A comparison of methods to assess the antimicrobial activity of nanoparticle combinations on bacterial cells

Abstract

Background: Bacterial cell quantification after exposure to antimicrobial compounds varies widely throughout industry and healthcare. Numerous methods are employed to quantify these antimicrobial effects. With increasing demand for new preventative methods for disease control, we aimed to compare and assess common analytical methods used to determine antimicrobial effects of novel nanoparticle combinations on two different pathogens.

Methods: Plate counts of total viable cells, flow cytometry (LIVE/DEAD BacLight viability assay) and qPCR (viability qPCR) were used to assess the antimicrobial activity of engineered nanoparticle combinations (NPCs) on Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria at different concentrations (0.05, 0.10 and 0.25 w/v%). Results were analysed using linear models to assess the effectiveness of different treatments.

Results: Strong antimicrobial effects of the three NPCs (AMNP0-2) on both pathogens could be quantified using the plate count method and flow cytometry. The plate count method showed a high log reduction (>8-log) for bacteria exposed to high NPC concentrations. We found similar antimicrobial results using the flow cytometry live/dead assay. Viability qPCR analysis of antimicrobial activity could not be quantified due to interference of NPCs with qPCR amplification.

Conclusion: Flow cytometry was determined to be the best method to measure antimicrobial activity of the novel NPCs due to high-throughput, rapid and quantifiable results.

Fig 1. Bar graph showing log reduction for a) P. aeruginosa and b) S. aureus for three AMNP nanoparticle composites (AMNP0, 1 and 2) at different NPC concentrations (0.05, 0.10 and 0.25 w/v%) with an antibody control (Ab control).

A log reduction of >8-log shows complete inhibition of bacterial growth. Data is expressed as mean (n = 3)-/+ SD.

Fig 2. Gating strategy used to determine ‘live’ populations of bacteria after exposure to NPCs (stained positive for SYTO9) and ‘dead’ populations of bacteria (stained positive for propidium iodide).

Bacterial populations were gated using positive and negative controls alongside FMO controls. (Gated using FlowJo V10, TreeStar).

Fig 3. Mean (n = 3) flow cytometry data for proportion of live and dead bacterial populations after exposure to NPCs (AMNP0, 1, 2 and antibiotic control (oxytetracycline)) with a) P. aeruginosa and b) S. aureus.

Proportions of live and dead were calculated using the total absolute cell count by the Guava easyCyte flow cytometer which was determined through gating of the live and dead cell populations (FlowJo V10, TreeStar).

Fig 4. Comparison to determine the effectiveness of qPCR-PMA and qPCR only to distinguish between live and dead cells.

P. aeruginosa (a) and S. aureus (b) were exposed to different concentrations of NPCs (0.05, 0.10 and 0.25 w/v %) for 24 hours and an Ab control. qPCR-only and qPCR-PMA was used to detect cell lysis via measurement of ΔCT. ΔCT values below 0 show a decrease in amplification (less DNA, dead cells) and ΔCT values above 0 show an increase in amplification (more DNA, live cells). Data expressed as mean (n = 3) -/+ SD.