Search Results (2,251)

Background: Intravascular catheters (ICs) are critical medical devices but require frequent replacement due to the risk of bacterial colonization, which can lead to bloodstream infections. This process causes patient discomfort and incurs significant health and economic costs. Aim: To evaluate the inhibitory activity of natural extracts as potential IC coatings to prevent colonization by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Thirty-six clinical MRSA isolates, obtained from ICs using the Maki technique, were tested. Three natural extracts were evaluated: garlic extract enriched in thiosulfinates (allicin: 7 mg/g), grape extract enriched in proanthocyanidins (92% proanthocyanidins), and propolis extract. Chlorhexidine gluconate (CHG) served as the bactericidal control. The minimum inhibitory concentration (MIC) was determined using the broth microdilution technique with optical density measurements and resazurin-based viability confirmation. The minimum bactericidal concentration (MBC) was assessed from viable cells in wells exceeding the MIC. Results: All tested extracts exhibited bacteriostatic activity against MRSA isolates. The grape extract demonstrated the lowest MIC₉₀ (3.125 mg/mL), followed by propolis extract (MIC₉₀ = 12.5 mg/mL) and garlic extract (MIC₉₀ = 50 mg/mL). Only the propolis extract showed bactericidal activity (MBC = 25 mg/mL). While CHG outperformed the natural extracts, their activity against MRSA suggests potential clinical utility. Conclusion: The natural extracts studied display promising bacteriostatic activity against MRSA isolates from ICs, with propolis extract additionally showing bactericidal effects. Although less potent than CHG, these extracts offer a potential alternative for combating multidrug-resistant pathogens in clinical settings, warranting further investigation for use as IC coatings. Full article

Background/Objectives: Chitosan-based films incorporating green-synthesized silver nanoparticles AgNPs) or copper oxide nanoparticles (CuONPs) were developed to compare their selective antimicrobial action for topical applications. While AgNPs are known for broad-spectrum activity, this study hypothesized that CuONPs would exhibit superior, targeted efficacy against the acne-associated bacterium Cutibacterium acnes. Methods: Nanoparticles were synthesized using Camellia sinensis extract and characterized. Antimicrobial activity was evaluated using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. Chitosan films containing AgNPs or CuONPs were further tested for selective antimicrobial activity and fibroblast cytocompatibility. Results: AgNPs showed strong activity against Escherichia coli and Staphylococcus aureus (MIC = 15 µg/mL) but were less effective against C. acnes (MIC = 125 µg/mL). In contrast, CuONPs demonstrated selective efficacy against C. acnes (MIC = 62 µg/mL; MBC = 125 µg/mL). When incorporated into chitosan films, AgNPs@CHI inhibited E. coli (35 mm halo) and S. aureus (30 mm), whereas CuONPs@CHI were selectively effective against C. acnes (45 mm). All films preserved fibroblast viability above the 70% ISO 10993-5 threshold. Conclusions: CuONPs@CHI films validated selective anti-C. acnes performance, highlighting their promise for targeted anti-acne therapies, while AgNPs@CHI films served as effective broad-spectrum antimicrobial barriers.revealed that AgNPs were potent against Escherichia coli and Staphylococcus aureus (MIC = 15 µg/mL) but less effective against C. acnes (MIC = 125 µg/mL). Conversely, CuONPs demonstrated a marked selective advantage against C. acnes (MIC = 62 µg/mL; MBC = 125 µg/mL). When incorporated into chitosan films, AgNPs@CHI films inhibited E. coli (35 mm halo) and S. aureus (30 mm), whereas CuONPs@CHI films were selectively effective only against C. acnes (45 mm), confirming the targeted performance. All films maintained fibroblast viability above the 70% ISO 10993-5 cytotoxicity threshold. These findings validate the selective action of CuONPs@CHI films, positioning them as a promising biomaterial for targeted anti-acne therapies, while AgNPs@CHI films serve as effective broad-spectrum antimicrobial barriers. Full article

