Search Results (39)

The fluorescent dyes 9-aminoacridine (9-AA) and acridine orange (AO) are known mutagens that induce frameshift mutations in cells by intercalating between DNA bases. However, these chemicals can also affect other cellular components, such as proteins. In this study, we tested the ability of 9-AA and AO to induce heat shock in bacteria using the following methods: lux-biosensors based on Escherichia coli cells with the luxCDABE genes transcriptionally fused to heat shock-specific inducible promoters, RT-qPCR, and nanoDSF. We demonstrated that acridine dyes not only induce mutagenesis but also cause heat shock in bacterial cells. AO significantly reduced the melting temperature of proteins and strongly activated σ^E- and σ³²-dependent promoters, but not PluxC, which is activated by elevated temperatures via a different mechanism. In contrast, 9-AA weakly denatured the proteins and induced the σ^E-dependent promoter; however, it activated the σ³²-dependent promoters and PluxC, supporting the hypothesis that the σ³² heat shock response system is activated via hairpin RNA denaturation by 9-AA. The study on the application of lux-biosensors was hampered by the high general toxicity and luminescence shielding effect of AO, and RT-qPCR’s sensitivity was insufficient for detection of the response to 9-AA. Thus, methodologically, it is justified to conduct a comprehensive study of substances that cause heat shock or affect bioluminescence by both RT-qPCR and lux-biosensors. Full article

Photodynamic inactivation and antimicrobial photodynamic therapy (PDI/APDT) based on the toxic properties of reactive oxygen species (ROS), which are generated by a number of photoexcited dyes, are promising for preventing and treating infections, especially those associated with drug-resistant pathogens. The negatively charged bacterial cell surface attracts polycationic photosensitizers, which contribute to the vulnerability of the bacterial plasma membrane to ROS. The integrity of the plasma membrane is critical for the viability of the bacterial cell. Polycationic phthalocyanines are regarded as promising photosensitizers due to their high quantum yields of ROS generation (mainly singlet oxygen), high extinction coefficients in the far-red spectral range, and low dark toxicity. For application in PDI/APDT, the wide range of possibilities of modifying the chemical structure of phthalocyanines is particularly valuable, especially by introducing various peripheral and non-peripheral substituents into the benzene rings. Depending on the type and location of such substituents, it is possible to obtain photosensitizers with different photophysical properties, photochemical activity, solubility in an aqueous medium, biocompatibility, and tropism for certain structures of photoinactivation targets. In this study, we tested novel water-soluble Zn (II) phthalocyanines bearing four 4-((diethylmethylammonium)methyl)phenoxy substituents with symmetric and asymmetric charge distributions for photodynamic antibacterial activity and compared them with those of water-soluble octacationic zinc octakis(cholinyl)phthalocyanine. The obtained results allow us to conclude that the studied tetracationic aryloxy-substituted Zn(II) phthalocyanines effectively bind to the oppositely charged cell wall of the Gram-negative bacteria E. coli. This finding is supported by data on bacteria’s zeta potential neutralization in the presence of phthalocyanine derivatives and fluorescence microscopy images of stained bacterial cells. Asymmetric substitution influences the aggregation and fluorescent characteristics but has little effect on the ability of the studied tetracationic phthalocyanines to sensitize the bioluminescent E. coli K12 TG1 strain. Both symmetric and asymmetric aryloxy-substituted phthalocyanines are no less effective in PDI than the water-soluble zinc octakis(cholinyl)phthalocyanine, a photosensitizer with proven antibacterial activity, and have significant potential for further studies as antibacterial photosensitizers. Full article

This study comprehensively evaluated the antimicrobial performance of copper ions against three bacterial species relevant to water systems: E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and S. epidermidis (ATCC 12228). Disinfection kinetics were determined at three copper concentrations (0.5, 1.5, and 3.3 mg/L) using the Gard model. E. coli exhibited the highest susceptibility, with inactivation rate constants of 0.63, 3.27, and 9.83, achieving complete inactivation at 3.3 mg/L. P. aeruginosa was the most resistant, showing values below 1.0 across all concentrations, while S. epidermidis displayed intermediate responses. Selected experiments further examined the influence of growth phase, temperature, and water chemistry. Exponential-phase cells were more sensitive than stationary-phase cultures, and higher temperatures (37 °C vs. 5 °C) significantly enhanced inactivation. Moderate bicarbonate (50 mg/L) improved bacterial removal by stabilizing soluble Cu²⁺ ions (2.60 lg reduction), whereas elevated calcium and magnesium (Ca²⁺ 100 mg/L, Mg²⁺ 50 mg/L) reduced effectiveness (≤2.10 lg reduction) through competitive interactions. In addition to culture-based methods, adenosine triphosphate (ATP) bioluminescence assays and flow cytometry (FCM) provided complementary insights, confirming early metabolic disruption and membrane damage prior to culturability loss in selected experiments. Full article

