Search Results (514)

The threats leading to the extinction of humanity accelerate the evolution and development of materials that are capable of providing conditions for preserving health and, implicitly, life. In our work, we developed drug delivery systems based on mesoporous silica which can deliver an antibiotic, bacitracin, in a more controlled manner. The synthesis of the FDU-12 was performed through a sol–gel method and alternatively functionalized with -NH₂ groups or with poly(N-acryloylmorpholine) chains. The loading of bacitracin was performed using the vacuum-assisted method we successfully used to load these mesoporous materials preferentially within the pores as proved by the TGA-DSC results. The release was performed in two types of simulated body fluid (SBF) and this process was evaluated with chromatographic method using UV detection. The obtained data were fitted in three mathematical models of kinetic drug release (Weibull model, Korsmeyer–Peppas model, and nonlinear regression). The antimicrobial evaluation demonstrated that bacitracin-loaded FDU-12 formulations exhibited strong activity against both reference and clinical Staphylococcus strains. At sub-inhibitory concentrations, all formulations significantly reduced microbial adherence and biofilm formation, although certain strain-dependent stimulatory effects were observed. Furthermore, exposure to sub-MIC levels modulated the production of soluble virulence factors (hemolysins, lipase, and amylase), in a formulation- and strain-dependent manner, underscoring the ability of surface-functionalized FDU-12 carriers to influence bacterial pathogenicity while enhancing antimicrobial efficacy. Full article

Volatile organic compounds produced by microbes are increasingly recognized as modulators of microbial interactions and mediators of both intra- and inter-kingdom communication. This study explored the possible ecophysiological roles of nine amides from Streptomyces sp. NP10 in quorum sensing (QS) and biofilm formation in Pseudomonas aeruginosa PAO1. GC-MS profiling, synthesis, spectral validation, and co-injection experiments confirmed compound identities. Notably, N-(3-methyl-2-butenyl)acetamide is reported as a new natural product and N-(2-methylbutyl)acetamide as a new Streptomyces-produced metabolite. At subinhibitory concentrations (250 μg/mL), most of the amides enhanced P. aeruginosa biofilm formation, with N-(2-methylbutyl)acetamide, N-(3-methyl-2-butenyl)acetamide, and 2-phenylacetamide showing the strongest effects. Simultaneously, these compounds suppressed QS by reducing the production of N-acyl homoserine lactones (AHLs) and 2-alkyl-4-quinolones (AHQs). Aliphatic acetamides preferentially inhibited short-chain AHLs, while N-acetyltyramine and 2-phenylacetamide mainly affected quinolone signaling. These opposing effects on QS and biofilm are consistent with the involvement of alternative regulatory circuits. Motility assays showed biofilm stimulation was not correlated with altered swarming or twitching. Cross-species assays revealed limited QS inhibition, with only N-acetyltryptamine reducing violacein production in Chromobacterium violaceum CV026. Most of the amides were non-cytotoxic at 100 μM (10.5–20.2 μg/mL), except for 2-phenylacetamide. Overall, these amides likely serve as microbial signals influencing QS and biofilm formation, offering leads for anti-virulence strategies. Full article

Porphyromonas gingivalis (P. gingivalis), a key periodontal pathogen, has been linked to atherosclerosis development. The clinical failure of antibiotics to improve cardiovascular outcomes necessitates alternative explanations. This study examines how sub-inhibitory concentrations of metronidazole affect the biogenesis and pathogenic potential of P. gingivalis extracellular vesicles (EVs) on human umbilical vein endothelial cells (HUVECs). EVs were isolated from both untreated bacteria (N-EVs) and those treated with sub-inhibitory concentrations of metronidazole (M-EVs) through ultracentrifugation. Characterization included transmission electron microscopy (TEM), nanoparticle tracking analysis, and Western blotting for virulence factors. HUVECs were evaluated using viability, migration, cell death assays, ROS detection, NF-κB activation imaging, and cytokine measurement. Sub-inhibitory concentrations of metronidazole increased EV production by 2.3-fold and enriched M-EVs with virulence factors (lipid A LPS, Kgp, RgpA). M-EVs demonstrated significantly stronger cytotoxicity, causing greater impairment of HUVEC viability and migration, alongside increased cell death. Mechanistically, M-EVs induced elevated mitochondrial and cellular ROS, promoting NF-κB activation and enhancing secretion of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6). Sub-inhibitory concentrations of metronidazole exacerbate endothelial injury by amplifying EV production and virulence factor loading in P. gingivalis, offering a mechanistic explanation for the limited cardiovascular benefits of antibiotic therapy in periodontitis patients. Full article

