Development of a high-throughput screening Queso Fresco model for evaluating antimicrobials

Sammanfattning: Listeria monocytogenes is a pathogen, sometimes occurring in fresh cheese, such as Queso Fresco. Antimicrobials could be utilized to prevent the presence of L. monocytogenes in this type of cheese. In order to be able to screen and assess several novel antimicrobials simultaneously in the future, the validated miniaturized laboratory-scale model for Queso Fresco (MLQF) was further developed into a high-throughput screening model (HTQF). This included studying and comparing the microbial behavior of L. monocytogenes 10403S (LM), L. monocytogenes 10403S expressing GFP (LM-GFP), and a cocktail of five L. monocytogenes (LM-COCKTAIL). Additionally, analysis of luminescent bacteria (LUX) was also part of the study, however, not luminescent L. monocytogenes. LM-GFP and LUX was studied due to their light-emitting properties, which enables an efficient screening with a fluorometer instead of performing time consuming plating. In the HTQF, the Queso Fresco was produced in 96-well microplates, with a size of 0.05 ± 0.0004 g/cheese. The strains and the model were evaluated with three different antimicrobial treatments; Lauric arginate, Ɛ-polylysine and a combination of these two. The aim for the combination of the antimicrobials was to have a variety in order to be able to see if the method is applicable in different scenarios. Preferably one antimicrobial without effect, one bacteriostatic, and a combination. The result showed that the engineered LM-GFP had similar growth behavior as the parental strain. This was true both with and without treatments. They both grew 4 log CFU/g under no antimicrobial treatment for 28 days. The LM-COCKTAIL was comparable to these two for the control cheese (no treatment), with a growth of 4 log CFU/g and for one out of three treatments (lauric arginate), also for 28 days. The microbial behavior of the LM-COCKTAIL in the Queso Fresco produced in 96-well microplates needs further investigation in order to conclude its correlation to the other L. monocytogenes strains in this model. The fluorescence measurements with the LM-GFP gave a result which was difficult to correlate to the viable count (VC) of the same strain. However, the emitted light and the VC from the LUX suggested a better correlation. In conclusion, based on the comparison of the result from the LM-GFP and the LUX, the luminescent reporter system seems to be preferable to the fluorescent in this HTQF model.