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Antibiotics have a wide application range in human and veterinary medicines. Being designed for pharmacological stability, most antibiotics are recalcitrant to biodegradation after ingestion and can be persistent in the environment. Antibiotic residues have been detected as contaminants in various environmental compartments where they cause human and environmental threats, notably with respect to the potential emergence and proliferation of antibiotic-resistant bacteria. An important component of managing environmental risk caused by antibiotics is to understand exposure of soil and water resources to their residues. One challenge is to gain knowledge on the fate of antibiotics in the ecosystem along the soil-water continuum, and on the collateral impact of antibiotics on environmental microorganisms responsible for crucially important ecosystem functions. In this context, the ANTIBIOTOX project aims at studying the environmental fate and impact of two antibiotics of the sulfonamide class of antibiotics, sulfamethazine (SMZ), and sulfamethoxazole (SMX).

In the context of the ecological sustainability of phosphorus, emerging evidence for the ubiquitous presence of polyphosphate-accumulating bacteria in natural environments invites efforts to reveal their roles in the biogeochemical cycle of phosphorus. This requires high-throughput methods to characterize their structure and dynamics in ecosystems. A promising strategy is to combine the staining of intracellular polyphosphate granules and their subsequent detection by flow cytometry, enabling rapid data acquisition. In this study, we evaluated the potential of this approach by testing various factors that could affect the efficiency and specificity of polyphosphate labeling. Most of our experiments were performed using the 4′,6-diamidino-2-phenylindole dye (DAPI). However, we also carried out a preliminary study using the synthetic fluorochrome JC-D7, a new selective fluorescent dye used for the specific labeling of endogenous polyphosphate in living cells. The assays were performed on Tetrasphaera elongata, a Gram-positive bacterium known to accumulate large amounts of intracellular polyphosphates. We also used six bacterial strains belonging to different phyla, in particular a Gram-negative bacterial strain belonging to the genus Pseudomonas, which is characterized by low levels of cellular polyphosphate. The potential of flow cytometry to quantify and sort polyphosphate-accumulating bacteria in complex environmental samples, including soil, freshwater and sediments, was also examined. Our tests provide useful information for the design of future experiments and highlight the potential pitfalls and limitations of detecting polyphosphate-accumulating bacteria using the cytometric approach. We also show that JC-D7 is a promising dye for achieving these objectives, particularly for enumerating polyphosphate-accumulating bacteria from environmental samples.

Abstract To evaluate the effects of hydrological variability on pesticide dissipation capacity by stream biofilms, we conducted a microcosm study. We exposed biofilms to short and frequent droughts (daily frequency), long and less frequent droughts (weekly frequency) and permanently immersed controls, prior to test their capacities to dissipate a cocktail of pesticides composed of tebuconazole, terbuthylazine, imidacloprid, glyphosate and its metabolite aminomethylphosphonic acid. A range of structural and functional descriptors of biofilms (algal and bacterial biomass, extracellular polymeric matrix (EPS) concentration, microbial respiration, phosphorus uptake and community-level physiological profiles) were measured to assess drought effects. In addition, various parameters were measured to characterise the dynamics of pesticide dissipation by biofilms in the different hydrological treatments (% dissipation, peak asymmetry, bioconcentration factor, among others). Results showed higher pesticide dissipation rates in biofilms exposed to short and frequent droughts, despite of their lower biomass and EPS concentration, compared to biofilms in immersed controls or exposed to long and less frequent droughts. High accumulation of hydrophobic pesticides (tebuconazole and terbuthylazine) was measured in biofilms despite the short exposure time (few minutes) in our open-flow microcosm approach. This research demonstrated the stream biofilms capacity to adsorb hydrophobic pesticides even in stressed drought environments.

