Expression construct for AvR2-V10.3. pDG389, which is a derivative of pETcoco, contains the entire R2 pyocin gene cluster (prf3 to prf24), with the exception of the tail fiber gene (prf15). The pyocin structural genes are transcribed to the right and the regulatory genes, prtR and prtN, are transcribed to the left. The plasmid also encodes an R2-V10.3 tail fiber fusion under the control of P_BAD. Pyocin production is induced with arabinose, which increases the plasmid copy number via augmented expression of the replication regulator TrfA. Arabinose also promotes expression of the R2-V10.3 fusion protein.

Recovery of E. coli O157:H7 from infected rabbits following treatment with various doses of AvR2-V10.3. For each litter of infected rabbits, half were treated with buffer alone (closed symbols) and half were treated with AvR2-V10.3 (open symbols) (L, M, and H doses are explained in the legend to Fig. 2). Tissue and stool were isolated 72 h after infection, and the number of E. coli O157:H7 cells was determined on selective medium. Symbols represent the mean and standard deviation for each group (n ≥ 6 rabbits per group). Data were analyzed using Student's t test. Mean recoveries in the ileum (P ≤ 0.01), colon (P ≤ 0.001), and stool (P ≤ 0.001) were significantly different at the high dose of AvR2-V10.3 only.

Prophylactic administration of AvR2-V10.3 prevents or mitigates symptoms of E. coli O157:H7 infection. (A) Schematic of the onset of diarrhea and timing of E. coli O157:H7 infection coupled with prophylactic treatment protocols. Low (L; 10¹¹ KU), medium (M; 2 × 10¹¹ KU), and high (H; 10¹² KU) doses of AvR2-V10.3 or R2 pyocin were administered at the indicated times (P) after inoculation of infant rabbits with E. coli O157:H7 at time zero. The appearance of fecal smears and diarrhea in treated rabbits was compared to that in infected rabbits treated with buffer alone. Signs of diarrhea were assessed daily. (B) Representative stools isolated at 3 days postinfection from E. coli O157:H7-infected rabbits treated with either 3 doses of 10¹² AvR2-V10.3 KU (left) or buffer alone (right). (C) Heterophil scores for mid- and distal colonic tissue isolated from E. coli O157:H7-infected rabbits treated with 10¹² KU of AvR2-V10.3 or with buffer. Each symbol represents the score for an individual rabbit, and each bar shows the median. Heterophil scores were analyzed using the Mann-Whitney U test in Prism software; the sums of ranks differed significantly (P ≤ 0.05) for the midcolons of animals treated with AvR2-V10.3 versus those that received buffer.

AvR2-V10.3 reduces diarrhea severity and intestinal bacterial load when administered after the onset of diarrhea. (A) Fecal contamination at necropsy (9 days postinfection) on E. coli O157:H7-infected rabbits treated with AvR2-10.3 or buffer daily after the onset of diarrhea. Images are representative of rabbits in each treatment group. (B) Histological abnormalities in the colons of rabbits treated with AvR2-10.3 or buffer. Representative images of hematoxylin-and-eosin-stained tissue are shown. The arrowhead shows mononuclear cells, and the asterisk shows edema. (C) Recovery of E. coli O157:H7 (log CFU g⁻¹) from intestinal tissues and stools of AvR2-V10.3- and buffer-treated rabbits. Each data point corresponds to colonization of a single animal, and each bar represents the mean value. ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05.