Search Results (521)

Salmonella, a major global foodborne pathogen, is a leading cause of salmonellosis. Quantitative detection of Salmonella provides a scientific basis for establishing microbiological criteria and conducting risk assessments. The plate count method remains the primary approach for bacterial quantification, whereas the most probable number (MPN) method is commonly used for detecting low levels of bacterial contamination. However, both methods are time-consuming and labor-intensive. Validated digital polymerase chain reaction (dPCR) techniques are emerging as promising alternatives because they enable rapid, absolute quantification with high specificity and sensitivity. Herein, we developed a novel droplet dPCR (ddPCR) assay for identifying and quantifying Salmonella using invA as the target. The assay demonstrated high specificity and sensitivity, with a limit of quantification of 1.1 × 10² colony-forming units/mL in meat samples. Furthermore, the log₁₀ values obtained via ddPCR and plate counting exhibited a strong linear relationship (R² > 0.99). Mathematical modeling of growth kinetics further confirmed a high correlation between plate count and ddPCR measurements (Pearson correlation coefficient: 0.996; calculated bias factor: 0.88). Collectively, these results indicate that ddPCR is a viable alternative to the MPN method and represents a powerful tool for the quantitative risk assessment of food safety. Full article

Quality control of drinking water is essential for safeguarding public health, particularly in densely populated urban environments. Environmental microbiological monitoring can complement conventional surveillance by providing deeper insights into the dissemination of pathogens and antimicrobial resistance genes within aquatic systems. In this study, we assessed the quality of wastewater and treated water from two urban water supply systems, representing the southern and northern regions of Porto Alegre, Rio Grande do Sul, Brazil, across four climatic seasons between 2024 and 2025. Fifteen water samples were analyzed, including raw water from Guaíba Lake and treated water collected from public distribution points. The Water Quality Index was calculated, microbiological indicators were quantified, and carbapenem resistance genes were detected using molecular assays. Most treated water samples complied with established bacteriological standards; however, the bla_OXA-48-like gene was recurrently detected in both wastewater and treated water. No resistance genes were identified during the summer, whereas the bla_VIM gene was detected exclusively in spring samples. The presence of carbapenem resistance genes in the absence of cultivable coliforms suggests the persistence of extracellular DNA or viable but non-culturable bacteria, highlighting limitations inherent to conventional microbiological monitoring. Integrating classical microbiological methods with molecular assays enables a more comprehensive assessment of water quality and strengthens evidence-based decision-making within a One Health framework. Full article

The global prevalence of antibiotic-resistant bacteria, such as Staphylococcus aureus, presents a substantial challenge to public health, necessitating the development of innovative therapeutic strategies to combat these infections. This study examined the synergistic effects of a biosurfactant (BS) derived from Lactiplantibacillus plantarum and a novel extract from Rosmarinus officinalis (RoME) obtained through supercritical CO₂ extraction against S. aureus sourced from the microbiology laboratory at King Salman Hospital in Ha’il, Saudi Arabia. Antibacterial efficacy was determined using minimum inhibitory concentration (MIC) assays, assessments of bacterial membrane damage, and qRT-PCR analysis of genes associated with antibiotic resistance. The findings revealed that the S. aureus strain exhibited resistance to multiple antibiotics with a resistance score of 0.44. RoME and BS demonstrated MICs of 0.125 mg/mL and 0.5 mg/mL, respectively. The assays indicated significant bacterial membrane damage and reduced expression of the norA, mdeA, and sel genes, which are implicated in resistance and virulence, respectively. The combination of BSs with plant extracts may provide innovative approaches for treating infections caused by multidrug-resistant bacteria, highlighting the potential of probiotic-derived BSs in combination with plant extracts. Full article