Microbacterium sp. C448, isolated from a soil regularly exposed to sulfamethazine (SMZ), can use various sulphonamide antibiotics as the sole carbon source for growth. The basis for the regulation of genes encoding the sulphonamide metabolism pathway, the dihydropteroate synthase sulphonamide target (folP), and the sulphonamide resistance (sul1) genes, is unknown in this organism. In the present study, the response of the transcriptome and proteome of Microbacterium sp. C448 following exposure to subtherapeutic (33 µM) or therapeutic (832 µM) SMZ concentrations was evaluated. Therapeutic concentration induced the highest sad expression and Sad production, consistent with the activity of SMZ degradation observed in cellulo. Following complete SMZ degradation, Sad production tended to return to the basal level observed prior to SMZ exposure. Transcriptomic and proteomic kinetics were concomitant for the resistance genes and proteins. The abundance of Sul1 protein, 100-fold more abundant than FolP protein, did not change in response to SMZ exposure. Moreover, non-targeted analyses highlighted the increase of a deaminase RidA and a putative sulphate exporter expression and production. These two novel factors involved in the 4-aminophenol metabolite degradation and the export of sulphate residues formed during SMZ degradation, respectively, provided new insights into Microbacterium sp. C448 SMZ detoxification process.

Leaf microbial communities possess a large panel of enzymes permitting the breakdown of leaf polymers as well as the transformation of organic xenobiotic compounds present in stream waters. This study aims to assess the potential of leaf microbial communities, exhibiting different exposure histories to pesticides (upstream versus downstream), to biotransform three maize herbicides (mesotrione, S-metolachlor, and nicosulfuron) in single and cocktail molecule exposures. The results showed a high dissipation of nicosulfuron (sulfonylurea herbicide) (from 29.1 ± 10.8% to 66 ± 16.2%, day 40) in both single and cocktail exposures, respectively, but not of mesotrione and S-metolachlor. The formation of nicosulfuron metabolites such as ASDM (2-(aminosulfonyl)-N,N-dimethyl-3-pyridinecarboxamide) and ADMP (2-amino-4,6-dimethoxypyrimidine) and the weak sorption (<0.4%) on the leaf matrix confirmed the transformation of this molecule by leaf microorganisms. In addition, the downstream communities showed a greater ability to transform nicosulfuron than the upstream communities suggesting that the exposure history to pesticides is an important parameter and can enhance the biotransformation potential of leaf microorganisms. After 40-day single exposure to nicosulfuron, the downstream communities were also those experiencing the greatest shifts in fungal and bacterial community diversity suggesting a potential adaptation of microorganisms to this herbicide. Our study emphasizes the importance of leaf microbial communities for herbicide biotransformation in polluted stream ecosystems where fungi could play a crucial role.

Microbial communities driving the nitrogen cycle contribute to ecosystem services such as crop production and air, soil, and water quality. The responses to herbicide stress of ammonia-oxidizing and ammonia-denitrifying microbial communities were investigated by an analysis of changes in structure-function relationships. Their potential activities, abundances (quantitative PCR), and genetic structure (denaturing gradient gel electrophoresis) were assessed in a microcosm experiment. The application rate (1 × FR, 0.45 μg g(-1) soil) of the mesotrione herbicide did not strongly affect soil N-nutrient dynamics or microbial community structure and abundances. Doses of the commercial product Callisto® (10 × FR and 100 × FR) or pure mesotrione (100 × FR) exceeding field rates induced short-term inhibition of nitrification and a lasting stimulation of denitrification. These effects could play a part in the increase in soil ammonium content and decrease in nitrate contents observed in treated soils. These functional impacts were mainly correlated with abundance shifts of ammonia-oxidizing Bacteria (AOB) and Archaea (AOA) or denitrifying bacteria. The sustained restoration of nitrification activity, from day 42 in the 100 × FR-treated soils, was likely promoted by changes in the community size and composition of AOB, which suggests a leading role, rather than AOA, for soil nitrification restoration after herbicide stress. This ecotoxicological community approach provides a nonesuch multiparameter assessment of responses of N-cycling microbial guilds to pesticide stress.