Rapid evaluation of water toxicity requires biological methods capable of detecting sub-lethal physiological changes without depending on chemical identification. Conventional microscopy-based bioassays are limited by low throughput and difficulties in observing small, transparent and fast-moving microorganisms. This study applies a laser-biospeckle, non-imaging microbioassay to assess the motility responses of Paramecium caudatum and Euglena gracilis exposed to two organic pollutants, trichloroacetic acid (TCAA) and acephate. Dynamic speckle patterns were recorded using a 638 nm laser diode (Thorlabs Inc., Tokyo, Japan) and a CCD camera (Gazo Co., Ltd., Tokyo, Japan) at 60 fps for 120 s. Correlation time, derived from temporal cross-correlation analysis, served as a quantitative indicator of motility. Exposure to TCAA (0.1–50 mg/L) produced strong concentration-dependent inhibition, with correlation time increasing up to 16-fold at 500× PL in P. caudatum (p < 0.01), whereas E. gracilis showed a delayed response, with significant inhibition only above 250× PL. In contrast, acephate exposure (0.036–3.6 mg/L) induced motility enhancement in both species, reflected by decreases in correlation time of up to 57% in P. caudatum and 40% in E. gracilis at 100× PL. Acute trends diminished after 24–48 h, indicating time-dependent physiological adaptation. These results demonstrate that biospeckled-derived correlation time sensitively captures both inhibitory and stimulatory behavioral responses, enabling real-time, high-throughput water toxicity screening without microscopic imaging. The method shows strong potential for integration into automated water-quality monitoring systems. Full article

Background: Nosocomial infections represent a significant clinical burden due to high morbidity, mortality and healthcare costs. Invasive fungal infections, particularly those caused by Candida species, are of growing concern due to increasing antifungal resistance, which limits therapeutic options and worsens patient outcomes. This study aimed to characterize the prevalence, species distribution, antifungal susceptibility profiles, and molecular mechanisms of resistance in clinical Candida isolates from hospitalized patients. Methods: A cross-sectional study was conducted involving 55 hospitalized patients, yielding 60 isolates from blood, secretions, fluids, and catheter tips. Species identification was performed using chromogenic media and confirmed by MALDI-TOF MS. Antifungal susceptibility testing followed CLSI M27-A4 broth microdilution guidelines for amphotericin B, fluconazole and 5-flucytosine. Gene expression of ERG2, ERG11 and MDR1 was evaluated by RT-qPCR after exposure to subinhibitory antifungal concentrations using the 2^−∆∆Ct method. Results:Candida albicans was the most frequent species, followed by Nakaseomyces glabratus, C. tropicalis and C. parapsilosis. Resistance varied among species, with elevated rates for fluconazole. ERG2 was notably overexpressed in amphotericin B-resistant isolates, while ERG11 and MDR1 showed species-dependent variation. Conclusions: Resistance mechanisms in Candida are species-specific and drug-dependent. Accurate species identification and understanding their molecular profiles are essential to guide targeted antifungal therapy and improve clinical outcomes. Full article

Aeromonas salmonicida is a major pathogen in aquaculture, and its ability to form biofilms contributes significantly to antibiotic resistance and chronic infections. This study investigated the effects of four antibiotics—ampicillin, amoxicillin, oxytetracycline, and doxycycline—at various concentrations on bacterial growth, biofilm formation, and gene expression related to antibiotic resistance and quorum sensing (QS) in two subspecies: A. salmonicida subsp. masoucida (ASM) and A. salmonicida subsp. salmonicida (ASS). Bacterial isolates from Atlantic salmon were identified using 16S rRNA and vapA gene sequencing. Growth inhibition was more pronounced in ASS than ASM under high antibiotic concentrations. Conversely, sub-inhibitory concentrations (sub-MICs) enhanced biofilm formation in both subspecies, particularly in ASM. PCR results showed that tetA and tetE resistance genes were present only in ASM. qRT-PCR analysis revealed that expression of QS-related genes (ahyI and ahyR) was generally downregulated under tetracycline treatment, while litR expression varied across antibiotic conditions and strains. Some isolates showed increased litR expression alongside elevated biofilm formation, suggesting involvement of additional regulatory mechanisms. These results highlight the potential for sub-MIC antibiotic exposure to promote biofilm development and modulate gene expression, emphasizing the need for careful antibiotic use in aquaculture and providing insight into alternative pathogen control strategies. Full article

