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.

The significance of metallic nanoparticles in the emerging, development and spread of antibiotic resistance.

An ever-increasing number of newly synthesised nanoparticles have a constantly expanding range of applications. The large-scale implementation of nanoparticles will inevitably lead to intentional or accidental contamination of various environments. Since the major benefit of using several metallic nanoparticles is antimicrobial activity, these emerging contaminants may have a potentially hazardous impact on the development and spread of antibiotic resistance - a challenge that threats infection therapy worldwide. Few studies underline that metallic nanoparticles may affect the emergence and evolution of resistance via mutations and horizontal transfer between different bacterial species. Due to the complexity of factors and mechanisms involved in disseminating antibiotic resistance, it is crucial to investigate if metallic nanoparticles play a significant role in this process through co-selection ability and pressure exerted on bacteria. The aim of this review is to summarise the current research on mutations and three main horizontal gene transfer modes facilitated by nanoparticles. Here, the current results in the field are presented, major knowledge gaps and the necessity for more environmentally relevant studies are discussed.

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.

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.

Application of Erythromycin and/or Raoultella sp. Strain MC3 Alters the Metabolic Activity of Soil Microbial Communities as Revealed by the Community Level Physiological Profiling Approach.

Erythromycin (EM), a macrolide antibiotic, by influencing the biodiversity of microorganisms, might change the catabolic activity of the entire soil microbial community. Hence, the goal of this study was to determine the metabolic biodiversity in soil treated with EM (1 and 10 mg/kg soil) using the community-level physiological profiling (CLPP) method during a 90-day experiment. In addition, the effect of soil inoculation with antibiotic-resistant Raoultella sp. strain MC3 on CLPP was evaluated. The resistance and resilience concept as well as multifactorial analysis of data was exploited to interpret the outcomes obtained. EM negatively affected the metabolic microbial activity, as indicated by the values of the CLPP indices, i.e., microbial activity expressed as the average well-color development (AWCD), substrate richness (R), the Shannon-Wiener (H) and evenness (E) indices and the AWCD values for the six groups of carbon substrate present in EcoPlates until 15 days. The introduction of strain MC3 into soil increased the degradative activity of soil microorganisms in comparison with non-inoculated control. In contrast, at the consecutive sampling days, an increase in the values of the CLPP parameters was observed, especially for EM-10 + MC3-treated soil. Considering the average values of the resistance index for all of the measurement days, the resistance of the CLPP indices and the AWCD values for carbon substrate groups were categorized as follows: E > H > R > AWCD and polymers > amino acids > carbohydrates > miscellaneous > amines > carboxylic acids. The obtained results suggest a low level of resistance of soil microorganisms to EM and/or strain MC3 at the beginning of the exposure time, but the microbial community exhibited the ability to recover its initial decrease in catabolic activity over the experimental period. Despite the short-term effects, the balance of the soil ecosystem may be disturbed.

Vancomycin and/or Multidrug-Resistant Citrobacter Freundii Altered the Metabolic Pattern of Soil Microbial Community.

Despite many studies, our knowledge on the impact of antibiotics and antibiotic-resistant bacteria on the metabolic activity of soil microbial communities is still limited. To ascertain this impact, the community level physiological profiles (CLPPs) and the activity of selected enzymes (dehydrogenase, urease, and phosphatases) in soils treated with vancomycin (VA) and/or multidrug resistant Citrobacter freundii were determined during a 90-day experiment. A multivariate analysis and the resistance (RS)/resilience (RL) concept were used to assess the potential of native microorganisms to maintain their catabolic activity under exposure of VA and/or a high level of C. freundii. In addition, the dissipation rate of VA was evaluated in non-sterile (nsS) and sterile (sS) soils. The results revealed a negative impact of VA on the metabolic activity of soil microorganisms on days 1, 15, and 30 as was showed by a decrease in the values of the CLPP indices (10-69%) and the enzyme activities (6-32%) for treated soils as compared to the control. These observations suggested a low initial resistance of soil microorganisms to VA and/or C. freundii but they were resilient in the long term. Considering the mean values of the RS index, the resistance of measured parameters was categorized in the following order: alkaline phosphatase (0.919) > acid phosphatase (0.899) > dehydrogenase (0.853) > the evenness index (0.840) > urease (0.833) > the Shannon-Wiener index (0.735) > substrate richness (0.485) > the AWCD (0.301). The dissipation process of VA was relatively fast and independent of the concentration used. The DT50 values for VA applied at both concentrations were about 16 days. In addition, the dissipation of VA in nsS was three times faster compared to the dissipation of antibiotic in sS. In conclusion, both CLPP and enzyme activities assays appeared to be useful tool for the determination of disturbances within soil microbial communities and used together may be helpful to understand the changes in their catabolic features. The entry of large quantities of VA and/or C. freundii into soil may temporarily change microbial activity thus pose a potential risk for soil functioning.

The age of developing diabetes and FTO polymorphisms (rs9939609, rs1421085, and rs9930506).

