Virulent bacteriophage for efficient biocontrol of Listeria monocytogenes in ready-to-eat foods

Abstract

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 x 10(3) CFU/g), phage added thereafter (3 x 10(6) to 3 x 10(8) PFU/g), and samples stored at 6 degrees C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20 degrees C) yielded similar results. In general, the application of more phage particles (3 x 10(8) PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log(10). In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.

FIG. 1.

Effect of phage A511 on growth of Listeria monocytogenes strains WSLC 1001 and Scott A in eight different RTE foods. Samples were spiked with bacteria (1 × 10³ CFU/g or ml), and phage A511 was applied (3 × 10⁸ PFU/g or ml) to the test samples approximately 1 h later. Samples were stored for 6 days at 6°C and monitored for bacterial counts at time points indicated. Closed circles, WSLC 1001 controls without phage; closed triangles, WSLC 1001 with A511; open circles, Scott A controls without phage; open triangles; Scott A with phage A511; n.d., none detected.

FIG. 2.

Effect of phage A511 on growth of L. monocytogenes Scott A over extended storage periods. Selected foods were spiked with bacteria (1 × 10³ CFU/g or ml), and A511 was applied (3 × 10⁸ PFU/g or ml) approximately 1 h later. Samples were then stored for up to 13 days at 6°C and monitored for bacterial counts at the time points indicated. Open circles, controls without phage; open triangles, samples with A511; n.d., none detected.

FIG. 3.

Effect of phage A511 on growth of L. monocytogenes WSLC 1001 during storage at elevated temperature. Selected foods were spiked with bacteria (1 × 10³ CFU/g or ml), and phage A511 was applied (3 × 10⁸ PFU/g or ml) approximately 1 h later. Food samples were stored for 6 days at 20°C and monitored for bacterial counts at the time points indicated. Closed circles, controls without phage; closed triangles, samples with A511; n.d., none detected.

FIG. 4.

Effects of different initial phage concentrations on growth inhibition of Listeria. Foods were spiked with L. monocytogenes WSLC 1001 (1 × 10³ CFU/g or ml), and phage A511 was applied 1 h later at three different final concentrations (3 × 10⁶, 3 × 10⁷, or 3 × 10⁸ PFU/g or ml). Samples were then stored for 6 days at 6°C and monitored for bacterial counts at the time points indicated. Closed circles, controls without phage; closed squares, 3 × 10⁶ PFU/g phage; closed diamonds, 3 × 10⁷ PFU/g phage; closed triangles, 3 × 10⁸ PFU/g phage; n.d., none detected.

FIG. 5.

Stability of phage A511 in different RTE foods (see figure insert) during storage for 6 days at 6°C. At the time points indicated, the PFU of A511 (added at 3 × 10⁸ PFU/g or ml) were determined directly from the food samples spiked with L. monocytogenes Scott A bacteria (1 × 10³ CFU/g or ml) (see Fig. 1).

FIG. 6.

Effect of phage P100 on growth of L. monocytogenes WSLC 1001 in four different RTE foods. Samples were spiked with the bacteria (1 × 10³ CFU/g or ml), and P100 was added (3 × 10⁸ PFU/g or ml) approximately 1 h later. Samples were stored for 6 days at 6°C and monitored for bacterial counts at the time points indicated. Closed circles, controls without phage; closed triangles, samples with P100; n.d., none detected.