\u3ci\u3eClostridioides Difficile\u3c/i\u3e Biofilm and Spore Production in Response to Antibiotics and Immune Stress

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. C. difficile, an anaerobic spore-forming Gram-positive pathogenic bacterium, is a major cause of hospital-acquired infections. C. difficile persists in the environment and spreads the infection to new hosts in the form of dormant spores and can persist within hosts as surface-attached biofilms. These studies investigate bacterial vegetative cell survival, biofilm formation, and sporulation in response to stress. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating immune system function and directly killing bacteria through membrane disruption and oxidative damage. HDPs produced naturally by animal immune systems are promising candidates to develop novel therapies for bacterial infection because they cause oxidative stress that damages multiple targets in bacterial cells, so it is difficult for bacteria to evolve resistance to these attacks. We investigate the C. difficile response to HDPs applied alone or in combination with antibiotics and to oxidative stresses similar to those caused by the human immune system. In our investigation of fishderived copper-binding HDPs known as piscidins applied to C. difficile in an anaerobic environment, we found that the interaction of piscidin and copper depends on environmental oxygen. While copper-binding increases piscidin potency in an aerobic environment, copper does not synergize with these peptides anaerobically. Piscidins suppress the proliferation of C. difficile by killing bacterial cells and strongly increase the efficacy of multiple classes of antibiotics when applied in combination. Piscidins disrupt the bacterial cell membrane and increase the uptake of exogenous substances. We find that piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. While extracellular stress can induce C. difficile to sporulate or form biofilm for protection, piscidins alone kill vegetative C. difficile cells without triggering spore formation and repress antibiotic-induced sporulation after combined treatments. Piscidins may stimulate more C. difficile biofilm formation at sub-inhibitory doses, so dosage will need to be carefully considered in any potential infection treatments using these peptides

Characterizing the Activity of Antimicrobial Peptides Against the Pathogenic Bacterium Clostridium Difficile in an Anaerobic Environment

Clostridium difficile is an anaerobic Gram-positive pathogen with high treatment costs and mortality and very high antibiotic tolerance. Antimicrobial host-defense peptides (HDPs) produced naturally by animal immune systems are promising candidates to develop novel therapies for bacterial infection because they cause oxidative stress that damages multiple targets in bacterial cells, so it is difficult for bacteria to evolve resistance to these attacks. Piscidins, fish-derived HDPs that can also form complexes with copper (Cu) to enhance their activities, are very active against multiple bacterial species in an aerobic environment. We examined their activity against C. difficile and other species in an anaerobic environment and found that the interaction of piscidins and copper is different in different oxygen environments. Piscidins are highly active against C. difficile and could be a good candidate for drug development

Characterizing the Activity of Antimicrobial Peptides Against the Pathogenic Bacterium Clostridium Difficile in Anaerobic Environment

Clostridium difficile is an anaerobic Gram-positive pathogen with high treatment costs and mortality and very high antibiotic tolerance. Antimicrobial host-defense peptides (HDPs) produced naturally by animal immune systems are promising candidates to develop novel therapies for bacterial infection because they cause oxidative stress that damages multiple targets in bacterial cells, so it is difficult for bacteria to evolve resistance to these attacks. Piscidins, fish-derived HDPs that can also form complexes with copper (Cu) to enhance their activities, are very active against multiple bacterial species in an aerobic environment. We examined their activity against C. difficile and other species in an anaerobic environment and found that the interaction of piscidins and copper is different in different oxygen environments. Piscidins are highly active against C. difficile and could be a good candidate for drug development.https://digitalcommons.odu.edu/sciences_achievement/1018/thumbnail.jp

\u3ci\u3eClostridioides difficile\u3c/i\u3e Spore Production in Response to Antibiotic and Immune Stress

Clostridioides (Clostridium) difficile, an anaerobic, spore-forming Gram-positive pathogenic bacterium, is a major cause of hospital-acquired infections and can persist as surface-attached biofilms for protection from antibiotic and immune stress. C. difficile can form biofilms as a single species or with other anaerobic intestinal bacteria. The environmental signals that cause individual cells to secrete toxins, form biofilms, or develop into spores that can spread the infection to new patients are unknown. In these studies, we investigate bacterial responses to different stress. Antimicrobial host-defense peptides (HDPs) produced by animal immune systems are promising candidates to develop novel therapies for bacterial infection because they cause oxidative stress that damages multiple targets in bacterial cells, so it is difficult for bacteria to evolve resistance to these attacks. We investigate antibiotic treatments, metal ions and sugars, and antimicrobial peptide treatments to determine how. C. difficile reacts to multiple environmental stresses like those from antibiotic treatment or the human immune system. In our investigation of C. difficile and HDPs in an anaerobic environment, we found that the interaction of piscidin and copper is different in different oxygen environments. Antibiotics and oxidative stresses from other sources cause the cells to form spores and/or biofilms to protect themselves, but piscidin kill vegetative C. difficile cells without triggering these protective responses. Piscidins are highly active against C. difficile and could be a good candidate for drug development.https://digitalcommons.odu.edu/gradposters2022_sciences/1016/thumbnail.jp

