Targeting the isolation of rumen microbes involved in trimethylamine metabolism using MALDI-TOF MS : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manawatū, New Zealand

Abstract

Trimethylamine (TMA, N(CH3)3) is produced in gut microbial systems, such as the human gut and the rumen, through the microbial degradation of dietary substrates like choline, betaine, and carnitine. The cultivation of microbes involved in TMA metabolism is of mutual interest for both human health and rumen fermentation studies. TMA is a precursor to trimethylamine N-oxide (TMAO), a metabolite correlated with disease, and TMA is also a precursor for methane (CH4) production in ruminants. Microbes that carry genes encoding TMA metabolism have been identified from gut microbial metagenomic sequence data. However, confirming TMA metabolism in individual microbes requires experimental validation using pure cultures. Using genes known to be involved in TMA metabolism, a list of candidate TMA-metabolising strains was identified from bacterial isolates within the Hungate1000 culture collection. The study aimed to isolate new microbes involved in TMA metabolism using the Bruker Biotyper® Matrix-Assisted Laser Desorption/Ionisation Time of-Flight Mass Spectrometry (MALDI-TOF MS) system as a high-throughput screening and identification tool. The MALDI Biotyper® database was expanded by creating mass spectral profiles (MSPs) from rumen-derived candidate strains implicated in TMA metabolism, to assist with the rapid identification of new isolates. Rumen and faecal samples were selectively enriched for microorganisms able to grow on the TMA precursors choline, betaine, and carnitine, and TMA production was confirmed in the choline enrichments. However, purified isolates in this study were unable to produce TMA. Isolated pure cultures were rapidly screened with the MALDI Biotyper® using the expanded targeted database, which resulted in identifications requiring further inclusion of MSPs from the rumen source. MALDI identifications were verified using 16S rRNA gene sequencing of the isolates.