Search Results (2,434)

Biofilm-mediated antimicrobial resistance remains a significant challenge for healthcare and patient safety. Currently, there are gaps in standardised methods for assessing antimicrobials against biofilm formations such as (1) assessment of initial bacterial attachment inhibition, as well as (2) assessment of antimicrobial compounds against both the external biofilm mass and biofilm-embedded metabolically active bacteria. The aim of this study is to address these gaps by combining several anti-biofilm techniques. In the procedure96-well anti-biofilm assessments were performed using plate well and lid peg growth surfaces so as to determine the effects of bioactive compounds (silver nitrate (AgNO₃), nisin, chitosan and zinc oxide nanopowder (ZnO)) on biofilm growth inhibition, formed biofilm reduction and bacterial attachment inhibition. These studies focused on the initial attachment stage against in vitro biofilms of P. aeruginosa and S. aureus. Effects were measured against biofilm mass using Crystal Violet (CV) staining, while embedded bacteria metabolic activity was measured using Resazurin. AgNO₃ exhibited significant inhibition and reduction against P. aeruginosa at all stages of biofilm development (p < 0.0001). AgNO₃ showed significant results against S. aureus during biofilm development and against the embedded, metabolically active population of established biofilms (p < 0.0001). Nisin showed significant inhibition against S. aureus biofilm populations (p < 0.0001). Chitosan showed significant increases in S. aureus biofilm formations following exposure, during initial attachment (p < 0.02), during biofilm growth (p < 0.0001) and against formed biofilm populations (p < 0.0001). ZnO showed significant increases during initial attachment exposure (p < 0.0001), but also exhibited growth inhibition (p < 0.0001) and biofilm reduction (p < 0.0001). Although variance in anti-biofilm efficacy was evident depending upon treatment used, Gram-staining phenotype and test growth surfaces, this combinational method offers potential for high throughput screening and for evaluating pipeline bioactives isolated from different environments for biofilm prevention, inhibition and removal. Additionally, this approach will help elucidate the relationship between bacteria of interest and biofilm mitigation. Full article

Objectives: This study aimed to isolate secondary metabolites from sea cucumber-associated fungus Trichoderma koningii KMM 4751, obtain their bromine derivatives, and investigate their antimicrobial and cytotoxic activities. Results: 3-Ethyl-4-hydroxy-6-methyl-2H-pyran-2-one (EHMP) was isolated from fungal extract. It was brominated, and a previously unreported 6-(bromomethyl)-3-ethyl-4-hydroxy-2H-pyran-2-one (Br-EHMP) was obtained. EHMP inhibited the formation of Candida albicans biofilms with an IC₅₀ of 49.3 µM, but Br-EHMP was less active. Simultaneously, bromination of EHMP significantly enhanced the inhibitory effect of Br-EHMP on Staphylococcus aureus growth and biofilm formation without increasing cytotoxicity to H9c2 cells. Br-EHMP at 10 μM can inhibit sortase A activity by near 30% in a cell-free assay. In silico molecular docking predicted the interaction of Br-EHMP with Cys184 in the sortase A active site. Conclusions: Br-EHMP emerges as a promising antibiofilm agent, and its mechanism involves sortase A inhibition. Full article

