Morphological and Genetic Diversity of Temperate Phages in Clostridium difficile

Abstract

Eight temperate phages were characterized after mitomycin C induction of six Clostridium difficile isolates corresponding to six distinct PCR ribotypes. The hypervirulent C. difficile strain responsible for a multiinstitutional outbreak (NAP1/027 or QCD-32g58) was among these prophage-containing strains. Observation of the crude lysates by transmission electron microscopy (TEM) revealed the presence of three phages with isometric capsids and long contractile tails (Myoviridae family), as well as five phages with long noncontractile tails (Siphoviridae family). TEM analyses also revealed the presence of a significant number of phage tail-like particles in all the lysates. Southern hybridization experiments with restricted prophage DNA showed that C. difficile phages belonging to the family Myoviridae are highly similar and most likely related to previously described prophages C2, C5, and CD119. On the other hand, members of the Siphoviridae phage family are more genetically divergent, suggesting that they originated from distantly related ancestors. Our data thus suggest that there are at least three genetically distinct groups of temperate phages in C. difficile; one group is composed of highly related myophages, and the other two groups are composed of more genetically heterogeneous siphophages. Finally, no gene homologous to genes encoding C. difficile toxins or toxin regulators could be identified in the genomes of these phages using DNA hybridization. Interestingly, each unique phage restriction profile correlated with a specific C. difficile PCR ribotype.

Prophage-Stimulated Toxin Production in Clostridium difficile NAP1/027 Lysogens

“…Three milliliters of an overnight C. difficile culture was centrifuged, and total genomic DNA was extracted using an Illustra bacterial genomic DNA extraction kit following the manufacturer's recommendations (GE Healthcare). PCR ribotyping was performed on an Eppendorf Mastercycler with 20 ng purified DNA and primers published by Bidet et al (3), with modifications described previously (12). Band patterns were analyzed and compared using GelComparII (Applied Maths).…”

“…Phage CD38-2 was isolated from a mitomycin C induction lysate (12). Three rounds of purification from single plaques were performed using the double agar overlay method (13) and 0.5 ml of a log-phase culture (optical density at 600 nm [OD 600 ] of 0.4) of C. difficile strain CD274 as the sensitive host.…”

“…In a previous study, we identified CD38-2, a temperate Siphoviridae phage induced from a C. difficile clinical isolate (12). Here, we provide the full characterization of this phage, including whole genome sequencing and phenotypic characterization of lysogens.…”

Lack of Association between Clinical Outcome of Clostridium difficile Infections, Strain Type, and Virulence-Associated Phenotypes

“…PCR-ribotyping was performed using primers CD16S-1F and CD23S-2R as described previously (3) (Table 1) with modifications described elsewhere (13). Patterns were compared using Gel Compar II (Applied Maths, Belgium), and profiles with Յ85% similarity using Pearson's correlation were considered different PCR ribotypes.…”

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

“…More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11). In addition, the multidrug-resistant C. difficile strain CD630 was found to harbor two highly related prophages (13,39) as part of its mosaic genome, where nearly 11% is made of mobile genetic elements.…”