
• ¹⁰Centre for the Studies of Animal Science, Institute of Agrarian and Agri-Food Sciences and Technologies, University of Oporto, Porto, Portugal

In this study, we assessed the genomic epidemiology, diversity and antimicrobial resistance (AMR) mechanisms of 61 Enterobacter spp. isolates recovered from interconnected human, animal, plant, and environmental reservoirs of a Portuguese Open Air Laboratory, during an annual longitudinal monitoring study. Whole-Genome Sequencing revealed nine Enterobacter species and 32 sequence types, including 16 novel ones, across nine different compartments (river water, wastewater, soil, manure, feed, air, farmers, pigs, wild animals), reflecting the diversity and ubiquity of Enterobacter species. Core-genome analysis revealed eight genetic clusters, suggesting clonal transmission across compartments. A comprehensive search revealed a diverse collection of 29 antibiotic resistance genes across all isolates. Notably, this study provides the first documentation of blaFRI-harbouring Enterobacterales in European environmental settings and the first to describe blaFRI, blaIMI and mcr-10 in Portugal. blaFRI-8 was detected in all E. vonholyi isolates (n = 17), located on four different IncFII(Yp) plasmids, and blaIMI-6 in an E. asburiae isolate, flanked by IS3 family transposases. Antibiotic susceptibility testing showed that E. vonholyi and the blaIMI-6-harbouring E. asburiae isolate were resistant to carbapenems. A mcr-10.1 gene was identified in an E. roggenkampii isolate on an IncFII(pECLA) plasmid. These plasmids exhibited high sequence similarity with global counterparts, indicating potential for horizontal gene transfer. Other antimicrobial resistance genes included qnrE1, sul1, and aadA2. Overall, this study emphasises the importance of Enterobacter species carrying mobile genetic elements as vectors for AMR and underscores the critical role of environmental compartments in its spread. Our findings also highlight the importance of adopting a One Health approach to fully understand AMR dynamics.