Antibiotic resistance is one of the major threats to public health in the twenty-first century. In this line of work, plants represent a priceless source of antimicrobial compounds since they house chemically different metabolites with a wide range of therapeutic applications. This study reports the bioactivity-guided fractionation, characterization, and evaluation of the efficacy of antimicrobial compounds from leaf acetone extracts of the traditional medicinal plant Terminalia catappa against bacterial clinical isolates and dermatophytes. The acetone extract of T. catappa was subjected to column chromatography for the separation and purification of the phytocompounds. The fractions were analyzed using a thin-layer chromatography–bioautography assay to detect the antimicrobial potency of the eluted compounds. The efficacy of the antimicrobial compounds was evaluated by the minimum inhibitory concentration, minimum bactericidal concentration, and minimum fungicidal concentration. Spectral characterization and structure elucidation of the compound were also achieved. The leaf acetone extract, when subjected to gradient elution by column chromatography, resulted in eight fractions. The fraction Fr-2 was subjected to thin-layer chromatographic elution, which resulted in the elution of phytocompound with Rf value of 0.50 and the phytocompound exhibited antimicrobial activity in the TLC–bioautography assay, and it was isolated in pure form and confirmed as Apigenin 7-O-ß-D-glucopyranoside. The compound exhibited significant inhibition of the clinical isolate Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus 1503 at 9.5 µg/mL. Dermatophytes, viz., Microsporum gypseum and Microsporum canis, were inhibited at 312 µg/mL. The present study successfully achieved the bioactivity-guided isolation and characterization of the flavone compound Apigenin 7-O-ß-D-glucopyranoside from T. catappa. Furthermore, the plant T. catappa represents a promising candidate for the exploration of antimicrobial compounds that could serve as potential plant-derived lead molecules for antimicrobial agents. Full article

This review addresses four controversial aspects of shot blasting in the surface treatment of titanium dental implants. Shot blasting, which involves the projection of abrasive particles onto the titanium surface, is widely used to achieve surface roughness that promotes osteoblastic activity and, consequently, high levels of osseointegration. The first issue examined is the effect of residual alumina particles that remain embedded in the titanium surface after blasting. It has been shown that these residues—typically not exceeding 8% of the surface—can actually enhance osseointegration and even exhibit mild bactericidal properties. The second issue concerns the use of titanium dioxide particles for blasting. Our findings indicate that due to its low abrasiveness, titanium dioxide produces minimal surface roughness and low surface energy, resulting in limited osteoblastic adhesion, inferior fatigue performance, and reduced osseointegration compared to alumina-blasted surfaces. The third topic focuses on the role of compressive residual stress induced by grit blasting. Residual stress contributes to increased surface hydrophilicity, enhancing osteoblast adhesion and mineralization, as evidenced by elevated alkaline phosphatase levels. Finally, the fourth issue involves the effect of acid etching following grit blasting. This treatment introduces microroughness superimposed on the macroroughness generated by grit blasting. In vivo studies demonstrate that grit blasting is the primary driver of osseointegration, while acid etching provides only a marginal improvement in bone–implant contact. Full article

The emergence of bacterial resistance has spurred an urgent need to develop effective alternatives to traditional antibiotics. Phenylethyl alcohol from plants exhibits potential antimicrobial properties, but its efficacy is limited due to its compromised dispersion in water and structural stability in ambient conditions. Herein, for the first time, a polymerization-induced self-assembly strategy was employed to obtain different morphological nanogels with phenylethyl alcohol moieties as hydrophobic cores through in situ reversible addition–fragmentation chain-transfer (RAFT) polymerization. The well-defined copolymers of PTEG_x-co-PPMA_y with controllable molecular weights and narrow polydispersity were confirmed by a combination of techniques. The generated phenylethyl alcohol-based nanogels demonstrated potent antibacterial activity, particularly PTEG₃₀-co-PPMA₇₀ with a one-dimensional linear architecture, which achieved a minimum inhibitory concentration of 62 μg mL⁻¹ against E. coli. SEM revealed membrane disruption as the bactericidal mechanism, highlighting enhanced efficacy against Gram-negative bacteria due to structural differences in cell envelopes. This study establishes a robust platform for designing phenylethyl alcohol-based nanogels with controllable structures toward achieving potent antimicrobial performance, offering a promising strategy for combating bacterial resistance while addressing the dilemma of conventional antibiotic drug systems. Full article

