Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid—Novel Approaches to Fight Food-Borne Pathogens

The common causes of community and hospital-acquired infections are Staphylococcus aureus and Staphylococcus epidermidis. One of the key factors enables pathogens to survive, colonize and proliferate in body is the ability to form biofilm. In this study investigated the antimicrobial, antibiofilm formation activities and time-kill assay of (+)-usnic acid against S. aureus and S. epidermidis. Antimicrobial activities were determined by broth microdilution method. The MIC values of (+)-usnic acid for S. aureus and S. epidermidis were 250 and 62.50 µg/ml, respectively. In addition, MBC values of (+)-usnic acid for both pathogens were more than 4 mg/ml. Furthermore, in time-kill assay showed that (+)-usnic acid has no bacteriostatic activity against S. aureus in all concentrations used (125, 250, 500 and 1000 µg/ml) whereas it has bacteriostatic activity against S. epidermidis only at 250 µg/ml after 12 h of incubation. (+)-Usnic acid also inhibited biofilm formation against S. aureus and S. epidermidis. The inhibitory effect on biofilm formation was concentration dependent manner at the concentration equal and greater than MIC values of each microorganism with statistical significance comparing with 5% DMSO (p < 0.05). For in vivo study, silkworms were utilized for efficacy testing of (+)-usnic acid. ED50 of (+)-usnic acid for S. aureus and S. epidermidis were more than 4.00 mg/ml (0.57mg/g of larva). (+)-Usnic acid may lose its therapeutic effect because of the influence of pharmacokinetic in silkworm model. In addition, silkworm infectious model can also be utilized as a screening tool for drugs discovery.

BackgroundCoagulase-negative staphylococci (CoNS) are gram-positive, aerobic, commensal bacteria found on the skin and mucous membranes, including the conjunctiva. Usnic acid (UA) is a dibenzofuran derivative isolated from lichens. This study aimed to investigate the effects of usnic acid on inhibition of ocular biofilm formation due to CoNS.Material/MethodsNine Staphylococcus epidermidis isolates, 5 Staphylococcus hominis isolates, 2 Staphylococcus saprophyticus isolates, and 1 Staphylococcus capitis and Staphylococcus lentus isolates were taken as test bacteria. They were inoculated into brain heart infusion broth and incubated for 24 hours at 35°C and activated. Antibiotic susceptibility was investigated by Kirby-Bauer disc diffusion method. Biofilm production was determined using the microtiter plate method and optical densitometry was measured at 570 nm using an automated microplate reader. Anti-biofilm activity of UA was determined by microtitration method and biofilm removal percentage was calculated.ResultsAll tested bacteria were found as high biofilm-producer strains; they were generally resistant to methicillin, but susceptible to vancomycin. UA inhibited the biofilm formation of S. epidermidis isolates, ranging from 5.7% to 81.5%. It inhibited the biofilm formation of S. saprophyticus and S. lentus by 73.3% and 74.3%, respectively. There was no effect of UA on mature biofilms of S. epidermidis 17.7H, S. epidermidis 15.41, S. hominis 9.3, S. hominis 17.2H, S. saprophyticus, and S. lentus.ConclusionsIt was determined that UA exerted anti-biofilm activity on some CoNS isolated from the ocular surface. Anti-biofilm activity was found to be higher even in strains that did not show antibacterial activity.

Usnic acid inhibits biofilm formation and virulent morphological traits of Candida albicans

Background/Aims: N-acetylcysteine (NAC) is a novel and promising agent with activity against bacterial biofilms. Human serum also inhibits biofilm formation by some bacteria. We tested whether the combination of NAC and human serum offers greater anti-biofilm activity than either agent alone. Methods: Microtiter plate assays and confocal laser scanning microscopy were used to evaluate bacterial biofilm formation in the presence of NAC and human serum. qPCR was used to examine expression of selected biofilm-associated genes. Extracellular matrix (ECM) was observed by transmission electron microscopy. The antioxidants GSH or ascorbic acid were used to replace NAC, and human transferrin, lactoferrin, or bovine serum albumin were used to replace serum proteins in biofilm formation assays. A rat central venous catheter model was developed to evaluate the effect of NAC on biofilm formation in vivo. Results: NAC and serum together increased biofilm formation by seven different bacterial strains. In Staphylococcus aureus, expression of genes for some global regulators and for genes in the ica-dependent pathway increased markedly. In Pseudomonas aeruginosa, transcription of las, the PQS quorum sensing (QS) systems, and the two-component system GacS/GacA increased significantly. ECM production by S. aureus and P. aeruginosa was also enhanced. The potentiation of biofilm formation is due mainly to interaction between NAC and transferrin. Intravenous administration of NAC increased colonization by S. aureus and P. aeruginosa on implanted catheters. Conclusions: NAC used intravenously or in the presence of blood increases bacterial biofilm formation rather than inhibits it.

