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 > or = 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 > or = 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.

FIG. 1.

The effect of oral phage treatment on E. coli O157:H7 in mice. Two groups of three mice (control 1 and phage 1) received a single oral dose of 10⁸ CFU E. coli O157:H7 ATCC 43894 on day −1. On days 0, 1, and 2, mice in phage treatment group 1 were given an oral dose of 10¹⁰ PFU of SH1 phage. Another two groups of three mice (control 2 and phage 2) received a single oral dose of 10⁸ CFU E. coli O157:H7 containing equal numbers of four strains (ATCC 43894, WSU180, WSU400, and WSU 588) on day −1. On days 0, 1, and 2 the treated mice were given oral doses of 10¹⁰ PFU of both SH1 and KH1. Untreated control mice received 100 μl of a mock bacterial lysate without phage. Data points below E+ indicate samples positive for E. coli O157:H7 only by an enrichment procedure and with total numbers <10 CFU/fecal pellet. Standard errors ranged from 0.5 log to 1.2 logs. Neg indicates RAMS cultures that were negative by both direct and enrichment culture procedures.

FIG. 2.

Effect of phage treatment on the number of E. coli O157:H7 CFU in steers. Five phage-treated steers (A) and five untreated control steers (B) received a single rectal application of E. coli O157:H7 (approximately 10⁶ CFU) containing equal amounts of four strains (ATCC 43894, WSU180, WSU400, and WSU588) on day −7. On days 0, 1, 2, and 4, each steer in the phage treatment group was given rectal applications of 8.1 × 10¹⁰ PFU of phages containing equal amounts of SH1 and KH1 in a 30-ml solution. In addition, starting on day 0, drinking water contained SH1 and KH1 (10⁶ PFU/ml). The control steers were given 30 ml mock lysate rectally on days 0, 1, 2, and 4, and the drinking water contained the mock lysate without phage. E+ indicates RAMS cultures positive for E. coli O157:H7 only by an enrichment procedure and thus contained <30 E. coli O157 CFU/swab. Neg indicates RAMS cultures that were negative by both direct and enrichment culture procedures. (C) Average numbers of total SH1 and KH1 phages in RAMS samples from phage-treated steers.

FIG. 3.

Digital photographs of representative E. coli O157 colonies from (A) control steers or mice and (B) phage-treated steers or mice. Colonies are on SMAC-CTVM plates incubated for 18 h at 37°C.