The objectives of this work were to compare the infrared spectra of bacterial endocarditis vegetation with those of native valvular tissue and the infrared spectra of vegetation bacterial masses with those of surrounding vegetation tissue. Streptococcal aortic endocarditis was induced in three rabbits. Vegetation slices were cryo-sectioned for study by Fourier transform infrared (FT-IR) microspectroscopy. Valvular apparatus, vegetation, and bacterial masses within the vegetation were localized on hematoxylin and eosin (H&E) stained contiguous slices. Infrared images of whole vegetations and images of bacterial masses were acquired with apertures set to 80 × 80 and 20 × 20 μm, respectively. Valvular apparatus and vegetation showed different infrared spectra, mainly in the amide I and amide II bands (1674–1518 cm<sup>−1</sup>), and at about 1450, 1400, 1340, 1280, 1240, 1200, 1080, and 1030 cm<sup>−1</sup>. Valvular collagen, elastin, and proteoglycans may explain these differences. Bacterial masses and surrounding vegetation showed different infrared patterns, mainly in the amide I and amide II bands and in the 1142–991 cm<sup>−1</sup> carbohydrate spectral range. Bacterial nucleic acids and polysaccharides may partly explain these differences. Study of experimental endocarditis vegetation using FT-IR microspectroscopy distinguishes (1) the vegetation from the valvular tissue, and (2) the bacterial masses from the surrounding tissue. This study demonstrates for the first time that FT-IR microspectroscopy is able to detect bacterial growth in infected tissue. FT-IR microspectroscopy appears to be a useful tool for investigation of the biochemical structure of endocarditis vegetation.

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