Matrix-assisted laser desorption/ionisation time of flight mass spectrometry: new applications in veterinary science

In the battle against bacterial infections, an important challenge is to identify the infectious agent as quickly and accurately as possible. Historically, techniques for identifying bacteria rely on laborious and time-consuming biochemical tests such as fermentation series and phenotypic characterisation, both of which are to some extent prone to misidentification.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a well-known and versatile mass spectrometry method which has been applied to specialised areas such as bacterial species identification and molecular imaging of tissue sections (MS imaging). The aim of this project was to investigate the utility of MALDI-TOF MS as a tool for bacterial identification in veterinary microbiology and diagnostics as well as an alternative to antibody-based methods for visualisation of biological molecules directly in tissue sections.

Compared to classical microbial identification methods MALDI-TOF MS promises to shorten accurate microbial identification workflows by several days, however, has been very scarcely applied in the veterinary setting. We investigated the use MALDI-TOF MS for the identification of microbial pathogens in production and wildlife animals (minks, otters, seals), for microbial diagnosis of subclinical and clinical pathogens involved in bovine mastitis and for visualisation of protein biomarker distribution in animal tissue originating from animals undergoing bacterial infection.

After amending (expanding) the spectral database with relevant veterinary bacterial species including bacterial variants corresponding to distinct colonisation loci the MALDI-TOF MS method was found to be a fully applicable to bacterial identification in veterinary science, representing a significant advance in the field as an excellent alternative to classical methods for bacterial identification.

The method allowed the identification of two bacterial species not previously reported in Denmark (Arcanobacterium phocae and Arcanobacetrium phocisimile). The method employing the expanded database made it possible to broadly and rapidly characterise the staphylococcal population of the bovine mastitic udder, although in order to achieve full bacterial coverage also in other animal species further expansions of the reference spectrum library may be needed.

Thus, a tool for the rapid and accurate bacterial identification of veterinary bacteria was updated and improved and shown to perform well in combination with a critical assessment based on microbial knowledge. The employment of the resulting expanded database will be of benefit the scientific community as a future research tool using the MALDI-TOF MSID technique to explore other microbial niches.

Based on our now well-developed database for human and veterinary opportunistic and pathogenic bacteria MALDI-TOF MSID can function as a tool to confirm the presence of microbials in bovine mastitis and a wide range of animals. Furthermore, from now on the reference spectra created in this thesis will be of benefit to the Dairy industry as weel as veterinarians attending factory farms and organic farms.

Applying the MALDI-TOF MSID technique will improve microbial identification and indirectly assist in reducing excessive use of antimicrobials. Importantly, the technology is also important as it allows rapid decisions on the use of narrow-spectrum antibiotics to be taken, substituting the use of less desired broad-spectrum antibiotics.

Furthermore through the work with MALDI TOF based imaging of tissue sections critical factors for the success of this application were identified, laying the ground for further work in this area.