Following the route of veterinary antibiotics tiamulin and tilmicosin from livestock farms to agricultural soils.

Veterinary antibiotics (VAs) are not completely metabolized in the animal body. Hence, when animal excretes are used as soil manures, VA residues are dispersed with potential implications for environmental quality and human health. We studied the persistence of tiamulin (TIA) and tilmicosin (TLM) along their route from pig administration to fecal excretion and to agricultural soils. TLM was detected in feces at levels folds higher (4.27-749.6 µg g-1) than TIA (0.55-5.99 µg g-1). Different administration regimes (feed or water) showed different excretion patterns and residual levels for TIA and TLM, respectively. TIA and TLM (0.5, 5 and 50 µg g-1) dissipated gradually from feces when stored at ambient conditions (DT50 5.85-35.9 and 23.5-49.8 days respectively), while they persisted longer during anaerobic digestion (DT90 >365 days) with biomethanation being adversely affected at VA levels > 5 µg g-1. When applied directly in soils, TLM was more persistent than TIA with soil fumigation extending their persistence suggesting microbial degradation, while soil application through feces increased their persistence, probably due to increased sorption to the fecal organic matter. The use of TIA- and TLM-contaminated feces as manures is expected to lead to VAs dispersal with unexplored consequences for the environment and human health.

Strong host-specific selection and over-dominance characterize arbuscular mycorrhizal fungal root colonizers of coastal sand dune plants of the Mediterranean region.

Sand dunes of the Mediterranean region constitute drought-stressed, low-fertility ecosystems. Arbuscular mycorrhizal fungi (AMF) are regarded as key components of their biota, that contribute to plant host adaptation and fitness. However, AMF community assembly rules in the roots of the psammophilous plants of coastal sand dunes have not been investigated. We studied the root colonizing AMF communities of four characteristic native plants of eastern Mediterranean coastal foredunes, in nine locations in Greece. Host-specificity (plant identity) was the major driver of AMF community assembly in the plant roots, while geographical distance between locations was not related to differences in the AMF communities. Additionally, colonizer AMF communities were characterized by over-dominance of a single operational taxanomic unit (OTUs), which was remarkably host-specific among locations. Wider dissimilarity in AMF communities was observed in small and disturbed (SD) sites compared to large and undisturbed (LU) sites, a trait that may be attributed to relaxed environmental filtering and facilitated AMF dispersal/immigration in SD sites from surrounding habitats. Overall, our results indicate that the assembly of root-colonizing AMF communities in the eastern Mediterranean sand dunes is characterized by strong biotic filtering (host identity), suggesting that co-adaptation processes may be more pronounced than previously proposed, under extreme environmental conditions.

Bioaugmentation of animal feces as a mean to mitigate environmental contamination with anthelmintic benzimidazoles.

Anthelmintics are used to control infestations of ruminants by gastrointestinal nematodes. The limited metabolism of anthelmintics in animals result in their excretion in feces. These could be piled up in the floor of livestock farms, constituting a point source of environmental contamination, or used as manures in agricultural soils where they persist or move to water bodies. Hence the removal of anthelmintics from feces could mitigate environmental contamination. We hypothesized that a thiabendazole-degrading bacterial consortium would also degrade other benzimidazole anthelmintics like albendazole, fenbendazole, ricobendazole, mebendazole and flubendazole. In liquid culture tests the consortium was more effective in degrading compounds with smaller benzimidazole substituents (thiabendazole, albendazole, ricobendazole), rather than benzimidazoles with bulky substituents (fenbendazole, flubendazole, mebendazole). We then explored the bioaugmentation capacity of the consortium in sheep feces fortified with 5 and 50 mg kg-1 of thiabendazole, albendazole and fenbendazole. Bioaugmentation enhanced the degradation of all compounds and its efficiency was accelerated upon fumigation of feces, in the absence of the indigenous fecal microbial community. The latter contributes to anthelmintics degradation as suggested by the significantly lower DT50 values in fumigated vs non-fumigated, non-bioaugmented feces. Overall, bioaugmentation could be an efficient means to reduce environmental exposure to recalcitrant anthelmintic benzimidazoles.