Background: Helicobacter pylori (H. pylori) is a major pathogen associated with a variety of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. As a natural bioactive product, propolis exhibits multifaceted and multi-mechanistic effects. Due to its immunomodulatory, anti-inflammatory, and antioxidant properties, propolis has emerged as a promising therapeutic alternative, offering an innovative approach to managing H. pylori infections and providing new insights into addressing antibiotic resistance. Methods: This comprehensive review, synthesizing data from PubMed, ScienceDirect, and SciFinder, examines the mechanisms by which propolis combats H. pylori. Results: Propolis has demonstrated significant antibacterial efficacy against H. pylori in both in vitro and in vivo models. Its multitargeted mechanisms of action include direct inhibition of bacterial growth, interference with the expression of virulence factors, suppression of virulence-associated enzymes and toxin activity, immunomodulation, and anti-inflammatory effects. These combined actions alleviate gastric mucosal inflammation and damage, reduce bacterial colonization, and promote mucosal healing through antioxidant and repair-promoting effects. Furthermore, propolis disrupts oral biofilms, restores the balance of the oral microbiome, and exerts bactericidal effects in the oral cavity. Synergistic interactions between propolis and conventional medications or other natural agents highlight its potential as an adjunctive therapy. Conclusions: Propolis demonstrates dual functionality by inhibiting the release of inflammatory mediators and suppressing H. pylori growth, highlighting its potential as an adjuvant therapeutic agent. However, clinical translation requires standardized quality control and higher-level clinical evidence. Future research should focus on validating its clinical efficacy and determining optimal dosing regimens, and exploring its role in reducing H. pylori recurrence. Full article

Purpose: The synthesis of nanoscale particles with antibacterial properties has garnered significant attention in pharmaceutical research, driven by the escalating threat of antibiotic-resistant bacteria. This study investigates the antibacterial efficacy of Zn–Co ferrite nanoparticles against virulent, antibiotic-resistant, and biofilm-forming strains of Escherichia coli. Methods: Three nanoparticle variants—S1 (Zn_0.7Co_0.3Fe₂O₄), S2 (Zn_0.5Co_0.5Fe₂O₄), and S3 (Zn_0.3Co_0.7Fe₂O₄)—were synthesized using the solution combustion method by systematically varying the Zn:Co molar ratio. The Scanning Electron Micrograph, X-ray diffraction analysis, Complementary Fourier-transform infrared, Minimum Inhibitory Concentration, and Minimum Bactericidal Concentration were performed. Results: The SEM spectroscopy study revealed distinct morphological differences as a function of the cobalt substitution level within the spinel ferrite matrix. At the highest level of cobalt substitution (Zn_0.3Co_0.7Fe₂O₄), the microstructure displayed significant irregularities, with enhanced agglomeration and a notably broader particle size distribution. X-ray diffraction analysis confirmed the formation of crystalline structures, with an average crystallite size of 12.65 nm. Complementary Fourier-transform infrared spectroscopy revealed characteristic absorption bands in the 400–600 cm⁻¹ range, indicative of the cubic spinel structure of the ferrite nanoparticles. The higher-frequency band was associated with metal–oxide stretching in the tetrahedral sites, while the lower-frequency band corresponded to stretching in the octahedral sites. The Minimum Inhibitory Concentration and Minimum Bactericidal Concentration assays revealed that Zn–Co ferrite nanoparticles possess potent antibacterial activity against virulent, antibiotic-resistant, and biofilm-forming strains of E. coli. Conclusion: Increasing the molar ratio of Zn to Co enhances the antibacterial activity of the nanoparticles. These findings suggest that Zn–Co ferrite nanoparticles could serve as a promising alternative to conventional antibacterial agents for combating multidrug-resistant pathogenic bacteria in the future. Full article

Persistent bacteria (PB) are a subpopulation of dormant cells that tolerate high antibiotic concentrations and cause chronic, hard-to-treat infections, posing a serious global health threat. In this study, the antibacterial efficacy of six cationic polymers, poly(hexamethylene guanidine) (PHMG), polyethyleneimines of different molecular weights, α-polylysine, ε-polylysine, and polyacrylamide, against persistent bacteria was systematically evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of these cationic polymers against susceptible and persistent methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and Escherichia coli (E. coli) were determined using a microbroth dilution method, while cytotoxicity to mouse fibroblast (L929) cells was assessed via MTT assay. PHMG demonstrated superior antibacterial activity, with MBC values as low as 2 μg/mL against persistent MSSA, markedly outperforming the other polymers tested. The key novelties of this work are (i) the first establishment of a cationic polymer library with diverse structural parameters for persistent bacteria clearance, offering a potential strategy for treating recalcitrant infections; and (ii) the elucidation of quantitative correlations between polymer charge density and hydrophobic chain segments with antimicrobial efficacy through structure–activity relationship analysis, providing a theoretical basis for the rational design of anti-persistent materials. Full article

