A Rapid Growth-Independent Antibiotic Resistance Detection Test by SYBR Green/Propidium Iodide Viability Assay

Abstract

Antibiotic-resistant bacteria have caused huge concerns and demand innovative approaches for their prompt detection. Current antimicrobial susceptibility tests (AST) rely on the growth of the organisms which takes 1–2 days for fast-growing organisms and several weeks for slow growing organisms. Here, we show for the first time the utility of the SYBR Green I/propidium iodide (PI) viability assay for rapidly identifying antibiotic resistance in less than 30 min for major, antibiotic-resistant, fast-growing bacteria, such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii for bactericidal and bacteriostatic agents and in 16 h for extremely rapid detection of drug resistance for isoniazid and pyrazinamide in slow-growing Mycobacterium tuberculosis. The SYBR Green I/PI assay generated rapid and robust results in concordance with traditional AST methods. This novel growth-independent methodology changes the concept of the current growth-based AST and may revolutionize current drug susceptibility testing for all cells of prokaryotic and eukaryotic origin and, subject to further clinical validation, may play a major role in saving lives and improving patient outcomes.

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

“…Further studies with more strains are needed to assess its potential as a rapid DST method. 122 EMB is included in both the first-line regimen and in the short MDR regimen. Several authors have pointed out discrepancies in results obtained in different media and further studies are needed to evaluate suitable CC for EMB.…”

“…More recently, an ultra‐rapid DST method by SYBR Green/propidium iodide viability assay was developed that could determine PZA resistance and other drug resistances in less than 24 h. This approach, used for other bacteria, seems applicable to detect resistance to PZA and other drugs in MTB. Further studies with more strains are needed to assess its potential as a rapid DST method …”

Identification of Genes Regulating Cell Death inStaphylococcus aureus

“…To design our assay, we utilized the heat-ramp assay that has been used to study cell death programs in yeast [17]. To determine viability, we employed both the traditional agar replica plating for visualization of viable growth on solid media but also stained cells with SYBR Green I/PI, a viability stain that can detect both live and dead cells [21, 22]. Using a cidA mutant [15] which has been shown to be death-resistant as a control and the parental strain of USA300 as a death sensitive control, we optimized the condition of our heat-ramp experiment to show the biggest difference between both the death-resistant and death-sensitive phenotypes based on agar plating and the live/dead ratio from viability staining with SYBR Green I/PI.…”

Supplementation of salvianic acid A to boar semen extender to improve seminal quality and antioxidant capacity

“…Sperm plasma membrane integrity and acrosome integrity were assessed at 0, 1, 3, and 5 days during liquid preservation of semen. Sperm plasma membrane integrity was assessed using combined SYBR‐14 and propidium iodide (PI) staining (Feng et al, ). Semen samples (0.1 ml) were placed in a 1.5 ml centrifuge tube in a 37°C water bath for 10 min.…”

“…Sperm plasma membrane integrity was assessed using combined SYBR-14 and propidium iodide (PI) staining (Feng et al, 2018).…”