Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes

Abstract

The ability of phages to specifically interact with and lyse their host bacteria makes them ideal antibacterial agents. The range of applications of bacteriophage can be extended by their immobilization on inert surfaces. A novel method for the oriented immobilization of bacteriophage has been developed. The method was based on charge differences between the bacteriophage head, which exhibits an overall net negative charge, and the tail fibers, which possess an overall net positive charge. Hence, the head would be more likely to attach to positively charged surfaces, leaving the tails free to capture and lyse bacteria. Cellulose membranes modified so that they had a positive surface charge were used as the support for phage immobilization. It was established that the number of infective phages immobilized on the positively charged cellulose membranes was significantly higher than that on unmodified membranes. Cocktails of phages active against Listeria or Escherichia coli immobilized on these membranes were shown to effectively control the growth of L. monocytogenes and E. coli O157:H7 in ready-to-eat and raw meat, respectively, under different storage temperatures and packaging conditions. The phage storage stability was investigated to further extend their industrial applications. It was shown that lyophilization can be used as a phage-drying method to maintain their infectivity on the newly developed bioactive materials. In conclusion, utilizing the charge difference between phage heads and tails provided a simple technique for oriented immobilization applicable to a wide range of phages and allowed the retention of infectivity.

Fig. 1.

Transmission electron microscope (TEM) image of T4 heads produced from T4 10⁻ mutants.

Fig. 2.

Bioluminescent signal from E. coli O157:H7 (amp::lux) cells grown with a starting inoculum of around 10⁵ CFU/ml in the presence of positively charged (a) and unmodified (b) cellulose membranes treated with different concentrations of E. coli phage cocktail. Phage-free membranes were used as the control. Data are the means from three independent replicate trials.

Fig. 3.

Effect of the immobilized Listeria phage cocktail on growth of Listeria monocytogenes C391 on RTE oven-roasted turkey breast incubated at 4°C under aerobic (a), modified atmospheric packaging (MAP) (b), and vacuum (c) conditions. Phage-free positively charged and unmodified cellulose membranes were used as controls. Data are the means from three independent replicate trials.

Fig. 4.

Effect of the immobilized E. coli phage cocktail on the growth of E. coli O157:H7 (amp::lux) C918 on raw beef incubated aerobically at 4°C. Phage-free positively charged and unmodified cellulose membranes were used as controls. Data are the means from three independent replicate trials.

Fig. 5.

Bioluminescence activity of E. coli O157:H7 (amp::lux) on the surface of raw beef incubated for 1 week at 10°C (a and b) and 4°C (c and d) and then at 30°C for 16 h. The inoculated meat samples were covered by immobilized E. coli phage cocktail on positively charged cellulose membranes (b and d) and phage-free unmodified cellulose membranes (a and c). The blue color represents a low degree of bioluminescence, while red represents the highest level of bioluminescence.

Fig. 6.

Effect of air drying at 25 and 37°C on the stability of phages of different morphotypes. Phages were exposed to air at 25 or 37°C until they were completely dry. The dried phage particles were reconstituted in phage buffer before counting the amount of active phages in the produced phage lysate. Data are the means from three independent replicate trials.