A Validation System for Selection of Bacteriophages against Shiga Toxin-Producing Escherichia coli Contamination

Abstract

Shiga toxin-producing Escherichia coli (STEC) can cause severe infections in humans, leading to serious diseases and dangerous complications, such as hemolytic-uremic syndrome. Although cattle are a major reservoir of STEC, the most commonly occurring source of human infections are food products (e.g., vegetables) contaminated with cow feces (often due to the use of natural fertilizers in agriculture). Since the use of antibiotics against STEC is controversial, other methods for protection of food against contaminations by these bacteria are required. Here, we propose a validation system for selection of bacteriophages against STEC contamination. As a model system, we have employed a STEC-specific bacteriophage vB_Eco4M-7 and the E. coli O157:H7 strain no. 86-24, bearing Shiga toxin-converting prophage ST2-8624 (Δstx2::cat gfp). When these bacteria were administered on the surface of sliced cucumber (as a model vegetable), significant decrease in number viable E. coli cells was observed after 6 h of incubation. No toxicity of vB_Eco4M-7 against mammalian cells (using the Balb/3T3 cell line as a model) was detected. A rapid decrease of optical density of STEC culture was demonstrated following addition of a vB_Eco4M-7 lysate. However, longer incubation of susceptible bacteria with this bacteriophage resulted in the appearance of phage-resistant cells which predominated in the culture after 24 h incubation. Interestingly, efficiency of selection of bacteria resistant to vB_Eco4M-7 was higher at higher multiplicity of infection (MOI); the highest efficiency was evident at MOI 10, while the lowest occurred at MOI 0.001. A similar phenomenon of selection of the phage-resistant bacteria was also observed in the experiment with the STEC-contaminated cucumber after 24 h incubation with phage lysate. On the other hand, bacteriophage vB_Eco4M-7 could efficiently develop in host bacterial cells, giving plaques at similar efficiency of plating at 37, 25 and 12 °C, indicating that it can destroy STEC cells at the range of temperatures commonly used for vegetable short-term storage. These results indicate that bacteriophage vB_Eco4M-7 may be considered for its use in food protection against STEC contamination; however, caution should be taken due to the phenomenon of the appearance of phage-resistant bacteria.

Synergistic Effects of Bacteriophage vB_Eco4-M7 and Selected Antibiotics on the Biofilm Formed by Shiga Toxin-Producing Escherichia coli

“…In our experiments, we have employed the vB_Eco4M-7 bacteriophage [ 15 , 16 ], and tested its effects on biofilms formed by a model STEC strain, named E. coli O157:H7 (ST2-8624) [ 15 ]. To prepare a bacteriophage cocktail, vB_Eco4M-7 was mixed with another STEC-infecting phage, ECML-117 which has been broadly investigated by other scientists previously, as a component of the phage cocktail [ 20 , 21 ] and by us, in a comparative study with vB_Eco4M-7 [ 15 ].…”

“…On the other hand, when monitoring the infection process of E. coli O157:H7 (ST2-8624) cells, the vB_Eco4M-7 phage was more efficient in killing bacteria than ECML-117 [ 15 ]. For this reason and for clarity of the presented results, we have decided to focus on the phage vb_Eco4M-7, isolated and characterized previously by us [ 15 , 16 ].…”

“…The E. coli O157:H7 (ST2-8624) strain, bearing the Shiga toxin-converting prophage ST2-8624 (Δ stx2 :: cat gfp ) [ 76 , 77 ], was the host of choice for phages vB_Eco4M-7 and ECML-117 [ 15 , 16 , 78 ]. The E. coli strain C600 [ 79 ] was used as the host for the titration of phage ST2-8624 [ 77 ].…”

“…To determine the phage titer in a viral stock, expressed in the number of plaque-forming units per ml (PFU/mL), the double agar layer assay was used [ 16 ]. Serial 10-fold dilutions of phage lysate were prepared in TM buffer (10 mM Tris-HCl, 10 mM MgSO4, pH 7.2; BioShop, Burlington, ON, Canada).…”

“…This phage, named vB_Eco4M-7, belongs to the Myoviridae family, displays a lytic mode of development, does not encode any known toxins, and does not cause induction of prophages in infected STEC cells [ 15 ]. This phage appeared to be effective in the protection of vegetables from contamination with STEC, and it was demonstrated to be non-toxic for mammalian cells [ 16 ]. Since other bacteriophages were reported previously to be effective in destroying bacterial cells included in biofilms [ 17 , 18 , 19 ], in this work, we have tested the effects of vB_Eco4M-7 used alone or in combination with another STEC-infecting phage, ECML-117 (forming together the phage cocktail) and/or different antibiotics (ciprofloxacin and rifampicin) on biofilm formed by STEC.…”