Escherichia coli (E. coli) is a ubiquitous opportunistic pathogen in nature and serves as an important reservoir for antibiotic resistance genes. The tet(X4) gene is a key determinant mediating tigecycline resistance. Although its core resistance mechanism, encoding a flavin-dependent monooxygenase, has been elucidated, the broader impact of the tet(X4) gene on the secondary regulatory networks of E. coli remains not fully understood. In recent years, multiple studies have indicated that the tet(X4) gene participates in pathways contributing to resistance to other antibiotics by regulating the expression of various genes. In this study, E. coli tet(X4) gene deletion and complementation strains were constructed to investigate the mechanisms by which the tet(X4) gene influences the growth characteristics and antibiotic resistance of E. coli. The minimum inhibitory concentrations (MICs) of 24 different antibiotics, as well as the degradation capacities of tetracycline and tigecycline, were determined for the wild-type, deletion, and complementation strains. In addition, a four-week starvation stress experiment was performed under both the presence and absence of sub-inhibitory concentrations of tigecycline, during which the bacterial growth curves, survival rates, and MIC variations were analyzed. Transcriptomic sequencing of the wild-type, deletion, and complementation strains identified 531 differentially expressed genes associated with ABC transporter activity, drug metabolism, and bacterial two-component systems. These findings provide reliable evidence for elucidating the mechanism by which the tet(X4) gene affects E. coli resistance, offering valuable insights into the prevention and control of tigecycline-resistant E. coli infections. Full article

Background/Objectives: Arnica montana L. is widely recognized for its diverse biological activities, including antimicrobial effects. This study aimed to evaluate the antimicrobial and antibiofilm activity of A. montana L. extracts against Acinetobacter baumannii, a pathogen of urgent public health concern due to its increasing antibiotic resistance and capacity for biofilm formation. Methods: The antimicrobial activity of ethanolic (EtE) and aqueous (AqE) extracts of A. montana flowers was evaluated via the broth microdilution method. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), and the MBC/MIC ratio were used. The effects of EtE on A. baumannii biofilm formation were assessed via a crystal violet assay. Additionally, transcriptional profiling of biofilm-associated genes following exposure to sub-MIC levels of the extract was conducted via RT-qPCR. Results: The anti-Acinetobacter activity of EtE was demonstrated (MIC = 234.4 and 468.75 µg/mL for A. baumannii ATCC BAA-3252 and ATCC 19606, respectively). The EtE exhibited bactericidal activity against both strains, whereas the AqE showed no activity. Additionally, EtE inhibited biofilm formation and significantly downregulated the expression of key biofilm-associated genes, including those of the csu operon and ompA. Conclusions: Arnica montana EtE demonstrated antimicrobial and antibiofilm activities against A. baumannii and inhibited biofilm development by suppressing the transcription of genes involved in pilus assembly and surface adherence, highlighting their essential role in biofilm formation. Full article

Background/Objectives: Staphylococcus aureus is a well-known opportunistic pathogen that causes a wide range of infections, from cutaneous blemishes to potentially fatal systemic diseases. The increasing prevalence of antibiotic-resistant bacteria highlights the critical need for alternative therapeutic methods that target virulence factors rather than growth. Methods: The antibacterial activity of 3-fluorocatechol (3-FC) against bacterial and fungal pathogens (e.g., Candida albicans) was determined by broth microdilution to establish the lowest inhibitory concentration. The antibiofilm impact of 3-FC against S. aureus was evaluated using crystal violet staining and viable colony counts, followed by scanning electron microscopy to visualize the biofilm architecture. The methanol extraction method was used to quantify staphyloxanthin synthesis in S. aureus cells. Furthermore, in silico molecular docking was used to evaluate 3-FC binding interactions and provide mechanistic insight into its impacts on S. aureus biofilms and virulence-associated factors. Results: Although the study showed that 3-FC exhibits weak antibacterial activity against S. aureus (MIC > 2048 µg/mL), it shows effective inhibition of up to 86.5% at sub-inhibitory doses during the initial stage of biofilm formation. The CFU enumeration also confirms the significant reduction of viable cell count of S. aureus in the presence of sub-MIC of 3-FC. The SEM analysis confirms disruption of the S. aureus biofilm architecture in the presence of a sub-MIC of 3-FC. Furthermore, the eradication of mature S. aureus biofilm at a sub-MIC dose of 3-FC was 60.6%. 3-FC significantly reduced staphyloxanthin formation, a vital antioxidant pigment that contributes to bacterial pathogenicity, with a maximal suppression of 66.3% at 2048 µg/mL. Molecular docking analyses provide further insight into the molecular basis of 3-FC activity, revealing strong binding affinities with numerous S. aureus virulence regulators and enzymes, suggesting interference with quorum-sensing, adhesion, and oxidative-stress response pathways. Conclusions: Collectively, our findings indicate that 3-FC has antibiofilm and antivirulence properties against S. aureus. Furthermore, this study suggests 3-FC as a viable structural scaffold for the development of a novel anti-infective agent to treat chronic staphylococcal infections. Full article