Background and purpose: Vulvovaginal candidiasis (VVC), caused by Candida albicans (C. albicans), is exacerbated by oxidative stress and uncontrolled inflammation. Pathogens like C. albicans generate reactive oxygen species (ROS) to enhance virulence, while host immune responses further amplify oxidative damage. This study investigates the antioxidant and antifungal properties of Hyssopus cuspidatus Boriss volatile extract (SXC), a traditional Uyghur medicinal herb, against fluconazole-resistant VVC. We hypothesize that SXC’s bioactive volatiles counteract pathogen-induced oxidative stress while inhibiting fungal growth and inflammation. Methods: GC-MS identified SXC’s major bioactive components, while broth microdilution assays determined minimum inhibitory concentrations (MICs) against bacterial/fungal pathogens, and synergistic interactions with amphotericin B (AmB) or fluconazole (FLC) were assessed via time–kill kinetics. Anti-biofilm activity was quantified using crystal violet/XTT assays, and in vitro studies evaluated SXC’s effects on C. albicans-induced cytotoxicity (LDH release in A431 cells) and inflammatory responses (cytokine production in LPS-stimulated RAW264.7 macrophages). A murine VVC model, employing estrogen-mediated pathogenesis and intravaginal C. albicans challenge, confirmed SXC’s in vivo effects. Immune modulation was assessed using ELISA and RT-qPCR targeting inflammatory and antioxidative stress mediators, while UPLC-MS was employed to profile metabolic perturbations in C. albicans. Results: Gas chromatography-mass spectrometry identified 10 key volatile components contributing to SXC’s activity. SXC exhibited broad-spectrum antimicrobial activity with MIC values ranging from 0.125–16 μL/mL against bacterial and fungal pathogens, including fluconazole-resistant Candida strains. Time–kill assays revealed that combinations of AmB-SXC and FLC-SXC achieved sustained synergistic bactericidal activity across all tested strains. Mechanistic studies revealed SXC’s dual antifungal actions: inhibition of C. albicans hyphal development and biofilm formation through downregulation of the Ras1-cAMP-Efg1 signaling pathway, and attenuation of riboflavin-mediated energy metabolism crucial for fungal proliferation. In the VVC model, SXC reduced vaginal fungal burden, alleviated clinical symptoms, and preserved vaginal epithelial integrity. Mechanistically, SXC modulated host immune responses by suppressing oxidative stress and pyroptosis through TLR4/NF-κB/NLRP3 pathway inhibition, evidenced by reduced caspase-1 activation and decreased pro-inflammatory cytokines (IL-1β, IL-6, TNF-α). Conclusions: SXC shows promise as a broad-spectrum natural antimicrobial against fungal pathogens. It inhibited C. albicans hyphal growth, adhesion, biofilm formation, and invasion in vitro, while reducing oxidative and preserving vaginal mucosal integrity in vivo. By disrupting fungal metabolic pathways and modulating host immune responses, SXC offers a novel approach to treating recurrent, drug-resistant VVC. Full article

Background/Objectives: There are many commercial mouthrinses, used for a variety of purposes, including antiseptic activity. The objective of this study was to determine the antibacterial activity of various TheraBreath™ oral rinses against the cariogenic bacterium, Streptococcus mutans, and saliva-derived microbial communities, and their antibiofilm activity against S. mutans in vitro biofilms. Methods: Bactericidal activity against planktonic S. mutans was assessed by colony counting after 30 and 2 min exposures to mouthrinses. Ten saliva samples were exposed to mouthrinses for 30 s and plated aerobically on blood agar and Mitis Salivarius agar. Mature biofilms of S. mutans were treated with mouthrinses for 15 min followed by fluorescent vitality staining and polysaccharide measurement, followed by crystal violet staining for measurement of total biofilm remaining. Statistical analysis was performed using Kruskal–Wallis with Dunn’s multiple comparisons test comparing all mean ranks (α = 0.05). Results: TheraBreath™ Fresh Breath, Healthy Smile, and Dry Mouth exhibited no significant antibacterial activity. TheraBreath™ Healthy Gums showed antibacterial activity against S. mutans and microbes from saliva samples similar to Listerine^® Naturals at all exposure times. Whitening Fresh Breath showed intermediate killing of S. mutans after 30 min in liquid but not after 2 min or against salivary microbes. Live/Dead fluorescence vitality staining showed that Healthy Gums and Whitening Fresh Breath had antibacterial activity against mature biofilms of S. mutans statistically similar to Listerine^® Naturals and Colgate^® Total; however, Whitening Fresh Breath did not have significant killing compared to PBS. Conclusions: TheraBreath™ Healthy Gums demonstrated similar antiseptic activity levels to other antiseptic-claiming commercial rinses. Whitening Fresh Breath was comparable but unable to kill in short exposure times. Full article

