Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

Abstract

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.