Trevor Lawley and colleagues report whole-genome sequencing of a large global collection of Clostridium difficile, the most common cause of healthcare-associated infection in the developed world. Their phylogenetic analysis traces the spread of this pathogen through healthcare-associated epidemics worldwide. Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance–conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

The microbiome dysbiosis caused by antibiotic treatment has been associated with both susceptibility to and relapse of Clostridium difficile infection (CDI). Bacteriophage (phage) therapy offers target specificity and dose amplification in situ , but few studies have focused on its use in CDI treatment. This mainly reflects the lack of strictly virulent phages that target this pathogen. While it is widely accepted that temperate phages are unsuitable for therapeutic purposes due to their transduction potential, analysis of seven C. difficile phages confirmed that this impact could be curtailed by the application of multiple phage types. Here, host range analysis of six myoviruses and one siphovirus was conducted on 80 strains representing 21 major epidemic and clinically severe ribotypes. The phages had complementary coverage, lysing 18 and 62 of the ribotypes and strains tested, respectively. Single-phage treatments of ribotype 076, 014/020, and 027 strains showed an initial reduction in the bacterial load followed by the emergence of phage-resistant colonies. However, these colonies remained susceptible to infection with an unrelated phage. In contrast, specific phage combinations caused the complete lysis of C. difficile in vitro and prevented the appearance of resistant/lysogenic clones. Using a hamster model, the oral delivery of optimized phage combinations resulted in reduced C. difficile colonization at 36 h postinfection. Interestingly, free phages were recovered from the bowel at this time. In a challenge model of the disease, phage treatment delayed the onset of symptoms by 33 h compared to the time of onset of symptoms in untreated animals. These data demonstrate the therapeutic potential of phage combinations to treat CDI.

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype. Hospital infections are increasingly being recognised as a major patient safety issue with the hospital environment providing a niche for the rapid evolution of microbial pathogens that are well adapted to infecting susceptible patients. The spore-forming Clostridium difficile is one such bacterium, which causes disease in patients undergoing antibiotic therapy. Since 2000, there has been a striking increase in C. difficile infections due to the emergence of hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Here we use a genetic approach to identify a factor linked to the development of hypervirulence in C. difficile. This study shows that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor contributing to the development of hypervirulence in epidemic isolates, presumably because it leads to significantly increased toxin production. These results have important implications for C. difficile pathogenesis since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype. This study has increased our understanding of how these new variant strains cause disease and why they are more harmful, which is critical for the development of improved strategies for preventing and treating these infections.

Chromophore-binding domains from plant and bacterial photoreceptor proteins have recently gathered increasing attention as new sources of genetically encoded fluorescent proteins (FPs). In particular, FPs based on the flavin-binding LOV (light, oxygen, or voltage sensing) domain offer advantages over green fluorescent protein (GFP) owing to their smaller size, pH and thermal stability, utility under anaerobic conditions and their ability to generate reactive oxygen species. This review focuses on the potential applications of this emerging class of fluorescent reporters, discusses the advantages and limitations of LOV-based FPs, whilst offering insights regarding the further development of this technology for bioimaging and photodynamic therapy.

Rare Clostridium difficile mutants lacking surface-layer protein A escape killing by Avidocin-CDs and display severe phenotypic defects that compromise virulence in animals.