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

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.IMPORTANCEListeria 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.

Keywords: Listeria monocytogenes; biofilm; extracellular polysaccharide; metabolomics; swarming motility.

Fig 1

(A) The antibacterial effect of Citrocin against L. monocytogenes (a 0.15 mg/mL Citrocin; b 0.15 mg/mL Citrocin after treatment at 121°C; c equal amount of PBS solution). (B) MBC detected by agar plate method. (C) Effect of Citrocin on biofilm formation. (D) Effect of Citrocin on biofilm elimination.

Fig 2

(A) Effect of Citrocin on the metabolic activity of biofilm. (B) Effect of Citrocin on the extracellular polysaccharides of biofilm. (C) Effect of Citrocin on swarming motility of L. monocytogenes. (D) The effect of the antimicrobial peptide Citrocin on the structure of L. monocytogenes. (*) represents P < 0.05; (**) represents P < 0.01.

Fig 3

(A) PCA score plots of L. monocytogenes biofilm. (B) OPLS-DA score plots in response to different Citrocin treatments. (C) Volcano plots showing the metabolite expression levels between CK, LP, and HP. Blue dots represent downregulated differentially expressed metabolites; red dots represent upregulated differentially expressed metabolites; and gray dots show nondifferentially expressed metabolites. CK, control; LP, 0.15 mg/mL Citrocin; HP, 0.3 mg/mL Citrocin.

Fig 4

(A) Heat map visualization of differential metabolites in L. monocytogenes under different concentrations of Citrocin. The blue and orange represent the low abundance and high abundance ratios, respectively. CK, the control group; LP, 0.15 mg/mL Citrocin; HP, 0.3 mg/mL Citrocin. (B) The classification of metabolites with VIP >1 and P < 0.05 in L. monocytogenes. (C) Pathway impact analysis between CK and HP. The size of the circle represents the degree of influence of the pathway, with larger circles denoting a stronger influence on the metabolic pathway. The colors of the circles represent −log P values for metabolic pathways, with darker colors indicating larger −log P values.

Fig 5

Changes in key metabolites mapped to metabolic pathways between CK vs. HP. The blue color indicates that metabolite content is significantly downregulated, and the red color indicates that the metabolite content is significantly upregulated.