Professor Achillefs Kapanidis

Abstract:

The rise of antimicrobial resistance (AMR) is one of the greatest public health challenges, already causing up to 1.2 million deaths annually and rising. Current culture-based turnaround times for bacterial identification in clinical samples and antimicrobial susceptibility testing (AST) are typically 18-24 h. We present a novel proof-of-concept methodological advance in susceptibility testing based on the deep-learning of single-cell specific morphological phenotypes directly associated with antimicrobial susceptibility in Escherichia coli. Our models can reliably (80% single-cell accuracy) classify untreated and treated susceptible cells for a lab-reference fully susceptible E. coli strain, across four antibiotics (ciprofloxacin, gentamicin, rifampicin and co-amoxiclav). For ciprofloxacin, we demonstrate our models reveal significant (p < 0.001) differences between bacterial cell populations affected and unaffected by antibiotic treatment, and show that given treatment with a fixed concentration of 10 mg/L over 30 min these phenotypic effects correlate with clinical susceptibility defined by established clinical breakpoints. Deploying our approach on cell populations from six E. coli strains obtained from human bloodstream infections with varying degrees of ciprofloxacin resistance and treated with a range of ciprofloxacin concentrations, we show single-cell phenotyping has the potential to provide equivalent information to growth-based AST assays, but in as little as 30 min.

Abstract:

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Abstract:

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.