The influence of ZnO nanoparticles on horizontal transfer of resistance genes in lab and soil conditions.

Antibiotic resistance is a severe problem that threatens the achievements of modern medicine. Metallic nanoparticles may promote the horizontal transfer of resistance genes due to their toxicity to bacterial cells and metal-induced co-selection mechanisms. In this study, we investigated the toxicity of ZnO nanoparticles to E. coli DH5α laboratory strain and the abundance of soil microbial community. Moreover, the influence of ZnO nanoparticles on resistance gene transfer in laboratory and soil conditions was evaluated. ZnO nanoparticles at concentrations up to 10 mg L-1 reduced the survival of E. coli cells by 14.6% and increased the transformation frequency by almost 1.8 fold. In soil, ZnO nanoparticles at a concentration of 1000 mg kg-1 affected the total abundance of bacteria, causing a decrease in the 16S rRNA gene copy number. We did not detect the presence of 11 target antibiotic resistance genes (sul1, sul2, imp2, imp5, blaCTX-M, ermB, mefA, strB, aadA1, tetA1, tetB), which confer resistance to five classes of antibiotics in soil treated with ZnO nanoparticles. No elevated conjugation frequency was observed in soil microbial communities treated with ZnO nanoparticles. However, the increase in czcA gene copies indicates the spread of genetic elements harbouring metal resistance. The data shows that metallic nanoparticles promote the spread of antibiotic and metal resistance genes. The broad implication of the present research is that the inevitable nanoparticles environmental pollution may lead to the further dissemination of antibiotic resistance and profoundly influence public health.

Key Properties of a Bioactive Ag-SiO2/TiO2 Coating on NiTi Shape Memory Alloy as Necessary at the Development of a New Class of Biomedical Materials.

Recent years have seen the dynamic development of methods for functionalizing the surface of implants using biomaterials that can mimic the physical and mechanical nature of native tissue, prevent the formation of bacterial biofilm, promote osteoconduction, and have the ability to sustain cell proliferation. One of the concepts for achieving this goal, which is presented in this work, is to functionalize the surface of NiTi shape memory alloy by an atypical glass-like nanocomposite that consists of SiO2-TiO2 with silver nanoparticles. However, determining the potential medical uses of bio(nano)coating prepared in this way requires an analysis of its surface roughness, tribology, or wettability, especially in the context of the commonly used reference coat-forming hydroxyapatite (HAp). According to our results, the surface roughness ranged between (112 ± 3) nm (Ag-SiO2)-(141 ± 5) nm (HAp), the water contact angle was in the range (74.8 ± 1.6)° (Ag-SiO2)-(70.6 ± 1.2)° (HAp), while the surface free energy was in the range of 45.4 mJ/m2 (Ag-SiO2)-46.8 mJ/m2 (HAp). The adhesive force and friction coefficient were determined to be 1.04 (Ag-SiO2)-1.14 (HAp) and 0.247 ± 0.012 (Ag-SiO2) and 0.397 ± 0.034 (HAp), respectively. The chemical data showed that the release of the metal, mainly Ni from the covered NiTi substrate or Ag from Ag-SiO2 coating had a negligible effect. It was revealed that the NiTi alloy that was coated with Ag-SiO2 did not favor the formation of E. coli or S. aureus biofilm compared to the HAp-coated alloy. Moreover, both approaches to surface functionalization indicated good viability of the normal human dermal fibroblast and osteoblast cells and confirmed the high osteoconductive features of the biomaterial. The similarities of both types of coat-forming materials indicate an excellent potential of the silver-silica composite as a new material for the functionalization of the surface of a biomaterial and the development of a new type of functionalized implants.

Land Spreading of Wastewaters from the Fruit-Packaging Industry and Potential Effects on Soil Microbes: Effects of the Antioxidant Ethoxyquin and Its Metabolites on Ammonia Oxidizers.

