Modeling the impact of urban and hospital eco-exposomes on antibiotic-resistance dynamics in wastewaters.

The emergence and selection of antibiotic resistance is a major public health problem worldwide. The presence of antibiotic-resistant bacteria (ARBs) in natural and anthropogenic environments threatens the sustainability of efforts to reduce resistance in human and animal populations. Here, we use mathematical modeling of the selective effect of antibiotics and contaminants on the dynamics of bacterial resistance in water to analyze longitudinal spatio-temporal data collected in hospital and urban wastewater between 2012 and 2015. Samples were collected monthly during the study period at four different sites in Haute-Savoie, France: hospital and urban wastewater, before and after water treatment plants. Three different categories of exposure variables were collected simultaneously: 1) heavy metals, 2) antibiotics and 3) surfactants for a total of 13 drugs/molecules; in parallel to the normalized abundance of 88 individual genes and mobile genetic elements, mostly conferring resistance to antibiotics. A simple hypothesis-driven model describing weekly antibiotic resistance gene (ARG) dynamics was proposed to fit the available data, assuming that normalized gene abundance is proportional to antibiotic resistant bacteria (ARB) populations in water. The detected compounds were found to influence the dynamics of 17 genes found at multiple sites. While mercury and vancomycin were associated with increased ARG and affected the dynamics of 10 and 12 identified genes respectively, surfactants antagonistically affected the dynamics of three genes. The models proposed here make it possible to analyze the relationship between the persistence of resistance genes in the aquatic environment and specific compounds associated with human activities from longitudinal data. Our analysis of French data over 2012-2015 identified mercury and vancomycin as co-selectors for some ARGs.

Do Microorganisms in Bathing Water in Guadeloupe (French West Indies) Have Resistance Genes?

Waterborne faecal contamination is a major public health concern. The main objectives of this study were to investigate faecal contamination and Escherichia coli (E. coli) antibiotic resistance in recreational fresh water from Guadeloupe and to characterise the microbiome and resistome composition in biofilms from submerged rocks. Significant faecal contamination was observed at 14 freshwater sites. E. coli predominated (62%), followed by Enterobacter cloacae (11%) and Acinetobacter spp. (11%). Of 152 E. coli isolated, none produced extended-spectrum beta-lactamases (ESBLs), but 7% showed resistance to streptomycin and 4% to tetracycline. Biofilm resistome analysis revealed clinically significant antibiotic-resistance genes (ARGs), including those coding for resistance to sulfonamides (sul1), carbapenems (blaKPC), and third-generation cephalosporins (blaCTX-M). Mobile genetic elements (MGEs) (intI1, intI2, intI3) linked to resistance to aminoglycosides, beta-lactams, tetracycline, as well as heavy metal resistance determinants (copA, cusF, czcA, merA) conferring resistance to copper, silver, cadmium, and mercury were also detected. Diverse bacterial phyla were found in biofilm samples, of which Proteobacteria, Bacteroidetes, Planctonomycetes, and Cyanobacteria were predominant. Despite the frequent presence of E. coli exceeding regulatory standards, the low levels of antibiotic-resistant bacteria in freshwater and of ARGs and MGEs in associated biofilms suggest limited antibiotic resistance in Guadeloupean recreational waters.

Simultaneous nitrogen and phosphorus removal through an integrated partial-denitrification/anammox process in a single UAFB system.

With the aim of obtaining enhanced nitrogen removal and phosphate recovery in mainstream sewage, we examined an integrated partial-denitrification/anaerobic ammonia oxidation (PD/A) process over a period of 189 days to accomplish this goal. An up-flow anaerobic fixed-bed reactor (UAFB) used in the integrated PD/A process was started up with anammox sludge inoculated and the influent composition controlled. Results showed that the system achieved a phosphorus removal efficiency of 82% when the influent concentration reached 12.0 mg/L. Batch tests demonstrated that stable and efficient removal of chemical oxygen demand (COD), nitrogen, and phosphorus was achieved at a COD/NO3--N ratio of 3.5. Scanning electron microscope (SEM) and X-ray diffraction (XRD) analysis indicated that hydroxyapatite was the main crystal in the biofilm. Furthermore, substrate variation along the axial length of UAFB indicated that partial denitrification and anammox primarily took place near the reactor's bottom. According to a microbiological examination, 0.4% of the PD/A process's microorganisms were anaerobic ammonia oxidizing bacteria (AnAOB). Ca. Brocadia, Ca. Kuenenia, and Ca. Jettenia served as the principal AnAOB generals in the system. Thauera, Candidatus Accumulibacter, Pseudomonas, and Acinetobacter, which together accounted for 27% of the denitrifying and phosphorus-accumulating bacteria, were helpful in advanced nutrient removal. Therefore, the combined PD/A process can be a different option in the future for sewage treatment to achieve contemporaneous nutrient removal.

