Virulence attenuation effect of medium- and long chain- fatty acids on Listeria monocytogenes

Introduction: Listeria monocytogenes is a significant food-borne pathogen, with multiple genes involved in its virulence. Medium- and long-chain free fatty acids (FAs) may inhibit pathogen growth and decrease virulence gene expression, in a species dependent manner.
Purpose: To investigate the impact of free fatty acids on the expression level of L. monocytogenes genes, in particular those contributing to virulence, using RNA sequencing.
Methods: Commercial free FAs including C16:1, C18:1, conjugated C18:2, C18:2, and C18:3 and FAs derived from beef fat including total beef fat, beef FA fractions enriched for mono-unsaturated fatty acids (UFAs) or poly-UFAs were used in this study. Log phase L. monocytogenes was exposed to FAs for 3 h at their respective minimum inhibitory concentrations. Total RNA was then extracted and subsequently subjected to rRNA depletion. The libraries were sequenced on an Illumina HiSeq4000 platform (100 bp paired-end). Good quality reads following quality-based filtering and trimming were mapped to the L. monocytogenes EGD-e reference genome assembly. Differential gene expression analyses were conducted using the ‘edgeR’ Bioconductor package in R. Ten confirmed virulence genes (prfA, mpl, InlA, InlB, inlC, plcA, plcB, actA, hly and hpt) were further analyzed. The experiment was conducted in triplicate.
Results: Principal component analysis based on gene counts showed separation of samples, with C18:1 and conjugated C18:2, and C16:1, C18:2 and C18:3 forming distinct clusters. Total beef fat, mono-UFA, and ploy-UFA fractions were closer to the former cluster. C18:1 did not have any effect on the 10 virulence genes and mpl was not affected by any of the treatments. Conjugated 18:2, C16:1, C18:2 and C18:3 significantly downregulated all remaining 9 virulence genes, while total beef fat, mono-UFA and poly-UFA downregulated 7, 5 and 6 of the virulence genes, respectively.
Significance: FAs including those from beef may be explored as an alternatives to antibiotics against L. monocytogenes to combat antimicrobial resistance.