Research Article Rapid Detection of Beta-Lactamases Genes among Enterobacterales in Urine Samples by Using Real-Time PCR Mariem Yengui, 1 Rahma Trabelsi, 1 Lamia Khannous, 1 Nour Elhouda Mathlouthi, 1 Mohd Adnan , 2 Arif Jamal Siddiqui , 2 Emira Noumi , 2,3 Mejdi Snoussi , 2,4 and Radhouane Gdoura 1 1 Research Laboratory of Environmental Toxicology-Microbiology and Health (LR17ES06), Faculty of Sciences, University of Sfax, Tunisia 2 Department of Biology, College of Science, Hail University, P.O. Box 2440, Hail 81451, Saudi Arabia 3 Laboratory of BioResources: Integrative Biology and Recovery, High Institute of Biotechnology-University of Monastir, Monastir 5000, Tunisia 4 Laboratory of Genetics, Biodiversity and Valorisation of BioResources, High Institute of Biotechnology-University of Monastir, Monastir 5000, Tunisia Correspondence should be addressed to Mejdi Snoussi; snmejdi@yahoo.fr Received 30 January 2022; Revised 1 June 2022; Accepted 27 June 2022; Published 8 August 2022 Academic Editor: Maria T. Mascellino Copyright © 2022 Mariem Yengui et al. This 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. The objective of this study was to develop and evaluate newly improved, rapid, and reliable strategies based on real-time PCR to detect the most frequent beta-lactamase genes recorded in clinical Enterobacterales strains, particularly in Tunisia (bla SHV12 , bla TEM , bla CTX-M-15 , bla CTX-M-9 , bla CMY-2 , bla OXA-48 , bla NDM-1 , and bla IMP ) directly from the urine. Following the design of primers for a specific gene pool and their validation, a series of real-time PCR reactions were performed to detect these genes in 78 urine samples showing high antibiotic resistance after culture and susceptibility testing. Assays were applied to DNA extracted from cultured bacteria and collected urine. qPCR results were compared for phenotypic sensitivity. qPCR results were similar regardless of whether cultures or urine were collected, with 100% sensitivity and specificity. Out of 78 multiresistant uropathogenic, strains of Enterobacterales (44 E. coli and 34 K. pneumoniae strains) show the presence of the genes of the bla group. In all, 44% E. coli and 36 of K. pneumoniae clinical strains harbored the bla group genes with 36.4%, 52.3%, 70.5%, 68.2%, 18.2%, and 4.5% of E. coli having bla SHV-12 , bla TEM , bla CTX-M 15 , bla CTX-M-9 , bla CMY-2 , and bla OXA-48 group genes, respectively, whereas 52.9%, 67.6%, 76.5%, 35.5%, 61.8, 14.7, and 1.28% of K. pneumoniae had bla SHV-12 , bla TEM , bla CTX-M 15 , bla CTX-M-9 , bla CMY-2 , bla OXA-48 , and bla NDM-1 group genes, respectively. The time required to have a result was 3 hours by real-time PCR and 2 to 3 days by the conventional method. Resistance genes of Gram-negative bacteria in urine, as well as cultured bacteria, were rapidly detected using qPCR techniques. These techniques will be used as rapid and cost- effective methods in the laboratory. Therefore, this test could be a good candidate to create real-time PCR kits for the detection of resistance genes directly from urine in clinical or epidemiological settings. 1. Introduction Urinary tract infections (UTIs) are among the most com- mon infectious diseases in humans and represent a real pub- lic health problem in terms of both their frequency and their difficulty of treatment. Enterobacterales is primarily respon- sible for UTIs. Escherichia coli is the main pathogen respon- sible for cystitis and pyelonephritis as well as other species of Enterobacterales, such as Proteus mirabilis and especially Klebsiella pneumoniae [1, 2]. Βeta-lactams are the antibiotics preferentially used against these Enterobacterales. The emergence and spread of these bacteria producing extended-spectrum β-lactamases (ESBLs) or carbapenemases, have become a concern. In fact, Hindawi BioMed Research International Volume 2022, Article ID 8612933, 11 pages https://doi.org/10.1155/2022/8612933