Bacteriophage Combinations Significantly Reduce Clostridium difficile Growth In Vitro and Proliferation In Vivo

Abstract

The microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ, but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reduced C. difficile colonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.

Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes

“…This method was also effectively used in humans to restore resilience against Clostridium difficile colonization. 16,17 A second approach that presents a more structured form of microbiome engineering involves genetically modifying selected microorganisms to provide them new functions that may benefit a host. 7,18−20 These methods could include the (i) introduction of new metabolic enzymes that are absent in the host and its microbiome consortia or (ii) incorporation of new small-molecule-generating biosynthetic genes that are responsible for the production of biomolecules not currently made in humans.…”

Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology

“…Both the genome organisation and particle morphology of CDKM9 and CDKM15 resemble that of the long tailed myoviruses ΦCD27, ΦCD505 and ΦMMP02. Interestingly, however, CDKM9 has the broadest reported host spectrum compared to the reported host ranges of other long tail myoviruses (ΦCD27, ΦCD505, ΦCD508, ΦCDHM2, ΦCDHM4, ΦCDHM5 and ΦCDHM6) as these infected 13% (4/30), 11% (5/47), 4/47, (28%) 22/80, 4/80 (5%), 20/80 (25%), (29%) 23/80 isolates, respectively [6,14,16]. Further, no other long tailed myoviruses have been shown to lyse R027 isolate [6,14,16,60], while siphoviruses infecting R027 strains have been identified [6,16,17] and the only other report in the literature of myoviruses with this ability are the medium myoviruses ΦCD481-1 and ΦCDHM3, both of which caused turbid plaques on a single strain of R027 each [6,16].…”

“…The DNA was pelleted by centrifugation at 21,000× g for 20 min and the pellet was washed once with 0.5 mL of 70% ethanol before resuspension in an elution buffer (5 mM TrisCl, pH 8.5). DNA quantity and quality were measured using a Nanodrop 2000 and Qubit Fluorimeter (Thermo Scientific, Loughborough, UK) as described here [6]. …”

“…The natural resistance of C. difficile to multiple antibiotics has prompted researchers to investigate phage therapy to combat CDI [6,7,8]. Several C. difficile phages from both the Myoviridae and Siphoviridae families have already been characterised, including by genome sequencing [9,10,11,12,13,14,15,16,17].…”

Microencapsulation of Clostridium difficile specific bacteriophages using microfluidic glass capillary devices for colon delivery using pH triggered release

“…difficile phages has limited research on phage therapy for CDI. A recent study [28] however, showed the potential of phages to treat CDI in a hamster model using combinations of C . difficile phages (a polyvalent cocktail containing between 2 to 5 phages covering a suitable host range was used; the phages delivered to the hamsters were given as simple phage suspensions and bicarbonate was administered to the animals prior to phage delivered orogastrically to reduce the stomach acidity).…”