Brazilian Journal of Microbiology Brazilian Journal of Microbiology
Braz J Microbiol 2018;49:801-7 - Vol. 49 Num.4 DOI: 10.1016/j.bjm.2018.04.012
Veterinary Microbiology
Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk
Juliana R. Barreiroa, Juliano L. Gonçalvesa, Rafaella Grenfellb, Renata F. Leitea, Luiz Julianob, Marcos V. Santosa,,
a Universidade de São Paulo (USP), Faculdade de Medicina Veterinária e Zootecnia, Departamento de Nutrição e Produção animal, Pirassununga, SP, Brazil
b Universidade Federal de São Paulo, Departamento de Biofísica, São Paulo, SP, Brazil
Received 09 August 2017, Accepted 16 April 2018
Abstract

The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n=120) were skimmed by centrifugation (10,000×g/10min) and pre-incubated at 37°C for 12h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Keywords
Milk, Bacteria, Mastitis, Mass spectrometry
Braz J Microbiol 2018;49:801-7 - Vol. 49 Num.4 DOI: 10.1016/j.bjm.2018.04.012