Characterization of temperate phages infecting Clostridium difficile isolates of human and animal origins

Abstract

Clostridium difficile is a Gram-positive pathogen infecting humans and animals. Recent studies suggest that animals could represent potential reservoirs of C. difficile that could then transfer to humans. Temperate phages contribute to the evolution of most bacteria, for example, by promoting the transduction of virulence, fitness, and antibiotic resistance genes. In C. difficile, little is known about their role, mainly because suitable propagating hosts and conditions are lacking. Here we report the isolation, propagation, and preliminary characterization of nine temperate phages from animal and human C. difficile isolates. Prophages were induced by UV light from 58 C. difficile isolates of animal and human origins. Using soft agar overlays with 27 different C. difficile test strains, we isolated and further propagated nine temperate phages: two from horse isolates (ΦCD481-1 and ΦCD481-2), three from dog isolates (ΦCD505, ΦCD506, and ΦCD508), and four from human isolates (ΦCD24-2, ΦCD111, ΦCD146, and ΦCD526). Two phages are members of the Siphoviridae family (ΦCD111 and ΦCD146), while the others are Myoviridae phages. Pulsed-field gel electrophoresis and restriction enzyme analyses showed that all of the phages had unique double-stranded DNA genomes of 30 to 60 kb. Phages induced from human C. difficile isolates, especially the members of the Siphoviridae family, had a broader host range than phages from animal C. difficile isolates. Nevertheless, most of the phages could infect both human and animal strains. Phage transduction of antibiotic resistance was recently reported in C. difficile. Our findings therefore call for further investigation of the potential risk of transduction between animal and human C. difficile isolates.

FIG 1

Phage morphology as observed by TEM after negative staining with uranyl acetate. The sizes of the capsids and tails were measured on five different particles, and the average values ± the standard deviations are reported below each phage. Note that all of the phages are not displayed on the same scale. The black bar represents 100 nm.

FIG 2

PFGE analysis of the genomic DNA of isolated phages. Undigested purified phage DNA was separated by PFGE and stained with ethidium bromide. The sizes of the mass marker fragments are indicated on the left.

FIG 3

Restriction enzyme analysis of isolated phages. Purified phage DNA was digested with HindIII, and fragments were separated on a 0.8% agarose gel and then stained with ethidium bromide (the inverted image is presented). The sizes of selected bands from the mass marker are indicated on the left.