Background: Bacterial skin infections in companion animals, particularly those involving multidrug-resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus, pose significant therapeutic challenges due to rising resistance and biofilm formation. While Piper betle is well-recognized in human medicine, data on its efficacy against veterinary isolates—and the comparative phytochemistry and potency of its extracts versus essential oils—remains scarce. Objective: This study evaluated the antimicrobial and antibiofilm efficacy of an ethanolic P. betle leaf extract compared to a panel of representative essential oils—including P. betle leaf oil, clove, tea tree, and plai—against 73 feline and canine clinical isolates. Methods: Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined via broth microdilution. Biofilm inhibition was assessed using crystal violet staining to determine the minimum biofilm inhibitory concentration (MBIC). Phytochemical profiles were characterized using gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–quadrupole time-of-flight mass spectrometry (LC–MS/MS-QTOF). Results: P. betle leaf extract exhibited superior anti-pseudomonad activity (mean MIC: 1.5 mg/mL; MBC: 1.9 mg/mL), demonstrating significantly greater potency than the tested essential oils. Among the essential oils, clove oil was the most effective against Staphylococcus strains (mean MIC: 0.8 mg/mL; MBC: 1.2 mg/mL). Despite 74.4% of P. aeruginosa and 90.0% of Staphylococcus spp. being strong biofilm producers, the P. betle extract demonstrated the highest inhibitory potency against P. aeruginosa (MBIC: 0.7 mg/mL) and, alongside clove oil, showed superior efficacy against Staphylococcus spp. (MBICs: 0.3 and 0.7 mg/mL, respectively). GC–MS analysis identified chavibetol (confirmed via standard spiking) and hydroxychavicol as the primary extract constituents. LC–MS/MS-QTOF profiling further revealed a prominent phenolic profile, including 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde. Comparative analysis suggests that while clove oil efficacy is primarily driven by high eugenol content, the broad-spectrum potency of the P. betle extract arises from a complex phenolic richness, specifically the synergistic presence of hydroxychavicol and chavibetol. Conclusions: These findings confirm the robust potential of P. betle extract as a promising plant-based antiseptic for managing biofilm-associated infections and mitigating antimicrobial resistance in veterinary medicine. Full article

Bacterial biofilms pose a major public health challenge by increasing the antimicrobial tolerance in pathogenic bacteria, thereby limiting the effect of medication-based treatment and promoting the development of antimicrobial resistance. Hence, there is a need to discover new molecules with the ability to prevent biofilm formation. We screened seven synthetic analogues of the breitfussin family of natural products for antimicrobial and antibiofilm activity using a broth microdilution and crystal violet method, respectively. Two compounds inhibited the growth of Gram-positive bacteria in their planktonic state at concentrations of 50 µM, of which one compound (2) demonstrated the ability to inhibit the biofilm formation of Staphylococcus epidermidis at sub-growth-inhibitory, low micromolar concentrations. Compound 2 did not inhibit biofilm growth in Staphylococcus aureus or Listeria monocytogenes, or the ability to eradicate pre-established biofilms. Initial Mode of Action (MoA) studies with compound 2 against S. epidermidis showed a modest impact on the cell surface hydrophobicity and early-stage adhesion to polystyrene. These findings highlight the breitfussin framework as a promising scaffold for the development of new antimicrobial and antibiofilm agents. Full article

Cationic antimicrobial peptides (AMPs) that can target multidrug-resistant pathogenic bacteria via multiple mechanisms are considered promising alternatives to antibiotics. Small (~9 kDa) highly acidic acyl carrier proteins (ACPs), which are a well-known cofactor protein in bacterial fatty acid synthesis (FAS), are a potential intracellular target for AMPs. A previous study has demonstrated that the human AMP LL-37 can bind to ACP and thereby affect FAS and the bacterial membrane integrity. In this work, we have investigated the interactions of different classes of AMPs and antibiofilm peptides (ABPs) with the ACPs of two pathogens. We first studied the folding characteristics of the two ACPs and found that Pseudomonas aeruginosa ACP (PaACP) is fully folded at neutral pH in the absence of divalent cations. On the other hand, the homologous Francisella novicida ACP (FnACP) is unfolded at low ionic strength, but it adopts a fully folded conformation after the addition of divalent cations such as Ca²⁺ or Mg²⁺. These distinct characteristics were shown to be related to a unique His residue that is involved in a stabilizing cation–π interaction. Subsequent biophysical SPR and NMR interaction studies reveal that cationic AMPs and ABPs such as LL-37, melittin, tritrpticin, indolicidin, puroindoline A, lactoferricin B and IDR-1018, but not F5W-magainin 2, can bind to both apo- and holo-ACPs. Binding of Arg-rich peptides is preferred over their Lys-rich analogs. Interestingly, all the peptides bind to holo-ACP with higher affinity than to apo-ACP, which lacks the functionally important phosphopantothenate group. NMR peak intensity perturbation data reveal that helix II of ACP, which is known to be directly involved in complex formation with bacterial FAS enzymes, acts as a common and main recognition site for the peptides. We propose that binding of AMPs and ABPs to this region of bacterial ACPs can directly block fatty acid synthesis and interfere in other ACP-dependent biosynthetic and regulatory events, which in turn could contribute to killing the bacteria and could also intervene in biofilm formation. Full article