Thiabendazole (TBZ), imazalil (IMZ), ortho-phenylphenol (OPP), diphenylamine (DPA), and ethoxyquin (EQ) are used in fruit-packaging plants (FPP) with the stipulation that wastewaters produced by their application would be depurated on site. However, no such treatment systems are currently in place, leading FPP to dispose of their effluents in agricultural land. We investigated the dissipation of those pesticides and their impact on soil microbes known to have a key role on ecosystem functioning. OPP and DPA showed limited persistence (50% dissipation time [DT50], 0.6 and 1.3 days) compared to TBZ and IMZ (DT50, 47.0 and 150.8 days). EQ was rapidly transformed to the short-lived quinone imine (QI) (major metabolite) and the more persistent 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite). EQ and OPP exerted significant inhibition of potential nitrification, with the effect of the former being more persistent. This was not reflected in the abundance (determined by quantitative PCR [qPCR]) of the amoA gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Considering the above discrepancy and the metabolic pattern of EQ, we further investigated the hypothesis that its metabolites and not only EQ were toxic to ammonia oxidizers. Potential nitrification, amoA gene abundance, and amoA gene transcripts of AOB and AOA showed that QI was probably responsible for the inhibition of nitrification. Our findings have serious ecological and practical implications for soil productivity and N conservation in agriculturally impacted ecosystems and stress the need to include metabolites and RNA-based methods when the soil microbial toxicity of pesticides is assessed.

Response of microbial communities from an apple orchard and grassland soils to the first-time application of the fungicide tetraconazole.

The aim of the study was to assess the impact of the triazole fungicide tetraconazole applied at the field rate (FR) and at ten-fold the FR (10FR) on microorganisms in orchard soil with a long-term history of fungicides application and in grassland soil that had not previously been treated with pesticides. To ascertain this impact, the microbial activity determined by fluorescein diacetate (FDA) hydrolysis, the culturable number of bacteria, fungi and tetraconazole-resistant fungi, and the phospholipid microbial biomass and the structural and functional biodiversity assessed by the PLFA and Biolog approaches, respectively, were examined under laboratory conditions during 28-day experiment. The response of soil microorganisms to the fungicide tetraconazole, which had never been used before in these soils, depended on the management of the soils. In apple orchard soil that had been treated with FR or 10FR tetraconazole, a decrease in microbial activity was still observed on the 28th day after the application of the fungicide. In contrast, a significant impact of tetraconazole on the number of bacteria was still observed at the end of experiment in grassland soil. Results of principal component analysis (PCA) indicated that the application of tetraconazole significantly changed the structure of the microbial communities in the orchard soil. In addition, analysis of the Biolog profiles revealed a decrease in the catabolic activity of the microbial communities in grassland soil that had been treated with tetraconazole at both rates over time. The evaluation of the structural and functional diversity of microbial communities using PCA appears to be the most valuable monitoring tool for assessing the impact of tetraconazole application on soil microorganisms.

Non-target impact of fungicide tetraconazole on microbial communities in soils with different agricultural management.

Effect of the fungicide tetraconazole on microbial community in silt loam soils from orchard with long history of triazole application and from grassland with no known history of fungicide usage was investigated. Triazole tetraconazole that had never been used on these soils before was applied at the field rate and at tenfold the FR. Response of microbial communities to tetraconazole was investigated during 28-day laboratory experiment by determination of changes in their biomass and structure (phospholipid fatty acids method-PLFA), activity (fluorescein diacetate hydrolysis-FDA) as well as changes in genetic (DGGE) and functional (Biolog) diversity. Obtained results indicated that the response of soil microorganisms to tetraconazole depended on the management of the soils. DGGE patterns revealed that both dosages of fungicide affected the structure of bacterial community and the impact on genetic diversity and richness was more prominent in orchard soil. Values of stress indices-the saturated/monounsaturated PLFAs ratio and the cyclo/monounsaturated precursors ratio, were almost twice as high and the Gram-negative/Gram-positive ratio was significantly lower in the orchard soil compared with the grassland soil. Results of principal component analysis of PLFA and Biolog profiles revealed significant impact of tetraconazole in orchard soil on day 28, whereas changes in these profiles obtained for grassland soil were insignificant or transient. Obtained results indicated that orchards soil seems to be more vulnerable to tetraconazole application compared to grassland soil. History of pesticide application and agricultural management should be taken into account in assessing of environmental impact of studied pesticides.

Nanopesticide risk assessment based on microbiome profiling - Community structure and functional potential as biomarkers in captan@ZnO35-45 nm and captan@SiO220-30 nm treated orchard soil.

