Search Results (322)

Ampicillin residues in animal-derived foods may cause allergic reactions and promote antimicrobial resistance in consumers; however, data on residue behavior in poultry remain limited. This study aimed to evaluate the depletion of ampicillin in muscle and skin plus fat of broiler chickens. Thirty birds were treated with ampicillin intramuscularly (20 mg kg⁻¹ every 24 h for three days) and sacrificed at 0.5, 1, 2, 5, and 9 days post-administration. Samples were analyzed by liquid chromatography coupled with tandem mass spectrometry, a method successfully validated according to Commission Implementing Regulation (EU) 2021/808, VICH GL49 and GL2. Quantification was performed by linear regression from matrix-matched calibration curves. Residue depletion was evaluated following the European Medicines Agency guidelines. Ampicillin residues in muscle were detected only during the first 24 h post-administration (6.50–8.48 µg kg⁻¹). Residues in skin plus fat remained detectable until day 5 post-administration (6.87–59.88 µg kg⁻¹). Based on this, the withdrawal period calculated for skin plus fat was 9 days considering EU maximum residue limit (MRL) and 19 days considering method limit of quantification, with 95% confidence. These results provide critical data on ampicillin residue kinetics under controlled experimental conditions, supporting risk assessments and the establishment of MRLs in broiler chickens by the Codex Alimentarius. Full article

A screening method based on dispersive solid-phase extraction (DSPE) coupled with ultra-performance liquid chromatography–quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was established for the analysis of non-edible substances in food. This method is applicable to a wide range of non-edible substances, including but not limited to antihypertensive, hypoglycemic, weight-loss, antimicrobial, antipyretic–analgesic, sedative–hypnotic, and antifatigue agents. Through systematic optimization of sample pretreatment and UPLC-Q-TOF-MS conditions, ultrasonic extraction with methanol followed by cleanup using 25 mg Primary Secondary Amine (PSA) and 50 mg C₁₈ was identified as the optimal procedure. The methodological validation demonstrated that all 38 quality control compounds exhibited excellent linear correlation coefficients (R² > 0.99) across a concentration range of 0.005~5.0 mg/kg. At three spiking levels, the mean recoveries and relative standard deviations (RSDs) in four matrices ranged from 67.79% to 110.93% and from 0.23% to 9.37%, respectively. The screening detection limits (SDLs) and limits of quantification (LOQs) were within the range of 0.003~0.5 mg/kg. A screening database comprising 390 substances was constructed. In addition, an identification strategy for the unknown structural analogues was established by summarizing the mass spectrometric fragmentation patterns of the phosphodiesterase-5 (PDE-5) inhibitor analogues. Applied to 110 batches of samples, the method screened 12 known non-edible substances and identified a new PDE-5 inhibitor analogue, phenyl 3-desethyl 3-propyl carbodenafil. The workflow integrates suspected screening using a comprehensive database with a non-targeted identification strategy for unknown analogues. Overall, this strategy is efficient, sensitive and accurate, providing a robust analytical platform for high-throughput screening and discovery of illegally added unknown substances in food. Full article

Background/Objectives: Recombinant poliovirus (PV) virus-like particle (VLP) antigens mimic the conformation of the surface proteins in native PVs (i.e., serotype-specific D-antigen epitopes). Since they lack genomes and are non-infectious, PV-VLPs offer the promise of a safer, next-generation polio vaccine compared to traditional inactivated (IPV) or attenuated live (OPV) vaccines. Sandwich D-antigen ELISA formats are commonly used to measure the in vitro potency values (relative D-antigen content, DU/mL) of unadjuvanted trivalent IPV antigens. If IPV is formulated with aluminum-salt adjuvants, however, a pretreatment step (i.e., adjuvant dissolution or antigen desorption) is required, which may compromise antigen integrity during sample handling. Methods: This work describes the development of three competitive ELISAs to measure the relative D-antigen content of aluminum-salt adjuvanted PV-VLPs (Types 1, 2, 3) without the need for pretreatment. Results: First, key assay parameters were established, including specificity, accuracy, precision, linearity, limit of quantification, and stability-indication. Next, preformulation characterization studies were performed with these methods including (1) rank-ordering the inherent thermal stability profiles of the PV-VLPs (Types 1 > 3 > 2) in-solution and adsorbed to an aluminum phosphate adjuvant (AdjuPhos™, AP) and (2) determining the effect of formulation variables on the thermal stability profiles of AP-adsorbed PV-VLPs including antimicrobial preservatives (thimerosal, 2-PE) and five different antigens present in pediatric combination vaccines (D, T, wP, Hib, Hep B). Conclusions: The development and application of three competitive D-antigen ELISAs were demonstrated, and future use in formulation and storage stability studies with the AP-adjuvanted, trivalent PV-VLPs (Types 1, 2, 3) is discussed with the long-term goal to develop a stable, efficacious, multi-dose, hexavalent combination vaccine presentation. Full article