We investigated the effects of vancomycin, estradiol, ethanol, and their combinations on the growth of mono- and binary-species biofilms of Lactobacillus paracasei and Staphylococcus aureus. It was found that vancomycin at a subinhibitory concentration of 0.001 µg/mL, estradiol, and ethanol acted antagonistically in all cases. This effect was observed across all strains studied. Furthermore, the effects of the active compounds were evident at population, cellular and molecular levels, and were reflected in changes to the count of colony-forming units (CFUs), gene expression, and the physiological and biochemical characteristics of cells (e.g., lipid composition of membranes and the extracellular matrix). Therefore, at subinhibitory concentrations of vancomycin in the medium, estradiol can modulate the antibiotic’s effect on biofilms, thereby regulating deeply microbial communities. Full article

This study evaluated the antagonistic activity of the cell-free supernatant of Lacticaseibacillus rhamnosus ATCC 9595 (Lcr-CFS) against Listeria monocytogenes, a major foodborne pathogen, that represents a challenge to food safety, due to its remarkable tolerance to environmental stresses and strong biofilm-forming ability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Lcr-CFS against L. monocytogenes were defined as 31.25 and 62.5 mg/mL, respectively. Time-kill assays revealed dose- and time-dependent bactericidal effects. At sub-MICs, Lcr-CFS significantly reduced L. monocytogenes biofilm formation, disrupted preformed biofilms and decreased cell viability (80.3–96.7%), effects that were confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and fluorescence microscopy. Transmission electron microscopy showed L. monocytogenes cell wall damage, cytoplasmic leakage, and morphological alterations consistent with bactericidal effects. Additionally, exposure to 1x and 2x MIC of Lcr-CFS induced reactive oxygen species (ROS) accumulation, indicating oxidative stress as part of the mechanism by which Lcr-CFS exerts its antimicrobial activity. Gene expression analysis revealed upregulation of stress and virulence-associated genes (sigB, prfA, degU, flaA, motA, hlyA, pclA, and actA) upon exposure to 0.5x MIC suggesting a complex cross-talk network between adaptive mechanisms and environmental stresses. Although L. monocytogenes initiates a stress response, it appears unable to counteract the damage induced by Lcr-CFS, resulting in cell death. These findings highlight the antimicrobial and anti-biofilm properties of Lcr-CFS against L. monocytogenes. Given its in vitro efficacy, Lcr-CFS emerges as a promising biocontrol agent to improve food safety by mitigating the persistence of L. monocytogenes in food processing settings. Full article

Mastitis is one of the major diseases affecting dairy cattle worldwide. Antibiotic therapy remains the most widely used treatment. However, its effectiveness has been compromised due to the selection of antibiotic-resistant and biofilm-producing pathogenic bacteria. This promotes the search for alternatives that increase the antibacterial and antibiofilm efficacy of antibiotics such ceftiofur (CFT). Nisin (N) and chitosan (CH) may possess these properties. The aim of this study was to evaluate whether N + CFT and CH + CFT combinations enhance the antibacterial activity of the antibiotic on Staphylococcus aureus associated with bovine mastitis, as well as its antibiofilm effect. Two clinical isolates of S. aureus (AMC-43 and AMC-48) and the reference strain ATCC 27543 resistant to CFT were used. Through the microdilution method in 96-well microplates, the combination of sub-inhibitory concentrations of N (320 µg/mL) and CH (400 µg/mL) with CFT (1, 2, 4, and 8 µg/mL) significantly reduced bacterial growth; however, the CH + CFT mixtures were the most efficient. The crystal violet staining method and live cell plating showed antibiofilm activity in biofilm synthesis and in the reduction in living bacterial cells located inside this preformed structure. These results highlight N and CH as potential agents for the prevention or control of bovine mastitis. Full article

