Antimicrobial Susceptibility Test with Plasmonic Imaging and Tracking of Single Bacterial Motions on Nanometer Scale

Abstract

Antimicrobial susceptibility tests (ASTs) are important for confirming susceptibility to empirical antibiotics and detecting resistance in bacterial isolates. Currently, most ASTs performed in clinical microbiology laboratories are based on bacterial culturing, which take days to complete for slowly growing microorganisms. A faster AST will reduce morbidity and mortality rates and help healthcare providers administer narrow spectrum antibiotics at the earliest possible treatment stage. We report the development of a nonculture-based AST using a plasmonic imaging and tracking (PIT) technology. We track the motion of individual bacterial cells tethered to a surface with nanometer (nm) precision and correlate the phenotypic motion with bacterial metabolism and antibiotic action. We show that antibiotic action significantly slows down bacterial motion, which can be quantified for development of a rapid phenotypic-based AST.

Current and emerging techniques for antibiotic susceptibility tests

“…A plasmonic imaging and tracking (PIT) technique has been used to track 3D motions of single bacterial cells associated with metabolic viability, thus leading to rapid AST 58, 59. The PIT setup is built on an inverted optical microscope, where light from a luminescence diode is directed onto the sensor chip made of gold-coated glass film with immobilized bacterial cells.…”

Simultaneous and quantitative monitoring of co-cultured Pseudomonas aeruginosa and Staphylococcus aureus with antibiotics on a diffusometric platform

“…Numerous techniques based on morphological analysis13, fluorescence intensity1415, asynchronous magnetic bead rotation1617, dielectrophoresis18, Raman-enhanced spectroscopy1920, atomic force microscopy21 and surface plasmon resonance imaging22 are developed to improve AST. These techniques use direct quantification factors (including counting by image analysis13, fluorescence intensity1415 and bacterial proliferation volume16) and indirect factors (including morphology1318 medium viscosity16, bacteria-disrupted secretion1920 and bacterial fluctuations originating from metabolism2122) to efficiently determine the antibiotic susceptibility profiles of bacteria.…”

“…These techniques use direct quantification factors (including counting by image analysis13, fluorescence intensity1415 and bacterial proliferation volume16) and indirect factors (including morphology1318 medium viscosity16, bacteria-disrupted secretion1920 and bacterial fluctuations originating from metabolism2122) to efficiently determine the antibiotic susceptibility profiles of bacteria. These techniques require a timeframe of 0.5–4 h to a complete an AST.…”

Identification and Antibiotic‐Susceptibility Profiling of Infectious Bacterial Agents: A Review of Current and Future Trends

“…In other higher resolution tools, multichannel test cassettes are used for real‐time observation with high‐resolution cameras, allowing the direct observation and measurement of bacterial growth. An example of such technology is the multiplexed automated digital microscopy (MADM) . A commercially available device using this technology is the Accelerate Pheno System (Accelerate Diagnostics) that can perform both identification and AST of bacteria and yeast and allows the diagnosis of mono and polymicrobial infections directly from blood‐cultures, dismissing the overnight sub‐culturing step.…”