Clostridium difficile infection (CDI), one of the most common hospital-acquired infections, is increasing in incidence and severity with the emergence and diffusion of hypervirulent strains. CDI is precipitated by antibiotic treatment that destroys the equilibrium of the gut microbiota. Human α-defensin 5 (HD5), the most abundant enteric antimicrobial peptide, is a key regulator of gut microbiota homeostasis, yet it is still unknown if C. difficile, which successfully evades killing by other host microbicidal peptides, is susceptible to HD5. We evaluated, by means of viability assay, fluorescence-activated cell sorter (FACS) analysis, and electron microscopy, the antimicrobial activities of α-defensins 1 and 5 against a panel of C. difficile strains encompassing the most prevalent epidemic and hypervirulent PCR ribotypes in Europe (012, 014/020, 106, 018, 027, and 078). Here we show that (i) concentrations of HD5 within the intestinal physiological range produced massive C. difficile cell killing; (ii) HD5 bactericidal activity was mediated by membrane depolarization and bacterial fragmentation with a pattern of damage peculiar to C. difficile bacilli, compared to commensals like Escherichia coli and Enterococcus faecalis; and (iii) unexpectedly, hypervirulent ribotypes were among the most susceptible to both defensins. These results support the notion that HD5, naturally present at very high concentrations in the mucosa of the small intestine, could indeed control the very early steps of CDI by killing C. difficile bacilli at their germination site. As a consequence, HD5 can be regarded as a good candidate for the containment of hypervirulent C. difficile strains, and it could be exploited in the therapy of CDI and relapsing C. difficile-associated disease.
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