Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

20-POS Board 20 DETECTING BACTERIA NANOSCALE INTRACELLULAR MOVEMENT USING SUB-CELLULAR FLUCTUATION IMAGING (SCFI) TECHNIQUE AS A RAPID ANTIBIOTIC SUSCEPTIBILITY TEST (AST)

Kanasanun Phonrat 1 ; Matthew B Avison 2 ; Massimo Antognozzi 1 ; 1 University of Bristol, School of Physics, Bristol, United Kingdom

2 University of Bristol, School of Cellular and Molecular Medicine, Bristol, United Kingdom The gold standard of an Antimicrobial Susceptibility Test (AST) takes 24-48 hours, leading to empirical prescription and mandating the misuse of antibiotics. A rapid AST is crucial to provide optimal treatment and prevent the spreading of antimicrobial resistance. The single-cell imaging technique, Sub-Cellular Fluctuation Imaging (SCFI), is capable of detecting intracellular fluctuations that can be linked to bacterial viability. The method is based on Total Internal Reflection Microscopy (TIRM) and does not require fluorescent labelling. In SCFI, a single bacterium is illuminated using an evanescent field and produces a changing scattering pattern representing the nanoscale motion within the cytoplasm. A 20-second video of a bacterium is recorded and analysed to quantify the SCFI-fluctuations. Here we demonstrate how SCFI fluctuations can be used as a biomarker indicating bacteria susceptibility to an antibiotic. We found that Escherichia coli (E. coli) DH1 treated with 100 µg/mL kanamycin showed significant lower SCFI-fluctuations than a kanamycin resistance isolate derived by E. coli DH1 (p-vale < 0.05, one-way ANOVA). We also found that the bacteriostatic trimethoprim in 20 µg/mL concentration caused a detectable reduction in SCFI-fluctuations in a susceptible E. coli suspension (p-vale < 0.05, one-way ANOVA). Furthermore, methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) exhibited a statistically significant difference in SCFI-fluctuations when exposed to a solution containing 100 µg/mL methicillin (p-vale < 0.05, one-way ANOVA). Additionally, we were able to monitor the SCFI-fluctuations in real-time and observed the effect of the antimicrobial polymyxin B (500 µg/mL) on a single E. coli bacterium. In this case, we observed a significant reduction (> 90%) in SCFI-fluctuations within 10 minutes. These results support the use of the SCFI technique to perform rapid AST on bacteria suspensions containing Gram-positive or Gram-negative bacteria. Further development of SCFI combining machine learning and automation will further reduce time and improve sensitivity.

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