Efficacy of an Optimised Bacteriophage Cocktail to Clear Clostridium difficile in a Batch Fermentation Model

Abstract

Clostridium difficile infection (CDI) is a major cause of infectious diarrhea. Conventional antibiotics are not universally effective for all ribotypes, and can trigger dysbiosis, resistance and recurrent infection. Thus, novel therapeutics are needed to replace and/or supplement the current antibiotics. Here, we describe the activity of an optimised 4-phage cocktail to clear cultures of a clinical ribotype 014/020 strain in fermentation vessels spiked with combined fecal slurries from four healthy volunteers. After 5 h, we observed ~6-log reductions in C. difficile abundance in the prophylaxis regimen and complete C. difficile eradication after 24 h following prophylactic or remedial regimens. Viability assays revealed that commensal enterococci, bifidobacteria, lactobacilli, total anaerobes, and enterobacteria were not affected by either regimens, but a ~2-log increase in the enterobacteria, lactobacilli, and total anaerobe abundance was seen in the phage-only-treated vessel compared to other treatments. The impact of the phage treatments on components of the microbiota was further assayed using metagenomic analysis. Together, our data supports the therapeutic application of our optimised phage cocktail to treat CDI. Also, the increase in specific commensals observed in the phage-treated control could prevent further colonisation of C. difficile, and thus provide protection from infection being able to establish.

Keywords: Clostridium difficile; Clostridium difficile infection; bacteriophages; in vitro fermentation model; microbiome; phage therapy.

Figure 1

Contributory culturable bacterial counts from each of the individual donors and final cumulative counts of each bacterium added to the fermentation vessels. The bacteria present in the fecal sample of each donor were determined by recovery on selective medium for each bacterial grouping, after which, the samples were mixed together in relatively equal amounts and used to prime the fermentation vessels. The data was analysed using GraphPad Prism 7. Error bars are SEMs of three biological replicates.

Figure 2

Impact of phage treatment on C. difficile and other components of the gut microbiota. The impact of phage treatment was ascertained by recovering the bacteria on selective media for (A) C. difficile; (B) bifidobacteria; (C) enterococci; (D) enterobacteria; (E) lactobacilli; (F) total anaerobes. The bacterial counts of the different treatment vessels and time points are presented. Black lines, vessel 1, untreated slurries; red lines, vessel 2, C. difficile control; green lines, vessel 3, phage-only-treated control; blue lines, vessel 4, prophylaxis regimen, and purple lines, vessel 5, remedial regimen. The data was analysed using GraphPad Prism 7. Error bars are SEMs of 3 biological replicates.

Figure 3

Analysis of the 10 most abundant taxa from Archea, Bacteria, and Viruses as ascertained by the metagenomics data. Total genomic DNA was extracted at 24 h time point from (A) vessel 1, untreated slurries; (B) vessel 2, C. difficile control; (C) vessel 3, phage-only-treated control; (D) vessel 4, prophylaxis regimen; (E) vessel 5, remedial regimen. The samples were prepared using NexteraXT sample preparation kit and sequenced on MiSeq platform using V2 (2 × 250 bp) chemistry. The resulting fastq files were trimmed with Sickle, and the metagenomes were assembled using megahit. An overview of the 10 most abundant taxa: Phyla (P), Classes (C), order (O), and family (F) are shown for each treatment vessel, as visualised using Pavian. The percent reads mapped to Archaea, Bacteria, and Viruses in the vessels at 24 h are shown in (Fi), (Fii), and (Fiii), respectively.