Nanoparticles Partially Restore Bacterial Susceptibility to Antibiotics.

The growing resistance of bacteria to antibiotics is one of the main public health problems nowadays. The influence of silver nanoparticle (AgNP) pretreatment of 220 cows with mastitis on the susceptibility of Staphylococcus epidermidis bacteria to 31 antibiotics was studied. The obtained results were compared with the previous results for Escherichia coli, Streptococcus dysgalactiae, and Staphylococcus aureus. For all four bacteria, an increase in susceptibility (9.5-21.2%) to 31 antibiotics after cow treatment with AgNPs was revealed, while after first-line antibiotic drug treatment as expected, the susceptibility decreased (11.3-27.3%). These effects were explained by (1) the increase in the contribution of isolates with efflux effect after antibiotic treatments and its decrease after AgNP treatment and (2) the changes in bacteria adhesion and anti-lysozyme activity after these treatments. The effect of the increasing antibacterial activity of antibiotics after AgNP treatment was the most pronounced in the case of E. coli and was minimal in the case of S. epidermidis. With AgNP treatment, the time of recovery decreased by 26.8-48.4% compared to the time of recovery after treatment with the first-line antibiotic drugs. The AgNP treatment allows for achieving the partial restoration of the activity of antibiotics.

Solution of the Drug Resistance Problem of Escherichia coli with Silver Nanoparticles: Efflux Effect and Susceptibility to 31 Antibiotics.

The current work is a continuation of our studies focused on the application of nanoparticles of metallic silver (AgNPs) to address the global problem of antibiotic resistance. In vivo, fieldwork was carried out with 200 breeding cows with serous mastitis. Ex vivo analyses showed that after the cow was treated with an antibiotic-containing drug DienomastTM, E. coli sensibility to 31 antibiotics decreased by 27.3%, but after treatment with AgNPs, it increased by 21.2%. This could be explained by the 8.9% increase in the portion of isolates showing an efflux effect after DienomastTM treatment, while treatment with Argovit-CTM resulted in a 16.0% drop. We verified the likeness of these results with our previous ones on S. aureus and Str. dysgalactiae isolates from mastitis cows processed with antibiotic-containing medicines and Argovit-CTM AgNPs. The obtained results contribute to the recent struggle to restore the efficiency of antibiotics and to preserve the wide range of antibiotics on the world market.

Silver nanoparticles induce a non-immunogenic tumor cell death.

Immunogenic cell death (ICD) is a form of cell death characterized by the release of danger signals required to trigger an adaptive immune response against tumor-associated antigens. Silver nanoparticles (AgNP) display anti-proliferative and cytotoxic effects in tumor cells, but it has not been previously studied whether AgNP act as an ICD inductor. The present study evaluated the in vitro release of calreticulin as a damage-associated molecular pattern (DAMP) associated with the cytotoxicity of AgNP and their in vivo anti-cancer effects. In vitro, mouse CT26 colon carcinoma and MCA205 fibrosarcoma cells were exposed to AgNP and then cell proliferation, adhesion, and release of calreticulin were determined. The results indicated there were time- and concentration-related anti-proliferative effects of AgNP in both the CT26 and MCA205 lines. Concurrently, changes in cell adhesion were detected mainly in the CT26 cells. Regarding DAMP detection, a significant increase in calreticulin was observed only in CT26 cells treated with doxorubicin and AgNP; however, no differences were found in the MCA205 cells. In vivo, the survival and growth of subcutaneous tumors were monitored after vaccination of mice with cell debris from tumor cells treated with AgNP or after intra-tumoral administration of AgNP to established tumors. Consequently, anti-tumoral prophylactic immunization with AgNP-dead cells failed to protect mice from tumor re-challenge; intra-tumor injection of AgNP did not induce a significant effect. In conclusion, there was a noticeable anti-tumoral effect of AgNP in vitro in both CT26 and MCA205 cell lines, accompanied by the release of calreticulin in CT26 cells. In vivo, immunization with cell debris derived from AgNP-treated tumor cells failed to induce a protective immune response in the cancer model mice. Clearly, further research is needed to determine if one could combine AgNP with other ICD inducers to improve the anti-tumor effect of these nanoparticles in vivo.

AgNPs Targeting the Drug Resistance Problem of Staphylococcus aureus: Susceptibility to Antibiotics and Efflux Effect.

