Within the same study site, microbial diversity significantly differs between these two types of communities. Pharmaceutical substances tend to accumulate either preferentially in periphyton or in the sediment compartment. This leads to very different exposure profiles for the microbial communities, depending on whether they develop in one compartment or the other. This phenomenon was recently demonstrated in the ANTIBIOTOOLS project, funded by PNREST Anses 2017 (2017/3 ABR/2235). The teams at UR RiverLy, UMR LEM, and UMR CARRTEL have extensive experience in sampling and studying these two types of biofilms across various aquatic ecosystems.

Experimental Strategy

The PharmOneHealth project combines artificial channel approaches to expose natural periphytic and sedimentary microbial communities to model compounds under controlled conditions for several weeks, along with in situ monitoring conducted in two rivers with different properties.

In situ monitoring

• Mercier river is located near Lyon in a rural area, within the municipalities of Pollionnay (approximately 3,000 inhabitants) and Grézieu-la-Varenne (around 6,000 inhabitants). It is associated with the Yzeron pilot site of the Field Observatory in Urban Hydrology (OTHU) of the ZABR. Characterized by diffuse pollution, it is a prime location for understanding the mechanisms of transportation and dispersion of contaminants in watercourses. Its geographical and ecological characteristics make it an ideal study site for exploring the complex interactions between human activities, such as livestock farming (primarily cattle, but also poultry, goats, and equestrian centers¹⁶), and intensive agriculture (mainly large-scale maize cultivation¹⁶), and aquatic ecosystems.Mercier river is studied by several research teams, including UR RiverLy and UMR LEM, which monitor the chemical and microbiological quality of the river, allowing for a precise assessment of its ecological status, or the Chante Ruisseau Association which compiles the contamination history. Under the ANR Chypster project (2021-2025) "Integrated biogeochemical, geographical and hydrological approaches to track sources of contaminants in mixed land-use watersheds," the MRU PACTE (Pauline Dusseux and Nicolas Robinet; Univ. Grenoble-Alpes) gathered information from the Chante Ruisseau Association regarding livestock farming practices and the use of phytosanitary products, antibiotics, and antiparasitics in this area. This information is made available to the PharmOneHealth project for a better understanding of the anthropogenic pressures exerted on this aquatic ecosystem.

• Tillet river, a tributary of Lake Bourget, is a watercourse of significant importance in the area in terms of aquatic ecology and water resource management. Associated with the Alpine Lakes Observatory (OLA), its watershed presents a diversity of landscapes, ranging from densely populated urban areas to regions with low anthropogenic pressure. Its downstream section crosses the city of Aix-les-Bains (~30,000 inhabitants) and is contaminated by a localized discharge (a channelized area draining a large part of the city) from domestic waste and thermal activity, without prior treatment. This discharge is a vector of chemical pollution (especially PCBs, monitored for over ten years by the MRU EDYTEM, and pharmaceutical substances), organic pollution (including microorganisms representative of domestic pollution), and thermal pollution (+5 to +10°C). This contamination poses a major challenge for preserving water quality in the area. In contrast, upstream section of the Tillet is located in an area with very low anthropogenic pressure, whether domestic or agricultural (see the Agence de l’eau RM&C-ZABR CommuSED 2017-19 project for details) ¹⁷.

Considering these two very different study sites allows testing scientific hypotheses in contrasted contexes, as well as the tools and approaches developed to enhance the robustness of the results obtained.

Studies in controled conditions

These experiments are conducted using artificial laboratory channels to study periphytic or sedimentary microbial communities under different simulated environmental conditions. The artificial channels allow for control over both the exposure in terms of chemical composition and pollution concentration, as well as the duration of exposure. The approaches in artificial channels target three model substances frequently detected in French aquatic environments, which seem to exhibit different distributions between periphytic and sedimentary compartments (although the amount of data on this topic is still limited to date): one antibiotic from the sulfonamide family (sulfamethoxazole), another from the quinolone family (ofloxacin), and a non-steroidal anti-inflammatory drug (diclofenac). The matrices used in these experiments (periphyton and sediment) will be derived from the Tillet, which exhibits a pronounced gradient of chemical contamination, with a minimally contaminated upstream section and a downstream section that collects all the contamination, over a very restricted geographical area, with particularly high accumulation in the sediment compartment¹⁸. This strategy will allow to adapt the origine of studied matrices (i.e., upstream or downstream) according to the objectives.

Physico-chemical context charactrisation (inculding exposition to pharmaceutical )

In the laboratory : the bioaccumulation and the influence of exposure levels on the microbial communities of the three targeted model substances (sulfamethoxazole, ofloxacin, and diclofenac), either alone or in mixtures, are analyzed in biofilms, water, and sediments using LC/MS-MS (Xevo® TQ-XS, Waters).

In situ : quantitative targeted analyses by LC/MS-MS will enable the study of contamination levels of about forty pharmaceutical substances in biofilms, water (integrative sampling over 15 days using POCIS - Polar Chemical Integrative Sampler), and surface sediments. The presence of a broader range of pharmaceutical substances and biocides will be assessed via suspected qualitative exploratory analyses via LC/QTOF (Xevo G2-S Q-TOF, Waters). Finally, chemical pressures due to pesticides and metallic elements, as well as PCBs in sediments, will be analyzed (data already known for the 2 sites through measurements conducted in past projects by the UR RiverLy team LAMA and the UMR EDYTEM (Emmanuel Naffrechoux, Univ. Savoie Mont-Blanc). A more comprehensive characterization of dissolved organic matter through analyses of dissolved organic carbon, UV-visible spectrophotometry, and size exclusion chromatography coupled with UV and fluorescence detectors (HPSEC-UV/Fluo) will help identify any other potential anthropogenic inputs.