Background/Objectives: Escherichia coli is the predominant pathogen in community-onset urinary tract infections (UTIs) requiring hospitalization. This study characterized antimicrobial resistance profiles, biofilm formation, extended-spectrum β-lactamase (ESBL) gene distribution, and phylogenetic background of E. coli isolates from hospitalized UTI patients in Western Mexico. Methods: Seventy isolates (September 2023–September 2024) underwent susceptibility testing (CLSI M100, 35th edition), multiplex PCR for blaTEM, blaCTX-M, and blaSHV genes, crystal violet biofilm quantification, and Clermont quadruplex PCR phylotyping. Associations were evaluated by Fisher’s exact test with Benjamini–Hochberg FDR (BH-FDR) correction. Results: ESBL phenotype and MDR were detected in 57.1% and 58.6% of isolates. After BH-FDR correction, ESBL production was significantly associated with amikacin (OR = 5.55; 95% CI: 1.80–18.74; q = 0.002) and TMP-SMX non-susceptibility (OR = 3.00; 95% CI: 1.02–9.23; q = 0.036); ciprofloxacin non-susceptibility was linked to MDR status (OR = 7.21; 95% CI: 1.28–75.66; q = 0.017) but not ESBL phenotype. Biofilm was detected in 77.1% of isolates. blaTEM predominated among ESBL producers (85.0%). Phylogroup B2 (51.4%) was inversely associated with recurrent UTI on both univariate (OR = 0.17; 95% CI: 0.03–0.73; p = 0.008) and adjusted analysis (adjusted OR = 0.19; 95% CI: 0.05–0.81; p = 0.025). Phylogroup C (22.9%) exhibited the highest MDR prevalence (81.3%) and the highest biofilm formation rate among phylogroups (87.5%). Conclusions: The high prevalence of ESBL-producing and MDR E. coli, combined with an unexpected predominance of blaTEM, reveals a distinctive local resistance landscape diverging from regional trends. The inverse association of phylogroup B2 with recurrence and TMP-SMX resistance reinforces the clinical value of phylogenetic surveillance in guiding UTI management strategies. Full article