Effects of organic loading rate and hydraulic retention time on bioaugmentation performance to tackle ammonia inhibition in anaerobic digestion.

Three continuously stirred-tank reactors fed with manure operating under high ammonia levels (5.0 g NH3-N L-1) and with increased organic loading rate (OLR), (2.09 R1, 3.02 R2 and 4.0 R3 g VS L-1 d-1), achieved through glucose amendment in R2 and R3, were inoculated with an ammonia-acclimatized microbial culture. Successful bioaugmentation was endured only in R2 and R3, both reactors characterized by high OLR, resulting in 19.6 and 24.5% increase in methane production, respectively. The high OLRs in these reactors favored the co-occurrence of the hydrogenotrophic (Methanobacteriaceae), methylotrophic (Methanomethylophilaceae) and aceticlastic methanogenic pathways. The latter was supported by the successful establishment of ammonium-tolerant Methanosarcina, prevailing in the inoculum. Oppositely in R1, the low OLR prevented the establishment of Methanosarcina, leading to an exclusive hydrogenotrophic methanogenesis and reduced methane production. HRT shortening resulted in limited effect on biomethane performance, indicating a well establishment of the introduced bioaugmentation culture in the reactors.

Factors Structuring the Epiphytic Archaeal and Fungal Communities in a Semi-arid Mediterranean Ecosystem.

The phyllosphere microbiome exerts a strong effect on plants' productivity, and its composition is determined by various factors. To date, most phyllosphere studies have focused on bacteria, while fungi and especially archaea have been overlooked. We studied the effects of plant host and season on the abundance and diversity of the epiphytic archaeal and fungal communities in a typical semi-arid Mediterranean ecosystem. We collected leaves in two largely contrasting seasons (summer and winter) from eight perennial species of varying attributes which could be grouped into the following: (i) high-canopy, evergreen sclerophyllοus shrubs with leathery leaves, and low-canopy, either semi-deciduous shrubs or non-woody perennials with non-leathery leaves, and (ii) aromatic and non-aromatic plants. We determined the abundance of epiphytic Crenarchaea, total fungi, Alternaria and Cladosporium (main airborne fungi) via q-PCR and the structure of the epiphytic archaeal and fungal communities via amplicon sequencing. We observed a strong seasonal effect with all microbial groups examined showing higher abundance in summer. Plant host and season were equally important determinants of the composition of the fungal community consisted mostly of Ascomycota, with Hypocreales dominating in winter and Capnodiales and Pleosporales in summer. In contrast, the archaeal community showed plant host driven patterns dominated by the Soil Crenarchaeotic Group (SCG) and Aenigmarchaeota. Plant habit and aromatic nature exhibited filtering effects only on the epiphytic fungal communities. Our study provides a first in-depth analysis of the key determinants shaping the phyllosphere archaeal and fungal communities of a semi-arid Mediterranean ecosystem.

Ammonia-induced inhibition of manure-based continuous biomethanation process under different organic loading rates and associated microbial community dynamics.

Three Continuously Stirred Tank Reactors (CTSRs) were operating at steady state conditions with Organic Loading Rates (OLR) of 2.09, 3.024 and 4.0 g VS L-1 d-1. Glucose was used as the sole factor for increasing the OLR, linking the increase of the OLR with the C/N ratio increase. The reactors were stressed by increasing the ammonia concentration to 5 g L-1 from 1.862 g L-1. The results showed elevating inhibition of the anaerobic process by increasing the C/N ratio just by increasing the OLR, under the high ammonia concentration. A different response of the bacterial and archaeal community under ammonia stressed conditions was also observed. Under the high ammonia concentration, hydrogen-depended methylotrophic was the dominant methanogenesis route at OLR of 2.09 g VS L-1d-1, while the hydrogenotrophic route was the dominant at the high OLR of 4 g VS L-1d-1, which coincided with high acetate and propionate concentrations.

