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.

Clostridium difficile infection (CDI) is a global health threat associated with high rates of morbidity and mortality. Conventional antibiotic CDI therapy can result in treatment failure and recurrent infection. C. difficile produces biofilms which contribute to its virulence and impair antimicrobial activity. Some bacteriophages (phages) can penetrate biofilms and thus could be developed to either replace or supplement antibiotics. Here, we determined the impact of a previously optimized 4-phage cocktail on C. difficile ribotype 014/020 biofilms, and additionally as adjunct to vancomycin treatment in Galleria mellonella larva CDI model. The phages were applied before or after biofilm establishment in vitro, and the impact was analyzed according to turbidity, viability counts and topography as observed using scanning electron and confocal microscopy. The infectivity profiles and efficacies of orally administered phages and/or vancomycin were ascertained by monitoring colonization levels and larval survival rates. Phages prevented biofilm formation, and penetrated established biofilms. A single phage application reduced colonization causing extended longevity in the remedial treatment and prevented disease in the prophylaxis group. Multiple phage doses significantly improved the larval remedial regimen, and this treatment is comparable to vancomycin and the combined treatments. Taken together, our data suggest that the phages significantly reduce C. difficile biofilms, and prevent colonization in the G. mellonella model when used alone or in combination with vancomycin. The phages appear to be highly promising therapeutics in the targeted eradication of CDI and the use of these models has revealed that prophylactic use could be a propitious therapeutic option.

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.

ABSTRACTProphages are encoded in most genomes of sequencedClostridium difficilestrains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity ofC. difficileprophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific toC. difficilemyoviruses and siphoviruses. Subsequent PCR analysis of 16 clinicalC. difficileribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups ofC. difficilephages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, includingClostridium perfringens,Clostridium botulinum, andBacillusspp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains ofC. difficileand correlates the molecular data to the morphological observation.

BackgroundThe hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027.Methodology/Principal FindingsWe induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species.Conclusion/SignificanceA diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as “critical” for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no “gold standard” for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows “phage synergy” to inform cocktail design.

Graphical abstract

The Galleria mellonella is an effective model for probing Clostridioides difficile interactions with phages. Despite valuable insights from this model, the larvae are not easily amenable to assessing detailed clinical responses to either bacteria or phages. Here, larval survival, colonisation and toxin levels were compared to expression profiles of 17 G. mellonella stress genes to monitor Clostridiodes difficile infection (CDI), and recuperation during phage therapy. The larvae were infected with a ribotype 014/020 isolate and treated with an optimised phage cocktail. Larvae treated prophylactically with phages and the phage-control larval group were protected, showing the highest survival, and low C. difficile colonisation and toxin rates, compared to co-infection, remedial and bacterial-control larval groups. Expression of growth (9) and reproduction (2) genes were enhanced within prophylaxis and phage-control larval groups compared to the co-infection, remedial and bacterial control groups. In contrast, expression of infection (2), humoral (1) and cellular (3) immunity genes declined in the prophylactic and phage-control groups but increased in the co-infection, remedial and bacterial control larvae. The molecular markers augment the survival, colonisation and toxin data and allow detailed monitoring of CDI and recovery. This data support the use of stress marker genes as tools to analyse clinical symptoms in this model.