Test Agreement among Biochemical Methods, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry, and 16S rRNA Sequencing for Identification of Microorganisms Isolated from Bovine Milk

ABSTRACT The objective of this prospective study was a blinded comparison of three methods for the identification of bacteria isolated on Columbia blood agar from milk samples of dairy cows. Basic biochemical testing, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA partial genome sequence analysis were compared for bacterial identification to the genus or species level. Milk samples submitted from a commercial dairy farm from recently calved cows or clinical mastitis cases were cultured, and 181 isolates were identified by biochemical testing, MALDI-TOF MS, and 16S rRNA sequence analysis (179 isolates; 2 isolates could not be recovered from storage). For Staphylococcus aureus and Escherichia coli, agreement was determined at the species level. For other microbes, agreement was determined at the genus level or at the group level for streptococcus-like organisms. The positive agreement among all 3 diagnostic methods was 94%, with 95% to 98% between each pair of methods. The overall (including negative agreement) agreement among all 3 methods ranged from 97% to 100%. MALDI-TOF MS is becoming more commonplace for the genus- and/or species-level identification of bacteria isolated from milk samples and, in some laboratories, has replaced conventional biochemical methods. The results of the present study suggest that when identifying pathogens at the genus or group level, conventional culture followed up with either secondary biochemical testing or MALDI-TOF MS is of practical value. For purposes of milk quality and udder health monitoring or research, any of the 3 methods is a valuable tool for genus-level identification of bacteria isolated from dairy cow milk.