Volatile organic compounds (VOCs) have garnered substantial research interest in recent years due to their biodegradability, low toxicity, and potent antimicrobial properties against various plant pathogens. As a typical herbivore-induced plant volatile (HIPV) elicited by Nilaparvata lugens (Brown planthopper, BPH), (E)-2-hexenal has been identified as a promising natural antimicrobial agent. In this study, we investigated the protective potential of (E)-2-hexenal against Rhizoctonia solani (R. solani) in rice, focusing on both its direct antifungal activity and host-mediated defense mechanisms. In vitro antifungal assays demonstrated that treatment with 100 μL/mL (E)-2-hexenal resulted in a 91.07% inhibition of R. solani mycelial growth after 48 h. Scanning electron microscopy (SEM) observation and chitinase activity analysis revealed that (E)-2-hexenal suppressed fungal growth by disrupting the structural integrity of the pathogen cell wall. Furthermore, 100 μL/mL (E)-2-hexenal effectively conferred protection to detached rice leaves. Whole-plant inoculation assays confirmed that (E)-2-hexenal pretreatment significantly alleviated disease symptoms and triggered systemic resistance in rice plants. Physiological and biochemical analyses showed that (E)-2-hexenal treatment enhanced the activities of defense-related enzymes, elevated hydrogen peroxide (H₂O₂) levels, and promoted the accumulation of defensive metabolites in rice leaves. HPLC-MS quantification further revealed significant increases in the endogenous levels of jasmonic acid (JA) and salicylic acid (SA). Transcriptomic KEGG pathway enrichment analysis indicated that differentially expressed genes (DEGs) were mainly involved in alpha-linolenic acid metabolism, diterpenoid biosynthesis, phenylpropanoid biosynthesis, plant–pathogen interaction, and plant hormone signal transduction. Collectively, these results suggest that (E)-2-hexenal enhances rice resistance to sheath blight disease via a dual-action mechanism: direct inhibition of fungal development and activation of host immune responses. Our findings highlight the potential application of (E)-2-hexenal and other VOCs in developing eco-friendly strategies for sustainable rice disease management. Full article

Background/Objectives: Klebsiella pneumoniae is a major pathogen involved in both acute and chronic infections, characterized by high incidence and significant clinical severity. Over the past decade, resistance to traditional antimicrobial treatments has risen rapidly, highlighting the urgent need for innovative approaches. Light-based antimicrobial strategies, including antimicrobial photodynamic therapy (aPDT), offer a promising approach for addressing drug-resistant bacteria. Combining two photosensitizers (PSs) with antibiotics synergistically enhances ROS generation and multi-target bacterial damage, achieving superior antimicrobial efficacy at reduced PS, light and antibiotic doses while limiting resistance development. We evaluated the efficacy of aPDT using the photosensitizers (PSs) methylene blue (MB) and Photodithazine (PDZ), either alone or in combination with the antibiotic ciprofloxacin (CIP), gentamicin (GEN), or ceftriaxone (CEF), against K. pneumoniae. Methods: Bacterial suspensions were treated with PDZ (25–200 µg/mL) and/or MB (5–20 µg/mL) in the presence of CIP (0.005–4 µg/mL), GEN (0.5–16 µg/mL), or CEF (0.5–16 µg/mL), followed by irradiation at either 15 J/cm² or 30 J/cm². Bacterial survival was assessed by colony-forming unit (CFU/mL) quantification. Results: The combined application of photosensitizers and antibiotics demonstrated a synergistic bactericidal effect against planktonic K. pneumoniae. The combined use of two PSs with antibiotics markedly reduced the antibiotic dose required to achieve a comparable bactericidal effect. Conclusions: This study highlights the potential of combining aPDT with conventional antibiotics as a promising strategy to combat drug-resistant infections, offering enhanced antimicrobial efficacy while allowing for reduced antibiotic dosages to achieve comparable therapeutic outcomes. Full article

Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa rank among the most challenging pathogens due to increasing prevalence of multidrug-resistant (MDR) strains. These pathogens pose major risks to public health and food safety, earning their inclusion on the World Health Organization (WHO) priority list of MDR bacteria. While available conventional antibiotics are becoming less effective, natural products from plant extracts offer promising alternative and synergetic effects that can restore efficacy and lower required doses. Their antimicrobial activity is attributed to phytochemicals such as phenolic compounds and terpenoids acting via membrane disruption, efflux pump inhibition, biofilm interference, and cell protein disruption. Furthermore, phytochemicals in essential oils, such as carvacrol, thymol, and cinnamaldehyde, also exhibit antimicrobial and antioxidant activities. Their broad antimicrobial effects extend shelf life and enhance food safety, making them effective natural alternatives to synthetic preservatives. Moreover, advances in extraction and characterization techniques, including green solvents, spectrometry and hyphenated chromatographic methods, have improved recovery, identification and quantification. In addition, artificial intelligence (AI) emerges as a transformative tool to accelerate discovery, optimize compound screening, and predict synergistic interactions. Notwithstanding these advances, challenges persist in standardization, bioavailability, and clinical translation. Further studies are needed to isolate active compounds, elucidate mechanisms of action, validate combined use with conventional antibiotics and overcome formulation, delivery, sensory and regulatory hurdles. This review examines current knowledge of opportunities and limitations of plant-based antimicrobials against MDR pathogens supported by advances in extraction, characterization, and AI. Full article