Nanoformulation should minimise the usage of pesticides and limit their environmental footprint. The risk assessment of two nanopesticides with fungicide captan as an active organic substance and ZnO35-45 nm or SiO220-30 nm as nanocarriers was evaluated using the non-target soil microorganisms as biomarkers. The first time for that kind of nanopesticides next-generation sequencing (NGS) of bacterial 16 S rRNA and fungal ITS region and metagenomics functional predictions (PICRUST2) was made to study structural and functional biodiversity. During a 100-day microcosm study in soil with pesticide application history, the effect of nanopesticides was compared to pure captan and both nanocarriers. Nanoagrochemicals affected microbial composition, especially Acidobacteria-6 class, and alpha diversity, but the observed effect was generally more substantial for pure captan. As for beta diversity, the negative impact was detected only in response to captan and still observed on day 100. Fungal community in the orchard soil showed only a decrease in phylogenetic diversity in captan set-up since day 30. PICRUST2 analysis confirmed several times lower impact of nanopesticides considering the abundance of functional pathways and genes encoding enzymes. Furthermore, the overall data indicated that using SiO220-30 nm as a nanocarrier speeds up a recovery process compared to ZnO35-45 nm.

Copper-oxide nanoparticles exert persistent changes in the structural and functional microbial diversity: A 60-day mesocosm study of zinc-oxide and copper-oxide nanoparticles in the soil-microorganism-nanoparticle system.

Recent advances in nanotechnology and development of nanoformulation methods, has enabled the emergence of precision farming - a novel farming method that involves nanopesticides and nanoferilizers. Zinc-oxide nanoparticles serve as a Zn source for plants, but they are also used as nanocarriers for other agents, whereas copper-oxide nanoparticles possess antifungal activity, but in some cases may also serve as a micronutrient providing Cu ions. Excessive application of metal-containing agents leads to their accumulation in soil, where they pose a threat to non-target soil organisms. In this study, soils obtained from the environment were amended with commercial zinc-oxide nanoparticles: Zn-OxNPs(10-30), and newly-synthesized copper-oxide nanoparticles: Cu-OxNPs(1-10). Nanoparticles (NPs) in 100 and 1000 mg kg-1 concentrations were added in separate set-ups, representing a soil-microorganism-nanoparticle system in a 60-day laboratory mesocosm experiment. To track environmental footprint of NPs on soil microorganisms, a Phospholipd Fatty Acid biomarker analysis was employed to study microbial community structure, whereas Community-Level Physiological Profiles of bacterial and fungal fractions were measured with Biolog Eco and FF microplates, respectively. The results revealed a prominent and persistent effects exerted by copper-containing nanoparticles on non-target microbial communities. A severe loss of Gram-positive bacteria was observed in conjunction with disturbances in bacterial and fungal CLPPs. These effects persisted till the end of a 60-day experiment, demonstrating detrimental rearrangements in microbial community structure and functions. The effects imposed by zinc-oxide NPs were less pronounced. As persistent changes were observed for newly synthesized Cu-containing NPs, this work stresses the need for obligatory testing of nanoparticle interactions with non-target microbial communities in long-term experiments, especially during the approval procedures of novel nano-substances. It also underlines the role of in-depth physical and chemical studies of NP-containing agents, which may be tweaked to mitigate the unwanted behavior of such substances in the environment and preselect their beneficial characteristics.

Land application of sewage sludge: Response of soil microbial communities and potential spread of antibiotic resistance.

The effect of land application of sewage sludge on soil microbial communities and the possible spread of antibiotic- and metal-resistant strains and resistance determinants were evaluated during a 720-day field experiment. Enzyme activities, the number of oligotrophic bacteria, the total number of bacteria (qPCR), functional diversity (BIOLOG) and genetic diversity (DGGE) were established. Antibiotic and metal resistance genes (ARGs, MRGs) were assessed, and the number of cultivable antibiotic- (ampicillin, tetracycline) and heavy metal- (Cd, Zn, Cu, Ni) resistant bacteria were monitored during the experiment. The application of 10 t ha-1 of sewage sludge to soil did not increase the organic matter content and caused only a temporary increase in the number of bacteria, as well as in the functional and structural biodiversity. In contrast to expectations, a general adverse effect on the tested microbial parameters was observed in the fertilized soil. The field experiment revealed a significant reduction in the activities of alkaline and acid phosphatases, urease and nitrification potential. Although sewage sludge was identified as the source of several ARGs and MRGs, these genes were not detected in the fertilized soil. The obtained results indicate that the effect of fertilization based on the recommended dose of sewage sludge was not achieved.

