Auranofin coated catheters inhibit bacterial and fungal biofilms in a murine subcutaneous model.

Microbe entry through catheter ports can lead to biofilm accumulation and complications from catheter-related bloodstream infection and ultimately require antimicrobial treatment and catheter replacement. Although strides have been made with microbial prevention by applying standardized antiseptic techniques during catheter implantation, both bacterial and fungal microbes can present health risks to already sick individuals. To reduce microbial adhesion, murine and human catheters were coated with polyurethane and auranofin using a dip coating method and compared to non-coated materials. Upon passage of fluid through the coated material in vitro, flow dynamics were not impacted. The unique antimicrobial properties of the coating material auranofin has shown inhibitory activity against bacteria such as Staphylococcus aureus and fungi such as Candida albicans. Auranofin coating on catheters at 10mg/mL reduced C. albicans accumulation in vitro from 2.0 x 108 to 7.8 x 105 CFU for mouse catheters and from 1.6 x 107 to 2.8 x 106 for human catheters, showing an impact to mature biofilms. Assessment of a dual microbe biofilm on auranofin-coated catheters resulted in a 2-log reduction in S. aureus and a 3-log reduction in C. albicans compared to uncoated catheters. In vivo assessment in a murine subcutaneous model demonstrated that catheters coated with 10 mg/mL auranofin reduced independent S. aureus and C. albicans accumulation by 4-log and 1-log, respectively, compared to non-coated catheters. In conclusion, the auranofin-coated catheters demonstrate proficiency at inhibiting multiple pathogens by decreasing S. aureus and C. albicans biofilm accumulation.

Antimicrobial Peptides and Small Molecules Targeting the Cell Membrane of Staphylococcus aureus.

Clinical management of Staphylococcus aureus infections presents a challenge due to the high incidence, considerable virulence, and emergence of drug resistance mechanisms. The treatment of drug-resistant strains, such as methicillin-resistant S. aureus (MRSA), is further complicated by the development of tolerance and persistence to antimicrobial agents in clinical use. To address these challenges, membrane disruptors, that are not generally considered during drug discovery for agents against S. aureus, should be explored. The cell membrane protects S. aureus from external stresses and antimicrobial agents, but membrane-targeting antimicrobial agents are probably less likely to promote bacterial resistance. Nontypical linear cationic antimicrobial peptides (AMPs), highly modified AMPs such as daptomycin (lipopeptide), bacitracin (cyclic peptide), and gramicidin S (cyclic peptide), are currently in clinical use. Recent studies have demonstrated that AMPs and small molecules can penetrate the cell membrane of S. aureus, inhibit phospholipid biosynthesis, or block the passage of solutes between the periplasm and the exterior of the cell. In addition to their primary mechanism of action (MOA) that targets the bacterial membrane, AMPs and small molecules may also impact bacteria through secondary mechanisms such as targeting the biofilm, and downregulating virulence genes of S. aureus. In this review, we discuss the current state of research into cell membrane-targeting AMPs and small molecules and their potential mechanisms of action against drug-resistant physiological forms of S. aureus, including persister cells and biofilms.

In Vitro and In Vivo Bactericidal and Antibiofilm Efficacy of Alpha Mangostin Against Staphylococcus aureus Persister Cells.

The formation of persister cells is associated with recalcitrance and infections. In this study, we examined the antimicrobial property of alpha mangostin, a natural xanthone molecule, against methicillin-resistant Staphylococcus aureus (MRSA) persisters and biofilm. The MIC of alpha mangostin against MRSA persisters was 2 µg/ml, and activity was mediated by causing membrane permeabilization within 30 min of exposure. The membrane activity of alpha mangostin was further studied by fast-killing kinetics of MRSA persiste r cells and found that the compound exhibited 99.99% bactericidal activity within 30 min. Furthermore, alpha mangostin disrupted established MRSA biofilms and inhibited bacterial attachment as biofilm formation. Alpha mangostin down-regulated genes associated with the formation of persister cells and biofilms, such as norA, norB, dnaK, groE, and mepR, ranging from 2 to 4-folds. Alpha mangostin at 16 µg/ml was non-toxic (> 95% cell survival) to liver-derived HepG2 and lung-derived A549 cells, similarly. Still, alpha mangostin exhibited 50% cell lysis of human RBC at 16 µg/ml. Interestingly, alpha mangostin was effective in vivo at increasing the survival up to 75% (p<0.0001) of Galleria mellonella larvae infected with MRSA persister for 120 h. In conclusion, we report that alpha mangostin is active against MRSA persisters and biofilms, and these data further our understanding of the antistaphylococcal activity and toxicity of this natural compound.