Biofilm formation constitutes a major factor in antibiotic treatment failure, shielding bacteria from drugs and promoting persistence. This study demonstrates that the anti-biofilm action of clove oil enhances the efficacy of fosfomycin against Escherichia coli O157:H7 (E. coli O157). Using a luxS-eGFP reporter system, it was found that clove oil inhibited E. coli O157 biofilm formation by up to 80% via suppression of the LuxS/AI-2 quorum sensing (QS) system and bacterial motility. Crucially, this disruption was shown to correlate with a strong synergistic effect when combined with fosfomycin in vitro. In a murine peritoneal infection model, the combination therapy demonstrated superior efficacy compared to monotherapy. Specifically, bacterial loads in the liver, spleen, and small intestine were significantly reduced, and histopathological damage was alleviated. Mechanistically, these effects were linked to the downregulation of the QS. These findings indicate that clove oil acts as a potent adjuvant to fosfomycin by disrupting biofilms, offering a promising strategy against systemic infections caused by E. coli O157. Full article

Background/Objectives: Biofilms are a form of defense that enables bacteria to withstand antibiotic pressure and demonstrate antibiotic resistance. It is crucial to develop anti-biofilm strategies in order to combat chronic and persistent multidrug-resistant (MDR) infections. Methods: In this study, we developed 3D biofilms of single-, dual-, and poly-species MDR ESKAPE components, including the pathogens P. aeruginosa S. aureus and K. pneumoniae, in CF Mu3Gel. We evaluated the efficacy of using a phage, a di-hetero phage cocktail or a poly-hetero phage cocktail in combination with ciprofloxacin to eliminate mature biofilm biomass after 72 h or one week in a single treatment. Results: The phage components mostly exhibited synergistic behavior when combined with ciprofloxacin and with each other in di- and poly-hetero-cocktails. The reduction in 72-h dual- and poly-species biofilms was one log higher than that of one-week biofilms treated with the phage–antibiotic combination. The greatest reductions were observed in the 72-h single-species biofilm with combination therapy, at 1.4–3.0 log. Reductions of 2.16 and 1.6 log were observed in the dual-species P. aeruginosa and S. aureus biofilm and the poly-species biofilm, respectively. Conclusions: This study examined how a single application of phages or phage cocktails, either alone or in combination with ciprofloxacin, impacted established biofilm models, and how this affected the proportion of microcolonies of different species within each model. These insights will facilitate the development of strategies for multiple follow-up treatments, as well as the reordering of phages, phage cocktails, and combinations with antibiotics, to improve outcomes. The 3D biofilm models developed here could be used to screen phages or phage cocktails either on their own or alongside other therapies. This would facilitate the application of in vitro findings to real physiological settings. Full article

Background/Objectives: Metabolic dormancy in biofilms leads to reduced drug efficacy in these communities. Different pharmacokinetics and adverse side effects complicate the simultaneous delivery of multiple drugs at appropriate concentrations to the infection site. This study aimed to develop chitosan-coated mesoporous silica nanoparticles loaded with curcumin and amphotericin B (CS@MSNs-Cur-AmB) and to evaluate their antibiofilm activity combined with antimicrobial photodynamic therapy (PDT) against Streptococcus mutans and Candida albicans dual-species biofilms. Methods: CS@MSNs-Cur-AmB were developed. The structure and morphology of the nanoparticles were evaluated using Fourier transform-infrared spectroscopy (FTIR), zeta potential, field emission scanning electron microscopy (FESEM), and thermogravimetric analysis (TGA). Cytotoxicity toward human gingival fibroblasts was assessed. Colony-forming units per milliliter (CFU/mL) were determined. The metabolic activity of biofilm-forming cells was measured using the tetrazolium (MTT) assay. Results: Physicochemical analyses confirmed the synthesis of CS@MSNs-Cur-AmB, revealing a particle size of 228 nm and thermal stability up to 600 °C. Cytotoxicity assays showed that CS@MSNs-Cur-AmB exhibited good biocompatibility (> 90%). CS@MSNs-Cur-AmB improved antimicrobial activity, which was further enhanced by blue light-emitting diode (LED) irradiation. CS@MSNs-Cur-AmB under LED irradiation showed the strongest effect, reducing metabolic activity to 27.74 ± 4.08% (1 W/cm², 1 min), p < 0.001). Conclusions: Formulating two drugs in nanocarrier systems may improve therapeutic efficacy by increasing local concentration and reducing systemic exposure. This offers an effective strategy for combating oral biofilms. Full article

