Whole Genome Sequencing of a Citrobacter freundii Strain Isolated from the Hospital Environment: An Extremely Multiresistant NDM-1 and VIM-48 Coproducing Isolate Raoudha Dziri, 1 Mert Ahmet Kus xkucu, 2 Amani Arfaoui, 1 Meha Fethi, 1 Safa Ifaoui, 1 Ridha Bellaaj, 3 Imene Ouzari, 1 Nes xe Saltog ˘ lu, 2 and Naouel Klibi 1 Citrobacter freundii has acquired resistance to several antimicrobial drugs, including last-resort antibiotics affecting, therefore, clinical efficacy and causing high rates of mortality. In this study, we investigate the whole genome sequence of a carbapenem-resistant C. freundii strain isolated from the hospital environment in Tunisia. A total of 210 samples were taken using sterile swabs, from inanimate surfaces, medical devices, and care staff, during the period extended between March and April 2019. After the microbiological analysis of samples and antimicrobial susceptibility testing, only one strain identified as C. freundii showing resistance to carbapenems was selected for the whole genome sequencing. The genome analysis revealed a high-level resistance to most antibiotics. Interestingly, we have noted the coexistence of bla NDM-1 and bla VIM-48 metallo-b-lactamase (MBL) encoding genes conferring resistance to carbapenems. Other b-lactamases encoding genes have also been detected, including bla TEM-1 , bla CMY-48 , and bla OXA-1 . Moreover, genes conferring resistance to aminoglyco- side [aac(3)-IId, ant(3†)-Ia, aadA, aac(6¢)-Ib], macrolide [mph(A)], sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(D)], chloramphenicol [cat(B3)], rifamycin (arr-3), and quinolone (qnrB) have been revealed. The multi-locus sequence typing analysis showed that this isolate could not be assigned to an existing sequence type (ST), but it is almost identical to ST22. The plasmid investigation revealed the presence of five plasmids belonging to diverse incompatibility groups (IncFII, IncHI1A, IncHI1B, IncN, and IncX3). To the best of our knowledge, our findings report the first detection of NDM-1 and VIM-48 coproducing C. freundii in Tunisia and the second detection in the world of the bla VIM-48 . Keywords: Citrobacter freundii, metallo-beta-lactamases, hospital environment, VIM-48, NDM-1 Introduction C itrobacter freundii is an opportunistic pathogen previously recognized as an environmental contami- nant. 1 This species is now involved in several nosocomial and community infections such as neonatal meningitis, pneumonia, gastroenteritis, and urinary tract infections. 2–4 Because of the high selection pressures in the hospital en- vironment, C. freundii can acquire several resistance genes, constituting, therefore, an important source of hospital in- fections. 5,6 This multiresistance is an emerging threat that could eventually lead to therapeutic deadlocks especially after the emergence of bacteria resistant to last-resort anti- biotics such as the case of carbapenems. 7 This class includes antibiotics with large spectrum bacteri- cidal activity and is kept as a last resort for the treatment of infections by multidrug-resistant bacteria. 8 They are effective against most Enterobacteriaceae species, including those that produce an ampC b-lactamases and extended-spectrum-b- lactamases. In fact, they are often used for the treatment of meningitis, pneumonia, intra-abdominal infections, gynecolog- ical infections, diabetic foot infection, and suspected or proven nosocomial infections with resistant gram-negative bacteria. 9–12 Unfortunately, the immoderate use of these antibiotics in the treatment of severe infections has led to the spread of resistant strains mainly through their acquisition of carba- penemase genes. 13 Several carbapenemase genes have so far been detected worldwide with a predominance of KPC, 1 Laboratory of Microorganisms and Active Biomolecules, Department of Biology, Faculty of Sciences of Tunis, University of Tunis El Manar, Tunis, Tunisia. 2 Department of Infectious Diseases and Clinical Microbiology, _ Istanbul University-Cerrahpas xa, _ Istanbul, Turkey. 3 Service of Hospital Hygiene and Environmental Protection, Military Hospital of Tunis, Tunis, Tunisia. MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2021 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2020.0417 1 Downloaded by 54.234.110.166 from www.liebertpub.com at 09/02/21. For personal use only.