Background: Standard test methods for evaluating the antibacterial performance of plastic (non-porous) and textile (porous) materials are accurate and reliable, but completing a standard assessment generally requires at least several days to a week. Well-trained and experienced technicians are also required to conduct the standard tests consistently and analyse the samples and test results systemically. These costs are often not favourable for the performance assurance of antimicrobial products in industrial production, nor for meeting the fast-return demands in research and development of antimicrobial materials nowadays. Methods: In this study, “Rapid Tests” are developed to evaluate the antibacterial activities of plastic and textile materials. Results: The assessment results from Rapid Tests for plastics and textiles are highly correlated to those from the ISO 22196 and the AATCC Test Method 100, respectively, whereas the evaluation operation can be completed within one day. Based on bioluminescence technology, colony-forming units of E. coli from the inoculated specimens are determined via luminometry. Antibacterial efficacy of the treated plastic and textile samples can be examined effectively. Conclusions: By analysing antimicrobial artificial leather samples composed of hydrophilic polyurethane polymer using Rapid Tests for plastics and textiles, the applicability and scope of these tests were remarkedly recognised and verified. Full article

There is currently a critical need for understanding how the response and activity of whole-cell bacterial reporters positioned in a complex biological or environmental matrix are impacted by the physicochemical properties of their micro-environment. Accordingly, a comprehensive analysis of the bioluminescence response of Cd(II)-inducible PzntA-luxCDABE Escherichia coli biosensors embedded in silica-based hydrogels is reported to decipher how metal bioavailability, cell photoactivity and ensuing light bioproduction are impacted by the hydrogel environment and the associated matrix effects. The analysis includes the account of (i) Cd speciation and accumulation in the host hydrogels, in connection with their reactivity and electrostatic properties, and (ii) the reduced bioavailability of resources for the biosensors confined (deep) inside the hydrogels. The measurements of the bioluminescence response of the Cd(II) inducible-lux biosensors in both hydrogels and free-floating cell suspensions are completed by those of the constitutive rrnB P1-luxCDABE E. coli so as to probe cell metabolic activity in these two situations. The approach contributes to unraveling the connections between the electrostatic hydrogel charge, the nutrient/metal bioavailabilities and the resulting Cd-triggered bioluminescence output. Biosensors are hosted in hydrogels with thickness varying between 0 mm (the free-floating cell situation) and 1.6 mm, and are exposed to total Cd concentrations from 0 to 400 nM. The partitioning of bioavailable metals at the hydrogel/solution interface following intertwined metal speciation, diffusion and Boltzmann electrostatic accumulation is addressed by stripping chronopotentiometry. In turn, we detail how the bioluminescence maxima generated by the Cd-responsive cells under all tested Cd concentration and hydrogel thickness conditions collapse remarkably well on a single plot featuring the dependence of bioluminescence on free Cd concentration at the individual cell level. Overall, the construction of this master curve integrates the contributions of key and often overlooked processes that govern the bioavailability properties of metals in 3D matrices. Accordingly, the work opens perspectives for quantitative and mechanistic monitoring of metals by biosensors in environmental systems like biofilms or sediments. Full article

Rainwater is susceptible to pollutants such as sulphur dioxide, nitrogen oxides, heavy metals, and particles, posing challenges to water quality protection and soil degradation, impacting ecosystems and agriculture. The study focuses on the effectiveness of combined ozonation and photocatalysis in improving physicochemical parameters and reducing toxic substances. Integrated analyses, including ecotoxicological assessments, evaluate the impact of treatment on actual rainwater samples. The results indicate significant reductions in color, heavy metals, and organic pollutants after treatment. Microbiological analyses reveal the inactivation of E. coli, which is crucial for safe water reuse. Ecotoxicity studies show no toxicity to crustaceans, but slight toxicity to algae and bioluminescence bacteria in post-treatment samples. Genotoxicity assessments indicate that there is no detectable DNA damage. Overall, the study highlights the complex nature of rainwater pollution and the efficacy of photocatalytic ozonation in reducing contaminants, underscoring the need for more research to ensure sustainable water resource management. Full article

