Validation of a Standard Luminescence Method for the Fast Determination of the Antimicrobial Activity of Nanoparticles in Escherichia coli

Abstract

The use of nanoparticles in multiple industries has raised concerned voices about the assessment of their toxicity/antimicrobial activity and the development of standardized handling protocols. Issues emerge during the antimicrobial assaying of multiple cargo, colorimetric, colloidal nanoformulations, as standard protocols often rely on visual evaluations, or optical density (OD) measurements, leading to high variance inhibitory concentrations (MIC). Thus, a fast, luminescence-based assay for the effective assessment of the antimicrobial activity of nanoparticles is herein reported, using the bioluminescence of an in-house E. coli ATCC® 8739TM construct with the pMV306G13 + Lux plasmid (E. coli Lux). The new strain’s sensitivity to ofloxacin as a standard antibiotic was confirmed, and the methodology robustness verified against multiple nanoparticles and colorimetric drugs. The reduction of incubation from 24 to only 8 h, and the sole use of luminescence (LUX490) to accurately determine and distinguish MIC50 and MIC90, are two main advantages of the method. By discarding OD measurements, one can avoid turbidity and color interferences when calculating bacterial growth. This approach is an important tool that contributes to the standardization of methods, reducing samples’ background interference and focusing on luminescence as a direct probe for bacterial metabolic activity, growth and, most importantly, the correct assessment of nanomaterials’ antimicrobial activity.

Preparation of graft copolymer of chitosan-poly ortho-toluidine for antibacterial properties

“…Using Equation (1) , the inhibition activity (I %) was calculated. I (%) = (OD control − OD sample )/OD control × 100% where OD sample and OD control are OD values at 600 nm of the bacterial supernatant without any treatment and the bacterial supernatant with treatment with different samples, respectively [ 19 ]. Fig.…”

Using Targeted Nano-Antibiotics to Improve Antibiotic Efficacy against Staphylococcus aureus Infections

“…In parallel, we employed a real-time bioluminescence measurement technique to evaluate the antimicrobial activity of nano-antibiotics [ 35 ]. The bioluminescent S. aureus Xen29 strain was generated via integrating the luciferase operon from P. luminescence into the bacterial chromosome [ 36 ].…”

“…Although the bioluminescence method may not fully replace conventional MBC and MBEC determination, it offers several advantages when antibiotic-loaded nano-carriers are evaluated. Indeed, conventional antimicrobial activity assays are susceptible to errors and exhibit a high level of variability, especially in the presence of colloidal nano-particles [ 35 ]. In liquid media, the presence of nano-particles can cause turbidity, making it challenging to accurately measure bacterial growth.…”