Microbial communities from subglacial water of naled ice bodies in the forefield of Werenskioldbreen, Svalbard.

We assessed the structure of microbial communities in the subglacial drainage system of the Werenskioldbreen glacier, Svalbard, which consists of three independent channels. Dome-shaped naled ice bodies that had been forming and releasing subglacial water in the glacial forefield during accumulations season were used to study glacial microbiome. We tested the hypothesis that the properties of the water transported by these channels are site-dependent and influence bacterial diversity. We therefore established the phylogenetic structure of the subglacial microbial communities using next generation sequencing (NGS) of the 16S rRNA gene and performed bioinformatics analyses. A total of 1409 OTUs (operational taxonomic units) belonged to 40 phyla; mostly Proteobacteria, Gracilibacteria, Bacteroidetes, Actinobacteria and Parcubacteria were identified. Sites located on the edge of Werenskioldbreen forefield (Angell, Kvisla) were mainly dominated by Betaproteobacteria. In the central site (Dusan) domination of Epsilonproteobacteria class was observed. Gracilibacteria (GN02) and Gammaproteobacteria represented the dominant taxa only in the sample Kvisla 2. Principal Coordinate Analysis (PCoA) of beta diversity revealed that phylogenetic profiles grouped in three different clusters according to the sampling site. Moreover, higher similarity of bacterial communities from Angell and Kvisla compared to Dusan was confirmed by cluster analysis and Venn diagrams. The highest alpha index values was measured in Dusan. Richness and phylogenetic diversity indices were significantly (p < .05) and positively correlated with pH values of subglacial water and negatively with concentration of Cl-, Br-, and NO3- anions. These anions negatively impacted the values of richness indices but positively correlated with abundance of some microbial phyla. Our results indicated that subglacial water from naled ice bodies offer the possibility to study the glacial microbiome. In the studied subglacial water, the microbial community structure was sampling site specific and dependent on the water properties, which in turn were probably influenced by the local bedrock composition.

An Organic-Inorganic Hybrid Nanocomposite as a Potential New Biological Agent.

To solve the problem of human diseases caused by a combination of genetic and environmental factors or by microorganisms, intense research to find completely new materials is required. One of the promising systems in this area is the silver-silica nanocomposites and their derivatives. Hence, silver and silver oxide nanoparticles that were homogeneously distributed within a silica carrier were fabricated. Their average size was d = (7.8 ± 0.3) nm. The organic polymers (carboxymethylcellulose (CMC) and sodium alginate (AS)) were added to improve the biological features of the nanocomposite. The first system was prepared as a silver chlorine salt combination that was immersed on a silica carrier with coagulated particles whose size was d = (44.1 ± 2.3) nm, which coexisted with metallic silver. The second system obtained was synergistically interacted metallic and oxidized silver nanoparticles that were distributed on a structurally defective silica network. Their average size was d = (6.6 ± 0.7) nm. Physicochemical and biological experiments showed that the tiny silver nanoparticles in Ag/SiO2 and Ag/SiO2@AS inhibited E. coli, P. aeruginosa, S. aureus, and L. plantarum's cell growth as well as caused a high anticancer effect. On the other hand, the massive silver nanoparticles of Ag/SiO2@CMC had a weaker antimicrobial effect, although they highly interacted against PANC-1. They also generated reactive oxygen species (ROS) as well as the induction of apoptosis via the p53-independent mechanism.

Sterile Capsule-Egg Cocoon Covering Constitutes an Antibacterial Barrier for Spider Parasteatoda tepidariorum Embryos.