Irrigation of radish (Raphanus sativus L.) with microcystin-enriched water holds low risk for plants and their associated rhizopheric and epiphytic microbiome.

Microcystins (MCs) are toxins produced during cyanobacterial blooms. They reach soil and translocated to plants through irrigation of agricultural land with water from MC-impacted freshwater systems. To date we have good understanding of MC effects on plants, but not for their effects on plant-associated microbiota. We tested the hypothesis that MC-LR, either alone or with other stressors present in the water of the Karla reservoir (a low ecological quality and MC-impacted freshwater system), would affect radish plants and their rhizospheric and phyllospheric microbiome. In this context a pot experiment was employed where radish plants were irrigated with tap water without MC-LR (control) or with 2 or 12 µg L-1 of pure MC-LR (MC2 and MC12), or water from the Karla reservoir amended (12 µg L-1) or not with MC-LR. We measured MC levels in plants and rhizospheric soil and we determined effects on (i) plant growth and physiology (ii) the nitrifying microorganisms via q-PCR, (ii) the diversity of bacterial and fungal rhizospheric and epiphytic communities via amplicon sequencing. MC-LR and/or Karla water treatments resulted in the accumulation of MC in taproot at levels (480-700 ng g-1) entailing possible health risks. MC did not affect plant growth or physiology and it did not impose a consistent inhibitory effect on soil nitrifiers. Karla water rather than MC-LR was the stronger determinant of the rhizospheric and epiphytic microbial communities, suggesting the presence of biotic or abiotic stressors, other than MC-LR, in the water of the Karla reservoir which affect microorganisms with a potential role (i.e. pathogens inhibition, methylotrophy) in the homeostasis of the plant-soil system. Overall, our findings suggest that MC-LR, when applied at environmentally relevant concentrations, is not expected to adversely affect the radish-microbiota system but might still pose risk for consumers' health.

The response of soil and phyllosphere microbial communities to repeated application of the fungicide iprodione: accelerated biodegradation or toxicity?

Pesticides interact with microorganisms in various ways with the outcome being negative or positive for the soil microbiota. Pesticides' effects on soil microorganisms have been studied extensively in soil but not in other pesticides-exposed microbial habitats like the phyllosphere. We tested the hypothesis that soil and phyllosphere support distinct microbial communities, but exhibit a similar response (accelerated biodegradation or toxicity) to repeated exposure to the fungicide iprodione. Pepper plants received four repeated foliage or soil applications of iprodione, which accelerated its degradation in soil (DT50_1st = 1.23 and DT50_4th = 0.48 days) and on plant leaves (DT50_1st > 365 and DT50_4th = 5.95 days). The composition of the epiphytic and soil bacterial and fungal communities, determined by amplicon sequencing, was significantly altered by iprodione. The archaeal epiphytic and soil communities responded differently; the former showed no response to iprodione. Three iprodione-degrading Paenarthrobacter strains were isolated from soil and phyllosphere. They hydrolyzed iprodione to 3,5-dichloraniline via the formation of 3,5-dichlorophenyl-carboxiamide and 3,5-dichlorophenylurea-acetate, a pathway shared by other soil-derived arthrobacters implying a phylogenetic specialization in iprodione biotransformation. Our results suggest that iprodione-repeated application could affect soil and epiphytic microbial communities with implications for the homeostasis of the plant-soil system and agricultural production.

Prebiotic supplementation over a cold season and during antibiotic treatment specifically modulates the gut microbiota composition of 3-6 year-old children.

