Antisense inhibition of RNA polymerase α subunit of Clostridioides difficile.

Clostridioides difficile, the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis, has emerged as a major enteric pathogen in recent years. Antibiotic treatment perturbs the gut microbiome homeostasis, which facilitates the colonization and proliferation of the pathogen in the host intestine. Paradoxically, the clinical repertoire for C. difficile infection includes the antibiotics vancomycin and/or fidaxomicin. The current therapies do not address the perturbed gut microbiome, which supports the recurrence of infection after cessation of antibiotic therapy. Peptide nucleic acids (PNAs) are novel alternatives to traditional antimicrobial therapy capable of forming strong and stable complexes with RNA and DNA, thus permitting targeted inhibition of specific genes. Here, we report a novel PNA that can target the RNA polymerase α subunit (rpoA) in C. difficile. The designed anti-rpoA construct inhibited clinical isolates of C. difficile (minimum inhibitory concentration values ranged between 4 and 8 µM) and exhibited bactericidal activity. Furthermore, silencing of the rpoA gene suppressed the expression of genes that encode virulence factors [toxin A (tcdA), toxin B (tcdB)] in C. difficile, and the gene that encodes the transcription factor stage 0 sporulation protein (spoOA). Interestingly, the efficacy of the designed PNA conjugate remained unaffected even when tested at different pH levels and against a high inoculum of the pathogen. The rpoA-TAT conjugate was very specific against C. difficile and did not inhibit members of the beneficial gut microflora. Taken altogether, our study confirms that the rpoA gene can be a promising narrow-spectrum therapeutic target to curb C. difficile infection. IMPORTANCE The widespread use of antibiotics can destroy beneficial intestinal microflora, opening the door for spores of Clostridioides difficile to run rampant in the digestive system, causing life-threatening diarrhea. Alternative approaches to target this deadly pathogen are urgently needed. We utilized targeted therapeutics called peptide nucleic acids (PNAs) to inhibit gene expression in C. difficile. Inhibition of the RNA polymerase α subunit gene (rpoA) by PNA was found to be lethal for C. difficile and could also disarm its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene did not impact healthy microflora. We also propose a novel approach to manipulate gene expression in C. difficile without the need for established genetic tools.

Discovery of 1,2-diaryl-3-oxopyrazolidin-4-carboxamides as a new class of MurA enzyme inhibitors and characterization of their antibacterial activity.

The cytoplasmic steps of peptidoglycan synthesis represent an important targeted pathway for development of new antibiotics. Herein, we report the synthesis of novel 3-oxopyrazolidin-4-carboxamide derivatives with variable amide side chains as potential antibacterial agents targeting MurA enzyme, the first committed enzyme in these cytosolic steps. Compounds 15 (isoindoline-1,3-dione-5-yl), 16 (4-(1H-pyrazol-4-yl)phenyl), 20 (5-cyanothiazol-2-yl), 21 and 31 (5-nitrothiazol-2-yl derivatives) exhibited the most potent MurA inhibition, with IC50 values of 9.8-12.2 µM. Compounds 15, 16 and 21 showed equipotent inhibition of the C115D MurA mutant developed by fosfomycin-resistant Escherichia coli. NMR binding studies revealed that some of the MurA residues targeted by 15 also interacted with fosfomycin, but not all, indicating an overlapping but not identical binding site. The antibacterial activity of the compounds against E. coli ΔtolC suggests that inhibition of MurA accounts for the observed effect on bacterial growth, considering that a few potent MurA inhibitors could not penetrate the bacterial outer membrane and were therefore inactive as proven by the bacterial cell uptake assay. The most promising compounds were also evaluated against a panel of Gram-positive bacteria. Remarkably, compounds 21 and 31 (MurA IC50 = 9.8 and 10.2 µM respectively) exhibited a potent activity against Clostridioides difficile strains with MIC values ranging from 0.125 to 1 µg/mL, and were also shown to be bactericidal with MBC values between 0.25 and 1 µg/mL. Furthermore, both compounds were shown to have a limited activity against human normal intestinal flora and showed high safety towards human colon cells (Caco-2) in vitro. The thiolactone derivative (compound 5) exhibited an interesting broad spectrum antibacterial activity despite its weak MurA inhibition. Altogether, the presented series provides a promising class of antibiotics that merits further investigation.

