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 (CD481-1 and CD481-2), three from dog isolates (CD505, CD506, and CD508), and four from human isolates (CD24-2, CD111, CD146, and CD526). Two phages are members of the Siphoviridae family (CD111 and CD146), 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.

Abundant and Diverse Clustered Regularly Interspaced Short Palindromic Repeat Spacers in Clostridium difficile Strains and Prophages Target Multiple Phage Types within This Pathogen

“…Published host range data available for C. difficile phages suggest that phage-host interactions do not depend solely on the CRISPR/Cas system (e.g., differences in absorption [49]). A host range analysis which included ϕCD38-2, ΦMMP04, and ΦMMP02, also used in the CRISPR analysis here, showed that ϕCD38-2 could infect strains CD196 and R20291 (50), but these strains have a spacer which identically matches to the sequence in its genome with an intact CCA motif. Although infective, the infection is reported on the lowest scoring, indicating that there may be predominantly lysogenic infection occurring.…”

Two Novel Myoviruses from the North of Iraq Reveal Insights into Clostridium difficile Phage Diversity and Biology

“…Both the genome organisation and particle morphology of CDKM9 and CDKM15 resemble that of the long tailed myoviruses ΦCD27, ΦCD505 and ΦMMP02. Interestingly, however, CDKM9 has the broadest reported host spectrum compared to the reported host ranges of other long tail myoviruses (ΦCD27, ΦCD505, ΦCD508, ΦCDHM2, ΦCDHM4, ΦCDHM5 and ΦCDHM6) as these infected 13% (4/30), 11% (5/47), 4/47, (28%) 22/80, 4/80 (5%), 20/80 (25%), (29%) 23/80 isolates, respectively [6,14,16]. Further, no other long tailed myoviruses have been shown to lyse R027 isolate [6,14,16,60], while siphoviruses infecting R027 strains have been identified [6,16,17] and the only other report in the literature of myoviruses with this ability are the medium myoviruses ΦCD481-1 and ΦCDHM3, both of which caused turbid plaques on a single strain of R027 each [6,16].…”

“…Several C. difficile phages from both the Myoviridae and Siphoviridae families have already been characterised, including by genome sequencing [9,10,11,12,13,14,15,16,17]. These phages are currently classified into two genera, phicd119virus [18] and the proposed phiMMP04virus [19] (both in the Myoviridae family), however there are other C. difficile phages that do not fall into either genera [13,16,17]. Previous attempts to classify C. difficile myoviruses suggested that they can be grouped according to their particle morphology (by tail length and capsid diameter).…”

Diversity and Evolution in the Genome of Clostridium difficile

“…Some phages have been shown to infect strains of multiple RTs, and phages have been recovered from human, animal, and environmental populations of C. difficile (131,132). As a result of the coevolution of phages and their hosts, many species of bacteria, including C. difficile, have developed ways to resist infection.…”

“…difficile carries a diverse collection of phages, including numerous members of the Siphoviridae and Myoviridae families, such as C2, MMP04, CD119, CDHM1, CD38-2, and CD27, ranging in size from 31 to 56 kbp with a GC content not dissimilar to that of the C. difficile genome (28 to 30%) (52,57,73,113,(131)(132)(133). Despite the absence of proven virulence factors in C. difficile phage genomes, there is increasing evidence that phages may play a role in C. difficile pathogenesis.…”