Oxidative stress contributes significantly to chronic disease burden, necessitating identification of accessible dietary antioxidant sources. Pigeon peas (Cajanus cajan L.) contain substantial bioactive compounds, yet most exist in bound forms with limited bioavailability. This study evaluated wild fermentation combined with systematic extraction optimization to enhance antioxidant recovery from pigeon peas. Seeds underwent wild fermentation in brine solution, followed by extraction under varying conditions (seven solvent systems, three temperatures, and three-time durations). Multiple complementary assays assessed antioxidant capacity (total phenolic content, DPPH radical scavenging, ferric reducing power, and ABTS activity). Fermentation substantially improved antioxidant properties across all parameters, with particularly pronounced effects on radical scavenging activities. Extraction optimization identified 70% methanol at 40 °C for 24 h as optimal, demonstrating marked improvements over conventional protocols. Strong intercorrelations among assays confirmed coordinated enhancement of multiple antioxidant mechanisms rather than isolated changes. The findings demonstrate that both biotechnological processing and analytical methodology critically influence antioxidant characterization in pigeon peas. This integrated approach offers practical guidance for developing antioxidant-rich functional foods, particularly relevant for resource-limited settings where pigeon peas serve as dietary staples. The study establishes foundation for translating fermentation technology into nutritional interventions, though further research addressing bioavailability, microbiological characterization, and bioactive compound identification remains essential. Full article

Microgreens have gained increasing popularity due to their cooking versatility, ease of cultivation, and high nutritional value. The use of alternative organic substrates, such as vermicompost and insect frass, offers a promising alternative to peat. This study has evaluated the integration of Tenebrio molitor and Hermetia illucens frass, along with vermicompost, in a microgreen production, while assaying several concentrations (25%, 50%, 75%, and 100%) as replacements by weight. After a preliminary assay aimed at determining the optimal frass and vermicompost levels, we assessed the agronomic, nutritional, and microbiological performances of microgreens. The preliminary results revealed phytotoxic effects of T. molitor frass, while the addition of H. illucens frass or vermicompost did not significantly impact microgreen production. In the second experiment, the interaction between plant species and substrate composition significantly influenced the leaf area, plant height, and mineral content. Partial replacement of peat with H. illucens frass or vermicompost enhanced leaf area and plant height, with a notable increase in iron content in the mizuna microgreens grown with H. illucens frass, compared to the control with peat. Additionally, microbiological safety was ensured, and a complete absence of Salmonella spp. and E. coli was observed in the plants, in accordance with European food safety regulations. Full article

The growing demand for sustainable cosmetic products and the rapid expansion of the pet care market have driven the search for environmentally friendly, safe, and effective alternatives to conventional formulations. In this study, an ecofriendly solid shampoo for pets was developed using exclusively natural ingredients and a microbial biosurfactant produced by Starmerella bombicola ATCC 22214 as a surface-active component. The biosurfactant was combined with renewable anionic and nonionic surfactants, conditioning agents, natural oils and butters, and minimal water content to obtain a compact, solid formulation. The shampoo was produced through a controlled multi-phase process and subsequently characterized by physicochemical, microbiological, toxicological, and performance analyses. The formulation exhibited stable pH values suitable for pet skin, low moisture content, absence of free alkalinity, high solid content, and satisfactory foaming capacity. Cleaning efficiency tests demonstrated effective removal of artificial sebum from pet fur while preserving softness and shine. Microbiological assays confirmed the absence of bacterial and fungal contamination, and toxicological evaluations revealed no cytotoxicity and low eye irritation potential. In addition, the shampoo showed 100% biodegradability and maintained physicochemical and organoleptic stability over six months of storage. Overall, the results demonstrate that the developed solid shampoo represents an innovative, safe, and biodegradable alternative that reduces water consumption and plastic packaging, contributing to sustainable development in the pet cosmetics sector. Full article

The rising global incidence of Candida parapsilosis infections is increasingly complicated by antifungal resistance, resulting in frequent therapeutic failure. This study investigated the potential of the natural compound catechin to enhance the efficacy of fluconazole through synergistic interaction. We evaluated the susceptibility of C. parapsilosis clinical isolates and a reference strain to combinations of catechin and fluconazole using standardized microbiological assays and molecular techniques. In vivo efficacy was assessed using the Galleria mellonella infection model. Mechanistic studies included the measurement of intracellular reactive oxygen species (ROS) production and plasma membrane permeability. Catechin alone caused growth retardation in all strains. However, the combination of catechin and fluconazole resulted in complete growth inhibition of the reference strain and significant growth reduction in azole-resistant clinical isolates. While the combination slightly increased intracellular ROS production, no significant changes in plasma membrane permeability or membrane potential were observed. Notably, catechin induced the expression of the resistance-associated genes CpTAC1 and CpCDR1B in resistant isolates. In vivo experiments demonstrated that catechin significantly reduced mortality in G. mellonella larvae infected with C. parapsilosis. These findings suggest that catechin is a promising candidate for developing synergistic antifungal therapies against resistant Candida species. Full article