The roots of Cannabis sativa L., historically overlooked, are gaining attention as a potential source of bioactive compounds with antioxidant, antimicrobial, and anti-inflammatory properties. While previous studies have focused on extractions using ethanol, water, or supercritical CO₂, the feasibility of edible oil-based extraction remains largely unexplored. This study evaluated the extraction of root compounds using hemp seed oil, MCT coconut oil, and grape seed oil at six temperatures (50–90 °C). Extracts were analyzed by GC–MS for compound identification and quantification, and antioxidant activity was assessed using the DPPH assay, ABTS test and β-carotene bleaching method, with results statistically evaluated by ANOVA. Friedelin was successfully extracted with all oils, with grape seed oil yielding the highest concentration (0.810 mg/g dry roots), achieving recoveries higher than those previously reported for ethanol-based extractions. All extracts demonstrated positive antioxidant activity, with grape seed oil, both alone and combined with extracts, showing higher values across the three methods. ANOVA revealed a significant effect of solvent type on both Friedelin concentration and antioxidant capacity. These results demonstrate that edible oils are effective solvents for extracting bioactive compounds from C. sativa roots, supporting their potential application in cosmetic or medicinal formulations. Full article

The high mortality rate from microbial infections underscores the need to discover new antimicrobials. This work produced antibacterial Chitosan biofilms with and without the incorporation of the ethanolic extract of Libidibia ferrea stem bark and its ethyl acetate and aqueous fractions. The extract and fractions were subjected to FTIR and ¹H NMR analysis. The biofilms were characterized by FTIR, scanning electron microscopy, thermogravimetry, and differential scanning calorimetry analysis. The ¹H NMR and FTIR data, as well as the colorimetric quantification of total phenolics, demonstrated the presence of phenolic compounds. Staphylococcus aureus and Bacillus cereus were the most susceptible bacteria for Chitosan/L. ferrea biofilms and fractions (growth inhibition zones values in the range of 10.8 ± 0.1 to 14.0 ± 0.1 mm, and minimum inhibitory or bactericidal concentration, MIC or MBC values of the fractions were in the range of 125 to 250 µg mL⁻¹. Only the fractions inhibited Pseudomonas aeruginosa (MIC = 250 µg mL⁻¹). Chitosan/L. ferrea biofilms exhibited efficient interactions between chitosan functional groups and secondary metabolites, good thermal stability, and increased rigidity in mechanical tests. This study reinforces the pharmacological potential of biodegradable Chitosan/L. ferrea biofilms as antibacterial agents biofilms. Full article

Background: Dental caries remains one of the most prevalent oral diseases worldwide, largely driven by the virulence of Streptococcus mutans. Although plant phenolics from green tea and pomegranate are known for their antimicrobial properties, their molecular mechanisms of action against key S. mutans virulence targets remain insufficiently characterized. Aim: This study investigated the antibacterial and anti-virulence properties of Viroelixir, a phenolic-rich formulation derived from green tea (Camellia sinensis) and pomegranate (Punica granatum), against S. mutans, with particular emphasis on predictive molecular docking interactions with critical virulence-associated proteins. Methods: Viroelixir phytochemical composition was characterized by LC–MS using a C18 reverse-phase column and negative electrospray ionization mode. Antibacterial activity was evaluated using growth kinetics, agar plating, and crystal violet assays. Acidogenicity, hemolytic activity, and biofilm formation were assessed using pH modulation, hemolysis assays, SEM, and biofilm biomass quantification. Virulence gene expression was analyzed by RT-qPCR. In silico molecular docking was performed to explore potential interactions between major LC–MS-supported phenolic constituents and S. mutans virulence proteins, including glucosyltransferase B (GtfB), LuxS, and SpaP. Biocompatibility was evaluated in human gingival epithelial cells. Results: The LC-MS analysis revealed a complex mixture of phenolic compounds consistent with catechins and ellagitannins. Compound identification was considered tentative and based on mass spectral range and chromatographic behavior. Viroelixir significantly inhibited S. mutans growth, acid production, hemolytic activity, and biofilm formation in a concentration-dependent manner. Key virulence genes were markedly downregulated. Docking analyses suggested stable binding of selected phenolics—particularly punicalagin, catechin, and epigallocatechin—within the active sites of GtfB, LuxS, and SpaP. Importantly, Viroelixir showed no cytotoxic effects on gingival epithelial cells. Conclusions: Viroelixir exerts potent antibacterial and anti-virulence effects against S. mutans through a multi-target mechanism combining transcriptional suppression and predictive molecular inhibition of virulence proteins, supporting its potential as a safe, natural therapeutic for caries prevention. Full article

