Multiplex MALDI-TOF-MS microbial identification could aid the identification of multiple microbial contaminants in food through proteome database search approach

Identification of microbial contaminants in food is a critical component of quality control effort during food production. Although multiple steps of the food production workflow need to be checked for microbial contaminants, the microbial load in the final product is perhaps the most important, and is the incision point at which regulatory agency conduct regular checks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a fast, accurate microbial identification method in the past decade, and has seen increasing use and adoption in the clinical and quality control laboratory. But, the method has been hampered by the need to isolate pure culture of the microbes such as through a spread plate on agar plates prior to instrumented analysis. This primer outlines an alternative workflow capable of multiplex analysis of multiple microbial species in food via MALDI-TOF MS through the use of a proteome database approach with ribosomal proteins as biomarkers of individual microbial species. More importantly, the method describes here profile the fittest microorganisms that could outcompete or eliminate other microbial species in food, and provides a good estimate of the pathogenicity potential of the food sample tested. Firstly, the food sampled is grounded or diluted in good proportion and serve as inoculum for liquid LB medium and brain heart infusion broth. Choice of rich LB medium is for enabling the growth of multiple microbial species. On the other hand, brain heart infusion broth serves to identify microbial species in the food sample that could grow in body fluids such as blood, and thus, pose a systemic infection risk. After cultivation at 37 ^oC and 230 rpm under aerobic conditions for 12 (LB medium) and 24 hrs (brain heart infusion broth) respectively, samples of the liquid cultures would be aliquoted for MALDI-TOF MS analysis after overlaid with suitable MALDI matrix. Noted here is the workflow does not require a lengthy cultivation step on agar plate through the spread plate technique to separate mixtures of microbes into individual colonies. Obtained mass spectrum of the sample would be a combination of mass peaks from different microbial species. Individual species could be identified by performing a proteome database search of a library of ribosomal proteins from different microbial species. As a first estimate, the above described approach could possibly identify 5 to 10 microbial species in a heavily contaminated food sample. But, it is to be noted that the results of this analysis reveals the fittest microorganisms in the sample, and not the entire ensemble of microbial species in the sample. Overall, direct analysis of liquid culture sample saves time needed for spread plate agar culture to isolate individual colonies for conventional MALDI-TOF MS analysis. However, the method is only possible with the use of a comprehensive ribosomal protein library of many microbial species to help perform the necessary proteome database search of the obtained multi-microbial species MALDI-TOF MS mass spectrum.