Background: Periodontitis is a chronic inflammatory disease characterized by dysbiotic biofilm formation, progressive destruction of periodontal tissues, and alveolar bone resorption. Conventional periodontal therapy primarily focuses on mechanical biofilm removal; however, adjunctive therapeutic approaches targeting host inflammatory responses and microbial activity have gained increasing attention. Cannabidiol (CBD), a non-psychoactive phytocannabinoid derived from Cannabis sativa, has demonstrated anti-inflammatory, antimicrobial, and immunomodulatory properties that may be relevant in periodontal disease management. Objective: This systematic review aimed to evaluate the available evidence regarding the potential role of CBD in modulating periodontal inflammation, microbial biofilms, and bone resorption processes. Methods: A systematic literature search was conducted in Web of Science, Cochrane, PubMed, Scopus, and Google Scholar. The review was conducted in accordance with PRISMA guidelines. Studies investigating the effects of CBD on periodontal inflammation, oral biofilms, or bone remodeling were included. Both preclinical (in vitro and animal) and clinical studies were considered. Results: Evidence from experimental studies consistently demonstrated that CBD modulates inflammatory signaling pathways, including inhibition of the TLR4/NF-κB pathway and a reduction in pro-inflammatory cytokine expression, but some results are contradictory. Animal studies reported reduced alveolar bone loss and decreased osteoclast activity following CBD administration. Several studies also demonstrated antimicrobial and antibiofilm effects of CBD against oral microorganisms. Conclusions: While preclinical evidence is promising, the current body of clinical data remains limited. Further well-designed randomized clinical trials are required to determine the efficacy, type of formulation, optimal dosing, and long-term safety of CBD as an adjunctive therapy in periodontal treatment. Full article

Background: Biofilm-associated urinary tract infections (UTIs) pose a significant therapeutic challenge due to the increased tolerance of biofilm-embedded bacteria to antimicrobial agents and the high risk of infection recurrence. The increasing prevalence of multidrug-resistant uropathogens necessitates the evaluation of alternative therapeutic strategies, including antibiotic combination therapy. This study aimed to assess the antibiofilm activity of selected antibiotics used individually and in combination against biofilms formed by clinically relevant uropathogens. Methods: Biofilms of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Enterococcus faecalis isolated from patients with UTIs were developed on polystyrene microtiter plates and exposed to ciprofloxacin, nitrofurantoin, amikacin, and imipenem applied as monotherapy and in combinations. Biofilm biomass reduction was quantified spectrophotometrically using crystal violet staining and expressed as a percentage relative to untreated controls. Results: Antibiotic monotherapy produced moderate reductions in biofilm biomass, with efficacy dependent on bacterial species and antibiotic concentration. In contrast, antibiotic combinations demonstrated enhanced antibiofilm activity. The ciprofloxacin–nitrofurantoin combination showed increased biofilm biomass reduction compared with monotherapy against P. aeruginosa and E. coli. The imipenem–amikacin combination reduced P. mirabilis biofilm biomass by over 80%. Conclusions: These findings suggest that rationally selected antibiotic combinations may represent a more effective strategy than monotherapy for controlling biofilm-associated UTIs. Full article