[Tracking transfers of resistance-carrying bacteria between animals, humans and the environment]. / Tracking des transferts des bactéries porteuses de résistances entre animal, homme et environnement.

The fight against antibiotic resistance must incorporate the "One Health" concept to be effective. This means having a holistic approach embracing the different ecosystems, human, animal, and environment. Transfers of resistance genes may exist between these three domains and different stresses related to the exposome may influence these transfers. Various targeted or pan-genomic molecular biology techniques can be used to better characterise the dissemination of bacterial clones and to identify exchanges of genes and mobile genetic elements between ecosystems.

La lutte contre la résistance aux antibiotiques doit intégrer le concept «  Une seule santé  ¼ pour être efficace. Ceci consiste à avoir une approche holistique embrassant les différents écosystèmes, homme, animal et environnement. Des transferts de gènes de résistance peuvent exister entre ces trois domaines et différents stress liés à l'exposome peuvent influencer ces transferts. Différentes techniques de biologie moléculaire ciblées ou pan-génomiques peuvent être mises en œuvre pour mieux caractériser les circulations de clones bactériens mais aussi pour identifier les échanges de gènes et d'éléments génétiques mobiles entre écosystèmes.

Hospital and urban wastewaters shape the matrix and active resistome of environmental biofilms.

Understanding the dynamics of antibiotic resistance gene (ARG) transfer and dissemination in natural environments remains challenging. Biofilms play a crucial role in bacterial survival and antimicrobial resistance (AMR) dissemination in natural environments, particularly in aquatic systems. This study focused on hospital and urban wastewater (WW) biofilms to investigate the potential for ARG dissemination through mobile genetic elements (MGEs). The analysis included assessing the biofilm extracellular polymeric substances (EPS), microbiota composition as well as metatranscriptomic profiling of the resistome and mobilome. We produced both in vitro and in situ biofilms and performed phenotypic and genomic analyses. In the in vitro setup, untreated urban and hospital WW was used to establish biofilm reactors, with ciprofloxacin added as a selective agent at minimal selective concentration. In the in situ setup, biofilms were developed directly in hospital and urban WW pipes. We first showed that a) the composition of EPS differed depending on the growth environment (in situ and in vitro) and the sampling origin (hospital vs urban WW) and that b) ciprofloxacin impacted the composition of the EPS. The metatranscriptomic approach showed that a) expression of several ARGs and MGEs increased upon adding ciprofloxacin for biofilms from hospital WW only and b) that the abundance and type of plasmids that carried individual or multiple ARGs varied depending on the WW origins of the biofilms. When the same plasmids were present in both, urban and hospital WW biofilms, they carried different ARGs.  We showed that hospital and urban wastewaters shaped the structure and active resistome of environmental biofilms, and we confirmed that hospital WW is an important hot spot for the dissemination and selection of antimicrobial resistance. Our study provides a comprehensive assessment of WW biofilms as crucial hotspots for ARG transfer. Hospital WW biofilms exhibited distinct characteristics, including higher eDNA abundance and expression levels of ARGs and MGEs, highlighting their role in antimicrobial resistance dissemination. These findings emphasize the importance of understanding the structural, ecological, functional, and genetic organization of biofilms in anthropized environments and their contribution to antibiotic resistance dynamics.

Life in an unsuspected antibiotics world: River biofilms.

