Early treatment with rifaximin during epileptogenesis reverses gut alterations and reduces seizure duration in a mouse model of acquired epilepsy.

The gut microbiota is altered in epilepsy and is emerging as a potential target for new therapies. We studied the effects of rifaximin, a gastrointestinal tract-specific antibiotic, on seizures and neuropathology and on alterations in the gut and its microbiota in a mouse model of temporal lobe epilepsy (TLE). Epilepsy was induced by intra-amygdala kainate injection causing status epilepticus (SE) in C57Bl6 adult male mice. Sham mice were injected with vehicle. Two cohorts of SE mice were fed a rifaximin-supplemented diet for 21 days, starting either at 24 h post-SE (early disease stage) or at day 51 post-SE (chronic disease stage). Corresponding groups of SE mice (one each disease stage) were fed a standard (control) diet. Cortical ECoG recording was done at each disease stage (24/7) for 21 days in all SE mice to measure the number and duration of spontaneous seizures during either rifaximin treatment or control diet. Then, epileptic mice ± rifaximin and respective sham mice were sacrificed and brain, gut and feces collected. Biospecimens were used for: (i) quantitative histological analysis of the gut structural and cellular components; (ii) markers of gut inflammation and intestinal barrier integrity by RTqPCR; (iii) 16S rRNA metagenomics analysis in feces. Hippocampal neuronal cell loss was assessed in epileptic mice killed in the early disease phase. Rifaximin administered for 21 days post-SE (early disease stage) reduced seizure duration (p < 0.01) and prevented hilar mossy cells loss in the hippocampus compared to epileptic mice fed a control diet. Epileptic mice fed a control diet showed a reduction of both villus height and villus height/crypt depth ratio (p < 0.01) and a decreased number of goblet cells (p < 0.01) in the duodenum, as well as increased macrophage (Iba1)-immunostaining in the jejunum (p < 0.05), compared to respective sham mice. Rifaximin's effect on seizures was associated with a reversal of gut structural and cellular changes, except for goblet cells which remained reduced. Seizure duration in epileptic mice was negatively correlated with the number of mossy cells (p < 0.01) and with villus height/crypt depth ratio (p < 0.05). Rifaximin-treated epileptic mice also showed increased tight junctions (occludin and ZO-1, p < 0.01) and decreased TNF mRNA expression (p < 0.01) in the duodenum compared to epileptic mice fed a control diet. Rifaximin administered for 21 days in chronic epileptic mice (chronic disease stage) did not change the number or duration of seizures compared to epileptic mice fed a control diet. Chronic epileptic mice fed a control diet showed an increased crypt depth (p < 0.05) and reduced villus height/crypt depth ratio (p < 0.01) compared to respective sham mice. Rifaximin treatment did not affect these intestinal changes. At both disease stages, rifaximin modified α- and ß-diversity in epileptic and sham mice compared to respective mice fed a control diet. The microbiota composition in epileptic mice, as well as the effects of rifaximin at the phylum, family and genus levels, depended on the stage of the disease. During the early disease phase, the abundance of specific taxa was positively correlated with seizure duration in epileptic mice. In conclusion, gut-related alterations reflecting a dysfunctional state, occur during epilepsy development in a TLE mouse model. A short-term treatment with rifaximin during the early phase of the disease, reduced seizure duration and neuropathology, and reversed some intestinal changes, strengthening the therapeutic effects of gut-based therapies in epilepsy.

Expanding the use of biobeds: Degradation and adsorption of pesticides contained in effluents from seed-coating, bulb disinfestation and fruit-packaging activities.

Agro-food industries that use pesticides constitute significant point sources for the contamination of natural water resources. Despite that, little is known about the treatment of their pesticide-contaminated effluents. Biobeds could be a possible solution for the depuration of these effluents. In this context, we explored the degradation and adsorption of pesticides used in seed-coating (carboxin (CBX), metalaxyl-M (MET-M), fluxapyroxad (FLX), fludioxonil (FLD)), bulb-dipping (chlorothalonil (CHT), thiabendazole (TBZ), FLD) and fruit-packaging activities (FLD) in a biomixture, used as biobed packing material, and in soil. The degradation of pesticides was tested individually and in mixtures relevant to their industrial use, while FLD was also tested at different concentrations (10, 20, and 150 mg kg-1) representing its use in the different industries. CBX, FLD, and CHT, when applied individually, and all other pesticides when applied in mixtures, degraded more rapidly in biomixture than in soil. In most cases pesticides application in mixtures retarded their degradation. This was more pronounced in soil than in biomixture, especially for MET-M and FLD. CHT had the most prominent inhibitory effect on the degradation of TBZ and FLD. FLD degradation showed a dose-dependent pattern (DT50 42.4 days at 10 mg kg-1 and 107.6 days at 150 mg kg-1). All pesticides showed higher adsorption affinity in the biomixture (Kf = 3.23-123.3 g mL-1) compared to soil (Kf = 1.15-31.2 g mL-1). We provide initial evidence for the potential of the tested biomixture to remove pesticides contained in effluents produced by different agro-industrial activities. Tests in full-scale biobeds packed with this biomixture will unravel their full depuration potential for the treatment of these agro-industrial effluents.

Effect of microplastic pollution on the gut microbiome of anecic and endogeic earthworms.