Acrylic resin is widely used in the fabrication of complete dentures, interacting significantly with the intraoral environment. However, complete dentures face challenges such as stability issues and biofilm accumulation. Glaze application is a common method to reduce surface porosity and microbial adhesion, but it also decreases surface wettability, potentially impairing salivary film formation essential for peripheral sealing. This study aimed to incorporate titanium dioxide and zinc oxide nanoparticles into the glaze applied to thermally activated acrylic resin (TAAR) via spray coating to enhance surface wettability and antifungal activity. Four groups were tested: G (TAAR + commercial glaze − control); AlG (TAAR + commercial glaze + aluminum oxide − roughness control); TiG (TAAR + commercial glaze + titanium dioxide); and ZnG (TAAR + commercial glaze + zinc oxide). Evaluations included flexural strength, color and translucency, surface analysis and antibiofilm activity against Candida albicans. Data were analyzed using one-way ANOVA. No statistically significant differences in mechanical strength (MPa) were observed (G: 108.54 ± 8.36; AlG: 113.60 ± 11.95; ZnG: 111.98 ± 9.27; TiG: 113.66 ± 10.41). Surface roughness significantly increased, and contact angle decreased, indicating improved wettability. Regardless of the antifungal activity no improvement was detected (G: 6.71 ± 0.10; AlG: 6.82 ± 0.08; ZnG: 6.72 ± 0.20; TiG: 6.66 ± 0.18). In conclusion, the incorporation of nanoparticles into the glaze improves the wettability of acrylic resin surfaces, potentially enhancing peripheral sealing and denture retention, which is beneficial for patients with reduced alveolar ridge height. Full article

Background/Objectives: Antimicrobial resistance, an evolutionarily entrenched microbial capacity amplified by extensive antibiotic exposure, has increased the burden of difficult-to-treat infections caused by priority pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. In this study, we assessed whether phytochemical-rich extracts from fully ripe Rosa canina pseudo-fruits (WF) and fully developed Colchicum autumnale flowers (CA) can provide combined antioxidant, antibacterial, and antibiofilm effects against multidrug-resistant clinical isolates. Methods: Plant materials were processed using seven extraction systems spanning non-polar to polar conditions (n-hexane, ethyl acetate, n-butanol, aqueous, 40% ethanol, 60% ethanol, and enzyme-assisted hydrolysis). Fractions were quantified for total phenolics, flavonoids, and tannins, evaluated for antioxidant capacity (DPPH and FRAP), tested for antibacterial activity (disc diffusion and MIC/MBC), and assessed for inhibition of early biofilm attachment. Differences among extraction methods and fractions were analyzed using standard comparative statistics (group comparisons across solvents/fractions), and relationships between chemical composition and bioactivity were examined using correlation-based analysis. Results: Extraction strategy emerged as the main determinant of bioactivity across endpoints. The WFE/ENZ fraction maximized phytochemical recovery (TPC 203.34 ± 11.55 mg GAE/g DW; TFC 35.67 ± 3.06 mg QE/g DW; TTC 53.00 ± 2.65 mg TAE/g DW) and showed strong antioxidant performance (DPPH IC₅₀ 33.60 ± 0.02 μg/mL; FRAP A₇₀₀ 1.90 ± 0.010 at 250 μg/mL). Antibacterial effects were strongest in polar fractions, particularly hydroethanolic and enzyme-assisted extracts, while n-hexane fractions were consistently weakest. Across eight clinical isolates and three reference strains, MIC values ranged from 0.04875 to 6.25 mg/mL for WF extracts and 0.0975–12.5 mg/mL for CA extracts. In the biofilm model, suppression of early attachment was most consistent for CAE/E60–ENZ and WFE/E40–E60–ENZ fractions. Conclusions: Correlation analysis indicated that antibacterial potency aligned primarily with flavonoid levels in R. canina pseudo-fruits and with tannin content in C. autumnale material. Overall, these results support hydroethanolic and enzyme-assisted extraction as rational strategies to enrich polyphenol-dense fractions with convergent antioxidant, antibacterial, and antibiofilm activity, reinforcing plant-derived matrices as a structured discovery space for developing complementary antimicrobial solutions beyond conventional antibiotics. Notably, this is among the first studies to evaluate the antibacterial potential of C. autumnale plant material in this context and to comprehensively assess R. canina pseudo-fruit extracts against multidrug-resistant clinical. Full article

