Diverse Temperate Bacteriophage Carriage in Clostridium difficile 027 Strains

The 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.We 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.A 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

‘Get in Early’; Biofilm and Wax Moth (Galleria mellonella) Models Reveal New Insights into the Therapeutic Potential of Clostridium difficile Bacteriophages

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

Evolutionary relationship of <i>Clostridium difficile</i> based on the myovirus capsid gene.

<p>The evolutionary history was inferred using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the p-distance method based on their amino acid sequences. All positions containing gaps and missing data were eliminated. The analysis involved 29 amino acid sequences of 10 (84L, 16L, 96L, 82L, 52L, 68L, 91L, 90L, 80L and 73L) representative isolates of the ribotype 027 subclades and seven other ribotypes (ribotypes 014, 005, 002, 020, 015 and 001). Five other sequences including CD196, CDR20291 and QCD-66c26 (ribotype 027), CD630 (ribotype 012) and phiC2 were obtained from <i>in-silico</i> PCR. Other sequences were obtained from NCBI searches. All sequences with 75% similarities were assigned into a subclade. Evolutionary analyses were conducted in MEGA5.</p

Phage carriage in 91 <i>C. difficile</i> 027 isolates in relation to their MLVA types.

*<p>, Phage tail-like particles.</p><p>Prophage carriage among the 91 <i>C. difficile</i> 027 isolates induced with mitomycin C or norfloxacin was correlated to their multiple-locus variable number tandem repeat analysis (MLVA) types. MLVA 1–15 yielded defective myoviruses with three exceptions in MLVA 12 (isolate 66L yielding a siphovirus F), MLVA 13 (isolate 96L yielding a myovirus A) and MLVA 15 (isolate 91L yielding a myovirus A) in addition to the defective myoviruses. MLVA 16 and 17 and 19–23 all yielded phage tail-like particles (PT-LPs) except in MLVA 16 with one isolate yielding myovirus E and another (isolate 36L) in MLVA 22 which yielded no phage under the experimental conditions. Among the three isolates examined in MLVA 18, two yielded siphoviruses and one (isolate 53L) yielded defective myoviruses.</p

Morphological diversity of temperate bacteriophages associated with 91 <i>C. difficile</i> 027 isolates used in this study.

<p>Isolates were induced using mitomycin C or norfloxacin at a final concentration of 3 µg/ml. Prophages from the induced filtered lysates were analysed using TEM. Bars ∼70 nm. Measurement was estimated by measuring 6 phages in each sample.</p

Phage carriage in 91 <i>C. difficile</i> 027 isolates in relation to their different pulsovar types.

*<p>, Phage tail-like particles.</p><p>Prophage carriage among the 91 <i>C. difficile</i> 027 induced using mitomycin C or norfloxacin was also correlated to their pulsovar types. Pulsovar types I and II yielded defective myoviruses with one exception in Pulsovar I (isolate 71L) which yielded only phage tail-like particles (PT-LPs). Pulsovar type III yielded siphoviruses. Pulsovar types IV and V yielded phage tail-like particles with ten exceptions in Pulsovar type IV and one in Pulsovar type V.</p