Microplastic (MP) pollution constitutes an emerging type of pollution threatening both aquatic and terrestrial ecosystems. The impact on aquatic ecosystems has been extensively studied, but the effect on terrestrial ecosystems and their inhabitants is mostly underexplored. In this study, we explored the effect of MP pollution on gut bacterial microbiome of endogeic (Aporrectodea caliginosa) and anecic (Lumbricus terrestris) earthworms. The experiments were performed in sandy soil with 0.2% of low-density polyethylene MPs (LDPE MPs). We observed that the endogeic earthworms had 100% survival, while anecic earthworms survived 25 days in the control (i.e. in absence of MPs) and 21 days in the treatment with LDPE MPs. The main driver of shifts in the diversity and composition of the bacterial communities in the gut of tested earthworms was the lifestyle of the worms, followed by the presence of MPs. The bacterial microbiome diversity was significantly different among the two types of earthworms, and the highest bacterial diversity was found in the gut of the endogeic earthworms. The effect of MPs on gut bacterial microbiome was clearly observed in the changes in the relative abundance of several phyla and families of the bacterial communities in both types of earthworms, although it was most evident in the anecic earthworms. The Actinobacteriota, Proteobacteria, and Firmicutes were the main groups enhanced in the MP treatments, suggesting enrichment of the bacterial communities with potential plastic degraders.

Using biobeds for the treatment of fungicide-contaminated effluents from various agro-food processing industries: Microbiome responses and mobile genetic element dynamics.

Agro-food processing industries generate large amounts of pesticide-contaminated effluents that pose a significant environmental threat if managed improperly. Biopurification systems like biobeds could be utilized for the depuration of these effluents although direct evidence for their efficiency are still lacking. We employed a column leaching experiment with pilot biobeds to (i) assess the depuration potential of biobeds against fungicide-contaminated effluents from seed-producing (carboxin, metalaxyl-M, fluxapyroxad), bulb-handling (thiabendazole, fludioxonil and chlorothalonil) and fruit-packaging (fludioxonil, imazalil) industries, (ii) to monitor microbial succession via amplicon sequencing and (iii) to determine the presence and dynamics of mobile genetic elements like intl1, IS1071, IncP-1 and IncP-1ε often associated with the transposition of pesticide-degrading genes. Biobeds could effectively retain (adsorbed but extractable with organic solvents) and dissipate (degraded and/or not extractable with organic solvents) the fungicides that were contained in the agro-industrial effluents with 93.1-99.98% removal efficiency in all cases. Lipophilic substances like fluxapyroxad were mostly retained in the biobed while more polar substances like metalaxyl-M and carboxin were mostly dissipated or showed higher leaching potential like metalaxyl-M. Biobeds supported a bacterial and fungal community that was not affected by fungicide application but showed clear temporal patterns in the different biobed horizons. This was most probably driven by the establishment of microaerophilic conditions upon water saturation of biobeds, as supported by the significant increase in the abundance of facultative or strict anaerobes like Chloroflexi/Anaerolinae, Acidibacter and Myxococcota. Wastewater application did not affect the dynamics of mobile genetic elements in biobeds whose abundance (intl1, IS1071, IncP-1ε) showed significant increases with time. Our findings suggest that biobeds could effectively decontaminate fungicide-contaminated effluents produced by agro-food industries and support a rather resilient microbial community.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil.

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L-1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L-1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

The Effects of Quinone Imine, a New Potent Nitrification Inhibitor, Dicyandiamide, and Nitrapyrin on Target and Off-Target Soil Microbiota.

Dicyandiamide (DCD) and nitrapyrin (NP) are nitrification inhibitors (NIs) used in agriculture for over 40 years. Recently, ethoxyquin (EQ) was proposed as a novel potential NI, acting through its derivative quinone imine (QI). Still, the specific activity of these NIs on the different groups of ammonia-oxidizing microorganisms (AOM), and mostly their effects on other soil microbiota remain unknown. We determined the impact of QI, and comparatively of DCD and NP, applied at two doses (regular versus high), on the function, diversity, and dynamics of target (AOM), functionally associated (nitrite-oxidizing bacteria-NOB), and off-target prokaryotic and fungal communities in two soils mainly differing in pH (5.4 versus 7.9). QI was equally effective to DCD but more effective than NP in inhibiting nitrification in the acidic soil, while in the alkaline soil QI was less efficient than DCD and NP. This was attributed to the higher activity of QI toward AOA prevailing in the acidic soil. All NIs induced significant effects on the composition of the AOB community in both soils, unlike AOA, which were less responsive. Beyond on-target effects, we noted an inhibitory effect of all NIs on the abundance of NOB in the alkaline soil, with Nitrobacter being more sensitive than Nitrospira. QI, unlike the other NIs, induced significant changes in the composition of the bacterial and fungal communities in both soils. Our findings have serious implications for the efficiency and future use of NIs on agriculture and provide unprecedented evidence for the potential off-target effects of NIs on soil microbiota. IMPORTANCE NIs could improve N use efficiency and decelerate N cycling. Still, we know little about their activity on the distinct AOM groups and about their effects on off-target soil microorganisms. Here, we studied the behavior of a new potent NI, QI, compared to established NIs. We show that (i) the variable efficacy of NIs across soils with different pH reflects differences in the inherent specific activity of the NIs to AOA and AOB; (ii) beyond AOM, NIs exhibit negative effects on other nitrifiers, like NOB; (iii) QI was the sole NI that significantly affected prokaryotic and fungal diversity. Our findings (i) highlight the need for novel NI strategies that consider the variable sensitivity of AOM groups to the different NIs (ii) identify QI as a potent AOA inhibitor, and (iii) stress the need for monitoring NIs' impact on off-target soil microorganisms to ensure sustainable N fertilizers use and soil ecosystem functioning.