Supplementing kindergarten children during a cold season with a prebiotic inulin-type fructans product with shorter and longer fructan chains has been shown to reduce febrile episodes requiring medical attention and to lower the incidence of sinusitis. These beneficial effects may be connected to the specific modulation of children's gut microbiota. By applying quantitative and qualitative microbiota analysis this study aimed at characterising the gut microbiota composition and at exploring effects of prebiotic intervention on the gut microbiota during a 24-weeks intervention and during antibiotic treatment in healthy children. The study was a randomised, placebo-controlled trial with 258 healthy children aged 3 to 6 years consuming 6 g/day prebiotic inulin-type fructans or maltodextrin. During the course of the study, faecal samples were collected and subject to targeted qPCR analysis and phylogenetic profiling by multiplexed high throughput sequencing of the prokaryotic 16S rRNA gene PCR amplicons. The microbiota composition of the cohort could be clustered into three distinct constellations (enterotypes). Prebiotic intake resulted in a selective modulation of the gut microbiota composition. Relative abundance of Bifidobacterium was significantly higher in the prebiotic group (n=104) compared to control group (n=105) and this effect was found for all three enterotypes. Antibiotic administration decreased the relative abundance of Bifidobacterium in both groups. Nonetheless, children of the prebiotic group receiving antibiotic treatment displayed significantly higher levels of Bifidobacterium than children receiving the placebo control. Prebiotic supplementation induced specific changes in the gut microbiota composition of children aged 3 to 6 years. Moreover, it attenuated antibiotic-induced disturbances in the gut microbiota composition as shown by higher relative abundance of bifidobacteria at the end of the antibiotic treatment in the prebiotic group. With the previously reported benefits on immune function, the study contributes to the evidence on the immune-modulating effects of prebiotics through gut microbiota modifications. The study was registered as NCT03241355 ( https://clinicaltrials.gov/show/NCT03241355 ).

Blame It on the Metabolite: 3,5-Dichloroaniline Rather than the Parent Compound Is Responsible for the Decreasing Diversity and Function of Soil Microorganisms.

Pesticides are key stressors of soil microorganisms with reciprocal effects on ecosystem functioning. These effects have been mainly attributed to the parent compounds, while the impact of their transformation products (TPs) has been largely overlooked. We assessed in a meadow soil (soil A) the transformation of iprodione and its toxicity in relation to (i) the abundance of functional microbial groups, (ii) the activity of key microbial enzymes, and (iii) the diversity of bacteria, fungi, and ammonia-oxidizing microorganisms (AOM) using amplicon sequencing. 3,5-Dichloroaniline (3,5-DCA), the main iprodione TP, was identified as a key explanatory factor for the persistent reduction in enzymatic activities and potential nitrification (PN) and for the observed structural changes in the bacterial and fungal communities. The abundances of certain bacterial (Actinobacteria, Hyphomicrobiaceae, Ilumatobacter, and Solirubrobacter) and fungal (Pichiaceae) groups were negatively correlated with 3,5-DCA. A subsequent study in a fallow agricultural soil (soil B) showed limited formation of 3,5-DCA, which concurred with the lack of effects on nitrification. Direct 3,5-DCA application in soil B induced a dose-dependent reduction of PN and NO3--N, which recovered with time. In vitro assays with terrestrial AOM verified the greater toxicity of 3,5-DCA over iprodione. "Candidatus Nitrosotalea sinensis" Nd2 was the most sensitive AOM to both compounds. Our findings build on previous evidence on the sensitivity of AOM to pesticides, reinforcing their potential utilization as indicators of the soil microbial toxicity of pesticides in pesticide environmental risk analysis and stressing the need to consider the contribution of TPs in the toxicity of pesticides on the soil microbial community.IMPORTANCE Pesticide toxicity on soil microorganisms is an emerging issue in pesticide risk assessment, dictated by the pivotal role of soil microorganisms in ecosystem services. However, the focus has traditionally been on parent compounds, while transformation products (TPs) are largely overlooked. We tested the hypothesis that TPs can be major contributors to the soil microbial toxicity of pesticides using iprodione and its main TP, 3,5-dichloroaniline, as model compounds. We demonstrated, by measuring functional and structural endpoints, that 3,5-dichloroaniline and not iprodione was associated with adverse effects on soil microorganisms, with nitrification being mostly affected. Pioneering in vitro assays with relevant ammonia-oxidizing bacteria and archaea verified the greater toxicity of 3,5-dichloroaniline. Our findings are expected to advance environmental risk assessment, highlighting the potential of ammonia-oxidizing microorganisms as indicators of the soil microbial toxicity of pesticides and stressing the need to consider the contribution of TPs to pesticide soil microbial toxicity.

