Measuring autofluorescence spectral signatures for detecting antibiotic-resistant bacteria using Bigfoot spectral flow cytometer

Application of flow cytometry to microbiology has been limited due to inadequate availability of bacterial-specific stains, expensive antibody-based fluorophores, ineffective stain cell permeability and challenges in differentiating bacterial cells from cell debris due to their similarity and their small size. Moreover, conventional staining methods require multiple washing steps, limiting sensitivity due to bacterial cells' significantly smaller volume compared to eukaryotic cells. Additionally, most flow cytometers are ill-equipped to handle pathogenic organisms. However, autofluorescence-based detection offers a promising alternative. By leveraging the inherent fluorescence of bacterial cells, the need for extensive washing steps is circumvented, reducing both time and cost. Research indicates that bacterial autofluorescence is primarily associated with specific metabolic components such as Flavin Adenine Dinucleotide (FAD) and Nicotinamide Adenine Dinucleotide (NAD).This study introduces a novel approach to differentiate between clinically isolated antibiotic-resistant and non-resistant bacteria using their autofluorescence spectral signatures. Employing the Bigfoot spectral cytometer, uniquely outfitted with a biosafety cabinet and multiple lasers and fluorescence detectors, bacterial autofluorescence signatures were captured. In a proof-of-concept experiment, E. coli and Salmonella sp. were subjected to gentamicin antibiotics, resulting in increased autofluorescence at distinct excitation and emission wavelengths compared to non-stressed bacteria. Similarly, Methicillin-resistant and susceptible Staphylococcus aureus (MRSA and MSSA) stressed with oxacillin exhibited differential autofluorescence responses, with MSSA showing increased autofluorescence after exposure to oxacillin, while MRSA did not display such changes. These findings suggest that antibiotic-resistant strains can be rapidly identified by monitoring changes in autofluorescence signatures within hours. The label-free, quantitative, and resistant-specific autofluorescence signatures obtained from the Bigfoot spectral flow cytometer hold promise for rapid detection of antibiotic-resistant strains.