APPLICATIONS OF BACTERIOPHAGES FOR THE CONTROL OF PATHOGENIC ESCHERICHIA COLI

Some Escherichia coli are characterized by virulence factors that can cause disease in humans. With the spread of antibiotic resistance genes in E. coli as well, the researchers are studying different solutions. One of the most promising alternatives to control pathogenic bacteria is the application of bacteriophages. The main purpose of this PhD work was to control pathogenic E. coli through the application of bacteriophages. Twenty phages were isolated from feces, sewage, and bedding material from livestock. The viral particles have been shown not to carry genes that encode for Shiga-toxins and intimin and have been therefore used against Shiga toxin-producing E. coli (STEC). No STEC showed resistance to all phages, but some strains revealed weak sensitivity. Among the most effective phages and based on their different RAPD (Random Amplification of Polymorphic DNA), three (which were used to make the “cocktail”), were used at different multiplicity of infection (MOI = 0,1, 1, and 10). Significant differences (p < 0,05) were reported in the mean values of optical density compared with the control. The best performance was obtained with the highest MOI. Additionally, the phage cocktail was tested on fresh cucumbers. The results showed a reduction in pathogenic E. coli of 1,97–2,01 log CFU/g at 25ºC and of 1,16–2,01 log CFU/g at 4ºC during 24 h. Bacteriophages alone or in a cocktail were used to prevent biofilm formation at 4 different MOI (1, 2, 10, 100). The crystal violet assay showed a 43,64% reduction in biofilm formation of the analyzed strains compared to the control. The most effective was a cocktail composed by six phages. This phage cocktail was also used in trials to remove already formed biofilm. The results showed no significant differences between the control and samples (p<0,05). The isolated phages were also used to control bacteria related to urinary tract infections (UTIs), after being screened for the presence of inducible prophage (17,43% have a prophage). The results indicated that at least one phage was effective against 262 out of 270 bacteria. Sequencing and TEM images showed that the bacteriophages used belonged to the Myoviridae family; no pathogenesis or lysogenesis related genes were found in phage genomes. Three STEC strains were studied for prophage release, by real time qPCR, after a stress related to cheese making process: addition of NaCl at 1, 1,5 and 2% w/v, lactic acid at 0,5, 1,5 and 3% (v/v), anaerobic condition, pasteurization, UV, and after exposure to ciprofloxacin, nalidixic acid and norfloxacin. Induction of the prophages showed that the addition of NaCl at 1,5 and 2% significantly increased the phages release compared to the control, while lactic acid addition at the three concentrations tested had a significant repressing effect on phage release. In conclusion, the positive results obtained in this work in the control of pathogenic E. coli, prevention of biofilm formation, together with the genetic characteristic of phages, suggest that the isolated phages could be used to improve food safety.