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 ؋ 10 3 CFU/g), phage added thereafter (3 ؋ 10 6 to 3 ؋ 10 8 PFU/g), and samples stored at 6°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°C) yielded similar results. In general, the application of more phage particles (3 ؋ 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.Listeria monocytogenes is an opportunistic human pathogen, widely distributed in the environment and transmitted to humans and animals via contaminated foods (50). The organism is well adapted to very different environmental conditions encountered in foods; it tolerates high levels of salt content (10 to 20%), can grow at pH values below 6, with low oxygen, and at temperatures down to 1°C (48). L. monocytogenes causes listeriosis, a severe disease which may result in septicemia, meningitis, encephalitis, or loss of the fetus during pregnancy (52). Although listeriosis is comparatively rare compared to other food-borne infections, the high mortality rate of 15 to 40% is of great concern (49, 52). Cases of sporadic and epidemic listeriosis are increasing (4, 6, 11), and it has been estimated that about 2,000 hospitalizations and 500 deaths occur annually in the United States as a result of the consumption of Listeria in foods (39). Although many foods can serve as vehicles for this pathogen, Listeria was often isolated from ready-to-eat (RTE) foods, such as milk and cheeses, cold-cut meats, smoked fish, seafood, and vegetables (45). RTE foods have been implicated in most of the major listeriosis outbreaks in the last 30 years (13,18,20,21,42,(45)(46)(47). Of particular concern is the fact that they are consumed directly, without a final bactericidal processing step. Since the preservation methods applicable to minimally processed RTE foods of...

Bacteriophage biocontrol ofListeria monocytogeneson soft ripened white mold and red-smear cheeses

“…28 Our results also correlate well with other studies using phage to target food-borne bacteria. [29][30][31] Efficacy of phage treatment appears to be highest at target cell concentrations at or below 100 cfu/cm 2 . However, it should be realized that this finding does not diminish its effectivity or affect its applicability in commercial cheese production, since the initial contamination levels actually found on soft-ripened cheese are usually very low.…”

“…Although the precise reasons for this phenotype are not clear, it is likely that the bacteria modified the specific surface carbohydrates responsible for phage attachment (Huber T and Loessner MJ, unpublished data). In other studies, no phage resistant Listeria emerged on phage-treated ready-to-eat foods over 6 to 13 days, 31 or red-smear cheese over 3 weeks. 24 Resistance to a specific phage added to food was also not observed with Salmonella Enteritidis on fresh-cut fruit during a 7 day period, 39 or Campylobacter jejuni on chicken skin after 10 days.…”

Effect of a bacteriophage cocktail in combination with modified atmosphere packaging in controlling Listeria monocytogenes on fresh-cut spinach

“…Supporting the results of this experiment, a recent study concluded that the same MA conditions used in this study in combination with an Escherichia coli O157:H7-specific lytic phage mixture resulted in lower E. coli O157:H7 populations on fresh-cut leafy greens compared to populations on phage-treated, inoculated samples packaged with atmospheric air (Boyacioglu et al, 2013). In a 2009 report, application of bacteriophage A511 to leafy greens reduced L. monocytogenes populations by ~2.5 log CFU/g on lettuce and cabbage samples during a 6-d storage period at 6°C (Guenther et al, 2009). Bacteriophage A511 was shown to be stable and active on lettuce and cabbage samples, showing only one log reduction during 6 d of storage at 6°C (Guenther et al, 2009).…”

“…In a 2009 report, application of bacteriophage A511 to leafy greens reduced L. monocytogenes populations by ~2.5 log CFU/g on lettuce and cabbage samples during a 6-d storage period at 6°C (Guenther et al, 2009). Bacteriophage A511 was shown to be stable and active on lettuce and cabbage samples, showing only one log reduction during 6 d of storage at 6°C (Guenther et al, 2009). Bacteriophage A511 reduced L. monocytogenes populations in cheese by levels ranging between 3 and 6 log CFU/cm 2 (depending on the initial L. monocytogenes populations) during the ripening process (11 d at 12°C, followed by another 11 d at 6°C) (Guenther and Loessner, 2011).…”

Phage-based biocontrol strategies to reduce foodborne pathogens in foods

“…The vegetables were spiked with bacteria (103 CFU/g), followed by addition of each phage to separate vegetable samples at concentrations of (3 x 106 to 3 x 108 PFU/g), and storage at 6°C for 6 d. The results indicated that both phages were able to reduce the concentrations of the L. monocytogenes strains by more than 2 logs in both the lettuce and cabbage, when compared with controls. 44 Others have combined the use of phage cocktails and other antimicrobials in an attempt to increase the efficacy of the phage treatment. Viazis et al 45 produced a phage cocktail (BEC8) of eight previously isolated phages that were shown to individually infect and lyse E. coli O157 and E. coli O26 strains with high efficiency.…”