INTRODUCTION: Type 2 diabetes (T2DM) is a common complex metabolic disorder that has a strong genetic predisposition. Fat mass and obesity-associated protein (FTO) is one of the genes of interest to us. Hypomethylation of a CpG site in the FTO gene was significantly associated with the risk of T2DM. The aim of the study was to find the answer to the question of whether the polymorphism changes of the FTO gene in the pathogenesis of type 2 diabetes are comparable in young, middle aged, and elderly people. MATERIAL AND METHODS: The study involved 282 consecutive patients with type 2 diabetes, who attended a primary healthcare clinic in Southern Poland. The study subjects were divided into three groups according to the age at which type 2 diabetes mellitus was diagnosed (> 40 years old, 40-60 years old, and > 60 years old). The genotyping of rs9939609, rs1421085, and rs9930506 FTO polymorphisms was conducted using TaqManPre-designed SNP Genotyping Assay. RESULTS: No statistically significant difference was shown between the examined FTO polymorphism (rs9939609, rs1421085, and rs9930506) distribution between the subjects diagnosed with diabetes < 40 years , 40-60 years, and > 60 years old. CONCLUSIONS: There were no statistically significant relationships between the different analysed anthropometric and other parameters and distribution of examined FTO polymorphisms (rs9939609 , rs1421085, and rs9930506). The age of diabetes was not affect by the tested FTO polymorphisms (rs9939609 , rs1421085, and rs9930506).

The urgent need for risk assessment on the antibiotic resistance spread via sewage sludge land application.

Sewage sludge is an ever-increasing by-product of the wastewater treatment process frequently used as a soil fertiliser. To control its quality and prevent any possible hazardous impact of fertilisation, some mandatory limits of heavy metal content have been established by the European Commission (Sewage Sludge Directive). However, since the implementation of the limits, new emerging contaminants have been reported worldwide. Regardless of the wastewater treatment process, sewage sludge contains antibiotics, antibiotic-resistant bacteria and antibiotic resistance genes, which can be released into the environment through its land application. Such a practice may even boost the dissemination and further development of antibiotic resistance phenomenon - already a global problem challenging modern medicine. Due to the growing pharmaceutical pollution in the environment, the time is ripe to assess the risk for the human and environmental health of sewage sludge land application in the context of antibiotic resistance spread. In this review we present the current knowledge in the field and we emphasise the necessity for more studies.

[Evaluation of the correlation between IL-1ß, IL-8, IFN-γ cytokine concentration in cervico-vaginal fluid and the risk of preterm delivery]. / Ocena korelacji miedzy stezeniem cytokin IL-1ß, IL-8, IFN-ß w wydzielinie szyjkowo-pochwowej, a ryzykiem wystapienia porodu przedwczesnego.

OBJECTIVES: The aim of the study was to evaluate IL-1ß, II-8, IFN-γ cytokine concentrations in cervico-vaginal fluid in patients with threatening preterm delivery. MATERIAL AND METHODS: The study group included 84 patients between 27 and 34 weeks of pregnancy admitted with symptoms of threatened preterm delivery. The cervico-vaginal fluid was taken from each patient qualified for the study and IL-1ß, II-8, IFN-γ concentration was analyzed by enzyme-linked immunosorbent assay (ELISA). The following factors were analyzed: maternal age and parity gestational age at admission for the examination and at delivery, sex of the newborn, Apgar evaluation at 1 and 5 minutes of life, umbilical blood pH, history of miscarriage or preterm delivery pre-pregnancy body mass index, and smoking. Their influence on the concentration of biochemical markers tested in patients at risk of preterm delivery was investigated. RESULTS: In the study group, patients who delivered preterm had significantly higher concentrations of IL-1ß and II-8 as compared to patients who delivered at term. Patients who delivered preterm more often had a history of a miscarriage and their newborns had lower birth weight, lower Apgar score, and lower pH of the umbilical blood. CONCLUSION: As far as factors significantly influencing cytokine concentrations are concerned, the level of IL-1ß and II-8 concentration is an independent predictor of preterm delivery in patients with threatened preterm labor. In the study group, the IFN-γ concentration did not significantly diversify patients who delivered preterm and at term.

Imidacloprid induces changes in the structure, genetic diversity and catabolic activity of soil microbial communities.

This is the first report describing the effect of imidacloprid applied at field rate (FR, 1 mg/kg of soil) and 10 times the FR (10*FR, 10 mg/kg of soil) on the structural, genetic and physiological diversity of soil bacterial community as determined by the phospholipid fatty acid (PLFA), the denaturing gradient gel electrophoresis (DGGE), and the community level physiological profile (CLPP) approaches. PLFA profiles showed that imidacloprid significantly shifted the microbial community structure and decreased the biomass of the total, bacterial and fungal PLFAs, however, this effect was transient at the FR dosage. The alterations in DGGE patterns caused by imidacloprid application, confirmed considerable changes in the overall richness and diversity of dominant bacteria. Although, as a result of imidacloprid application, the metabolic activity of microbial communities was generally lower, the richness and functional biodiversity of the soil microbial community were not negatively affected. In general, the analysis of the variance indicated that the measured parameters were significantly affected by treatment and the incubation time, however, the incubation time effect explained most of the observed variance. Imidacloprid degradation and the appearance of some new bands in DGGE profiles suggest the evolution of bacteria capable of degrading imidacloprid among indigenous microflora.