Diverse Temperate Bacteriophage Carriage in Clostridium difficile 027 Strains

Abstract

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.

Novel High-Molecular-Weight, R-Type Bacteriocins of Clostridium difficile

“…It appears that these particles are widespread among the species. In addition, a recent study involving a collection of ribotype 027 C. difficile isolates found that they produced phage tail-like particles that morphologically resembled diffocins similar to our 027 diffocin producer, CD16 (27). Further work is needed to determine if diffocins from all 027 strains have identical killing spectra.…”

“…difficile strains produce R-type bacteriocins. After exposure to mitomycin C, many strains of C. difficile have been found by electron microscopy to produce particles with phage tail-like morphology (9,27,32) (Fig. 1).…”

Characterization of Temperate Phages Infecting Clostridium difficile Isolates of Human and Animal Origins

“…We were able to further propagate them to high titers and characterize them by TEM, PFGE, restriction profiling, and host range analysis. Although several prophages have been induced from clinical and environmental C. difficile isolates (17)(18)(19)(20)(21), there is a dearth of genomic and functional data on this group of phages and one main reason for that is the lack of suitable propagating hosts and conditions, which are essential for further characterization. It is also worth mentioning that, to our knowledge, strictly lytic (i.e., virulent) phages in- …”

“…Several putative prophages can be identified by bioinformatic analyses of C. difficile whole-genome sequences available in public repositories. Several prophages and phage tail-like particles have also been induced from C. difficile lysogens by using UV, mitomycin C, and other antibiotics, and some of these phages have been partially characterized by electron microscopy, PFGE, and restriction profiling (17)(18)(19)(20)(21)(22). Most phages are members of the Myoviridae family, i.e., phages with nonflexible and contractile tails, whereas very few are members of the Siphoviridae family, i.e., phages with long, flexible, and noncontractile tails (23).…”

Metabolite profiling of Clostridium difficile ribotypes using small molecular weight volatile organic compounds

“…All ribotypes, except R076, were clinical samples isolated from stool specimens collected from patients infected with C. difficile at the UHL Leicester hospitals (in descending order of number of cases detected). All cultures were isolated in the Department of Infection, Immunity, and Inflammation at the University of Leicester using standard procedures (Nale et al 2012). Ribotypes were determined according to standard protocols (Indra et al 2008) with reference to the strains categorized as part of the C. difficile ribotyping network (Fawley and Wilcox, pers comm.).…”