Identificação de leveduras isoladas de leite de vacas com mastite por meio de técnicas espectroscópicas

Abstract

The rapid and accurate identification of microorganisms present in milk poses a challenge for diagnostic and research laboratories. Spectroscopic techniques represent an innovative alternative for identifying microorganisms because they use a minimal amount of biomass and little or no reagents, reducing the time for microbiological identification. The aim of this work was to use three spectroscopic techniques, MALDI-TOF mass spectrometry, Fourier transform infrared spectroscopy (FTIR-ATR) and Raman spectroscopy to identify yeasts isolated from the milk of cows with mastitis. The experiment consisted in isolating eleven yeasts from milk samples taken from the milk quarters of dairy cows. The microorganisms were previously isolated on Brain and Heart Infusion Agar–BHI and incubated at 35°C for 24 hours. Analyses were performed on the LT Microflex instrument (Bruker Daltonics) in duplicate. FTIR-ATR analyses were performed in triplicate on the VERTEX-70 FT-MIR instrument (Bruker). The spectra obtained were converted to text files using OPUS 6.5.97 software and Origin Pro 8. Raman measurements were performed with the Raman device (WITEC alpha 300 AR). Through MALDI-TOF MS using Flexcontrol 3.3 Software the yeasts were identified in the genera Candida, Pichia, Diutina, Cryptococcus, Kluyveromyces and Saccharomyces. Using the FTIR-ATR spectra, the peaks of the samples could be compared to characterize them according to their functional chemical groups. Principal component analysis (PCA) allowed to identify the similarities and differences between the yeasts by looking at the groupings. Raman spectroscopy was used to obtain single images and spectra of each sample, allowing the typical bands in these spectra to be assigned to cellular functional chemical groups, complementing analysis by FTIR – ATR. PCA of the spectra from FTIR-ATR and Raman spectroscopy performed with the software PAST, version 4.13 showed the grouping of yeasts by similarities in cellular constituents and also by similarities in species. In summary, this work demonstrates alternative, efficient, and accurate ways to characterize and differentiate yeasts species using mass spectrometry technique as well as optical spectroscopy methods.