Optimizing identification of Mycoplasma bovis by MALDI-TOF MS

Fast and accurate identification of Mycoplasma bovis in cattle samples is of great importance for rational treatment and control of pneumonia, arthritis and mastitis. However, which growth conditions will allow the fastest identification of M. bovis with MALDI-TOF MS remains unclear. Therefore, growth conditions and incubation time were investigated to optimize identification of M. bovis with MALDI-TOF MS and an in-house library was constructed. Nine different M. bovis strains were inoculated in triplicate in three liquid media (B1-3). Basic broth (B1) consisted of pleuropneumonia-like organism broth, enriched with 25% horse serum and 0.7% yeast extract. B2 and B3 were additionally supplemented with 0.5% pyruvate or 520 μg/mL ampicillin, respectively. Protein extraction was performed after 24, 48, 72, 96 and 120 h of incubation (37 °C, 5% CO₂) and processed with Autoflex III smartbeam. Identification scores ≥1.7 were interpreted as reliable. The present study showed reliable identification of M. bovis with MALDI-TOF MS as early as 24 h after inoculation, and in broth supplemented with pyruvate, up to 120 h after inoculation. Serial dilutions showed improved survival of M. bovis in broth with pyruvate. The addition of ampicillin to prevent contamination, did not impair identification of M. bovis and state-of-the-art in-house libraries contributed to higher identification scores for M. bovis with MALDI-TOF MS.