Waterborne bacteria that naturally live in biofilms are continuously exposed to pharmaceutical residues, regularly released into the freshwater environment. At the source level, the discharge of antibiotics into rivers has already been repeatedly linked to the development of antimicrobial resistance. But what about biofilms away from the discharge point? Two rivers, with sites subject to dispersed contamination of medium intensity, were studied as typical representatives of high- and middle-income countries. The biofilms developed on rocks indigenous to rivers are perfectly representative of environmental exposure. Our results show that away from the hotspots, the amount of antibiotics in the biofilms studied favours the maintenance and enrichment of existing resistant strains as well as the selection of new resistant mutants, and these favourable conditions remain over a period of time. Thus, in this type of river, the environmental risk of selection pressure is not only present downstream of urbanized areas but is also possible upstream and far downstream of wastewater treatment plant discharges. Despite this, correlation analysis found no strong positive correlation between antibiotic concentrations and the abundance of measured integrons and their corresponding resistance genes. Nevertheless, this work highlights the need to consider the risks of antibiotics beyond hotspots as well.

Resistome Diversity and Dissemination of WHO Priority Antibiotic Resistant Pathogens in Lebanese Estuaries.

Anthropogenic pressure is known to be a key driver of antimicrobial resistance (AMR) dissemination in the environment. Especially in lower income countries, with poor infrastructure, the level of AMR dissemination is high. Therefore, we assessed the levels and diversity of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Lebanese rivers at estuaries' sites (n = 72) of the Mediterranean Sea in spring 2017 and winter 2018. METHODS: A combined approach using culture techniques and high throughput qPCR were applied to identify ARB and ARGs in rivers along the Lebanese coast. RESULTS: Multidrug-resistant Gram-negative (Enterobacterales and Pseudomonas spp.) and Gram-positive bacterial pathogens were isolated. Levels of ARGs were highest in the winter campaign and areas with high anthropogenic activities and population growth with an influx of refugees. CONCLUSION: Qualitative analysis of ARB and the analysis of the Lebanese estuaries' resistome revealed critical levels of contamination with pathogenic bacteria and provided significant information about the spread of ARGs in anthropogenically impacted estuaries.

River Biofilms Microbiome and Resistome Responses to Wastewater Treatment Plant Effluents Containing Antibiotics.

Continuous exposure to low concentrations of antibiotics (sub-minimal inhibitory concentration: sub-MIC) is thought to lead to the development of antimicrobial resistance (AMR) in the environmental microbiota. However, the relationship between antibiotic exposure and resistance selection in environmental bacterial communities is still poorly understood and unproven. Therefore, we measured the concentration of twenty antibiotics, resistome quality, and analyzed the taxonomic composition of microorganisms in river biofilms collected upstream (UPS) and downstream (DWS) (at the point of discharge) from the wastewater treatment plant (WWTP) of Poitiers (France). The results of statistical analysis showed that the antibiotic content, resistome, and microbiome composition in biofilms collected UPS were statistically different from that collected DWS. According to Procrustes analysis, microbial community composition and antibiotics content may be determinants of antibiotic resistance genes (ARGs) composition in samples collected DWS. However, network analysis showed that the occurrence and concentration of antibiotics measured in biofilms did not correlate with the occurrence and abundance of antibiotic resistance genes and mobile genetic elements. In addition, network analysis suggested patterns of co-occurrence between several ARGs and three classes of bacteria/algae: Bacteroidetes incertae sedis, Cyanobacteria/Chloroplast, and Nitrospira, in biofilm collected UPS. The absence of a direct effect of antibiotics on the selection of resistance genes in the collected samples suggests that the emergence of antibiotic resistance is probably not only due to the presence of antibiotics but is a more complex process involving the cumulative effect of the interaction between the bacterial communities (biotic) and the abiotic matrix. Nevertheless, this study confirms that WWTP is an important reservoir of various ARGs, and additional efforts and legislation with clearly defined concentration limits for antibiotics and resistance determinants in WWTP effluents are needed to prevent their spread and persistence in the environment.

SIPIBEL observatory: Data on usual pollutants (solids, organic matter, nutrients, ions) and micropollutants (pharmaceuticals, surfactants, metals), biological and ecotoxicity indicators in hospital and urban wastewater, in treated effluent and sludge from wastewater treatment plant, and in surface and groundwater.