Abstract To evaluate the effects of hydrological variability on pesticide dissipation capacity by stream biofilms, we conducted a microcosm study. We exposed biofilms to short and frequent droughts (daily frequency), long and less frequent droughts (weekly frequency) and permanently immersed controls, prior to test their capacities to dissipate a cocktail of pesticides composed of tebuconazole, terbuthylazine, imidacloprid, glyphosate and its metabolite aminomethylphosphonic acid. A range of structural and functional descriptors of biofilms (algal and bacterial biomass, extracellular polymeric matrix (EPS) concentration, microbial respiration, phosphorus uptake and community-level physiological profiles) were measured to assess drought effects. In addition, various parameters were measured to characterise the dynamics of pesticide dissipation by biofilms in the different hydrological treatments (% dissipation, peak asymmetry, bioconcentration factor, among others). Results showed higher pesticide dissipation rates in biofilms exposed to short and frequent droughts, despite of their lower biomass and EPS concentration, compared to biofilms in immersed controls or exposed to long and less frequent droughts. High accumulation of hydrophobic pesticides (tebuconazole and terbuthylazine) was measured in biofilms despite the short exposure time (few minutes) in our open-flow microcosm approach. This research demonstrated the stream biofilms capacity to adsorb hydrophobic pesticides even in stressed drought environments.

The bacterial community structure of a diuron-degrading enrichment culture from lotic surface water samples was analyzed and the diuron-degrading strains were selected using a series of techniques combining temporal temperature gradient gel electrophoresis (TTGE) of 16 S rDNA gene V1-V3 variable regions, isolation of strains on agar plates, colony hybridization methods, and biodegradation assays. The TTGE fingerprints revealed that diuron had a strong impact on bacterial community structure and highlighted both diuron-sensitive and diuron-adapted bacterial strains. Two bacterial strains, designated IB78 and IB93 and identified as belonging to Pseudomonas sp. and Stenotrophomonas sp., were isolated and shown to degrade diuron in pure resting cells in a first-order kinetic reaction during the first 24 h of incubation with no 3,4-DCA detected. The percentages of degradation varied from 25% to 60% for IB78 and 20% to 65% for IB93 and for a diuron concentration range from 20 mg/L to 2 mg/L, respectively. It is interesting to note that diuron was less degraded by single isolates than by mixed resting cells, thereby underlining a cumulative effect between these two strains. To the best of our knowledge, this is the first report of diuron-degrading strains isolated from lotic surface water.

. We recently demonstrated in a microcosm study using PCR-TTGE analysis that a realistic diuron exposure (10 μg/L) could directly and indirectly affect the diversity of a natural riverine bacterial community. Here we extended our first approach by identifying predominant bacterial phylotypes in diuron and control microcosms by sequencing and phylogenetic analysis of bands excised from the previously obtained PCR-TTGE gel.We found a sharp difference between phylotypes obtained from the two types of microcosm. Those that appeared or were maintained only in treated microcosms were mainly γ-Proteobacteria, especially the Pseudomonadaceae family, the Verrucomicrobia and the Gemmatimonadetes. In contrast, phylotypes that appeared only in control microcosms belonged to the Chlamydiae. Methodological aspects related to biases encountered after sequence retrieval from fingerprint gels are also discussed.

In order to reduce the amounts of pesticides used, and thereby their associated risks, new generations of less environmentally dangerous molecules with lower weight are currently being used in the mixtures sprayed on crops. Few studies have been made, however, to analyse their impact on the soil, and more particularly on the microorganisms living in the soil which maintain the essential functions of this ecosystem. By taking a microcosmic approach, we were able to assess the impact of the maize herbicides “cocktail” Mesotrione and S-metolachlor on global soil microbial activity, biomass, and structures, by using the formulated compounds, respectively, Callisto and Dual Gold (both registered brands of Syngenta). Our results highlighted a synergetic effect in “cocktail” microcosms resulting in an increase in the Mesotrione herbicide dissipation time and in an impact on the microbial community at onefold field rate equally to more than a single herbicide used at tenfold field rate.