Droplet microfluidics enables high-throughput, compartmentalized reactions using minimal reagent volumes, but most implementations rely on precision-fabricated chips and external pumping systems that limit portability and accessibility. Here, we present a handheld vibrational droplet generator that repurposes a consumer electric toothbrush and a modified disposable pipette tip to produce nearly monodisperse water-in-oil droplets without microfluidic channels or syringe pumps. The device is powered by the toothbrush’s built-in motor and controlled by a simple 3D-printed adapter and adjustable counterweight that tune the vibration amplitude transmitted to the pipette tip. By varying the aperture of the pipette tip, droplets with diameters from ~100–300 µm were generated at rates of ~100 droplets s⁻¹. Image analysis revealed narrow size distributions with coefficients of variation below 5% in typical operating conditions. We further demonstrate proof-of-concept applications in digital loop-mediated isothermal amplification (LAMP) and microbiological colony-forming unit (CFU) assays. A commercial feline parvovirus (FPV) kit manufactured by Beyotime Biotechnology Co., Ltd. (Shanghai, China), three template concentrations yielded emulsified reaction droplets that remained stable at 65 °C for 45 min and produced distinct fractions of fluorescent-positive droplets, allowing estimation of template concentration via a Poisson model. In a second set of experiments, the device was used as a droplet-based spreader to dispense diluted Escherichia coli suspensions onto LB agar plates, achieving uniform colony distributions across the plate at different dilution factors. The proposed handheld vibrational generator is inexpensive, easy to assemble from off-the-shelf components, and minimizes dead volume and cross-contamination because only the pipette tip contacts the sample. Although the current prototype still exhibits device-to-device variability and moving droplets in open containers complicate real-time imaging, these results indicate that toothbrush-based vibrational actuation can provide a practical and scalable route toward “lab-in-hand” droplet assays in resource-limited or educational settings. Full article

Background: Smoking alters oral ecological balance, yet its influence on posterior teeth restored with lithium disilicate endocrowns is insufficiently documented. This study assessed the clinical and microbiological impact of smoking on the peri-coronal environment of endocrown-restored teeth, using an age-stratified approach to evaluate cumulative effects. Methods: A cross-sectional study was conducted on 100 adults, equally divided into smokers and non-smokers. Salivary pH, papillary bleeding index, and plaque index were clinically recorded. Subgingival samples collected from endocrown-restored posterior teeth were analyzed using a polymerase chain reaction (PCR) assay targeting major periodontal pathogens. Age-related variation in clinical and microbiological parameters was examined using one-way analysis of variance (ANOVA), followed by Tukey’s HSD post hoc test. Results: Smokers showed consistently lower salivary pH and higher plaque accumulation across all age groups. Gingival bleeding was reduced in younger smokers but increased in older individuals. Microbiological analysis identified markedly elevated levels of orange-complex organisms in smokers, including Prevotella intermedia and Fusobacterium nucleatum. Clinically, endocrowns in smokers presented more frequent marginal degradation, localized inflammation, and early signs of recurrent caries. These effects intensified with age. Conclusions: Smoking adversely modifies the peri-coronal biological environment of lithium disilicate endocrowns by increasing acidity, promoting plaque maturation, and supporting dysbiotic microbial communities. Age further amplifies these changes. Considering smoking status and patient age during treatment planning may improve long-term restorative outcomes. Full article

