Understanding Clostridium Difficile and the Bacteriophages from the Environment

Clostridium difficile is an important nosocomial pathogen that causes significant human morbidity and mortality. The environment is a natural reservoir for C. difficile where it is found in soils, rivers, and other natural settings. It is not clear whether these strains are active, or present as a result of human contamination. To address this, the physiological characterisation of isogenic pairs was tested to determine if strains from the environment had different properties to those derived from a clinical setting. Clinical strains were less motile but produced more toxins and spores than environmental strains. Little is known about environmental strains of C. difficile outside Europe, the USA, and Australia. To explore new areas, C. difficile and phages were isolated from environmental samples collected in Kurdistan in northern Iraq. Sixty-one strains were isolated and classified into 9 ribotypes including a new ribotype, R691. The two strains examined from dominant ribotype R604, were non-toxigenic and related to each other but distinct from most described clades of C. difficile. Evidence for phages playing important roles in environmental C. difficile dynamics can be seen from the large diverse prophage carriage within strains, and the extensive CRISPR system. Seventeen new phages were isolated and shown to infect ribotype 078 for which few phages exist. Host-range analysis showed that these phages can infect most clinically relevant ribotypes including the novel observation that two myoviruses can infect R027. Genome analysis of three phages revealed that they are distinct but related to known C. difficile phages. Interestingly CDKM15 is the first phage isolated to have an active CRISPR system. This study suggests that the natural environment is a potential reservoir for genetically diverse C. difficile strains and phages that are regionally structured. This could play a role in the emergence of new strains in hospitals.