Developing and optimizing bacteriophage treatment to control enterohemorrhagic Escherichia coli on fresh produce

Abstract

Bacteriophages are potentially useful in controlling foodborne pathogens on minimally processed products since phage application is a non-destructive treatment. The purpose of this study was to evaluate the efficacy of a newly isolated environmental bacteriophage against enterohemorrhagic Escherichia coli on fresh produce, and optimize the treatment with consideration for potential application. Seven anti E. coli O157:H7 EDL933 bacteriophages were isolated from various sources; the most promising was isolated from municipal wastewater. This isolate (designated as E. coli phage OSY-SP) was propagated with the host, in a growth medium, to a titer of 10(8) PFU/ml. Before inoculation into fresh produce, E. coli phage OSY-SP was incubated with the host bacterium, spent medium was filter-sterilized, and the resulting crude lysate was used as a source of phage inocula for preliminary experiments. For optimized testing, phage in the crude lysate was purified by ultra-centrifugation and resuspension in phosphate-buffered saline. Efficacy of phage treatments was determined as a function of fresh produce type (cut green pepper or spinach leaves), treatment time (2 or 5min rinsing), and temperature of holding treated produce (4°C, 25°, or a combination of both temperatures). Cut green pepper was treated with UV light, to eliminate background microbiota, then spot-inoculated with E. coli O157:H7 EDL933 on cut edges, and the inoculum was allowed to dry. Because of its susceptibility to damage, baby spinach leaves were not subjected to a decontamination treatment. These leaves were inoculated with the green fluorescent protein-labeled E. coli O157:H7 B6-914 to facilitate inoculum enumeration in the presence of background microbiota. Phage suspension was applied to the inoculated fresh produce that was subsequently held for three days under variable storage conditions. The optimized phage treatment decreased the populations of pathogenic E. coli by 2.4-3.0logCFU/g on cut green pepper (5-min rinse) and 3.4-3.5logCFU/g on spinach leaves (2-min rinse), during 72h storage. The majority of this decline was caused by the antimicrobial action of the phage. These findings suggest the utility of bacteriophage to selectively control pathogens on fresh produce.

Characterization of Lytic Bacteriophages Infecting Multidrug-Resistant Shiga Toxigenic Atypical Escherichia coli O177 Strains Isolated From Cattle Feces

“…These characteristics were similar to those reported for E. coli O157-, Listeria-, Pseudomonas-, Salmonella-, and Vibrio-specific phages (23,32,33). From a biocontrol point of view, strictly lytic phages with high titers are considered as ideal candidates for biocontrol application (4,34).…”

Differential Effects of Physical and Chemical Factors on Infectivity of Three Coliphages used as Water Quality Indicator

“…Almost all of the phages isolated from mammalian feces have been to Myoviridae family and are associated with the lytic effect in E.coli (Yang et al, 2010). In another study, six lytic bacteriophages which infected enterotoxigenic E.coli strains produced similar plaques that were clear and medium-sized, ranging from 1.5 to 3.5 mm in diameter (Snyder, 2012). On the other hand, electron microscopic examination showed that both phages ECP1and ECP2 were classified as members belonging to the Podoviridae family and phage ECP3 classified as member belonging to the Siphoviridae family.…”

Prophage induction reduces Shiga toxin producing Escherichia coli (STEC) and Salmonella enterica on tomatoes and spinach: A model study

“…The use of phages to control bacterial foodborne pathogens has previously been demonstrated by spraying virulent phage cocktails on food, including fresh produce. For example, a recent study, in which phages were used to control STEC O157:H7 on fresh produce at 4 C and 25 C, reported a 2.4e3.0 log CFU/g reduction on cut green peppers and a 3.4e3.5 log CFU/g reduction on spinach leaves (Snyder, Perry, & Yousef, 2016). A different group examined the effectiveness of phage cocktails introduced in packaging materials to control L. monocytogenes on cantaloupes or E. coli O104:H4 on alfalfa sprouts (Lone et al, 2016).…”