Research Article Molecular Characterization of Gene-Mediated Resistance and Susceptibility of ESKAPE Clinical Isolates to Cistus monspeliensis L. and Cistus salviifolius L. Extracts Imane Zalegh , 1 Mohammed Bourhia , 2 Khalid Zerouali , 3,4 Khalid Katfy, 3 Kaotar Nayme, 5 Farid Khallouki, 6 Ihssane Benzaarate, 7 Ahmad Mohammad Salamatullah , 8 Abdulhakeem Alzahrani , 8 Hiba-Allah Nafidi, 9 Mohamed Akssira , 1 and Rajaa Ait Mhand 1 1 RU Microbiology, Biomolecules and Biotechnology, Laboratory of Chemistry-Physics and Biotechnologies of Biomolecules and Materials, FST Mohammedia, Hassan II University of Casablanca, Casablanca, Morocco 2 Higher Institute of Nursing Professions and Technical Health, Laayoune 70000, Morocco 3 Laboratory of Microbiology-CHU Ibn Rochd Casablanca, Casablanca, Morocco 4 Laboratory of Microbiology, Faculty of Medicine and Pharmacy, University Hassan II Casablanca, Casablanca, Morocco 5 Molecular Bacteriology Laboratory, Institute Pasteur Du Marocco, 1 Place Louis Pasteur, Casablanca 20360, Morocco 6 Biology Department, FSTE, University Moulay Ismail, BP. 609, Errachidia 52000, Morocco 7 Microbiology, Health and Environment Team Research, Department of Biology, Faculty of Sciences, Chouaib Doukkali University, Route Ben Maachou, El Jadida 24000, Morocco 8 Department of Food Science & Nutrition, College of Food and Agricultural Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia 9 Department of Food Science, Faculty of Agricultural and Food Sciences, 2325 University Street Quebec City, Quebec City G1V 0A6, Canada Correspondence should be addressed to Imane Zalegh; zalegh.imane@gmail.com Received 10 May 2022; Accepted 18 August 2022; Published 27 September 2022 Academic Editor: Srinivasan Ramanathan Copyright © 2022 Imane Zalegh et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. Multidrug resistance (MDR) and extensively drug-resistant (XDR) are now the biggest threats to human beings. Alternative antimicrobial regimens to conventional antibiotic paradigms are extensively searched. Although Cistus extracts have long been used for infections in traditional folk medicines around the world, their efficacy against resistant bacteria still needs to be elucidated. We aim to investigate the antibiotic susceptibility profiles of clinical strains Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae (acronym “ESKAPE”), and their resistance mechanisms by PCR, as well as their sensitivity to C. monspeliensis (CM) and C. salviifolius (CS) methanol extracts and their fractions. Methods. Antibiotic susceptibility profile and resistance mechanism were done by antibiogram and PCR. Fractions of CM and CS were obtained using maceration and Soxhlet; their antibacterial activities were evaluated by determining inhibition zone diameter (IZD), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Results. Results revealed that all strains were XDR except S. aureus, which was MDR. e PCR indicates the presence of gene- mediated resistance (bla CTX-M , bla SHV , bla OXA-48 , bla NDM , bla OXA-51 , bla OXA-58 , bla IMP , bla VIM , and bla mecA ). Also, maceration was slightly better for bioactivity preservation. Overall, the extracts of CM (IZD  20 mm, MIC  0.01 mg/mL) were more active than those of CS. All extracts inhibited MRSA (methicillin-resistant Staphylococcus aureus) and ERV (Enterococcus faecium Vancomycin- Resistant) with interesting MICs. e ethyl acetate fraction manifested great efficacy against all strains. Monoterpene hydrocarbons and sesquiterpenes oxygenated were the chemical classes of compounds dominating the analyzed fractions. Viridiflorol was the major compound in ethyl acetate fractions of 59.84% and 70.77% for CM and CS, respectively. Conclusions. e superior activity of extracts to conventional antibiotics was seen for the first time in the pathogens group, and their bactericidal effect could be a promising alternative for developing clinical antibacterial agents against MDR and XDR ESKAPE bacteria. Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2022, Article ID 7467279, 16 pages https://doi.org/10.1155/2022/7467279