Design and Evaluation of Short Bovine Lactoferrin-Derived Antimicrobial Peptides against Multidrug-Resistant Enterococcus faecium.

Enterococcus faecium has become an important drug-resistant nosocomial pathogen because of widespread antibiotic abuse. We developed short and chemically simple antimicrobial peptides (AMPs) with a selective amino acid composition, fixed charge, and hydrophobicity ratio based on the core antimicrobial motif of bovine lactoferrin (LfcinB6). Among these peptides, 5L and 6L (both 12 residues long) demonstrated a narrow spectrum and high antibacterial activity against drug-resistant E. faecium isolates with a minimal inhibitory concentration (MIC) that ranged from 4-16 µg/mL. At 32 µg/mL, peptides 5L and 6L inhibited E. faecium strain C68 biofilm formation by 90% and disrupted established biofilms by 75%. At 40 µg/mL, 5L reduced 1 × 107E. faecium persister cells by 3 logs within 120 min of exposure, whereas 6L eliminated all persister cells within 60 min. At 0.5× MIC, 5L and 6L significantly downregulated the expression of a crucial biofilm gene ace by 8 folds (p = 0.02) and 4 folds (p = 0.01), respectively. At 32 µg/mL, peptides 5L and 6L both depolarized the E. faecium membrane, increased fluidity, and eventually ruptured the membrane. Physiologically, 5L (at 8 µg/mL) altered the tricarboxylic acid cycle, glutathione, and purine metabolism. Interestingly, in an ex vivo model of porcine skin infection, compared to no treatment, 5L (at 10× MIC) effectively eliminated all 1 × 106 exponential (p = 0.0045) and persister E. faecium cells (p = 0.0002). In conclusion, the study outlines a roadmap for developing narrow-spectrum selective AMPs and presents peptide 5L as a potential therapeutic candidate to be explored against E. faecium.

A systematic review on toxicity assessment of persistent emerging pollutants (EPs) and associated microplastics (MPs) in the environment using the Hydra animal model.

Emerging pollutants (EPs) are causative for teratogenic and reproductive effects. EPs are detected in all the environmental matrices at higher levels. A suitable model for aquatic toxicity assessment is Hydra, because of morphological, behavioral, reproductive (sexual and asexual), and biochemical changes. Many researchers have used Hydra for toxicity assessment of organic chemicals (BPA), heavy metals, pharmaceuticals, nanomaterials and microplastics. Various Hydra species were used for environmental toxicity studies; however H. magnipapillata was predominantly used due to the availability of its genome and proteome sequences. Teratogenic and reproductive changes in Hydra are species specific. Teratogenic effects were studied using sterozoom dissecting microscope, acridine orange (AO) and 4',6-diamidino-2-phenylindole (DPAI) staining. Reactive oxygen species (ROS) generation by EPs had been understood by the Dichlorodihydrofluorescein Diacetate (DCFDA) staining and comet assay. Multiple advanced techniques would aid to understand the effects at molecular level, such as real-time PCR, rapid amplification of cDNA end- PCR. EPs modulated the major antioxidant enzyme levels, therefore, defense mechanism was affected by the higher generation of reactive oxygen species. Genome sequencing helps to know the mode of action of pollutants, role of enzymes in detoxification, defense genes and stress responsive genes. Molecular techniques were used to obtain the information for evolutionary changes of genes and modulation of gene expression by EPs.

Thioredoxin Reductase Is a Valid Target for Antimicrobial Therapeutic Development Against Gram-Positive Bacteria.