Coexistence of organisms and pathogens has resulted in the evolution of efficient antimicrobial defense, especially at the embryonic stage. This investigation aimed to substantiate the hypothesis that the layers of silk in a spider cocoon play a role in the immunity of the embryos against microorganisms present in the external environment. A two-step interdisciplinary attempt has been made. First, the eggs and empty cocoons of the spider Parasteatoda tepidariorum were incubated on lysogeny broth agar media for 3 d. In the samples of eggs, no growth of bacteria was detected. This indicated that the eggs inside cocoons were sterile. Therefore, in the second step, the cocoons and egg surface were analyzed using SEM, TEM, and LM. The obtained images demonstrated that both inner and outer layers of the silk are built of threads of the same diameter, set in an irregular manner, and randomly clustered into groups. The threads in the outer layer were packed more densely than in the inner one. TEM analysis revealed threads of two types of fibrils and their arrangement. The resultant thread tangle of the cocoon, possibly correlated with the ultrastructure of the fibers, seems to be an example of a structure-function relationship playing a crucial ecoimmunological role in spider embryonic development.

Toward the Development of an Innovative Implant: NiTi Alloy Functionalized by Multifunctional ß-TCP+Ag/SiO2 Coatings.

In recent years, one of the more important and costly problems of modern medicine is the need to replace or supplement organs in order to improve the quality of human life. In this field, promising solutions seem to have been implants which are based on NiTi alloys with shape memory effects. Unfortunately, this material is susceptible to the corrosion and release of toxic nickel to the human organism. Hence, its application as a long-term material is strongly limited. Therefore, this paper presents a new solution which should help to improve the functionality of the NiTi alloy and elongate its medical stability to use. The idea was focused on functionalization of the implant surface by a biocompatible, multifunctional coating without any impact on the features of the substrate, i.e., the martensitic transformation responsible for shape memory effects. For this purpose, we prepared a colloidal suspension, composed of ß-TCP (particle size ∼450 nm) and the Ag/SiO2 nanocomposite which due to the electrophoretic deposition (EPD) led to the formation of structurally atypical calcium phosphosilicate coating. Those biomaterials formed a crack-free coating, adhering well to the NiTi surface when distributed over the entire surface, with low concentration of metallic and oxide silver (<3 at. %). At the same time, the coat-forming materials had resulted in the growth of a Gram-negative bacterial biofilm. Additionally, the additive of the silver-silica composite enhances cell proliferation, effectively a few times higher than commonly used coat-forming materials (e.g., pure ß-TCP).

Rhizospheric Bacterial Strain Brevibacterium casei MH8a Colonizes Plant Tissues and Enhances Cd, Zn, Cu Phytoextraction by White Mustard.

Environmental pollution by heavy metals has become a serious problem in the world. Phytoextraction, which is one of the plant-based technologies, has attracted the most attention for the bioremediation of soils polluted with these contaminants. The aim of this study was to determine whether the multiple-tolerant bacterium, Brevibacterium casei MH8a isolated from the heavy metal-contaminated rhizosphere soil of Sinapis alba L., is able to promote plant growth and enhance Cd, Zn, and Cu uptake by white mustard under laboratory conditions. Additionally, the ability of the rifampicin-resistant spontaneous mutant of MH8a to colonize plant tissues and its mechanisms of plant growth promotion were also examined. In order to assess the ecological consequences of bioaugmentation on autochthonous bacteria, the phospholipid fatty acid (PLFA) analysis was used. The MH8a strain exhibited the ability to produce ammonia, 1-amino-cyclopropane-1-carboxylic acid deaminase, indole 3-acetic acid and HCN but was not able to solubilize inorganic phosphate and produce siderophores. Introduction of MH8a into soil significantly increased S. alba biomass and the accumulation of Cd (208%), Zn (86%), and Cu (39%) in plant shoots in comparison with those grown in non-inoculated soil. Introduced into the soil, MH8a was able to enter the plant and was found in the roots and leaves of inoculated plants thus indicating its endophytic features. PLFA analysis revealed that the MH8a that was introduced into soil had a temporary influence on the structure of the autochthonous bacterial communities. The plant growth-promoting features of the MH8a strain and its ability to enhance the metal uptake by white mustard and its long-term survival in soil as well as its temporary impact on autochthonous microorganisms make the strain a suitable candidate for the promotion of plant growth and the efficiency of phytoextraction.