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| Author(s): |
Juliana Regina Barreiro
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Pirassununga. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD) |
| Defense date: | 2015-02-26 |
| Examining board members: |
Marcos Veiga dos Santos;
Nilson Roberti Benites;
Carlos Humberto Corassin;
Maria de Fatima Martins;
Luiz Alberto Beraldo de Moraes
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| Advisor: | Marcos Veiga dos Santos |
| Abstract | |
The purpose of the present study was to evaluate the technique of mass spectrometry by desorption / ionization assisted laser array time-of-flight (MALDI-TOF MS) for the direct identification in milk samples (no microbiological culture) of mastitis causing bacteria. Therefore, we carried out two experiments (1 and 2). In experiment 1, we determined the diagnostic sensitivity of MALDI-TOF MS technique for the direct identification in milk samples of Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli. Experimental contamination of S. aureus, S. uberis, S. agalactiae, S. dysgalactiae and E. coli in samples of milk to a concentration of 103-109 cfu/mL were performed. The contaminated milk samples were processed using kit Maldi Sepsityper® (Bruker Daltonics) and subjected to bacterial lysis protocol for analysis by MALDI-TOF MS. Mass spectra were collected in the mass range of 2000-20000 m/z and were analyzed by MALDI Biotyper 3.0 software (Bruker Daltonics) with default settings to obtain bacterial identification. Direct identification of mastitis causing pathogens from milk samples was possible at ≥106 cfu/mL for S. aureus, ≥107 cfu/mL for E. coli and ≥108 cfu/mL for S. agalactiae, S. dysgalactiae and S. uberis. In experiment 2, the objective was to evaluate the effect of pre-incubation of milk samples from mammary quarters with subclinical mastitis on the effectiveness of identification without cultivation, of mastitis-causing pathogens by MALDI-TOF MS. We selected mammary quarter milk samples from all lactating cows on two dairy herds. The milk samples were subjected to analyzes of: a) microbiological culture; b) pre-incubation followed by identification by mass spectrometry directly from milk; c) total bacterial count (TBC). Flow cytometry was used to determine TBC and; to directly identify the mastitis-causing pathogens from milk, fat was separated by centrifugation (10,000 Xg for 10 minutes) and; samples were pre-incubated at 37°C for 12 hours. Subsequently, the skim milk samples were submitted to kit Maldi Sepsityper® (Bruker Daltonics) and to the bacterial lysis protocol for analysis by MALDI-TOF MS. A total of 810 milk samples were analyzed by microbiological culture (reference method), of which 347 showed bacterial growth. Considering all culture positive samples 305 were identified as agents of interest in the direct identification by MALDI-TOF MS method: coagulase negative staphylococci (n = 191), S. aureus (n = 31), S. agalactiae (n = 42), S. uberis (n = 37) and S. dysgalactiae (n = 4). Therefore, 305 samples were directly identified by MALDI-TOF MS, which presented low sensitivity when compared to microbiological culture (Reference method): coagulase-negative staphylococci (14.08%), S. agalactiae (15.25 %), S. uberis (1.69%), S. aureus (6.12%) and S. dysgalactiae (0%). Pre-incubation of milk samples did not increase the sensitivity of the MALDI-TOF MS method directly identify mastitis-causing microorganisms. (AU) | |
| FAPESP's process: | 11/14456-0 - Rapid identification of microorganisms causing mastitis by mass spectrometry |
| Grantee: | Juliana Regina Barreiro |
| Support Opportunities: | Scholarships in Brazil - Doctorate |