Effect of Bacteriophages against Biofilms of Escherichia coli on Food Processing Surfaces

Abstract

The bacterial adhesion to food processing surfaces is a threat to human health, as these surfaces can serve as reservoirs of pathogenic bacteria. Escherichia coli is an easily biofilm-forming bacterium involved in surface contamination that can lead to the cross-contamination of food. Despite the application of disinfection protocols, contamination through food processing surfaces continues to occur. Hence, new, effective, and sustainable alternative approaches are needed. Bacteriophages (or simply phages), viruses that only infect bacteria, have proven to be effective in reducing biofilms. Here, phage phT4A was applied to prevent and reduce E. coli biofilm on plastic and stainless steel surfaces at 25 °C. The biofilm formation capacity of phage-resistant and sensitive bacteria, after treatment, was also evaluated. The inactivation effectiveness of phage phT4A was surface-dependent, showing higher inactivation on plastic surfaces. Maximum reductions in E. coli biofilm of 5.5 and 4.0 log colony-forming units (CFU)/cm² after 6 h of incubation on plastic and stainless steel, respectively, were observed. In the prevention assays, phage prevented biofilm formation in 3.2 log CFU/cm² after 12 h. Although the emergence of phage-resistant bacteria has been observed during phage treatment, phage-resistant bacteria had a lower biofilm formation capacity compared to phage-sensitive bacteria. Overall, the results suggest that phages may have applicability as surface disinfectants against pathogenic bacteria, but further studies are needed to validate these findings using phT4A under different environmental conditions and on different materials.

Keywords: Escherichia coli; bacterial biofilm; bacteriophages; food safety; plastic; stainless steel; surface decontamination.

Figure 1

Observation of the biofilms formed by the different strains after 24 h of incubation under an inverted microscope (MOTIC AE31). (a) E. coli ATCC 13706, with 200× magnification; (b) E. coli ATCC 25922, with 100× magnification.

Figure 2

Effect of phT4A phage on the reduction of E. coli ATCC 13706 biofilms on plastic during 24 h at MOI 10. (a) bacterial concentration; (b) phage titer. MOI, multiplicity of infection; CFU, colony-forming units; PFU, plaque-forming units. The values shown in both graphs are the average of three independent assays, and the error bars represent the standard deviation.

Figure 3

Effect of phT4A phage on the reduction of E. coli ATCC 13706 biofilms on stainless steel during 24 h at MOI 10. (a) bacterial concentration; (b) phage titer. MOI, multiplicity of infection; CFU, colony-forming units; PFU, plaque-forming units. The values shown in both graphs are the average of three independent assays, and the error bars represent the standard deviation.

Figure 4

Evaluation of phage phT4A efficacy in preventing E. coli ATCC 13706 biofilm formation on plastic during 48 h. CFU, colony-forming units. The values shown in the graph are the average of three independent assays, and the error bars represent the standard deviation.

Figure 5

Assessment of the biofilm formation capacity of resistant and sensitive bacteria to phT4A phage during 24 h of incubation. R1 and R2, bacteria resistant to phT4A phage after contact with phage; S1 and S2, bacteria sensitive to phT4A phage after contact with phage; Bacteria control, bacteria without contact with phage phT4A; CFU, colony-forming units. The values shown in the graph are the average of three independent tests, and the error bars represent the standard deviation.

Figure 6

Observation of the biofilm formation on plastic by different groups of bacteria (phage-resistant, phage-sensitive, and control), with 24 h of incubation, under an inverted microscope (MOTIC AE31) with 200× magnification. R1 and R2, Bacteria resistant to phT4A phage after contact with phage; S1 and S2, Bacteria sensitive to phT4A phage after contact with phage; BC1 and BC2, Bacteria without contact with the phage phT4A.