Rapid Detection of Beta-Lactamases Genes among Enterobacterales in Urine Samples by Using Real-Time PCR

Abstract

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 (blaSHV12, blaTEM, blaCTX-M-15, blaCTX-M-9, blaCMY-2, blaOXA-48, blaNDM-1, and blaIMP) 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 blaSHV-12, blaTEM, blaCTX-M 15, blaCTX-M-9, blaCMY-2, and blaOXA-48 group genes, respectively, whereas 52.9%, 67.6%, 76.5%, 35.5%, 61.8, 14.7, and 1.28% of K. pneumoniae had blaSHV-12, blaTEM, blaCTX-M 15, blaCTX-M-9, blaCMY-2, blaOXA-48, and blaNDM-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.

Using Quantitative Polymerase Chain Reaction (qPCR) to Identify a Myriad of Carbapenemase Genes in Fresh Cow Dung in Bangladesh

“…We looked into the possibility of identifying significant genes for antibiotic resistance in cow dung samples without culture. Our qPCR system has the advantage of detecting the target ARGs with 100% sensitivity and 100% specificity, as affirmed by earlier studies [36,37].…”

A Multiplex Recombinase‐Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC‐2, and blaNDM‐1 Genes in Klebsiella pneumoniae

“…Colonies obtained from blood plates were suspended in 250 μL of sterile distilled water and heated at 100°C for 10 min. After centrifugation at 15,000 rpm for 5 min, the supernatant containing the harvested DNA was collected and stored at −20°C for use [ 20 ].…”

Levels of Different Microbial Groups on Inert Surfaces of Poultry Slaughterhouses: Identification Using Matrix-Assisted Laser Desorption Ionization Time-of-Flight and Detection of Extended-Spectrum Beta-Lactamase- and Carbapenemase-Producing Enterobacteria

“…The spread of AMR poses a serious threat to public health, as the effective treatment of infections is hampered, leading to increased costs for the health system and increased mortality [14,15]. Beta-lactams are the antibiotics of choice for the treatment of many food-borne infections, so it is essential to know and monitor the prevalence of extended-spectrum beta-lactamases (ESBL) in bacteria of food origin [16]. The prevalence of these enzymes in Enterobacteriaceae isolates from broilers has been studied in many European countries at the abattoir level and has provided further evidence of chicken meat as a potential zoonotic source of ESBL-producing bacteria [17].…”