Bioprotective Effect of Lactococcus piscium CNCM I-4031 Against Listeria monocytogenes Growth and Virulence

Abstract

Listeria monocytogenes is a Gram-positive pathogen occurring in many refrigerated ready-to-eat foods. It is responsible for foodborne listeriosis, a rare but severe disease with a high mortality rate (20-30%). Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp and in a chemically defined medium using a cell-to-cell contact-dependent mechanism. To characterize this inhibition further, the effect of L. piscium was tested on a collection of 42 L. monocytogenes strains. All strains were inhibited but had different sensitivities. The effect of the initial concentration of the protective and the target bacteria revealed that the inhibition always occurred when L. piscium had reached its maximum population density, whatever the initial concentration of the protective bacteria. Viewed by scanning electron microscopy, L. monocytogenes cell shape and surface appeared modified in co-culture with L. piscium CNCM I-4031. Lastly, L. monocytogenes virulence, evaluated by a plaque-forming assay on the HT-29 cell line, was reduced after cell pre-treatment by the protective bacteria. In conclusion, the bioprotective effect of L. piscium toward L. monocytogenes growth and virulence was demonstrated, and a hypothesis for the inhibition mechanism is put forward.

Keywords: Lactococcus piscium; Listeria monocytogenes; biopreservation; cell ratio; co-culture; scanning electron microscopy; virulence.

FIGURE 1

Inhibition of 42 L. monocytogenes strains after a 30-h culture with L. piscium CNCM I-4031 in MSMA.

FIGURE 2

Final concentration of 42 L. monocytogenes strains in pure and mixed culture with L. piscium CNCM I-4031, after 30 h in MSMA medium.

FIGURE 3

Growth of L. piscium CNCM I-4031 (■) and L. monocytogenes RF191 () in pure culture (solid line) and in co-culture (dotted line) in MSMA at 26°C. Initial concentrations of L. piscium/L. monocytogenes: (A) 10^3/10³ CFU/ml, (B) 10⁵/10³ CFU/ml, and (C) 10⁶/10⁶ CFU/ml.

FIGURE 4

L. piscium CNCM I-4031 and L. monocytogenes RF191 cells in pure and co-culture viewed using scanning electron microscopy on polycarbonate membranes. Magnification: × 30,000. (A) L. piscium in pure culture (1) in Elliker; (2) in MSMA, 24 h at 26°C. (B) L. monocytogenes RF191 in pure culture (1) in mBHI; (2) in MSMA, 24 h at 26°C. (C) Co-culture of L. piscium CNCM I-4031 and L. monocytogenes RF191 in liquid MSMA. (D) Co-culture of L. piscium CNCM I-4031 and L. monocytogenes RF191 on MSMA plate.

FIGURE 5

L. monocytogenes plaque-forming assay (PFA) using HT-29 epithelial cells infected with strains ScottA and RF191 directly or after 1 h of pre-incubation with L. piscium CNCM I-4031. Plaques were counted in HT-29 cells infected with 10⁵ UFC/ml of L. monocytogenes. No plaque was observed when L. piscium CNCM I-4031 was inoculated alone. Error bars indicate standard deviations from at least three independent experiments. ^a,b,cRepresent groups determined with LSD test, a same letter indicates values not significantly different (p-value < 0.05 by one way-ANOVA).