Growth in a Biofilm Sensitizes \u3ci\u3eCutibacterium acnes\u3c/i\u3e to Nanosecond Pulsed Electric Fields

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells

How Oxygen Availability Affects the Antimicrobial Efficacy of Host Defense Peptides: Lessons Learned from Studying the Copper-Binding Peptides Piscidins 1 and 3

The development of new therapeutic options against Clostridioides difficile (C. difficile) infection is a critical public health concern, as the causative bacterium is highly resistant to multiple classes of antibiotics. Antimicrobial host-defense peptides (HDPs) are highly effective at simultaneously modulating the immune system function and directly killing bacteria through membrane disruption and oxidative damage. The copper-binding HDPs piscidin 1 and piscidin 3 have previously shown potent antimicrobial activity against a number of Gram-negative and Gram-positive bacterial species but have never been investigated in an anaerobic environment. Synergy between piscidins and metal ions increases bacterial killing aerobically. Here, we performed growth inhibition and time-kill assays against C. difficile showing that both piscidins suppress proliferation of C. difficile by killing bacterial cells. Microscopy experiments show that the peptides accumulate at sites of membrane curvature. We find that both piscidins are effective against epidemic C. difficile strains that are highly resistant to other stresses. Notably, copper does not enhance piscidin activity against C. difficile. Thus, while antimicrobial activity of piscidin peptides is conserved in aerobic and anaerobic settings, the peptide–copper interaction depends on environmental oxygen to achieve its maximum potency. The development of pharmaceuticals from HDPs such as piscidin will necessitate consideration of oxygen levels in the targeted tissue

Unique Features of Alarmone Metabolism in \u3ci\u3eClostridioides difficile\u3c/i\u3e

The “magic spot” alarmones (pp)pGpp, previously implicated in Clostridioides difficile antibiotic survival, are synthesized by the RelA-SpoT homolog (RSH) of C. difficile (RSHCd) and RelQCd. These enzymes are transcriptionally activated by diverse environmental stresses. RSHCd has previously been reported to synthesize ppGpp, but in this study, we found that both clostridial enzymes exclusively synthesize pGpp. While direct synthesis of pGpp from a GMP substrate, and (p)ppGpp hydrolysis into pGpp by NUDIX hydrolases, have previously been reported, there is no precedent for a bacterium synthesizing pGpp exclusively. Hydrolysis of the 5′ phosphate or pyrophosphate from GDP or GTP substrates is necessary for activity by the clostridial enzymes, neither of which can utilize GMP as a substrate. Both enzymes are remarkably insensitive to the size of their metal ion cofactor, tolerating a broad array of metals that do not allow activity in (pp)pGpp synthetases from other organisms. It is clear that while C. difficile utilizes alarmone signaling, its mechanisms of alarmone synthesis are not directly homologous to those in more completely characterized organisms

Characterizing the Activity of Antimicrobial Peptides Against the Pathogenic Bacterium \u3ci\u3eClostridium difficile\u3c/i\u3e in an Anaerobic Environment

In our lab we study oxidative stress response of Clostridioides difficile in relation to biofilm formation. Oxidative stress is the imbalance between oxidation and anti-oxidation in the bacterial system. Biofilm are extracellular matrices produced by the bacteria. C. difficile forms biofilm one of the phenotypic expression response to oxidative stress. We use peptides and metals as the major source of oxidative stress. Metals like copper and silver which are already known for their antimicrobial effect, showed stimulation of biofilm at sub-inhibitory concentrations while we notice no difference in biofilm formation when exposed to magnesium. Piscidin one of the host defense peptides (HDPs) produced by the innate immune system combats pathogens entering the host body by inducing oxidative stress. The peptide did not show significant biofilm stimulation suggesting that the response of the bacteria to different sources of oxidative stress may vary and this is worthy of more investigations

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C. difficile to clinically relevant antibiotics tested at sublethal concentrations. Both peptides bind to Cu2+ using an amino terminal copper and nickel binding motif. Here, we investigate the two peptides in the apo and holo states as antibiotic adjuvants against an epidemic strain of C. difficile. We find that the presence of the peptides leads to lower doses of metronidazole, vancomycin, and fidaxomicin to kill C. difficile. The activity of metronidazole, which targets DNA, is enhanced by a factor of 32 when combined with p3, previously shown to bind and condense DNA. Conversely, the activity of vancomycin, which acts at bacterial cell walls, is enhanced 64-fold when combined with membrane-active p1-Cu2+. As shown through microscopy monitoring the permeabilization of membranes of C. difficile cells and vesicle mimics of their membranes, the adjuvant effect of p1 and p3 in the apo and holo states is consistent with a mechanism of action where the peptides enable greater antibiotic penetration through the cell membrane to increase their bioavailability. The variations in effects obtained with the different forms of the peptides reveal that while all piscidins generally sensitize C. difficile to antibiotics, co-treatments can be optimized in accordance with the underlying mechanism of action of the peptides and antibiotics. Overall, this study highlights the potential of antimicrobial peptides as antibiotic adjuvants to increase the lethality of currently approved antibiotic dosages, reducing the risk of incomplete treatments and ensuing drug resistance