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Rapid identification of microorganisms by mastitis causing mass spectrometry

Grant number: 11/15815-4
Support type:Regular Research Grants
Duration: January 01, 2012 - June 30, 2014
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Marcos Veiga dos Santos
Grantee:Marcos Veiga dos Santos
Home Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Assoc. researchers:Juliana Regina Barreiro

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 a 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)

Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
BARREIRO, JULIANA R.; GONCALVES, JULIANO L.; GRENFELL, RAFAELLA; LEITE, RENATA F.; JULIANO, LUIZ; SANTOS, V, MARCOS. Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk. Brazilian Journal of Microbiology, v. 49, n. 4, p. 801-807, OCT-DEC 2018. Web of Science Citations: 2.
BARREIRO, JULIANA REGINA; GONCALVES, JULIANO LEONEL; CAMPOS BRAGA, PATRICIA APARECIDA; DIBBERN, ALINE GERATO; EBERLIN, MARCOS NOGUEIRA; DOS SANTOS, MARCOS VEIGA. Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry. JOURNAL OF DAIRY SCIENCE, v. 100, n. 4, p. 2928-2934, APR 2017. Web of Science Citations: 13.

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