Probiotics: insights and new opportunities for Clostridioides difficile intervention.

Clostridioides difficile infection (CDI) is a life-threatening disease caused by the Gram-positive, opportunistic intestinal pathogen C. difficile. Despite the availability of antimicrobial drugs to treat CDI, such as vancomycin, metronidazole, and fidaxomicin, recurrence of infection remains a significant clinical challenge. The use of live commensal microorganisms, or probiotics, is one of the most investigated non-antibiotic therapeutic options to balance gastrointestinal (GI) microbiota and subsequently tackle dysbiosis. In this review, we will discuss major commensal probiotic strains that have the potential to prevent and/or treat CDI and its recurrence, reassess the efficacy of probiotics supplementation as a CDI intervention, delve into lessons learned from probiotic modulation of the immune system, explore avenues like genome-scale metabolic network reconstructions, genome sequencing, and multi-omics to identify novel strains and understand their functionality, and discuss the current regulatory framework, challenges, and future directions.

Discovery of a novel natural product inhibitor of Clostridioides difficile with potent activity in vitro and in vivo.

Clostridioides difficile infection is a global health threat and remains the primary cause of hospital-acquired infections worldwide. The burgeoning incidence and severity of infections coupled with high rates of recurrence have created an urgent need for novel therapeutics. Here, we report a novel natural product scaffold as a potential anticlostridial lead with antivirulence properties and potent activity both in vitro and in vivo. A whole cell phenotypic screening of 1,000 purified natural products identified 6 compounds with potent activity against C. difficile (minimum inhibitory concentration (MIC) range from 0.03 to 2 µg/ml). All these 6 compounds were non-toxic to human colorectal cells. The natural product compounds also inhibited the production of key toxins, TcdA and TcdB, the key virulence determinants of C. difficile infection pathology. Additionally, the compounds exhibited rapid bactericidal activity and were superior to the standard-of-care antibiotic vancomycin, in reducing a high inoculum of C. difficile in vitro. Furthermore, a murine model of C. difficile infection revealed that compound NP-003875 conferred 100% protection to the infected mice from clinical manifestations of the disease. Collectively, the current study lays the foundation for further investigation of the natural product NP-003875 as a potential therapeutic choice for C. difficile infection.

High-throughput screening identifies a novel natural product-inspired scaffold capable of inhibiting Clostridioides difficile in vitro.

Clostridioides difficile is an enteric pathogen responsible for causing debilitating diarrhea, mostly in hospitalized patients. The bacterium exploits on microbial dysbiosis induced by the use of antibiotics to establish infection that ranges from mild watery diarrhea to pseudomembranous colitis. The increased prevalence of the disease accompanied by exacerbated comorbidity and the paucity of anticlostridial drugs that can tackle recurrence entails novel therapeutic options. Here, we report new lead molecules with potent anticlostridial activity from the AnalytiCon NATx library featuring natural product-inspired or natural product-derived small molecules. A high-throughput whole-cell-based screening of 5000 synthetic compounds from the AnalytiCon NATx library helped us identify 10 compounds capable of inhibiting the pathogen. Out of these 10 hits, we found 3 compounds with potent activity against C. difficile (MIC = 0.5-2 µg/ml). Interestingly, these compounds had minimal to no effect on the indigenous intestinal microbial species tested, unlike the standard-of-care antibiotics vancomycin and fidaxomicin. Further in vitro investigation revealed that the compounds were nontoxic to Caco-2 cell line. Given their potent anticlostridial activity, natural product-inspired scaffolds may suggest potential avenues that can address the unmet needs in preventing C. difficile mediated disease.

