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

Data_Sheet_1_Combining citizen science and molecular diagnostic methods to investigate the prevalence of Borrelia burgdorferi s.l. and Borrelia miyamotoi in tick pools across Great Britain.DOCX

Lyme disease is the most common tick-borne disease and is caused by a group of bacteria known as Borrelia burgdorferi sensu lato (s.l.) complex. Sharing the same genus as B. burgdorferi, Borrelia miyamotoi is a distinct genotype that causes relapsing fever disease. This emerging tick-borne disease is increasingly becoming a concern in public health. To investigate the prevalence of B. burgdorferi s.l. and B. miyamotoi in ticks first, we developed a PCR (Bmer-qPCR) that targets the phage terminase large subunit (terL) gene carried by B. miyamotoi. A similar approach had been used successfully in developing Ter-qPCR for detecting B. burgdorferi s.l. The terL protein functions as an enzyme in packaging phage DNA. Analytical validation of the Bmer-qPCR confirmed its specificity, efficiency and sensitivity. Second, we designed a citizen science-based approach to detect 838 ticks collected from numerous sites across Great Britain. Finally, we applied Bmer-qPCR and Ter-qPCR to 153 tick pools and revealed that the prevalence of B. burgdorferi s.l. and B. miyamotoi was dependent on their geographical locations, i.e. Scotland showed a higher rate of B. burgdorferi s.l. and lower rate of B. miyamotoi carriage as compared to those of the England data. A pattern of diminishing rate of B. miyamotoi carriage from southern England to northern Scotland was visible. Together, the citizen science-based approach provided an estimation of the carriage rate of B. burgdorferi s.l. and B. miyamotoi in tick pools and a potential spreading pattern of B. miyamotoi from the south to the north of Great Britain. Our findings underscore the power of combining citizen science with the molecular diagnostic method to reveal hidden pattern of pathogen-host-environment interplay. Our approach can provide a powerful tool to elucidate the ecology of tick-borne diseases and may offer guidance for pathogen control initiatives. In an era of limited resources, monitoring pathogens requires both field and laboratory support. Citizen science approaches provide a method to empower the public for sample collection. Coupling citizen science approaches with laboratory diagnostic tests can make real-time monitoring of pathogen distribution and prevalence possible.</p

Data_Sheet_2_Combining citizen science and molecular diagnostic methods to investigate the prevalence of Borrelia burgdorferi s.l. and Borrelia miyamotoi in tick pools across Great Britain.XLSX

Lyme disease is the most common tick-borne disease and is caused by a group of bacteria known as Borrelia burgdorferi sensu lato (s.l.) complex. Sharing the same genus as B. burgdorferi, Borrelia miyamotoi is a distinct genotype that causes relapsing fever disease. This emerging tick-borne disease is increasingly becoming a concern in public health. To investigate the prevalence of B. burgdorferi s.l. and B. miyamotoi in ticks first, we developed a PCR (Bmer-qPCR) that targets the phage terminase large subunit (terL) gene carried by B. miyamotoi. A similar approach had been used successfully in developing Ter-qPCR for detecting B. burgdorferi s.l. The terL protein functions as an enzyme in packaging phage DNA. Analytical validation of the Bmer-qPCR confirmed its specificity, efficiency and sensitivity. Second, we designed a citizen science-based approach to detect 838 ticks collected from numerous sites across Great Britain. Finally, we applied Bmer-qPCR and Ter-qPCR to 153 tick pools and revealed that the prevalence of B. burgdorferi s.l. and B. miyamotoi was dependent on their geographical locations, i.e. Scotland showed a higher rate of B. burgdorferi s.l. and lower rate of B. miyamotoi carriage as compared to those of the England data. A pattern of diminishing rate of B. miyamotoi carriage from southern England to northern Scotland was visible. Together, the citizen science-based approach provided an estimation of the carriage rate of B. burgdorferi s.l. and B. miyamotoi in tick pools and a potential spreading pattern of B. miyamotoi from the south to the north of Great Britain. Our findings underscore the power of combining citizen science with the molecular diagnostic method to reveal hidden pattern of pathogen-host-environment interplay. Our approach can provide a powerful tool to elucidate the ecology of tick-borne diseases and may offer guidance for pathogen control initiatives. In an era of limited resources, monitoring pathogens requires both field and laboratory support. Citizen science approaches provide a method to empower the public for sample collection. Coupling citizen science approaches with laboratory diagnostic tests can make real-time monitoring of pathogen distribution and prevalence possible.</p

‘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

The effects of MSH2 deficiency on spontaneous and radiation-induced mutation rates in the mouse germline

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of mismatch repair deficient Msh2 knock-out mice. Spontaneous mutation rates in homozygous Msh2-/- males were significantly higher than those in isogenic wild-type (Msh2+/+) and heterozygous (Msh2+/-) mice. In contrast, the irradiated Msh2-/- mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated Msh2+/+ and Msh2+/- animals. Considering these data and the results of other publications, we propose that the Msh2-deficient mice possess a mutator phenotype in their germline and somatic tissues while the loss of a single Msh2 allele does not affect the stability of heterozygotes

Paternal Exposure to Ethylnitrosourea Results in Transgenerational Genomic Instability in Mice

Paternal Exposure to Ethylnitrosourea Results in Transgenerational Genomic Instability in Mic

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