Chronic wounds remain locked in persistent inflammation with high microbial burden, demanding dressings that suppress infection without sacrificing biocompatibility. Bacterial nanocellulose (BNC) is an attractive matrix due to its biocompatibility, nanofibrillar architecture, and moisture retention, but it lacks antimicrobial activity. Here, we engineer BNC membranes post-functionalized with functionalized graphite (f-Gr; predominantly graphitic with residual surface groups) and/or niobium pentoxide (Nb₂O₅), and evaluate four groups: BNC (matrix control), BNC/Nb₂O₅, BNC/f-Gr, and BNC/f-Gr/Nb₂O₅. Physicochemical analyses (Raman and Voigt fitting, FTIR-ATR, XRD, and SEM) confirm a graphitic carbon phase and physical incorporation of the modifiers into the BNC network, with a noticeable shift in the hydration/polarity profile—more evident in the presence of f-Gr. In standardized microbiological assays, BNC/f-Gr promoted a moderate, contact-dependent reduction in bacterial proliferation, particularly against Staphylococcus aureus, whereas BNC/Nb₂O₅ behaved similarly to pristine BNC under the tested conditions. The combined f-Gr/Nb₂O₅ formulation showed an intermediate antimicrobial response, with no clear synergy beyond f-Gr alone. Cytotoxicity assays indicated cytocompatibility for BNC, BNC/f-Gr, and BNC/Nb₂O₅; the combined group displayed a slight reduction that remained within acceptable limits. Overall, BNC/f-Gr emerges as the most promising antimicrobial dressing, while Nb₂O₅ did not significantly enhance antimicrobial performance under the tested conditions and warrants further optimization regarding loading and distribution. Full article

Background/Objectives: Colistin (polymyxin E) has re-emerged as a last-hope treatment against MDR Gram-negative pathogens due to the development of extensively drug-resistant Gram-negative bacteria. Unfortunately, rapid global resistance towards colistin has emerged, which represents a major public health concern. In this context (CBD), a lipophilic molecule derived from Cannabis sativa, exhibits antimicrobial activity mainly against Gram-positive bacteria but is generally ineffective against Gram-negative species. However, synergistic antibacterial activity between CBD and polymyxin B has been reported. The objective of this work is to analyze the colistin–CBD synergy against clinically relevant Gram-negative isolates displaying diverse mechanisms of colistin resistance and to explore the basis of the possible mechanism of action involved in the first steps of this synergy. Methods: Microbiological assays, minimal inhibitory concentration, cell culture, synergy tests by checker board and time kill, biofilm inhibition evaluation by crystal violet and MTT, SEM (scanning electron microscopy), molecules interaction analysis by nuclear magnetic resonance (NMR). Results: The colistin–CBD combination displayed synergy in colistin resistant Gram-negative bacteria and also disrupted preformed biofilms and killed bacteria within them. Time-kill assays revealed rapid bactericidal activity and SEM showed mild surface alterations on bacterial outer membranes after sublethal colistin monotherapy. Furthermore, a series of sequential treatment assays on colistin-resistant Escherichia coli showed that simultaneous exposure to both compounds was required for activity, as introducing a washing step between treatments abolished the antibacterial effect. In order to obtain deeper insight into this mechanism, NMR analyses were performed, revealing specific molecular interactions between CBD and colistin molecules. Conclusions: These results provide evidence for the first time that both molecules engage through a specific and structurally meaningful interaction and only display synergy when acting together on colistin-resistant bacteria. Full article

Free-living amoebae (FLA) are protozoa ubiquitous in nature, isolated from a variety of environments worldwide. In addition to their natural distribution, some species have been found to be pathogenic to humans. In the present study, FLA presence was evaluated and characterized at the molecular level from different water and soil samples in Fuerteventura Island, Canary Islands, Spain. A total of 31 samples were analyzed by culture and molecular assays (q-PCR and PCR). Moreover, the microbiological quality of the water samples was examined as required by current legislation and international standards. The obtained data revealed that the genus Acanthamoeba was the most prevalent genus of FLA in soil samples and the species Vermamoeba vermiformis was the most isolated in water samples collected from Fuerteventura by culture and molecular assays, q-PCR, and conventional PCR/Sanger sequencing. On the other hand, a microbiological analysis revealed heterogeneous contamination patterns. Escherichia coli was detected in several samples, with some exhibiting high counts while others showed no presence. Salmonella spp. appeared in multiple samples, particularly FTVW1, FTVW9, and FTVW13, whereas Shigella spp. was only found in one sample (FTVW1). Moreover, q-PCR detection offers advantages such as reduced detection time and cost. In addition, culture was proven to be more effective for confirming FLA viability and isolating a greater variety of FLA. Overall, the occurrence of potentially pathogenic free-living amoebae in habitats related to the human population, as reported in the present study, supports the relevance of FLA as a potential health threat to humans. Full article

