Prophylactic and therapeutic effects of isolated farm bacteriophages in lamb diarrhea induced by Escherichia coli

If antimicrobial treatment of infections by multidrug-resistant bacteria fails, the use of bacteriophages comes into focus. This study was a proof of concept (POC) and investigated if carefully selected E.coli bacteriophages (Phi IA38) are superior to routine antibiotic post-infection treatment of E. coli-induced secretory diarrhea in neonatal lambs. Furthermore, the prophylactic application of Phi IA-38 was also investigated in the same trial. For this aim, a Phi IA-38 was isolated from sewage samples from a small sheep farm. The isolate had broad lytic activity against various E. coli strains, and its genome contains no antibiotic resistance or virulent genes. For the clinical part, 36 Awassi lambs (20 males, 16 females), aged 1-7 days and weighing 3.2-5.7 kg, were randomly allocated to four experimental groups comprising three treatment groups and one control group. Briefly, group 1 was the control group that was not infected nor received bacteriophages; group 2 was infected and treated 24 h later with a combination of procaine penicillin and dihydrostreptomycin; group 3 was treated prophylactically 12 h before infection, and group 4 was infected and treated 1 day after infection. A single dose of E. coli K99 (0.1 mL; 10' CFU/mL) was given orally for infection. Lambs intramuscularly received 0.1 mL of phage Phi IA-38 (10' PFU/mL; MOI:1) for 3 days for phage therapy. Next to routine hematology and clinical biochemistry, blood concentrations of Serum amyloid A (SAA), Haptoglobin (Hpt), immunoglobulin M (IgM), and immunoglobulin G (IgG) were determined to estimate the magnitude of inflammatory and general immune responses. None of the lambs died. All other lambs had mild to severe diarrhea except for the control group. SAA concentrations in the animals of Group 2 were increased at all time points after day 1, whereas the Hpt levels had decreased on day 10 in Group 4 (P < 0.05). IgM levels on day 7 were higher in phage-treated Groups 3 and 4, compared to the negative control and the pen-strep-treated other groups (P < 0.05). The IgG levels were significantly increased only in the animals of Group 3 on the 7th and 10th days (P <0.05). SAA concentrations in the animals treated with antibiotics (Group 2) were increased at all time points after day 1. In the Phi IA-therapeutically treated animals (Group 4), Hpt levels were already lowest and had significantly (p < 0.05) decreased by day 10. IgM levels on day 7 were higher in the prophylactic (Group 3) and therapeutic Phi IA-38- treated groups (Group 4) compared to the negative control and the pen-strep-treated groups (P < 0.05). The IgG levels were significantly increased only in the animals that had received the bacteriophages prophylactically before infection on the 7th and 10th days (P < 0.05). Prophylactic and therapeutic IA-38 treatment reduced significantly (p < 0.05) E. coli K99 counts in the feces of infected lambs, similar to the antibiotic treatment. However, complete E. coli K 99 elimination was not reached by day 10. In conclusion, we showed the POC for Phi IA-38 therapy, but dosing and treatment duration were insufficient to eliminate all E. coli K99. Further studies are warranted to optimize the elimination of multi-resistant Enterotoxigenic E. coli (ETEC) infection of the intestinal tract of neonatal lambs. If antimicrobial treatment of infections by multidrug-resistant bacteria fails, the use of bacteriophages comes into focus. This study was a proof of concept (POC) and investigated if carefully selected E.coli bacteriophages (Phi IA38) are superior to routine antibiotic post-infection treatment of E. coli-induced secretory diarrhea in neonatal lambs. Furthermore, the prophylactic application of Phi IA-38 was also investigated in the same trial. For this aim, a Phi IA-38 was isolated from sewage samples from a small sheep farm. The isolate had broad lytic activity against various E. coli strains, and its genome contains no antibiotic resistance or virulent genes. For the clinical part, 36 Awassi lambs (20 males, 16 females), aged 1-7 days and weighing 3.2-5.7 kg, were randomly allocated to four experimental groups comprising three treatment groups and one control group. Briefly, group 1 was the control group that was not infected nor received bacteriophages; group 2 was infected and treated 24 h later with a combination of procaine penicillin and dihydrostreptomycin; group 3 was treated prophylactically 12 h before infection, and group 4 was infected and treated 1 day after infection. A single dose of E. coli K99 (0.1 mL; 10' CFU/mL) was given orally for infection. Lambs intramuscularly received 0.1 mL of phage Phi IA-38 (10' PFU/mL; MOI:1) for 3 days for phage therapy. Next to routine hematology and clinical biochemistry, blood concentrations of Serum amyloid A (SAA), Haptoglobin (Hpt), immunoglobulin M (IgM), and immunoglobulin G (IgG) were determined to estimate the magnitude of inflammatory and general immune responses. None of the lambs died. All other lambs had mild to severe diarrhea except for the control group. SAA concentrations in the animals of Group 2 were increased at all time points after day 1, whereas the Hpt levels had decreased on day 10 in Group 4 (P < 0.05). IgM levels on day 7 were higher in phage-treated Groups 3 and 4, compared to the negative control and the pen-strep-treated other groups (P < 0.05). The IgG levels were significantly increased only in the animals of Group 3 on the 7th and 10th days (P <0.05). SAA concentrations in the animals treated with antibiotics (Group 2) were increased at all time points after day 1. In the Phi IA-therapeutically treated animals (Group 4), Hpt levels were already lowest and had significantly (p < 0.05) decreased by day 10. IgM levels on day 7 were higher in the prophylactic (Group 3) and therapeutic Phi IA-38- treated groups (Group 4) compared to the negative control and the pen-strep-treated groups (P < 0.05). The IgG levels were significantly increased only in the animals that had received the bacteriophages prophylactically before infection on the 7th and 10th days (P < 0.05). Prophylactic and therapeutic IA-38 treatment reduced significantly (p < 0.05) E. coli K99 counts in the feces of infected lambs, similar to the antibiotic treatment. However, complete E. coli K 99 elimination was not reached by day 10. In conclusion, we showed the POC for Phi IA-38 therapy, but dosing and treatment duration were insufficient to eliminate all E. coli K99. Further studies are warranted to optimize the elimination of multi-resistant Enterotoxigenic E. coli (ETEC) infection of the intestinal tract of neonatal lambs.

Erciyes Univ
Veterinärmedizinische Universität Wien
Erciyes University