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The genus Enterobacter belongs to the ESKAPE group, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. This group is characterized by the development of resistance to various antibiotics. In recent years, Enterobacter cloacae (E. cloacae) has emerged as a clinically important pathogen responsible for a wide range of healthcare-associated illnesses. Identifying Enterobacter species can be challenging due to their similar phenotypic characteristics. The emergence of multidrug-resistant E. cloacae is also a significant problem in healthcare settings. Therefore, our study aimed to identify and differentiate E. cloacae using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a fast and precise proteomic analytical technique. We also tested hospital-acquired E. cloacae isolates that produce Extended-spectrum beta-lactamases (ESBL) against commonly used antibiotics for treating urinary tract infections (UTIs). We used a total of 189 E. cloacae isolates from 2300 urine samples of patients with UTIs in our investigation. We employed culturing techniques, as well as the BD Phoenix™ automated identification system (Becton, Dickinson) and Analytical Profile Index (API) system for the biochemical identification of E. cloacae isolates. We used the MALDI Biotyper (MBT) device for peptide mass fingerprinting analysis of all isolates. We utilized the single peak intensities and Principal Component Analysis (PCA) created by MBT Compass software to discriminate and cluster the E. cloacae isolates. Additionally, we evaluated the sensitivity and resistance of ESBL-E. cloacae isolates using the Kirby Bauer method. Out of the 189 E. cloacae isolates, the BD Phoenix system correctly identified 180 (95.24%) isolates, while the API system correctly identified 165 (87.30%) isolates. However, the MBT accurately identified 185 (98.95%) isolates with a score of 2.00 or higher. PCA positively discriminated the identified E. cloacae isolates into one group, and prominent peaks were noticed between 4230 mass-to-charge ratio (m/z) and 8500 m/z. The ESBL-E. cloacae isolates exhibited a higher degree of resistance to ampicillin, amoxicillin-clavulanate, cephalothin, cefuroxime, and cefoxitin. Several isolates were susceptible to carbapenems (meropenem, imipenem, and ertapenem); however, potential future resistance against carbapenems should be taken into consideration. In conclusion, MALDI-TOF MS is a powerful and precise technology that can be routinely used to recognize and differentiate various pathogens in clinical samples. Additionally, the growing antimicrobial resistance of this bacterium may pose a significant risk to human health. [ABSTRACT FROM AUTHOR]