# Nontargeted metabolomics analysis to unravel the anti-biofilm mechanism of Citrocin on Listeria monocytogenes

## TL;DR

This study shows that Citrocin, an antimicrobial peptide, effectively inhibits Listeria monocytogenes biofilms and disrupts key metabolic pathways.

## Contribution

The paper presents a novel nontargeted metabolomics analysis of Citrocin's anti-biofilm mechanism on L. monocytogenes.

## Key findings

- Citrocin significantly inhibits L. monocytogenes growth with a MIC of 0.075 mg/mL and MBC of 0.15 mg/mL.
- Metabolomics analysis revealed 36 differentially expressed metabolites, mainly amino acids and organic acids.
- Citrocin enriched energy and amino acid metabolic pathways like TCA cycle and arginine biosynthesis.

## Abstract

Listeria monocytogenes biofilm formation is an important cause of cross-contamination in food processing. Citrinin is a potential broad-spectrum antimicrobial peptide. However, the effects of Citrocin on L. monocytogenes and its biofilm, as well as the associated mechanisms, remain to be explored. In this study, we evaluated the anti-biofilm effect of the antimicrobial peptide Citrocin on the foodborne pathogen L. monocytogenes and analyzed its anti-biofilm mechanism from the perspectives of swarming motility, extracellular polysaccharide production, and metabolite level changes. The results showed that Citrocin had a significant inhibitory effect on the growth of L. monocytogenes, with a minimum inhibitory concentration (MIC) of 0.075 mg/mL and a minimum bactericidal concentration (MBC) of 0.15 mg/mL. Citrocin at concentrations of MIC, 2 × MIC, and 4 × MIC could prevent biofilm formation and remove established biofilms. Metabolomics analysis revealed that Citrocin at 0.3 mg/mL caused a significant differential expression of metabolites in biofilms, up- and downregulating 23 and 13 metabolites, consisting mainly of amino acids, organic acids, and fatty acids, respectively. In addition, Citrocin significantly enriched energy and amino acid metabolic pathways, including alanine, glutamate, aspartate metabolism, TCA cycle, and arginine biosynthesis. This work provides potential biofilm regulation strategies and serves as a theoretical basis for the prevention and treatment of listeriosis.

Listeria monocytogenes biofilm formation is an important cause of cross-contamination during food processing. We found that Citrocin, an antimicrobial peptide that is widely used in animal feed, has good antimicrobial and anti-biofilm effects against L. monocytogenes. We preliminarily explored the anti-biofilm mechanism of Citrocin in terms of swarming motility, extracellular polysaccharide production, and metabolomics. Our work demonstrated that Citrocin is an excellent antimicrobial agent, which is important for the control of food cross-contamination and the preventive treatment of listeriosis.