Objectives: To evaluate the in vitro antimicrobial activity of TiAB, a compound based on silver-bound titanium dioxide, against clinical isolates from dermatological infections. Methods: We tested 155 strains clinically isolated from ulcers and skin infections, including MRSA, ESBL-producing Enterobacterales, and P. aeruginosa. MIC and MBC values were determined using broth microdilution according to CLSI guidelines. Time-kill assays were performed at 0.5×, 1×, and 2× MIC. Median values were used to describe susceptibility profiles. Results: TiAB exhibited strong bactericidal activity against Gram-negative bacteria, including ESBL-positive E. coli and K. pneumoniae, with complete killing at 2× MIC (4–8%) within 4–8 h. Gram-positive pathogens exhibited higher MICs (≥8%) and limited response within 24 h; however, extending exposure to 48 h resulted in enhanced activity. Conclusions: TiAB exhibited in vitro bactericidal activity with median MIC values ranging from 1% to 2% (w/v) against Gram-negative clinical isolates such as E. coli and P. aeruginosa, and 2% to 4% against Gram-positive strains including MRSA. Time-kill assays confirmed ≥3 log₁₀ CFU/mL reductions for Gram-negative bacteria at 2× MIC within 24 h. These results suggest TiAB’s potential as a topical antimicrobial agent, though further in vivo studies are needed to validate its safety and efficacy. Full article

Background/Objectives: The global increase in multidrug-resistant (MDR) bacterial infections underscores the urgent need for effective and sustainable antimicrobial alternatives. This study investigates the antimicrobial activity of exometabolite-based formulations (ExAFs), derived from the cell-free supernatants (CFS) of native lactic acid bacteria (LAB) applied individually or in combination thereof, against MDR-Escherichia coli strain L1PEag1. Methods: Fourteen ExAFs were screened for inhibitory activity using time–kill assays, and structural damage to bacterial cells was assessed via scanning and transmission electron microscopy (SEM/TEM). The most potent formulation was further characterized by liquid chromatography–tandem mass spectrometry (LC–MS/MS) employing a Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH) approach for untargeted metabolite profiling. Results: Among the tested formulations, E10, comprising CFS from Weissella cibaria UTNGt21O, exhibited the strongest inhibitory activity (zone of inhibition: 17.12 ± 0.22 mm), followed by E1 (CFS from Lactiplantibacillus plantarum Gt28L and Lactiplantibacillus plantarum Gt2, 3:1 v/v) and E2 (Gt28L CFS + EPS from Gt2, 3:1 v/v). Time–kill assays demonstrated rapid, dose-dependent bactericidal activity: E1 and E10 achieved >98% reduction in viable counts within 2–3 h, at 1× MIC, while E2 sustained 98.24% inhibition over 18 h, at 0.25× MIC. SEM and TEM revealed pronounced ultrastructural damage, including membrane disruption, cytoplasmic condensation, and intracellular disintegration, consistent with a membrane-targeting mode of action. Metabolomic profiling of E10 identified 22 bioactive metabolites, including lincomycin, the proline-rich peptide Val–Leu–Pro–Val–Pro–Gln, multiple flavonoids, and loperamide. Several compounds shared structural similarity with ribosomally synthesized and post-translationally modified peptides (RiPPs), including lanthipeptides and lassopeptides, suggesting a multifaceted antimicrobial mechanism. Conclusions: These findings position ExAFs, particularly E10, as promising, peptide-rich, bio-based antimicrobial candidates for food safety or therapeutic applications. The co-occurrence of RiPP analogs and secondary metabolites in the formulation suggests the potential for complementary or multi-modal bactericidal effects, positioning these compounds as promising eco-friendly alternatives for combating MDR pathogens. Full article