Bacterial infections represent a serious and global threat in modern medicine; thus, it is very important to rapidly detect pathogenic bacteria, such as Escherichia coli (E. coli) O157:H7. Once treatments are delayed after the commencement of symptoms, the patient’s health quickly deteriorates. Hence, real-time detection and monitoring of infectious agents are highly critical in early diagnosis for correct treatment and safeguarding public health. To detect these pathogenic bacteria, many approaches have been applied by the biosensors community, for example, widely-used polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), culture-based method, and adenosine triphosphate (ATP) bioluminescence. However, these approaches have drawbacks, such as time-consumption, expensive equipment, and being labor-intensive, making it critical to develop ultra-sensitive and highly selective detection. The microfluidic platform based on surface plasmon resonance (SPR), electrochemical sensing, and rolling circle amplification (RCA) offers proper alternatives capable of supplementing the technological gap for pathogen detection. Note that the microfluidic biochip allows to develop rapid, sensitive, portable, and point-of-care (POC) diagnostic tools. This review focuses on recent studies regarding accurate and rapid detection of E. coli O157:H7, with an emphasis on POC methods and devices that complement microfluidic systems. We also examine the efficient whole-body detection by employing antimicrobial peptides (AMPs), which has attracted growing attention in many applications. Full article

In this work, we present an analysis of the antibacterial activity of TiS₃ nanostructures in water and 0.9% NaCl solution suspensions. TiS₃ nanoribbons 1–10 µm long, 100–300 nm wide, and less than 100 nm thick were produced by the direct reaction of pure titanium powder with elemental sulphur in a quartz tube sealed under vacuum. For the toxicity test of a bioluminescent strain of E. coli we used concentrations from 1 to 0.0001 g L⁻¹ and also studied fresh suspensions and suspensions left for 24 h. The strongest toxic effect was observed in freshly prepared water solutions where the luminescence of bacteria decreased by more than 75%. When saline solution was substituted for water or when the solutions were stored for 24 h it resulted in a considerable decrease in the TiS₃ antibacterial effect. The toxicity of TiS₃ in water exceeded the toxicity of the reference TiO₂ nanoparticles, though when saline solution was used instead of water the opposite results were observed. In addition, we did not find a relationship between the antibacterial activity of water suspensions of nanoribbons and the stability of their colloidal systems, which indicates an insignificant contribution to the toxicity of aggregation processes. In 0.9% NaCl solution suspensions, toxicity increased in proportion to the increase in the zeta potential. We suppose that the noted specificity of toxicity is associated with the emission of hydrogen sulphide molecules from the surface of nanoribbons, which, depending on the concentration, can either decrease or increase oxidative stress, which is considered the key mechanism of nanomaterial cytotoxicity. However, the exact underlying mechanisms need further investigation. Thus, we have shown an important role of the dispersion medium and the period of storage in the antibacterial activity of TiS₃ nanoribbons. Our results could be used in nanotoxicological studies of other two-dimensional nanomaterials, and for the development of novel antibacterial substances and other biomedical applications of this two-dimensional material. Full article

Ozone is a triatomic allotropic modification of oxygen with very high oxidation potential and strong antimicrobial properties, and can be used as a disinfecting agent. The aim of this work was to investigate the effectiveness of gaseous ozone in reducing the number of bacteria and the total biomass of E. coli biofilm using different methods of quantification and detection. Biofilm of all tested clinical isolates and standard strain was grown on ceramic tiles with dimensions of 1.0 × 1.0 cm over 24 h. These plates were then treated with gaseous ozone for 1 h. After washing, CFU/cm² was determined, ATP bioluminescence was measured with a luminometer, and the total biomass reduction was measured after crystal-violet staining. Gaseous ozone proved to be very effective in destroying the created bacterial biofilm on ceramic tiles. Treatment caused a reduction in total bacteria number of up to 2.00 log₁₀CFU/cm², followed by a reduction in total biomass of up to 61.40%. Inhibition rates varied from 35.80% to 99.41%, depending on the method of quantification used. All methods used in this study are effective in determining the anti-biofilm activity of gaseous ozone, but more research is needed. Full article

The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL₁₄-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL₁₄ which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL₁₄ containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES. Full article

Street food outlets are characterised by poor microbiological quality of the food and poor hygiene practices that pose a risk to consumer health. The aim of the study was to evaluate the hygiene of surfaces in food trucks (FT) using the reference method together with alternatives such as Petrifilm^TM and the bioluminescence method. TVC, S. aureus, Enterobacteriaceae, E. coli, L. monocytogenes, and Salmonella spp. were assessed. The material for the study consisted of swabs and prints taken from five surfaces (refrigeration, knife, cutting board, serving board, and working board) in 20 food trucks in Poland. In 13 food trucks, the visual assessment of hygiene was very good or good, but in 6 FTs, TVC was found to exceed log 3 CFU/100 cm² on various surfaces. The assessment of surface hygiene using various methods in the food trucks did not demonstrate the substitutability of culture methods. Petrifilm^TM tests were shown to be a convenient and reliable tool for the monitoring of mobile catering hygiene. No correlation was found between the subjective visual method and the measurement of adenosine 5-triphosphate. In order to reduce the risk of food infections caused by bacteria in food trucks, it is important to introduce detailed requirements for the hygiene practices used in food trucks, including techniques for monitoring the cleanliness of surfaces coming into contact with food, in particular cutting boards and work surfaces. Efforts should be focused on introducing mandatory, certified training for food truck personnel in the field of microbiological hazards, appropriate methods of hygienisation, and hygiene monitoring. Full article