Disinfectant tolerance in bacteria may be related to exposure to subinhibitory concentrations of disinfectants, which may activate efflux pumps capable of expelling antimicrobial compounds. The aim of this study was to evaluate the impact of disinfection on the presence of efflux pump genes and the resistance profile of Escherichia coli from commercial laying farms employing different disinfection protocols. The emrE, qacE, qacEΔ1, qacH, sugE(c), ydgE, ydgF, and class 1 integron (intl1) genes were investigated using PCR. Susceptibility to 17 antibiotics was assessed, including β-lactams, fluoroquinolones, aminoglycosides, and tetracyclines. Disinfectant exposure was significantly associated with higher frequencies of qacE and qacH, and a reduced frequency of ydgF. Moreover, resistance to ampicillin, trimethoprim–sulfamethoxazole, and doxycycline was significantly more frequent in E. coli isolated from chickens exposed to disinfectants. These findings indicate that disinfectant use can select for E. coli carrying efflux pump genes and resistance genes, favoring the survival and dissemination of tolerant and resistant strains in poultry production. Continuous monitoring and the development of disinfection strategies that minimize selective pressures are crucial for limiting the spread of antimicrobial resistance at the animal–human–environment interface. Full article

This study aimed to evaluate the antibacterial activity of Cistus salviifolius L. and Helichrysum stoechas (L.) DC extracts against S. aureus, including methicillin-resistant S. aureus (MRSA) strains. To this end, assays were conducted to assess killing kinetics, antibiotic combination effects, modulatory effects on ethidium bromide, inhibition of quorum sensing, and biofilm formation. H. stoechas extract demonstrated the strongest activity, with MIC values ranging from 7.8 to 62.5 µg/mL. When combined with antibiotics such as ampicillin, ciprofloxacin, or vancomycin, the extracts of C. salviifolius and H. stoechas predominantly exhibited synergistic (FICI value ≤ 0.5) or additive effects (0.5 < FICI ≤ 1), with some combinations resensitizing resistant strains. The aerial parts of C. salviifolius displayed modulatory effects on ethidium bromide MIC, reducing the concentration from 32 to 8 µg/mL, suggesting efflux pump inhibitory activity. In addition, this extract displayed slight quorum-sensing inhibition at a concentration of 125 µg/mL. Moreover, C. salviifolius and H. stoechas extracts inhibit the formation of biofilm by S. aureus strains, even at subinhibitory concentrations (0.5× and 0.25× MIC). The presence of compounds such as myricetin 3 O-galactoside, catechin derivatives, gallic acid, kaempferol, and chlorogenic acid in the extracts may contribute to their anti-Staphylococcus activity. These results demonstrated the dual antimicrobial and antivirulence potential of C. salviifolius and H. stoechas extracts, highlighting their promise as therapeutic agents or adjuvants against S. aureus. These extracts can be promising candidates for further studies on the development of novel strategies targeting multiple pathogenic pathways. Full article

Anisomycin, a ribotoxic compound, is an efficient inhibitor of eukaryotic translation: at toxic concentrations, it interferes with the function of ribosomal peptidyl transferase, blocks protein synthesis, and ultimately leads to apoptosis. The process is accompanied by the activation of various cellular stress mechanisms. Subinhibitory anysomycin concentrations, in contrast, do not inhibit translation and cause apoptosis, but still activate certain stress pathways. The present study aimed to compare the signaling effects of toxic (1 µg/mL) and non-toxic (10 ng/mL) anisomycin treatment in PC12 cells. In addition, the role of the p53 tumor suppressor protein in these processes was explored, using a PC12 cell line expressing a dominant inhibitory p53 protein. Apoptosis-mediating events (PKR cleavage; eIF2α phosphorylation; activation of caspase 3, 8, and 9 enzymes) were caused by high, but not low, anisomycin concentration in a p53-dependent manner. MAPK pathways (JNK, p38 MAPK, ERK) were stimulated by non-toxic anisomycin treatment, with a more complex p53 involvement. The apoptotic response of cells appeared to be supported by exosomal paracrine signaling. Full article