Single-cell scattering and auto-fluorescence-based fast antibiotic susceptibility testing for gram-negative and gram-positive bacteria.

In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both Escherichia coli (Gram-negative) and Staphylococcus epidermidis (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the mecA+ Staphylococcus (i.e., resistant) strains treated with oxacillin.

Fast Antibiotic Susceptibility Testing via Raman Microspectrometry on Single Bacteria: An MRSA Case Study.

Despite recent advances in molecular diagnostics, ultrafast determination of the antibiotic susceptibility phenotype of pathogenic microorganisms is still a major challenge of in vitro diagnostics (IVD) of infectious diseases. Raman microspectroscopy has been proposed as a means to achieve this goal. Previous studies have shown that susceptibility phenotyping could be done through Raman analysis of microbial cells, either in large clusters or down to the single-cell level in the case of Gram-negative rods. Gram-positive cocci such as Staphylococcus aureus pose several challenges due to their size and their different metabolic and chemical characteristics. Using a tailored automated single-cell Raman spectrometer and a previously proposed sample preparation protocol, we acquired and analyzed 9429 S. aureus single cells belonging to three cefoxitin-resistant strains and two susceptible strains during their incubation in the presence of various concentrations of cefoxitin. We observed an effect on S. aureus spectra that is weaker than what was detected on previous bacteria/drug combinations, with a higher cell-to-cell response variability and an important impact of incubation conditions on the phenotypic resistance of a given strain. Overall, the proposed protocol was able to correlate strains' phenotype with a specific modification of the spectra using majority votes. We, hence, confirm that our previous results on single-cell Raman antibiotic susceptibility testing can be extended to the S. aureus case and further clarify potential limitations and development requirements of this approach in the move toward industrial applications.

Achieving magnification smaller than 1 in lensless microscopy by illumination with a convergent wavefront.

Lensless or lensfree microscopy is now available commercially. With these new microscopes, it is possible to record images in contact mode with a magnification of 1 or in holographic mode with a magnification larger than 1. In this Letter, we present an original setup that allows us to record the image of an object whose surface is larger than that of the image sensor without placing an optical component between the object and the image sensor.