Inhibition of Listeria monocytogenes by Phage Lytic Enzymes Displayed on Tailored Bionanoparticles

Abstract

The high mortality rate associated with Listeria monocytogenes and its ability to adapt to the harsh conditions employed in food processing has ensured that this pathogen remains a serious problem in the ready-to-eat food sector. Bacteriophage-derived enzymes can be applied as biocontrol agents to target specific foodborne pathogens. We investigated the ability of a listeriophage endolysin and derivatives thereof, fused to polyhydroxyalkanoate bionanoparticles (PHA_BNPs), to lyse and inhibit the growth of L. monocytogenes. Turbidity reduction assays confirmed the lysis of L. monocytogenes cells at 37 °C upon addition of the tailored BNPs. The application of BNPs also resulted in the growth inhibition of L. monocytogenes. BNPs displaying only the amidase domain of the phage endolysin were more effective at inhibiting growth under laboratory conditions (37 °C, 3 × 10⁷ CFU/mL) than BNPs displaying the full-length endolysin (89% vs. 83% inhibition). Under conditions that better represent those found in food processing environments (22 °C, 1 × 10³ CFU/mL), BNPs displaying the full-length endolysin demonstrated a greater inhibitory effect compared to BNPs displaying only the amidase domain (61% vs. 54% inhibition). Our results demonstrate proof-of-concept that tailored BNPs displaying recombinant listeriophage enzymes are active inhibitors of L. monocytogenes.

Keywords: BNPs; Listeria monocytogenes; amidase; bacteriophage; bionanoparticles; endolysin.

Figure 1

Experiment 1A: turbidity reduction assays performed at 37 °C using 1 × 10⁷ CFU/mL L. monocytogenes 473 (serotype 4e). The data have been adjusted according to Equation (1). L. monocytogenes strain 473 was inoculated into TSB containing PHA_lysin293_BNPs (pink symbols) (n = 4), PHA_amidase293_BNPs (black symbols) (n = 4), L. mono + PHA_BNP control (green symbols) (n = 4), and L. mono-PHA_BNPs (blue symbols) (n = 4). Absorbance at OD 600 nm was measured at 0, 30, 60, 90, 120, 150 and 180 min.

Figure 2

Experiment 1B: growth inhibition assays at 37 °C using 1 × 10⁷ CFU/mL L. monocytogenes 473 (serotype 4e). L. monocytogenes strain 473 was inoculated into TSB containing PHA_lysin293_BNPs (pink symbols) (n = 4), PHA_amidase293_BNPs (black symbols) (n = 4), L. mono + PHA_BNP control (green symbols) (n = 4), and L. mono-PHA_BNPs (blue symbols) (n = 4). Cells were incubated at 37 °C and samples taken for plating on Listeria Chromogenic Agar at 0, 30, 60, 90, 120, 150 and 180 min. The figure depicts total counts of L. monocytogenes.

Figure 3

Experiment 2B: growth inhibition assays at 22 °C using 1 × 10³ CFU/mL L. monocytogenes 473 (serotype 4e). L. monocytogenes strain 473 was inoculated into TSB containing PHA_lysin293_BNPs (pink symbols) (n = 4), PHA_amidase293_BNPs (black symbols) (n = 4), L. mono + PHA_BNP control (green symbols) (n = 4), and L. mono-PHA_BNPs (blue symbols) (n = 4). Cells were incubated at 22 °C and samples taken for plating on Listeria Chromogenic Agar at 0, 30, 60, 90, 120, 150 and 180 min. The figure depicts total counts of L. monocytogenes.