Synergistic effect of kanamycin and amikacin with setomimycin on biofilm formation inhibition of Listeria monocytogenes

Bacteria in biofilms are more resistant to antibacterial agents than bacteria in planktonic form. Hence, antibacterial agents should be able to eradicate biofilms to ensure the best outcomes. Little is known about how well many antibacterial agents can disrupt biofilms. In this study, we compared sodium lauryl sulfate (SDS), rhamnolipids (RHL), and N-acetylcysteine (NAC) for their ability to eradicate mature biofilms and inhibit new biofilm formation against Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans. SDS and RHL effectively inhibited formation of five bacterial biofilms in a dose-dependent manner, even at concentrations below the minimal inhibitory concentrations (MICs), suggesting that their antibiofilm activities are unrelated to their antibacterial activities. In contrast, NAC at certain concentrations promoted biofilm formation by all bacteria except P. aeruginosa, whereas at supra-MIC concentrations, it inhibited biofilm formation against the four bacteria, suggesting that its antibiofilm activity depends on its antibacterial activity. NAC was ineffective at eradicating mature H. pylori biofilms, and it actually promoted their formation at concentrations >10 mg/mL. Our results suggest that RHL is superior at eradicating biofilms of H. pylori, E. coli, and S. mutans; SDS is more effective against S. aureus biofilms; and NAC is more effective against P. aeruginosa biofilms. Our results may help determine which antibiofilm agents are effective against certain bacterial strains and develop agents effective against specific bacterial threats.

Preparation of novel antimicrobial polymer colloids based on (+)-usnic acid and poly(vinylbenzyl chloride)

Three secondary metabolites of lichens - usnic acid, atranorin and fumarprotocetraric acid - were evaluated for their in vitro antibacterial and antibiofilm activities against three strains each of methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MRSA) from cystic fibrosis patients. Antibacterial activity was assessed by broth microdilution, while antibiofilm activity was evaluated by spectrophotometry or viable count. Usnic acid was significantly more active than atranorin against planktonic cells, while fumarprotocetraric acid exhibited no activity. Atranorin was the most effective in counteracting adhesion to polystyrene, although usnic acid was more active against MRSA. Usnic acid and atranorin showed comparable activity against biofilm formation, although atranorin was more active against MRSA. Usnic acid was significantly more active than atranorin against preformed biofilms. Secondary metabolites of lichens may be considered to be 'lead compounds' for the development of novel molecules for the treatment of S. aureus infections in cystic fibrosis patients.

In modern medicine, artificial devices are used for repair or replacement of damaged parts of the body, delivery of drugs, and monitoring the status of critically ill patients. However, artificial surfaces are often susceptible to colonization by bacteria and fungi. Once microorganisms have adhered to the surface, they can form biofilms, resulting in highly resistant local or systemic infections. At this time, the evidence suggests that (+)-usnic acid, a secondary lichen metabolite, possesses antimicrobial activity against a number of planktonic gram-positive bacteria, including Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium. Since lichens are surface-attached communities that produce antibiotics, including usnic acid, to protect themselves from colonization by other bacteria, we hypothesized that the mode of action of usnic acid may be utilized in the control of medical biofilms. We loaded (+)-usnic acid into modified polyurethane and quantitatively assessed the capacity of (+)-usnic acid to control biofilm formation by either S. aureus or Pseudomonas aeruginosa under laminar flow conditions by using image analysis. (+)-Usnic acid-loaded polymers did not inhibit the initial attachment of S. aureus cells, but killing the attached cells resulted in the inhibition of biofilm. Interestingly, although P. aeruginosa biofilms did form on the surface of (+)-usnic acid-loaded polymer, the morphology of the biofilm was altered, possibly indicating that (+)-usnic acid interfered with signaling pathways.