The search for novel natural resources, such as extracts from algae and plant for use as reductants and capping agents for the synthesis of nanoparticles, may be appealing to medicine and nanotechnology. This study aimed to use Malva parviflora fruit extract as a novel source for the green synthesis of silver nanoparticles (AgNPs) and to evaluate their characterization. The results of biosynthesized AgNP characterization using multiple techniques, such as UV–Vis spectroscopy, scanning electron microscopy (SEM), FTIR analysis, and zeta potential (ZP), demonstrated that M. parviflora AgNPs exhibit a peak at 477 nm; possess needle-like and nanorod morphology with diameters ranging from 156.08 to 258.41 nm; contain –OH, C=O, C-C stretching from phenyl groups, and carbohydrates, pyranoid ring, and amide functional groups; and have a zeta potential of −21.2 mV. Moreover, the antibacterial activity of the M. parviflora AgNPs was assessed against two multidrug-resistant strains, including Staphylococcus aureus MRSA and Escherichia coli ESBL, with inhibition zones of 20.33 ± 0.88 mm and 13.33 ± 0.33 mm, respectively. The minimum bactericidal concentration (MBC) was 1.56 µg/mL for both. SEM revealed structural damage to the treated bacterial cells, and RAPD-PCR confirmed these genetic alterations. Additionally, M. parviflora AgNPs showed antioxidant activity (IC₅₀ = 0.68 mg/mL), 69% protein denaturation inhibition, and cytotoxic effects on MCF-7 breast cancer cells at concentrations above 100 µg/mL. These findings suggest that M. parviflora-based AgNPs are safe and effective for antimicrobial and biomedical applications, such as coatings for implanted medical devices, to prevent biofilm formation and facilitate drug delivery. Full article

Combating antibiotic resistance is critically significant for global public health. The development of new antibacterial nanomaterial is a promising way to do this. In this study, a bottom-up approach was employed to fabricate antibacterial carbon dots (ACDs). During the synthesis, quaternary ammonium function groups with long alkyl chains were successfully grafted on ACDs’ surfaces. The obtained ACDs exhibited potent inhibitory against methicillin-resistant Staphylococcus aureus (MRSA) bacteria with minimum inhibitory concentrations of 2.5 µg/mL. Crucially, 2.5 µg/mL of ACDs could inhibit the growth of MRSA for as long as 72 h, which highlighted their long-term activity. Mechanistic investigations revealed that ACDs exerted bactericidal effects for MRSA bacteria primarily through disrupting the cell wall/membrane, destroying cell membrane potential, inducing the generation of excessive ROS, and triggering the leakage of nucleic acids and intracellular components. In sum, this work provided a kind of ACD with high efficiency and long-term antibacterial activity, offering promising potential for combating drug-resistant bacterial infections. Full article

Background: Antimicrobial resistance is a growing global health concern that requires multiple strategies to be tackled effectively. While the discovery of new antimicrobial molecules is essential, the repurposing of existing compounds also plays a significant role. Standard methods to evaluate antimicrobial efficacy, regulated by the Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), such as the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), are available. However, several potential antimicrobics show interference with these standard methods, resulting in underestimated activity and their premature dismissal from further studies. This work compares reference methods in evaluating different compounds with unique physico-chemical characteristics. We aim to demonstrate that combining different susceptibility tests is mandatory for a successful preclinical screening of antimicrobial compounds. Methods: A selection of substances including natural extracts, both free and in the form of nanocomposites with fumed silica, ionic liquids, ozonated oils, commercial and pure antibiotics, was tested using broth microdilution, disk diffusion, and agar dilution. These methods were chosen following EUCAST and CLSI guidelines, and comparisons were made to evaluate their applicability and limitations for non-conventional substances. Results: The study highlighted significant variability in the outcomes depending on the method used, especially for substances with intrinsic properties such as high viscosity, poor solubility, or specific interactions with the testing medium. In several cases, the use of a single standard method failed to accurately reflect the real antimicrobial activity, leading to potential misinterpretation of effectiveness. Conclusions: A combined methodological approach is recommended to overcome the limitations of individual techniques. The integration of multiple reference methods offers a more accurate screening strategy for identifying and characterizing new and repurposed antimicrobials. Full article