The present work presents translational research with application of AgNPs targeting the global drug resistance problem. In vivo fieldwork was carried out with 400 breeding farm cows sick with a serous mastitis. Ex vivo results revealed that after cow treatment with LactobayTM (a mixture of antibiotic drugs) the susceptibility to 31 antibiotics of S. aureus isolates from cow breast secretion decreased by 25%, while after treatment with Argovit-CTM silver nanoparticles S. aureus susceptibility increased by 11%. The portion of isolates with an efflux effect leading to elimination of antibiotics from S. aureus after Lactobay-treatment resulted in a 15% increase, while Argovit-C-treatment led to a 17.5% decrease. The obtained results showed that mastitis treatments with Argovit-CTM AgNPs can partially restore the activity of antibiotics towards S. aureus and shorten the duration of mastitis treatment by 33%.

Search for Effective Approaches to Fight Microorganisms Causing High Losses in Agriculture: Application of P. lilacinum Metabolites and Mycosynthesised Silver Nanoparticles.

The manuscript presents the first report to produce silver nanoparticles (AgNPs) using soil-inhabiting Purpureocillium lilacinum fungus cell filtrate as a promising fungicide and nematicide on two microorganisms causing high economic losses in agriculture. METHODS: A fungus biomass was used as a reducing and stabilising agent in the process of NPs synthesis and then characterisation done by SEM, TEM, UV-Vis. Finally, the antimicrobial activity of the synthesised AgNPs was determined. RESULTS: Synthesised AgNPs with a spherical and quasi-spherical shape with an average diameter of 50 nm were effective to inhibit A. flavus fungi and M. incognita root knot nematode, which are extremely pathogenic for plants. Application of the AgNPs led to 85% reduction of proliferation of A. flavus, to a 4-fold decrease of hatching of M. incognita plant-parasite juveniles from eggs, and to a 9-fold increase of M. incognita nematode mortality. CONCLUSIONS: Biosynthesised AgNPs can be used as an effective fungicide and nematicide for food safety and security and improvement of agricultural production, but further agricultural field trials are required to observe their effect on environment and other factors.

Antibacterial and Antifungal Studies of Biosynthesized Silver Nanoparticles against Plant Parasitic Nematode Meloidogyne incognita, Plant Pathogens Ralstonia solanacearum and Fusarium oxysporum.

The possibility of using silver nanoparticles (AgNPs) to enhance the plants growth, crop production, and control of plant diseases is currently being researched. One of the most effective approaches for the production of AgNPs is green synthesis. Herein, we report a green and phytogenic synthesis of AgNPs by using aqueous extract of strawberry waste (solid waste after fruit juice extraction) as a novel bioresource, which is a non-hazardous and inexpensive that can act as a reducing, capping, and stabilizing agent. Successful biosynthesis of AgNPs was monitored by UV-visible spectroscopy showing a surface plasmon resonance (SPR) peak at ~415 nm. The X-ray diffraction studies confirm the face-centered cubic crystalline AgNPs. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques confirm the rectangular shape with an average size of ~55 nm. The antibacterial and antifungal efficacy and inhibitory impact of the biosynthesized AgNPs were tested against nematode, Meloidogyne incognita, plant pathogenic bacterium, Ralstonia solanacearum and fungus, Fusarium oxysporum. These results confirm that biosynthesized AgNPs can significantly control these plant pathogens.

Green synthesis of silver nanoparticles using Lysiloma acapulcensis exhibit high-antimicrobial activity.

