Cationic Homopolymers Inhibit Spore and Vegetative Cell Growth of Clostridioides difficile.

A wide range of synthetic polymers have been explored for antimicrobial activity. These materials usually contain both cationic and hydrophobic subunits because these two characteristics are prominent among host-defense peptides. Here, we describe a series of nylon-3 polymers containing only cationic subunits and their evaluation against the gastrointestinal, spore-forming pathogen Clostridioides difficile. Despite their highly hydrophilic nature, these homopolymers showed efficacy against both the vegetative and spore forms of the bacterium, including an impact on C. difficile spore germination. The polymer designated P34 demonstrated the greatest efficacy against C. difficile strains, along with low propensities to lyse human red blood cells or intestinal epithelial cells. To gain insight into the mechanism of P34 action, we evaluated several cell-surface mutant strains of C. difficile to determine the impacts on growth, viability, and cell morphology. The results suggest that P34 interacts with the cell wall, resulting in severe cell bending and death in a concentration-dependent manner. The unexpected finding that nylon-3 polymers composed entirely of cationic subunits display significant activities toward C. difficile should expand the range of other polymers considered for antibacterial applications.

Regulation and Anaerobic Function of the Clostridioides difficile ß-Lactamase.

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to ß-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to ß-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of ß-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a ß-lactamase, which is a common ß-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficilebla operon that encodes a lipoprotein of unknown function and a ß-lactamase that was greatly induced in response to several classes of ß-lactam antibiotics. An in-frame deletion of the operon abolished ß-lactamase activity in C. difficile strain 630Δerm and resulted in decreased resistance to the ß-lactam ampicillin. We found that the activity of this ß-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to ß-lactams. These results provide support for the function of a ß-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile ß-lactamase activity.

.Clostridioides difficile.

Clostridioides difficile is a spore-forming, anaerobic, intestinal pathogen that causes severe diarrhea that can lead to death. In 2011, C. difficile infected ∼500000 people in the USA and killed ∼29000 people. C. difficile infection (CDI) is the most common healthcare-related infection in the USA, leading to increased healthcare costs of $4.8 billion. This pathogen transmits via the oral-fecal route as a highly contagious and resilient spore. Upon exposure to primary bile acids in the intestine, C. difficile germinates, and in the absence of colonization resistance from the normal microbiota, the bacterium colonizes the colon and produces toxins. These toxins inhibit actin polymerization in host cells, leading to cell death. C. difficile cells can then sporulate in the intestine and exit the body via diarrheal shedding. In culture, sporulation is induced at stationary phase in a nutrient-limiting environment, but the intestinal triggers of sporulation are still unknown.