Candida albicans is the causal agent of invasive candidiasis, which might be lethal in immunocompromised patients. Biofilm formation is considered a key virulence factor of C. albicans and is associated with its elevated resistance to antifungals. C. albicans and bacteria like E. coli are frequently found to form mixed biofilms on biotic or abiotic surfaces, rendering them more refractory to existing antifungals. To investigate how E. coli endogenous indole interplaying with exogenous IAA exerts modulatory effects on dual-species biofilm with C. albicans, an E. coli strain deficient in the indole biosynthetic gene tnaA was constructed, and the enzyme TnaA inhibitor was administered to block the indole production in E. coli monoculture and/or E. coli–C. albicans dual culture. Phenotypic assay revealed that indole deficiency attenuated E. coli mono-species biofilm by 12% (tnaA∆ versus WT E. coli), and the lack of indole in the E. coli cell-free culture filtrate abolished the ability to promote C. albicans biofilms, evidenced by the fact that the treatment with WT E. coli culture supernatants exhibited a 1.7-fold promotive effect, while treatment with tnaA∆ displayed no significant difference from the broth control towards C. albicans biofilms. Furthermore, impaired E. coli indole production might disrupt E. coli–C. albicans biofilm, as examined by confocal laser scanning microscopy (CLSM). Moreover, indole-3-acetic acid (IAA) was found to exhibit more potent biofilm-modulatory activity than indole by CLSM imaging with dual biofilms of WT E. coli–C. albicans, in contrast to those of E. coli tnaA∆–C. albicans post-supplemented with exogenous IAA. This study provides evidence for indole as a signaling molecule mediating bacterial–fungal communication during mixed-biofilm formation. Indole and its derivatives, particularly in combination with existing antifungals, have potential in the development of anti-biofilm strategies to eradicate refractory fungal infections. Full article

The persistence of Mycobacterium tuberculosis within biofilm-like structures underscores the need for alternative drug discovery strategies aimed at resistance mechanisms. Medicinal plants provide a rich source of chemically diverse compounds with broad biological activities, including potential antimycobacterial properties. This study investigated acetone stem extracts from Buddleja saligna, Combretum hereroense, and Olea europaea subsp. africana for their phytochemical composition, antioxidant capacity, and antimycobacterial activity against planktonic and biofilm forms of Mycobacterium smegmatis. Phytochemical profiles were analyzed using liquid chromatography–mass spectrometry and quantified through colorimetric assays. Antioxidant activity was assessed using DPPH radical scavenging and ferric reducing power assays, while antimycobacterial effects at MIC and sub-MIC levels were determined through microdilution and growth kinetic assays. Phytochemical composition and concentrations varied among extracts, with B. saligna exhibiting the highest levels of tannins (287.18 ± 0.19 mgGAE/g extract) and flavonoids (16.48 ± 0.05 mgQE/g extract) and showing the strongest antioxidant activity (17.66 ± 5.396 and 399.1 ± 3.717 µg/mL). C. hereroense had a notable antimycobacterial activity with an MIC of 0.16 mg/mL followed by B. saligna and O. europaea subsp. afriana with MIC values of 0.31 and 0.63 mg/mL, respectively. All extracts significantly inhibited early biofilm formation by over 80% at sub-MICs. However, the mature biofilms and sliding motility were less susceptible to the extracts. Overall, the results confirm the antioxidant and antimycobacterial potential of the selected plant extracts, while highlighting challenges in targeting mycobacterial biofilms. Full article

Bovine mastitis is a major infectious disease in dairy cattle, causing significant economic losses and compromising animal health and milk quality worldwide. Among its etiological agents, Staphylococcus aureus is a key contagious pathogen due to its ability to establish persistent intramammary infections and evade host immune responses and antimicrobial therapy. This review summarizes current knowledge on the epidemiology, pathogenesis, clinical presentation, diagnosis, and control of S. aureus in bovine mastitis. Particular emphasis is placed on virulence mechanisms, including adhesion, intracellular persistence, biofilm formation, and immune evasion, which contribute to chronic and recurrent infections. The increasing prevalence of antimicrobial resistance, including methicillin-resistant and multidrug-resistant strains, is highlighted as a major challenge limiting treatment efficacy and posing risks within a One Health context. The review also discusses emerging alternative therapies and innovative control strategies, such as anti-biofilm approaches, immunomodulation, and improved diagnostics, aimed at reducing antimicrobial use. Advances in molecular and point-of-care diagnostic tools are considered for their role in early detection and targeted interventions. Overall, effective control of S. aureus mastitis requires integrated strategies combining prudent antimicrobial use, alternative therapies, improved hygiene, and a multidisciplinary One Health approach. Full article