The rhenium(I) tricarbonyl complex fac-[Re(CO)₃(5,6-epoxy-5,6-dihydro-1,10-phenanthroline)Br] (ReL) has previously demonstrated promising luminescent properties, enabling its direct application as a probe for walled cells such as Candida albicans and Salmonella enterica. In this new study, we present a significantly expanded and comprehensive characterization of ReL, incorporating a wide range of experimental and computational techniques not previously reported. These include variable-temperature ¹H and ¹³C NMR spectroscopy, CH-COSY, single-crystal X-ray diffraction, Hirshfeld surface analysis, DFT calculations, Fukui functions, non-covalent interaction (NCI) indices, and electrochemical profiling. Structural analysis confirmed a pseudo-octahedral geometry with the bromide ligand positioned cis to the epoxy group. NMR data revealed the coexistence of cis and trans isomers in solution, with the trans form being slightly more stable. DFT calculations and aromaticity descriptors indicated minimal electronic differences between isomers, supporting their unified treatment in subsequent analyses. Electrochemical studies revealed two oxidation and two reduction events, consistent with ECE and EEC mechanisms, including a Re(I) → Re(0) transition at −1.50 V vs. SCE. Theoretical redox potentials showed strong agreement with experimental data. Biological assays revealed a dose-dependent cytotoxic effect on HeLa cells, contrasting with previously reported low toxicity in microbial systems. These findings, combined with ReL’s luminescent and antimicrobial properties, underscore its multifunctional nature and highlight its potential as a bioactive and imaging agent for advanced therapeutic and microbiological applications. Full article

Ballast water (BW) is a major pathway for the spread of invasive microorganisms and pathogens, posing significant ecological and public health risks. The International Maritime Organization (IMO) has established strict discharge standards, yet routine monitoring remains limited, and no reliable onboard test is currently available to assist crews in verifying BW quality before discharge. This study presents the development of a rapid, portable method for onboard microbiological assessment of BW, based on potentiometric detection and biosensors engineered with the Bioelectric Recognition Assay (BERA). Two complementary approaches were evaluated: (i) direct potentiometric measurements of contaminated and non-contaminated samples, which confirmed the feasibility of detecting microbial presence but were restricted by high detection limits, and (ii) development of biosensors specifically engineered for Escherichia coli and Enterococcus spp. to improve specificity and lower the limit of detection (LOD). Results demonstrated successful detection of both microorganisms, with performance characteristics of 83.3% sensitivity and 81.9% accuracy for Enterococcus spp. (LOD: 10² CFU 100 mL⁻¹), and 89.8% sensitivity and 85.1% accuracy for Escherichia coli (LOD: 250 CFU 100 mL⁻¹). These findings underscore the potential of biosensor-based systems as practical, crew-operated tools for early warning and real-time monitoring of ballast water quality, supporting compliance with IMO standards and contributing to safer, more sustainable maritime operations. Full article

Early bacterial infections (EBI) remain a major cause of morbidity and mortality after liver transplantation (LT). This study aimed to characterize their incidence, microbiological spectrum, risk factors, and clinical impact in an Eastern European cohort. We retrospectively analyzed 64 adult LT recipients from a tertiary center, focusing on perioperative parameters, infection profiles, and postoperative outcomes. EBI, defined as infections occurring within 30 days after transplantation and diagnosed according to CDC/NHSN criteria, occurred in 48.4% of patients. Gram-negative bacilli predominated (73.7%), mainly Pseudomonas aeruginosa (34.2%) and Klebsiella pneumoniae (15.8%), while Clostridioides difficile (CDI) accounted for 18.4% of confirmed cases diagnosed by stool toxin assay. Infected patients had greater intraoperative blood loss (median 6500 mL vs. 5000 mL, p = 0.036) and required more transfusions. The higher infection rate in our cohort may also be related to longer surgical duration. All deaths within the first postoperative year occurred among infected recipients, yet overall 30-day (7.8%) and 1-year (14.1%) mortality rates remained within the range reported internationally, suggesting that timely diagnosis and adequate management limited the impact of infections on survival. These findings emphasize the importance of infection prevention, optimized transfusion and bleeding control, and tailored antibiotic prophylaxis based on local microbiological patterns after LT. Full article