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.

Mutation induction in directly exposed cells is currently regarded as the main component of the genetic risk of ionizing radiation for humans. However, recent data on the transgenerational increases in mutation rates in the offspring of irradiated parents indicate that the genetic risk could be greater than predicted previously. Here, we have analysed transgenerational changes in mutation rates and DNA damage in the germline and somatic tissues of non-exposed first-generation offspring of irradiated inbred male CBA/Ca and BALB/c mice. Mutation rates at an expanded simple tandem repeat DNA locus and a proteincoding gene (hprt) were significantly elevated in both the germline (sperm) and somatic tissues of all the offspring of irradiated males. The transgenerational changes in mutation rates were attributed to the presence of a persistent subset of endogenous DNA lesions (double-and singlestrand breaks), measured by the phosphorylated form of histone H2AX (c-H2AX) and alkaline Comet assays. Such remarkable transgenerational destabilization of the F 1 genome may have important implications for cancer aetiology and genetic risk estimates. Our data also provide important clues on the still unknown mechanisms of radiation-induced genomic instability.

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence-based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8-oxo-2P-deoxyguanosine (8-oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8-oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8-oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8-oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non-scavenging antioxidant effect.z 1998 Federation of European Biochemical Societies.

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.

Recent data show that the effects of ionising radiation are not restricted to the directly exposed parental germ cells, but can also manifest in their non-exposed offspring, resulting in elevated mutation rates and cancer predisposition. The mechanisms underlying these transgenerational changes remain poorly understood. One of the most important steps in elucidating these mechanisms is to investigate the initial cellular events that trigger genomic instability. Here we have analysed the effects of paternal treatment by ethylnitrosourea, an alkylating agent which is known to form specific types of DNA adducts, on the transgenerational effects in the first-generation (F 1 ) offspring of exposed CBA/Ca and BALB/c male mice. Mutation rates at two expanded simple tandem repeat loci were significantly elevated in the F 1 germline of both strains. Pre-and post-meiotic exposures resulted in similar increases in mutation rate in the F 1 germline. Within each strain mutation rates were equally elevated in the germline of male and female F 1 offspring of the directly exposed males. The results of our study suggest that transgenerational instability is not attributed to a specific sub-set of DNA lesions, such as double strand breaks, and is most probably triggered by a stress-like response to a generalised DNA damage.2

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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.