Hyperthermia induces therapeutic effectiveness and potentiates adjuvant therapy with non-targeted and targeted drugs in an in vitro model of human malignant melanoma.

In the present study, we have aimed to characterize the intrinsic, extrinsic and ER-mediated apoptotic induction by hyperthermia in an in vitro model of human malignant melanoma and furthermore, to evaluate its therapeutic effectiveness in an adjuvant therapeutic setting characterized by combinational treatments with non-targeted (Dacarbazine & Temozolomide) and targeted (Dabrafenib & Vemurafenib) drugs. Overall, our data showed that both low (43 °C) and high (45 °C) hyperthermic exposures were capable of inducing cell death by activating all apoptotic pathways but in a rather distinct manner. More specifically, low hyperthermia induced extrinsic and intrinsic apoptotic pathways both of which activated caspase 6 only as opposed to high hyperthermia which was mediated by the combined effects of caspases 3, 7 and 6. Furthermore, significant involvement of the ER was evident (under both hyperthermic conditions) suggesting its role in regulating apoptosis via activation of CHOP. Our data revealed that while low hyperthermia activated IRE-1 and ATF6 only, high hyperthermia induced activation of PERK as well suggesting that ultimately these ER stress sensors can lead to the induction of CHOP via different pathways of transmitted signals. Finally, combinational treatment protocols revealed an effect of hyperthermia in potentiating the therapeutic effectiveness of non-targeted as well as targeted drugs utilized in the clinical setting. Overall, our findings support evidence into hyperthermia's therapeutic potential in treating human malignant melanoma by elucidating the underlying mechanisms of its complex apoptotic induction.

Assessment of the impact of three pesticides on microbial dynamics and functions in a lab-to-field experimental approach.

The toxicity of pesticides on soil microorganisms is as an emerging area of concern. Novel and well-standardized tools could be now used to provide a robust assessment of the ecotoxicity of pesticides on soil microorganisms. We followed a tiered lab-to-field approach to assess the toxicity of three pesticides, widely used at EU level, (chlorpyrifos (CHL), isoproturon (IPU) and tebuconazole (TBZ)) on (i) the abundance of 11 microbial taxa and 8 functional microbial groups via q-PCR and (ii) the activity of enzymes involved in biogeochemical cycles via fluorometric analysis. Correlation of microbial measurements with the concentration of pesticides, and their transformation products (TPs) in soil enabled the identification of the compounds driving the effects observed. At lab tests (×1, ×2 and ×10 the recommended dose), CHL and TBZ significantly reduced the relative abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) which recovered by the end of the study, while all pesticides induced a persistent reduction in the relative abundance of sulfur-oxidizing bacteria (SOB). The two demethylated metabolites of IPU (MD-IPU and DD-IPU) adversely affected P-cycling enzymes and leucine aminopeptidase (Leu). At field tests (×1, ×2 and ×5 the recommended dose), a persistent reduction on the relative abundance of AOA was induced by all pesticides, but only CHL and its hydrolysis product 3,5,6 trichloro-2-pyridynol (TCP) soil levels were negatively correlated with AOA relative abundance. Our findings suggest that ammonia-oxidizing microorganisms constitute the most responsive microbial group to pesticides and could be potential candidates for inclusion in pesticide risk assessment.