Metal–polysaccharides have recently raised significant interest due to their multifunctional bioactivities. The antimicrobial activity of a complex of Cu₂O with the sulfated polysaccharide (PS) of the marine red microalga Porphyridium sp. was previously attributed to spikes formed on the complex surface (roughness). This hypothesis was further examined here using other Cu–PS complexes (i.e., monovalent-Cu₂O, CuCl and divalent-CuO, CuCl₂). The nanostructure parameters of the monovalent complexes, namely, longer spikes (1000 nm) and greater density (2000–5000 spikes/µm²) were found to be related to the superior inhibition of microbial growth and viability and biofilm formation. When Escherichia coli TV1061, used as a bioluminescent test organism, was exposed to the monovalent Cu–PS complexes, enhanced bioluminescence accumulation was observed, probably due to membrane perforation by the spikes on the surface of the complexes and consequent cytoplasmic leakage. In addition, differences were found in the surface chemistry of the monovalent and divalent Cu–PS complexes, with the monovalent Cu–PS complexes exhibiting greater stability (ζ-potential, FTIR spectra, and leaching out), which could be related to spike formation. This study thus supports our hypothesis that the spikes protruding from the monovalent Cu–PS surfaces, as characterized by their aspect ratio, are responsible for the antimicrobial and antibiofilm activities of the complexes. Full article

Currently, world population faces an episode where bacteria are becoming resistant to antibiotics, and it is crucial to find alternatives and new molecules to fight these microorganisms. Photodynamic inactivation of microorganisms has been pointed out as an alternative to conventional therapies. This work describes the synthesis of benzoporphyrins derivatives bearing triazolyl groups and of the analogues with pyridyl units under Heck coupling conditions. The benzoporphyrin derivatives containing pyridyl groups were further quaternized with iodomethane and 1-iodopentane to evaluate the influence of alkyl chain size on their photoinactivation ability. The biological studies towards Gram-negative Escherichia coli showed that the tetracationic benzoporphyrins can efficiently inactivate this bacterium. Full article

Whole-cell bacterial sensors are used in medical/environmental applications to detect chemicals, and to assess medium toxicity or stress. Non-specific constitutive biosensors generally serve the latter purpose, whereas chemical detection is performed with biosensors involving a specific chemical-inducible promoter. Herein, we show that functioning principles of specific and non-specific whole-cell biosensors are not exclusive as both can probe modulations of cell metabolic activity under stressing conditions. The demonstration is based on (i) time-resolved measurements of bioluminescence produced by constitutive rrnB P1-luxCDABE Escherichia coli biosensor in media differing with respect to carbon source, (ii) theoretical reconstruction of the measured signals using a here-reported theory for bioluminescence generated by constitutive cells, (iii) comparison between time-dependent cell photoactivity (reflecting metabolic activity) retrieved by theory with that we reported recently for cadmium-inducible PzntA-luxCDABE E. coli in media of similar compositions. Whereas signals of constitutive and non-constitutive biosensors differ in terms of shape, amplitude and peak number depending on nutritional medium conditions, analysis highlights the features shared by their respective cell photoactivity patterns mediated by the interplay between stringent response and catabolite repressions. The work advocates for the benefits of a theoretical interpretation for the time-dependent response of biosensors to unravel metabolic and physicochemical contributions to the bioluminescence signal. Full article

Bacterial contamination is an important factor causing food security issues. Among the bacteria, Escherichia coli is one of the main pathogens of food-borne microorganisms. However, traditional bacterial detection approaches cannot meet the requirements of real-time and on-site detection. Thus, it is of great significance to develop a rapid and accurate detection of bacteria in food to ensure food safety and safeguard human health. The pathogen heat-treatment module was designed in this paper based on the techniques including nanoprobe, pathogen heat-treatment, graphene transparent electrode (GTE), and adenosine triphosphate (ATP) bioluminescence technology. The system mainly consists of two parts: one is the optical detection unit; the other is the data processing unit. And it can quickly and automatically detect the number of bacterial colonies in food such as milk etc. The system uses not only the probe to capture and enrich E. coli by antigen-antibody interaction but also the heat treatment to increase the amount of ATP released from bacterial cells within five minutes. To enhance the detecting accuracy and sensitivity, the electric field generated by GTE is adopted in the system to enrich ATP. Compared to the other conventional methods, the linear correlation coefficient of the system can be reached 0.975, and the system meets the design requirements. Under the optimal experimental conditions, the detection can be completed within 25 min, and the detectable concentration of bacteria is in the range of 3.1 × 10¹–10⁶ CFU/mL. This system satisfies the demands of a fast and on-site inspection. Full article