Control of enterohemorrhagic Escherichia coli using bacteriophages

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply.

The objective of the first study was to isolate, identify and characterize a diverse collection of lytic bacteriophages capable of infecting EHEC serotypes O26, O111, and O157. Phages were isolated from dairy and feedlot manure using EHEC O157, O26, and O111 strains as hosts. Plaques were purified and screened against additional strains (14, O157; 10, O26; 10, O111) using the efficiency of plating method (EOP). Phage CEV2 and five other phages previously isolated were able to lyse all 14 O157 EHEC strains with EOP values consistently above 0.001. Two phages isolated from fecal slurry from dairy and feedlot cattle were highly effective against strains of E. coli O157, through EOP tests, and against O26 through spot tests, but not O111. Bacterial challenges against high titers of four E. coli O157 strains suggested that a mixture of the 8 most effective phages was just as effective as or more than each individual phage. This collection of phages can be grouped and potentially used as an antimicrobial cocktail to inactivate O157 and O26 serotypes.

The objective of the second study was to determine the effect of the bacteriophage cocktail, BEC8, on the viability of a mixture of EHEC O157:H7 strains applied on surfaces of materials representative of food processing plants. Sterile stainless steel chips (SSC), ceramic tile chips (CTC), and high density polyethylene chips (HDPEC) were used. The EHEC O157:H7 strains used were EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on surfaces and dried. EHEC cells were placed at high, medium, and low inoculum levels (10 6 , 10 5 , and 10 4 CFU/mL, respectively). Appropriate controls and BEC8 (approx. 106 PFU/mL) were applied on treated surfaces. The surfaces were incubated at 4, 12, room temperature (RT), and 37°C. EHEC survival was determined using standard plate count on TS agar. No survivors were detected after BEC8 treatment at a low inoculum level at the following incubation conditions: 37oC for 10 min and RT after 1 h on SSC and CTC; 12°C after 10 min on SSC, 1 h for CTC, and 24 h for HDPEC. These results indicated that the phage cocktail was effective within an hour against low levels of the EHEC mixture at RT on all 3 hard surfaces.

The objective of the third study was to determine the effect of the bacteriophage cocktail, BEC8, on its own and in combination with the essential oil trans -cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were nalidixic acid resistance mutants of EK27, ATCC 43895, and 472. The methods used were similar to the second study. The leaves were incubated at 4, 8, RT, and 37oC in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid containing Sorbitol MacConkey agar. No survivors were detected when treated with BEC8 or TC separately at low inoculum level after 24 h at RT on lettuce and spinach. However, when the EHEC inoculum size and/or incubation temperature increased, the efficacy of BEC8 and TC decreased. When the two treatments were combined, no survivors were detected after 10 min at all temperatures on lettuce and spinach. These results indicated that the phage cocktail and TC combination was highly effective against EHEC on leafy greens.