The scientific community is exploiting the use of silver nanoparticles (AgNPs) in nanomedicine and other AgNPs combination like with biomaterials to reduce microbial contamination. In the field of nanomedicine and biomaterials, AgNPs are used as an antimicrobial agent. One of the most effective approaches for the production of AgNPs is green synthesis. Lysiloma acapulcensis (L. acapulcensis) is a perennial tree used in traditional medicine in Mexico. This tree contains abundant antimicrobial compounds. In the context of antimicrobial activity, the use of L. acapulcensis extracts can reduce silver to AgNPs and enhance its antimicrobial activity. In this work, we demonstrate such antimicrobial activity effect employing green synthesized AgNPs with L. acapulcensis. The FTIR and LC-MS results showed the presence of chemical groups that could act as either (i) reducing agents stabilizing the AgNPs or (ii) antimicrobial capping agents enhancing antimicrobial properties of AgNPs. The synthesized AgNPs with L. acapulcensis were crystalline with a spherical and quasi-spherical shape with diameters from 1.2 to 62 nm with an average size diameter of 5 nm. The disk diffusion method shows the magnitude of the susceptibility over four pathogenic microorganisms of clinical interest. The antimicrobial potency obtained was as follows: E. coli ≥ S. aureus ≥ P. aeruginosa > C. albicans. The results showed that green synthesized (biogenic) AgNPs possess higher antimicrobial potency than chemically produced AgNPs. The obtained results confirm a more significant antimicrobial effect of the biogenic AgNPs maintaining low-cytotoxicity than the AgNPs produced chemically.

Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays.

In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARec provides a limit of detection (LOD) of 3.1 µg/L, PP1Rec and PP2AWild provide LODs of 0.6 and 0.5 µg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 µg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC50) values ranging from 1.4 to 359.3 µg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.

Inhibition equivalency factors for dinophysistoxin-1 and dinophysistoxin-2 in protein phosphatase assays: applicability to the analysis of shellfish samples and comparison with LC-MS/MS.

The protein phosphatase inhibition assay (PPIA) is a well-known strategy for the determination of diarrheic shellfish poisoning (DSP) lipophilic toxins, which deserves better characterization and understanding to be used as a routine screening tool in monitoring programs. In this work, the applicability of two PPIAs to the determination of okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2), and their acyl ester derivatives in shellfish has been investigated. The inhibitory potencies of the DSP toxins on a recombinant and a wild PP2A have been determined, allowing the establishment of inhibition equivalency factors (IEFs) (1.1 and 0.9 for DTX-1, and 0.4 and 0.6 for DTX-2, for recombinant and wild PP2A, respectively). The PPIAs have been applied to the determination of OA equivalent contents in spiked and naturally contaminated shellfish samples. Results have been compared to those obtained by LC-MS/MS analysis, after application of the IEFs, showing good agreements.

Magnetic particle-based enzyme assays and immunoassays for microcystins: from colorimetric to electrochemical detection.

In this work, magnetic particles (MPs) are used as supports for the immobilization of biorecognition molecules for the detection of microcystins (MCs). In one approach, a recombinant protein phosphatase 1 (PP1) has been conjugated to MPs via coordination chemistry, and MC-LR detection has been based on the inhibition of the enzyme activity. In the other approach, a monoclonal antibody (mAb) against MC-LR has been conjugated to protein G-coated MPs, and a direct competitive enzyme-linked immunoparticle assay (ELIPA) has been then performed. Conjugation of biomolecules to MPs has been first checked, and after optimization, MC detection has been performed. The colorimetric PPIA with PP1-MP and the best ELIPA strategy have provided limits of detection (LOD) of 7.4 and 3.9 µg/L of MC-LR, respectively. The electrochemical ELIPA has decreased the LOD to 0.4 µg/L, value below the guideline recommended by the World Health Organisation (WHO). The approaches have been applied to the analysis of a cyanobacterial culture and a natural bloom, and MC equivalent contents have been compared to those obtained by conventional assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results have demonstrated the viability of the use of MPs as biomolecule immobilization supports in biotechnological tools for MCs monitoring.

Novel nanobiotechnological concepts in electrochemical biosensors for the analysis of toxins.

This article gives an overview of the biosensors for the analysis of mycotoxins, marine toxins and cyanobacterial toxins, describing in depth the electrochemical biosensors that incorporate nanobiotechnological concepts. Firstly, it presents tailor-designed biomolecules, such as recombinant enzymes, recombinant antibody fragments and aptamers as novel biorecognition elements in biosensors. It also reviews the use of metallic nanoparticles (NPs) and carbon nanotubes (CNTs) aiming at improving the electrochemical transduction strategies. Finally, the exploitation of magnetic particles (MPs) as immobilisation carriers in flow-systems and the development of arrays are also described. The incorporation of these nanobiotechnological concepts provides with electrochemical biosensors with superior analytical performance in terms of specificity, sensitivity, stability and analysis time.

Conjugation of genetically engineered protein phosphatases to magnetic particles for okadaic acid detection.

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3µg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2µg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1µg/L).