The implementation of stringent regulatory policies for foodborne pathogens necessitates ultra-sensitive analytical methods. Digital detection, characterized by absolute quantification and tolerance to complex matrices, serves as a robust approach for food safety monitoring. This review summarizes recent advances in digital detection for foodborne pathogens, including nucleic acid amplification-based platforms such as droplet digital PCR and digital isothermal amplification, as well as emerging preamplification-free approaches based on enzyme-mediated signal conversion, functional nanomaterials, and microfluidic devices. We also profile the applications of digital detection technologies for achieving highly specific and accurate detection of foodborne pathogens and discuss their capabilities in viable bacteria quantification, antimicrobial resistance analysis, and multiplex detection. We finally discuss emerging trends, including partition-free digital detection and artificial intelligence-assisted analysis. These advances are expected to promote the development of intelligent and data-driven food safety surveillance strategies. Full article

Phenolic compounds are plant-derived antioxidants crucial for human health and food preservation. Their bioactive potential including anti-inflammatory, antimicrobial, and anti-carcinogenic properties makes them a vital focus in nutritional, pharmaceutical, and agricultural research. This review critically evaluates the methodologies for their extraction, detection, and quantification to accurately assess antioxidant activity. Oxidative stress in biological systems and food matrices necessitates accurate analytical methodologies for assessing antioxidant behavior, which include both in vitro, in vivo and ex vivo approaches. Sample pretreatment and extraction techniques are critical for reliable analysis and vary depending on the matrix, compound polarity, and target phenolic subclass. We compare conventional extraction techniques (Soxhlet, maceration) with advanced methods like ultrasound-assisted, microwave-assisted, and supercritical fluid extraction. Detection methods reviewed include spectrophotometric assays (e.g., DPPH, FRAP, ORAC), electrochemical sensors, and chromatographic techniques (e.g., HPLC, HPLC−MS). While each method has distinct advantages, a lack of standardization remains the primary challenge, driven by variations in protocols and the vast chemical diversity of phenolics. This review underscores the critical need for integrated, standardized approaches to ensure the accurate and comparable evaluation of antioxidant activity in research and industry. Full article

Phenyllactic acid (PLA), a natural antimicrobial metabolite produced by lactic acid bacteria (LAB), has emerged as a key compound for biopreservation in food systems. The aims of this review are to summarize the main findings on LAB-producing strains, the effects of primary PLA precursors, the impacts of culture conditions on PLA production, antimicrobial activity, mechanisms of action, quantification and analysis methods, food applications, regulatory status, and the challenges in PLA production and applications. In this review, the quorum sensing role in PLA production and multi-omics strain improvement was revised. Applications in dairy, bakery, fruits, vegetables, meat, and fish products as well as active packaging are analyzed, demonstrating their effectiveness in controlling microbial spoilage and pathogens while preserving sensory quality. Its broad-spectrum antifungal and antibacterial activities make it particularly attractive as a clean-label alternative to synthetic preservatives, contributing to both food safety and extended shelf life. Finally, current limitations and future research needs are outlined, particularly in optimizing PLA production and establishing its role as a sustainable and effective tool for food safety management. Full article

