Rapid identification of microorganisms causing mastitis by mass spectrometry

Abstract

The aim of this study is to evaluate the technique of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify directly (without in vitro culture) of bacteria present in milk samples. Two experiments will be undertaken: 1) Thirty isolates of each pathogen causing mastitis will be used (Staphylococcus aureus, coagulase negative Staphylococcus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Corynebacterium sp.), identified by standard microbiological, and ATCC for each pathogen, except for group coagulase negative Staphylococcus, totaling 186 isolates. To determine the concentration required for bacterial identification by MALDI-TOF MS in samples of milk will be produced at an experimental infection, where samples of sterile milk will be infected with the isolates to obtain concentrations from 103 to 109 cfu / ml and subsequently apply the identification protocol by direct MALDI-TOF MS methodology. 2) Three dairy herds in the region of Pirassununga/SP will be selected for the performance of three individual milk samples from all lactating cows from each herd. The samples from Experiment 1 and 2 will undergo the analysis: standard microbiological culture, analysis by mass spectrometry through bacterial colonies and directly from milk, and total bacterial count (TBC). Milk samples are subjected to flow cytometry and the recovery protocol mastitis-causing bacteria, they will be analyzed by MALDI-TOF MS spectrometer using an Autoflex III (Bruker Daltonics, Billerica, USA). The spectra obtained are analyzed by MALDI biotype 2.0 (Bruker Daltonics) with default settings to obtain bacterial identification. This study is expected to develop a rapid, sensitive and affordable for the identification of microorganisms that cause mastitis in milk samples; using the methodology will allow the segregation of infected animals or indication for treatment of mastitis most appropriate way depending on the type of causative agent. (AU)