Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

Abstract

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios >10 2 terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10 10 PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be >10 2 . In addition, phages were maintained at 10 6 PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.Since its association with disease in 1982 (33), Escherichia coli O157:H7 has become a worldwide threat to public health and is one of today's most troubling food-borne pathogens. Human illness with E. coli O157:H7 ranges from self-limited watery diarrhea and hemorrhagic colitis to life-threatening manifestations such as the hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. Cattle and sheep, important domestic ruminants, are the primary reservoirs for this human pathogen and are the most common sources for foodborne, waterborne, and direct-animal-contact infections (9-11, 18, 48, 51). In contrast to humans, the carrier ruminants remain healthy, and E. coli serotype O157:H7 is a transient member of the ruminant gastrointestinal flora. Persistence of E. coli O157:H7 carriage in naturally and experimentally infected animals varies from days to months (6,8,12,13,19,35,38).Successful strategies to control or reduce the carriage and prevalence of E. coli O157:H7 in live ruminants would reduce the risk of human exposure to this pathogen. There is currently no reliable intervention or animal vaccine available to curb carriage of E. coli O157:H7. Probiotics aimed at creating an intestinal microenvironment inhibitory to E. coli O157:H7 have been tested, but without consistent success (14,22,28,29,50). A lytic bacteriophage that would specifically target the E. coli O157 serotype is another appealing approach because phage therapy has been successful in animal trials against a broad range of bacterial pathogen...

Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduceEscherichia coliO157

“…22 The use of such phages to target a variety of gastrointestinal pathogens is one potential control strategy being examined by a number of researchers, but in vivo attempts targeting E. coli O157:H7 have yielded mixed results to date. [23][24][25][26][27][28] Although phages have long been known to play a key role in the gastrointestinal ecosystem, very few systematic studies of their prevalence, distribution, variety and importance in cattle or other food animals have been undertaken. This lack of basic knowledge is being addressed as researchers have begun to consider the use of bacteriophages in both pre-and post-harvest pathogen reduction strategies.…”

Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 : H7

“…Previous studies resulted in isolating several lytic O157 : H7 coliphages such as PP01, e11/2, e4/1c, CEV1, KH1, KH4, KH5, SH1, AKFV33, rv5, wV8, AR1, SFP10 and CBA120 (Kudva et al, 1999;Kutter et al, 2011;Liao et al, 2011;Morita et al, 2002;Niu et al, 2012;O'Flynn et al, 2004;Park et al, 2012;Raya et al, 2006;Sheng et al, 2006;Villegas et al, 2009). Some of them lyse both E. coli O157 : H7 and other pathogens of the family Enterobacteriaceae.…”

“…Longer bacterial killing times in milk could be due to the presence of other interacting proteins, components and ions which can affect adsorption of phage to the host. From the O157 phages, several such as CEV1, CEV2, KH1, SH1, e11/2 and e4/1c were tested in animals (Raya et al, 2006(Raya et al, , 2011Rivas et al, 2010;Sheng et al, 2006).…”

Bacteriophage biocontrol of foodborne pathogens

“…Also, a dose of 10 8 PFU/mL eradicated the test strains from a liquid culture medium contaminated with both high and low concentrations (10 6 and 10 2 CFU mL −1 ) of the bacterial cells Zuber et al 2008 In experimentally contaminated infant formula phage concentration of 10 9 PFU/ml was the most effective and able to completely eradicate the target organism Kim et al 2007 and reduced levels of C. coli and C. jejuni in feces by 2 log CFU/g. Studies have also been conducted on cattle to reduce fecal shedding of E. coli O157:H7 (Sheng et al 2006). Many other trials have been conducted with success for reducing intestinal colonization and fecal shedding of E. coli, Salmonella and Campylobacter (Greer 2005).…”