Background: Cancer patients are highly susceptible to microbial infections due to immune suppression, necessitating therapeutic strategies that integrate anticancer efficacy with effective antimicrobial intervention. Chalcone-derived nitrogen-fused heterocycles represent a promising platform for developing multi-target agents with relevance to antimicrobial drug delivery, particularly for localized infections. Methods: A series of chalcone-based pyrazoline-thiadiazole nitrogen-fused azole hybrids was synthesized via thiosemicarbohydrazide-functionalized intermediates and fully characterized. Antiproliferative activity was evaluated against MCF-7, HepG-2, HeLa, and HCT-116 cell lines, alongside selectivity toward WI-38 normal fibroblasts. Antibacterial, antibiofilm, and in vivo efficacy were assessed against methicillin-resistant Staphylococcus aureus (MRSA USA300) and Acinetobacter baumannii AB5057. Mechanistic investigations included cell-cycle analysis, apoptosis assays, ERK2, RIPK3, p53, BAX/Bcl-2 quantification, DNA gyrase inhibition, molecular docking, molecular dynamics simulations, and density functional theory calculations. Results: Compound 13 exhibited potent cytotoxicity, particularly against MCF-7 (IC₅₀ = 3.87 ± 0.2 µM), outperforming doxorubicin (IC₅₀ = 4.17 ± 0.2 µM), with high selectivity indices (SI = 10.7 for MCF-7). Mechanistically, compound 13 induced G₂/M arrest (40.16% vs. 14.15% control), increased apoptosis to 32.89%, up-regulated ERK2 (3.17-fold), RIPK3 (11.97-fold), and p53 (3.54-fold), and markedly increased the BAX/Bcl-2 ratio (~42-fold). Compounds 7 and 13 displayed bactericidal activity against MRSA and A. baumannii (MIC/MBC = 10 mg/mL), potent antibiofilm effects, and significant in vivo efficacy in an MRSA skin infection model. Compound 13 reduced bacterial load by ~5 log units, outperforming vancomycin. DNA gyrase inhibition (IC₅₀ = 17.10 ± 0.17 µM) and computational studies supported target engagement. Conclusions: Pyrazoline-thiadiazole-based nitrogen-fused azole hybrids, particularly compound 13, demonstrated quantifiable anticancer and antimicrobial efficacy with strong in vivo validation, supporting their potential as multi-target candidates relevant to antimicrobial drug delivery in infection-prone cancer patients. Full article

Chitooligosaccharides (COS) are short-chain chitosan derivatives with a wide range of biomedical, agricultural, and environmental applications, including antimicrobial therapy, wound healing, and pollutant removal. Reliable quantification of COS is essential but currently relies on high-performance liquid chromatography, mass spectrometry, or capillary electrophoresis, which require costly equipment, complex sample preparation, and are unsuitable for routine or on-site applications. This study reports a rapid, solvent-free, colorimetric assay for COS based on the reaction of 5% aqueous ninhydrin with free amino groups in McIlvaine buffer. The assay was optimized using glucosamine as a model analyte, yielding maximal sensitivity at pH 7.0. The chromophore generated (Ruhemann’s purple) remained stable for over 120 min after reaction, allowing measurements to be taken without strict time constraints. Calibration was linear from 0.4 to 2.2 mM (R² = 0.9926), with low limits of detection (0.006 mM) and quantification (0.018 mM). Increasing absorbance with COS polymerization degree (DP1–DP6) demonstrates specificity for free amino groups, while N-acetyl glucosamine showed a negligible response. Furthermore, the assay was successfully adapted for solid-phase detection on ninhydrin-pretreated filter paper and nitrocellulose, with enhanced sensitivity. This simple, efficient, and low-cost method provides an accessible alternative to instrumental techniques, supporting COS monitoring in laboratory workflows and enabling portable applications in biomedicine, agriculture, and environmental diagnostics. Full article