ß,γ-Diaryl α-methylene-γ-butyrolactones as potent antibacterials against methicillin-resistant Staphylococcus aureus.

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) synthesized via allylboration or allylindation reactions were screened against methicillin-resistant Staphylococcus aureus (MRSA) USA300. Unlike natural AMGBLs, such as parthenolide, synthetic analogs bearing aryl moieties at the ß- and γ-positions are potent against MRSA. The most potent molecules were comparable to vancomycin and linezolid, the drugs of the last resort for MRSA infections, in their effectiveness with minimum inhibitory concentrations (MICs) ranging from 3.0 to 5.2 µM. These lactones also exhibited potent antibacterial activity against other clinically important multidrug-resistant Gram-positive bacteria (except enterococci), while also showing high tolerability to mammalian cells. Several of these molecules surpassed vancomycin in their rapid killing of the high MRSA inoculum (2 h vs 12 h) in a standard time-kill kinetics assay, with compounds 1l and 1m significantly reducing the intracellular burden of MRSA by about 98-99%, at low concentrations. Additionally, the compounds surpassed vancomycin in inhibiting staphylococcal protease production, indicating that synthetic methylene lactones warrant further investigations as promising anti-MRSA candidates.

Screening of Natural Products and Approved Oncology Drug Libraries for Activity against Clostridioides difficile.

Clostridioides difficile is the most common cause of healthcare-associated diarrhea. Infection of the gastrointestinal tract with this Gram-positive, obligate anaerobe can lead to potentially life-threatening conditions in the antibiotic-treated populace. New therapeutics are urgently needed to treat this infection and prevent its recurrence. Here, we screened two libraries from the National Cancer Institute, namely, the natural product set III library (117 compounds) and the approved oncology drugs set V library (114 compounds), against C. difficile. In the two libraries screened, 17 compounds from the natural product set III library and 7 compounds from the approved oncology drugs set V library were found to exhibit anticlostridial activity. The most potent FDA-approved drugs (mitomycin C and mithramycin A) and a promising natural product (aureomycin) were further screened against 20 clinical isolates of C. difficile. The anticancer drugs, mitomycin C (MIC50 = 0.25 µg/ml) and mithramycin A (MIC50 = 0.015 µg/ml), and the naturally derived tetracycline derivative, aureomycin (MIC50 = 0.06 µg/ml), exhibited potent activity against C. difficile strains. Mithramycin A and aureomycin were further found to inhibit toxin production by this pathogen. Given their efficacy, these compounds can provide a quick supplement to current treatment to address the unmet needs in treating C. difficile infection and preventing its recurrence.

Nanosecond electric pulses rapidly enhance the inactivation of Gram-negative bacteria using Gram-positive antibiotics.

Physically disrupting microorganism membranes to enable antibiotics to overcome resistance mechanisms that inhibit or excrete antibiotics has great potential for reducing antibiotic doses and rendering resistance mechanisms inert. We demonstrate the synergistic inactivation of a Gram-positive (Staphylococcus aureus) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria by combining 222 30 kV/cm electric pulses (EPs) or 500 20 kV/cm EPs with 300-ns EP duration with various antibiotics with different mechanisms of action is demonstrated. Doses of antibiotics that produced no inactivation in 10 min of exposure in solution with bacteria induced several log reductions under the influence of nanosecond EPs. Combining 2 µg/L or 20 µg/mL of rifampicin with the 30 kV/cm EPs enhanced Staphylococcus aureus inactivation compared with EPs alone, while only a few of the other combinations demonstrated improvement. Combining 2 µg/L or 20 µg/mL of mupirocin or rifampicin with either EP train enhanced E. coli inactivation compared with EPs alone. Combining 2 µg/L or 20 µg/mL of erythromycin or vancomycin with the 30 kV/cm EPs enhanced E. coli inactivation compared with EPs alone. These results indicate that EPs can make Gram-positive antibiotics efficient for inactivating Gram-negative bacteria with future studies required to optimize EP parameters for other antibiotics and Gram-negative bacteria.