The evolution of multi-resistant bacteria poses a major threat to our healthcare system worldwide. To address this serious public health issue, the speed and sensitivity of state-of-the-art antibiotic susceptibility testing (AST) such as broth dilution and disc diffusion methods need to be significantly improved to provide better patient care. With the ultimate goal of developing a fast and reliable alternative that outperforms current state-of-the-art procedures, a high-throughput, generally– in terms of bug drug combinations – applicable analytical protocol was developed employing Raman spectroscopy as non-destructive analysis tool to monitor the deuterium uptake of metabolically active bacteria and additionally extract chemical-specific information for bacteria and/or contaminant identification. Our AST is based on two reference strains representing Gram-negative (E. coli) and Gram-positive bacteria (E. faecalis), treated with a total of four different antibiotics. Apart from high sensitivity and specificity, time is the most crucial parameter in clinical diagnosis. Hence, bulk analysis of highly concentrated samples was favored over a single-cell approach to allow timeefficient, straight-forward sample preparation and investigation. The developed AST also comprises a preincubation step (bug-drug incubation prior to the addition of D2O containing medium) which is shown to be key for the development of a reliable test comprising Gram-positive and Gram-negative bacteria. 52 clinical isolates typical for urinary tract infection causing pathogens were investigated in a semi-automated setting showing good agreement with state-of-the-art analytics.
We propose a new method for biochemical sensing using photoacoustic (PA) excitation of gold nanoparticles (GNPs) to achieve real-time detection of transient biomarkers. Our approach is based on the effect of a particle coating on nonlinear PA signal generation combined with our recently-developed serial PA tomographic imaging method. We have shown the ability to image the three-dimensional spatial distribution of GNPs that produce nonlinear PA signal with respect to fluence. If the occurrence of this nonlinearity is made dependent on the presence of transient biochemical markers, for example through the degradation of a nonlinearity-quenching particle coating, our serial PA tomographic imaging method can be extended for real-time three-dimensional in situ imaging of these biomarkers. Our early-stage proof-ofconcept experimental results presented here show that coating GNPs that exhibit nonlinear PA signal generation behavior is an effective method to remove the nonlinear effect. The coefficient of determination (R2 ) of a linear fit to the PA signal as a function of fluence can be effectively used to differentiate the coated GNPs and the GNPs without coating. This differentiation can also be achieved using the second order coefficient to a quadratic fit to the PA signal vs. fluence data.
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