Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100

Abstract

When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered.

Keywords: L. monocytogenes; ListexTM P100; bacteriophage P100; environmental influence; host–phage interaction; resistance.

FIGURE 1

Infectivity of P100 stored in smear water and SM buffer at 4°C (A) and 10°C (B) for 117 days. Smear water was either untreated (as obtained from the dairy plant), autoclaved or centrifuged in order to remove bacterial or eukaryotic (e.g., yeast) cells. Further, L. monocytogenes EGDe was added to one smear water sample. Experiments were done twice in duplicates and mean values and standard deviations were shown.

FIGURE 2

Adsorption tests performed over 60 min indicating attachment (decreasing number of free phages) of phage P100 to L. monocytogenes in TSB containing NaCl (A), Lutensol AO 7 (B), TSB adjusted to pH values 3–11 (C), smear water and Fraser (D), and TSB containing SDS (E). All experiments were done twice and in duplicates and mean values and standard deviations were shown.

FIGURE 3

Adsorption tests performed over 6 h in TSB media containing NaCl (A), adjusted to different pH values (B) and containing the detergents Lutensol AO 7 (C) or SDS (D). Graphs show the number of free (unattached extracellular) phages. All experiments were carried out twice and in duplicates and mean values and standard deviations are shown.

FIGURE 4

Growth curves of uninfected (A) and infected (B,C; MOI = 10) L. monocytogenes in TSB and NaCl containing TSB media. Bacteria concentrations at the beginning of the infections were 10⁷ CFU/ml (A,B) and 10⁶ CFU/ml (C). All NaCl concentrations (0–2 M) were tested. For the sake of clarity only the curves of growing bacteria were demonstrated while the others on base line level were not depicted. Moreover, one of four independent experiments is representatively shown.

FIGURE 5

Growth of uninfected (A) and infected (B,C; MOI = 10) L. monocytogenes in TSB adjusted to different pH values. The bacteria concentrations at the beginning of the infections were 10⁷ CFU/ml (A) and (B) and 10⁶ CFU/ml (C). All pH values (4–11) were tested. For the sake of clarity only the curves of growing bacteria were demonstrated while the others on base line level were not depicted. Moreover, one of four independent experiments is representatively shown.

FIGURE 6

Growth of uninfected (A) and infected (B,C; MOI = 10) L. monocytogenes in TSB containing different Lutensol AO 7 concentrations. Bacteria concentrations at the beginning of the infections were 10⁷ CFU/ml (A,B) and 10⁶ CFU/ml (C). All concentrations (0–5%) were tested. For the sake of clarity only the curves of growing bacteria were demonstrated while the others on base line level were not depicted. Moreover, one of four independent experiments is representatively shown.

FIGURE 7

Long-term infection experiment using different MOIs and temperatures for incubation. Growth of infected and non-infected L. monocytogenes was monitored at 4°C (A), 10°C (B), and 20°C (C).