Usnic acid induces apoptosis in human gastric cancer cells through ROS generation and DNA damage and causes up-regulation of DNA-PKcs and γ-H2A.X phosphorylation

Group A Streptococci (GAS) are involved in a number of life threatening diseases and biofilm formation by these pathogens are considered as an important virulence determinant as it mediates antibiotic resistance among them. In the present study, we have explored the ability of (+)-usnic acid, a lichen secondary metabolite, as an antibiofilm agent against four serotypes of Streptococcus pyogenes causing pharyngitis. Usnic acid inhibited the biofilms of M serotypes M56, st38, M89 efficiently and the biofilm of M74 to a lesser extent. Confocal imaging of the treated samples showed that usnic acid reduced the biomass of the biofilms when compared to that of the control. Fourier Transfer Infrared (FT-IR) spectroscopy indicated that usnic acid reduced the cellular components (proteins and fatty acids) of the biofilms. Interestingly, the FT-IR spectrum further revealed that usnic acid probably acted upon the fatty acids of the biofilms as evident from the disappearance of a peak at 2,455-2,100cm(-1) when compared to the control only in serotypes M56, st38 and M89 but not in M74. The present study shows, for the first time, that usnic acid can act as an effective antibiofilm agent against GAS.

Protecting surfaces from bacterial colonization and biofilm development is an important challenge for the medical sector, particularly when it comes to biomedical devices and implants that spend longer periods in contact with the human body. A particularly difficult challenge is ensuring long-term protection, which is usually attempted by ensuring sustained release of antibacterial compounds loaded onto various coatings. Graphene have a considerable potential to reversibly interact water insoluble molecules, which makes them promising cargo systems for sustained release of such compounds. In this study, we developed graphene coatings that act as carriers capable of sustained release of usnic acid (UA), and hence enable long-term protection of surfaces against colonization by bacterial pathogens Staphylococcus aureus and Staphylococcus epidermidis. Our coatings exhibited several features that made them particularly effective for antibiofilm protection: (i) UA was successfully integrated with the graphene material, (ii) a steady release of UA was documented, (iii) steady UA release ensured strong inhibition of bacterial biofilm formation. Interestingly, even after the initial burst release of UA, the second phase of steady release was sufficient to block bacterial colonization. Based on these results, we propose that graphene coatings loaded with UA can serve as effective antibiofilm protection of biomedical surfaces.

The usnic acid derivative peziculone targets cell walls of Gram-positive bacteria revealed by high-throughput CRISPRi-seq analysis

Antimicrobial resistance (AMR) poses a significant global public health threat, endangering both human and animal health. In clinical environments, AMR often undermines the effectiveness of antibacterial treatments, underscoring the urgent need to discover and develop new antibacterial agents or alternatives to antibiotics. Usnic acid, a secondary metabolite derived from lichens, has emerged as a promising candidate owing to its diverse pharmacological properties, which include antibacterial, immune-regulating, antiaging, and anti-inflammatory activities. Extensive research has shown that usnic acid exhibits strong direct antibacterial effects against Gram-positive bacteria and acts as an antimicrobial adjuvant to enhance the therapeutic efficacy of antibiotic drugs against Gram-negative pathogens. Its mechanisms of action are multifaceted, encompassing the inhibition of RNA, DNA, and protein synthesis; suppression of bacterial efflux pump protein expression and membrane-localized drug-resistant enzyme activity; disruption of cell membrane integrity and metabolic homeostasis; and reduction of virulence factor production and biofilm formation. Despite its potential, the clinical application of usnic acid as an antibacterial agent faces significant challenges including poor aqueous solubility, low bioavailability, and dose-dependent toxicity. To overcome these limitations, nanodelivery systems such as liposomes and polymeric nanoparticles have been developed to enhance solubility, improve targeted delivery, and reduce toxicity, thereby expanding its therapeutic potential. Structural modification can also enhance the antibacterial activity and address solubility issues. This review systematically consolidates current knowledge on usnic acid's antibacterial properties, molecular mechanisms, and combinatorial therapies. It critically evaluates advancements in nanoformulation strategies, assesses safety and toxicity profiles, and identifies obstacles to its development as a clinically viable antibacterial agent. By addressing these aspects, this review aims to provide actionable insights, foster interdisciplinary dialogue, and catalyze further innovation in leveraging this natural product to combat AMR.

Antibiofilm mechanism of mouse gastrointestinal stimulation against Vibrio parahaemolyticus under bile salt culture.