Molecular approaches

The diversity of periphytic and sedimentary microbial communities will be studied through several approaches: (i) at the level of bacterial genera via amplicon sequence analysis, (ii) at the species level via qPCR and ddPCR, and (iii) at the level of genetic determinants representing health concerns, namely virulence genes (referred to as "virulence gene loads") and GRA. DNA extraction from all samples (periphyton and sediment) will be conducted by UMR CARRTEL. This partner will perform Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes on all DNA extracts (the Illumina MiSeq sequencing will be subcontracted to Fasteris, Switzerland). Obtained sequences will then be assembled into operational taxonomic units or amplicon sequence variants (OTUs/ASVs)¹⁹ and compared to sequences available in public databases to affiliate taxa present in the analyzed communities. Bio-statistical analysis of the dataset will evaluate changes in bacterial community structure due to environmental constraints, as previously done in other consortium works, and study correlations with qPCR/ddPCR data.

qPCR and ddPCR analyses of MST (microbial source tracking) targets for the origin of fecal contamination (performed by UMR LEM) and GRA (performed by UMR Agroecology) will be carried out according to well-established protocols by these partners (see consortium publication including ^{20, 21} but also ²²). These PCR screens will be supplemented by the development of a new qPCR/ddPCR screening kit by the LEM-BPOE team to monitor families of virulence genes and thus study their enrichment in biofilms contaminated with pharmaceutical products. These screens will target genes encoding toxins but also adhesins, secretion pathways (T2SS, T3SS, T5SS), extracellular enzymes including lipases and proteases, as well as PAIs (pathogenicity islands) and other vectors such as integrative plasmids and ICEs associated with the mobility of these genetic determinants through transfer via, among others, the type IV²³ secretion system and certain transposons. These vir gene screens will evaluate the co-occurrence of these genes with (i) certain physico-chemical parameters such as organic matter or certain pharmaceutical coumpounds but also (ii) with certain pathogenic species that can also serve as shuttles in gene transfers such as Aeromonas hydrophila and A. caviae, and Pseudomonas aeruginosa, and others²⁴.

Pollution-Induced Community Tolerance (PICT) aproach

PICT approach has been used for over 15 years within the RiverLy-EMA team to characterize adaptation of periphytic and sedimentary microbial communities to various pesticides²⁵ and metal contaminants²⁶ and to assess in situ ecological impact of chemical pressure in the context of bioindication effort²⁷. This approach measures the tolerance of communities-as-a-whole to chemical contaminants through toxicity tests oh these contaminants (alone or in mixtures), using microbial functional parameters (such as photosynthesis, growth, respiration, extracellular enzymatic activities, etc.) as effect descriptors. Recent methodological developments, carried out within the framework of ANTIBIOTOOLS project²⁸, have established specific measurement protocols for the tolerance of periphytic and sedimentary communities to different pharmaceutical substances. In PharmOneHealth, it will thus be possible to deploy the PICT approach using toxicity tests via the measurement of beta-glucosidase enzymatic activity (ofloxacin and sulfamethoxazole), photosynthetic activity (diclofenac), or algal growth (sulfamethoxazole). Existing protocols (or those under development) will allow for the consideration of other types of substances during in situ monitoring (e.g., paracetamol, atenolol, sulfamethazine, erythromycin, etc.), notably to test the relevance of this approach in biosurveillance efforts in the context of pharmaceutical substance contamination.

Cultural approaches

Cultural approach is necessary for studying antibiograms, mechanisms involved in resistance, particularly efflux mechanisms (which sequencing approaches do not allow), or comparing transcriptomes of isolates exposed to different pollutants. In this context, the partnership between MRU Agroecology and the Bacteriology Laboratory of the University Hospital of Dijon will be responsible for isolating and characterizing certain Gram-negative bacilli from the ESKAPE group (Enterobacter, Klebsiella, Pseudomonas aeruginosa) and bacteria of the genus Achromobacter from samples collected from in situ devices^{18,28,29,30,31}. Achromobacter bacteria have been studied since 1995 at the Bacteriology Laboratory of the University Hospital of Dijon due to their emergence in nosocomial infections and among patients with cystic fibrosis³². The mode of patient contamination and the factors favoring this emergence remain unknown. These bacteria exhibit natural multidrug resistance to various classes of antibiotics and frequently acquire resistance during chronic colonizations. The laboratory's theme focuses on both clinical strains (epidemiology in patients and study of innate or acquired resistance mechanisms, frequently related to efflux pumps)^22,29,33 and environmental strains (development of selective media and identification techniques for detecting environmental strains and genotypic comparison with clinical strains)³⁴. In the context of approaches in artificial channels, the impact of the three model substances will be assessed on the evolution of resistance in Achromobacter bacteria. In situ, the goal will be to assess the prevalence of Enterobacter, Klebsiella, Pseudomonas aeruginosa, and Achromobacter, as well as their resistance phenotypes, based on the characteristics of the different sites studied.

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