There is a drought of new antibacterial compounds that exploit novel targets. Thioredoxin reductase (TrxR) from the Gram-positive bacterial antioxidant thioredoxin system has emerged from multiple screening efforts as a potential target for auranofin, ebselen, shikonin, and allicin. Auranofin serves as the most encouraging proof of concept drug, demonstrating TrxR inhibition can result in bactericidal effects and inhibit Gram-positive bacteria in both planktonic and biofilm states. Minimal inhibitory concentrations are on par or lower than gold standard medications, even among drug resistant isolates. Importantly, existing drug resistance mechanisms that challenge treatment of infections like Staphylococcus aureus do not confer resistance to TrxR targeting compounds. The observed inhibition by multiple compounds and inability to generate a bacterial genetic mutant demonstrate TrxR appears to play an essential role in Gram-positive bacteria. These findings suggest TrxR can be exploited further for drug development. Examining the interaction between TrxR and these proof of concept compounds illustrates that compounds representing a new antimicrobial class can be developed to directly interact and inhibit the validated target.

Novel Cecropin-4 Derived Peptides against Methicillin-Resistant Staphylococcus aureus.

Increasing microbial resistance, coupled with a lack of new antimicrobial discovery, has led researchers to refocus on antimicrobial peptides (AMPs) as novel therapeutic candidates. Significantly, the less toxic cecropins have gained widespread attention for potential antibacterial agent development. However, the narrow activity spectrum and long sequence remain the primary limitations of this approach. In this study, we truncated and modified cecropin 4 (41 amino acids) by varying the charge and hydrophobicity balance to obtain smaller AMPs. The derivative peptide C18 (16 amino acids) demonstrated high antibacterial activity against Gram-negative and Gram-positive bacteria, as well as yeasts. Moreover, C18 demonstrated a minimal inhibitory concentration (MIC) of 4 µg/mL against the methicillin-resistant Staphylococcus aureus (MRSA) and showed synergy with daptomycin with a fractional inhibition concentration index (FICI) value of 0.313. Similar to traditional cecropins, C18 altered the membrane potential, increased fluidity, and caused membrane breakage at 32 µg/mL. Importantly, C18 eliminated 99% persisters at 10 × MIC within 20 min and reduced the biofilm adherence by ~40% and 35% at 32 and 16 µg/mL. Besides, C18 possessed a strong binding ability with DNA at 7.8 µM and down-regulated the expression of virulence factor genes like agrA, fnb-A, and clf-1 by more than 5-fold (p < 0.05). Interestingly, in the Galleria mellonella model, C18 rescued more than 80% of larva infected with the MRSA throughout 120-h post-infection at a single dose of 8 mg/kg (p < 0.05). In conclusion, this study provides a reference for the transformation of cecropin to derive small peptides and presents C18 as an attractive therapeutic candidate to be developed to treat severe MRSA infections.

Characterization of Five Novel Anti-MRSA Compounds Identified Using a Whole-Animal Caenorhabditis elegans/Galleria mellonella Sequential-Screening Approach.

There is a significant need to combat the growing challenge of antibacterial drug resistance. We have previously developed a whole-animal dual-screening platform that first used the nematode Caenorhabditis elegans, to identify low-toxicity antibacterial hits in a high-throughput format. The hits were then evaluated in the wax moth caterpillar Galleria mellonella infection model to confirm efficacy and low toxicity at a whole animal level. This multi-host approach is a powerful tool for revealing compounds that show antibacterial effects and relatively low toxicity at the whole organism level. This paper reports the use of the multi-host approach to identify and validate five new anti-staphylococcal compounds: (1) 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol(PPT), (2) (1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylate dihydrochloride(NNC), (3) 4,5,6,7-tetrabromobenzotriazole (TBB), (4) 3-[2-[2-chloro-4-[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl] benzoic acid(GW4064), and (5) N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino] benzamide(PD198306). The compounds reduced the severity of methicillin-resistant Staphylococcus aureus (MRSA, strain MW2) infections in both C. elegans and G. mellonella and showed minimal inhibitory concentrations (MICs) in the range of 2-8 µg/mL. Compounds NNC, PPT, and TBB permeabilized MRSA-MW2 cells to SYTOX green, suggesting that they target bacterial membranes. Compound TBB showed synergistic activity with doxycycline and oxacillin against MRSA-MW2, and compounds PPT, NNC, GW4064, and PD198306 synergized with doxycycline, polymyxin-B, gentamicin, and erythromycin, respectively. The study demonstrates the utility of the multi-host approach with follow-up hit characterization for prioritizing anti-MRSA compounds for further evaluation.