A bacteriophage cocktail targeting Escherichia coli reduces E. coli in simulated gut conditions, while preserving a non-targeted representative commensal normal microbiota

Abstract

Antibiotics offer an efficient means for managing diseases caused by bacterial pathogens. However, antibiotics are typically broad spectrum and they can indiscriminately kill beneficial microbes in body habitats such as the gut, deleteriously affecting the commensal gut microbiota. In addition, many bacteria have developed or are developing resistance to antibiotics, which complicates treatment and creates significant challenges in clinical medicine. Therefore, there is a real and urgent medical need to develop alternative antimicrobial approaches that will kill specific problem-causing bacteria without disturbing a normal, and often beneficial, gut microbiota. One such potential alternative approach is the use of lytic bacteriophages for managing bacterial infections, including those caused by multidrug-resistant pathogens. In the present study, we comparatively analysed the efficacy of a bacteriophage cocktail targeting Escherichia coli with that of a broad-spectrum antibiotic (ciprofloxacin) using an in vitro model of the small intestine. The parameters examined included (i) the impact on a specific, pre-chosen targeted E. coli strain, and (ii) the impact on a selected non-targeted bacterial population, which was chosen to represent a defined microbial consortium typical of a healthy small intestine. During these studies, we also examined stability of bacteriophages against various pH and bile concentrations commonly found in the intestinal tract of humans. The bacteriophage cocktail was slightly more stable in the simulated duodenum conditions compared to the simulated ileum (0.12 vs. 0.58 log decrease in phage titers, respectively). It was equally effective as ciprofloxacin in reducing E. coli in the simulated gut conditions (2-3 log reduction), but had much milder (none) impact on the commensal, non-targeted bacteria compared to the antibiotic.

Keywords: antibiotic; in vitro; persistence; phage; small intestine.

Figure 1.

Host range of the Ec17B153DK1 component phages against 408 E. coli isolates. Host range and interaction network of the three monophages (ECML-122, ECML-363, ECML-359) included the Ec17B153DK1 phage cocktail. Phages are depicted as large pink circles. Results shown are for 408 susceptible E. coli strains (i.e., 199 resistant strains were not included in the chart for simplicity). Each small circle is an E. coli isolate. The gray lines connecting each phage to bacterial strains indicate the ability to infect and kill that given E. coli strain based on the Spot Test assay. Bacterial strains killed by more than one phage are grouped towards the canter of the chart. The graph was generated using proprietary PhageSelector™ program developed by Intralytix, Inc.

Figure 2.

Lytic activity of the bacteriophage cocktail against targeted bacteria in the TSI model. Testing the lytic activity of the phage cocktail in two feeding conditions (fasted, fed). E. coli culture was added to the reactors at the ileum stage (ca. 7 log CFU/mL). The data shown are for the ileum compartment. The dotted line indicates the number of E. coli before treatment with the bacteriophage cocktail. All experiments were performed in triplicates (n = 3). *P < 0.05 – significant. **P < 0.01 – highly significant.

Figure 3.

Impact of ciprofloxacin and bacteriophage cocktail on simulated small intestine microbiota in fasted (A) and fed (B) conditions (7 different bacterial spp., Table 1) in the TSI model. Survival of different bacteria species from the simulated small intestinal microbiota was tested on four different culturing media: Violet Red Bile Agar (VRB) for enumeration of E. coli, M17 Agar (M17) for enumeration of Streptococcus sp., MacConkey Agar (MCC) for enumeration of E. faecalis, and Gifu Anaerobic Agar (GAM) where all species from small intestinal consortium can be cultivated. All experiments were performed in duplicate (n = 2). *P < 0.05 – significant (difference between bacteriophage or ciprofloxacin treatment compared to control samples). **P < 0.01 – highly significant (difference between bacteriophage or ciprofloxacin treatment compared to control samples).