In-situ measurements of bacteria resistance to antimicrobial agents employing leaky mode sub-wavelength diffraction grating

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The research is dedicated to the real-time studies detecting wall changes of Gram-positive bacteria Staphyloccoccus aureus using different amounts of antimicrobial agent benzylpenicillin (Benzylpenicillinum natricum–500,000 International Units/ml). The optical method based on excitation of the leaky mode in a sub-wavelength diffraction grating was employed. The proposed optical method is sensitive to the effective refractive index changes in vicinity of the sub-wavelength diffraction grating and it was used to study efficiency of the antimicrobial agent in situ. The bacterial solution (2.1×109 colony forming unit/ml S. aureus in 0.9% saline solution) was placed in a temperature-controlled measurement cell. Different amounts of benzylpenicillin were injected in the measurement cell and changes of the optical signature were monitored in vitro. The results obtained employing the optical method were verified comparing with the ones obtained employing standard microbiological tests and bacteria visualization by scanning electron microscopy (SEM). Samples for the microbiological tests and SEM analysis were taken from the liquid cell after injection of the antimicrobial agent. From the SEM micrographs, it was obtained that the cell wall morphological structure has been visibly changed after 0.2ml and more of benzylpenicillin was applied. Changes in the optical signature of the proposed optical method in real time, i.e. the resonant wavelength shift and decrease in reflectivity, of the analyzed suspensions were addressed to the antimicrobial agent impact on the cell walls.