The Bellecombe pilot site - SIPIBEL - was created in 2010 in order to study the characterisation, treatability and impacts of hospital effluents in an urban wastewater treatment plant. This pilot site is composed of: i) the Alpes Léman hospital (CHAL), opened in February 2012, ii) the Bellecombe wastewater treatment plant, with two separate treatment lines allowing to fully separate the hospital wastewater and the urban wastewater, and iii) the Arve River as the receiving water body and a tributary of the Rhône River and the Geneva aquifer. The database includes in total 48 439 values measured on 961 samples (raw and treated hospital and urban wastewater, activated sludge in aeration tanks, dried sludge after dewatering, river and groundwater, and a few additional campaigns in aerobic and anaerobic sewers) with 44 455 physico-chemistry values (including 15 pharmaceuticals and 14 related transformation products, biocides compounds, metals, organic micropollutants), 2 193 bioassay values (ecotoxicity), 1 679 microbiology values (including microorganisms and antibioresistance indicators) and 112 hydrobiology values.

Environmental contamination in a high-income country (France) by antibiotics, antibiotic-resistant bacteria, and antibiotic resistance genes: Status and possible causes.

Antimicrobial resistance (AMR) is a major global public health concern, shared by a large number of human and animal health actors. Within the framework of a One Health approach, actions should be implemented in the environmental realm, as well as the human and animal realms. The Government of France commissioned a report to provide policy and decision makers with an evidential basis for recommending or taking future actions to mitigate AMR in the environment. We first examined the mechanisms that underlie the emergence and persistence of antimicrobial resistance in the environment. This report drew up an inventory of the contamination of aquatic and terrestrial environments by AMR and antibiotics, anticipating that the findings will be representative of some other high-income countries. Effluents of wastewater treatment plants were identified as the major source of contamination on French territory, with spreading of organic waste products as a more diffuse and incidental contamination of aquatic environments. A limitation of this review is the heterogeneity of available data in space and time, as well as the lack of data for certain sources. Comparing the French Measured Environmental Concentrations (MECs) with predicted no effect concentrations (PNECs), fluoroquinolones and trimethoprim were identified as representing high and medium risk of favoring the selection of resistant bacteria in treated wastewater and in the most contaminated rivers. All other antibiotic molecules analyzed (erythromycin, clarithromycin, azithromycin, tetracycline) were at low risk of resistance selection in those environments. However, the heterogeneity of the data available impairs their full exploitation. Consequently, we listed indicators to survey AMR and antibiotics in the environment and recommended the harmonization of sampling strategies and endpoints for analyses. Finally, the objectives and methods used for the present work could comprise a useful example for how national authorities of countries sharing common socio-geographic characteristics with France could seek to better understand and define the environmental dimension of AMR in their particular settings.

Role of pollution on the selection of antibiotic resistance and bacterial pathogens in the environment.

There is evidence that human activity causes pollution that contributes to an enhanced selection of bacterial pathogens in the environment. In this review, we consider how environmental pollution can favour the selection of bacterial pathogens in the environment. We specifically discuss pollutants released into the environment by human activities (mainly human waste) that are associated with the selection for genetic features in environmental bacterial populations that lead to the emergence of bacterial pathogens. Finally, we also identify key pollutants that are associated with antibiotic resistance and discuss possibilities of how to prevent their release into the environment.

Stability and nutrients removal performance of a Phanerochaete chrysosporium-based aerobic granular sludge process by step-feeding and multi A/O conditions.

A Phanerochaete chrysosporium-based aerobic granular sludge (PC-AGS) was developed by inoculating fungal mycelial pellets into a lab-scale aerobic granular sequencing batch reactor (AGSBR). A strategy using step-anaerobic feeding coupled with multi A/O conditions was adopted. The results showed that the removal efficiencies for total phosphorus (TP) and total inorganic nitrogen (TIN) were 94.56 ± 2.92% and 75.20 ± 7.74%, respectively, under relatively low aeration time. Compared with original AGS, the content of extracellular proteins for PC-AGS obviously increased from 18.61 to 41.44 mg/g MLSS by the end of phase I. Moreover, the mature granules had a larger size and better stability during the 100 days operation. Furthermore, the analysis of microbial diversity detected many key functional groups in PC-AGS granules that were beneficial to nutrients removal. This work demonstrated that the addition of fungal pellets not only enhanced the removal performance, but also improved the stability of the AGS system.

Ecotoxicity and antibiotic resistance of wastewater during transport in an urban sewage network.

Urban wastewater (UWW) management usually entails biological and physicochemical monitoring due to its potential impact on the quality of the receiving environment. A major component of a sewage system is the pipe network leading the water to the treatment plant. Up to now, few studies have been conducted on the diverse phenomena that may affect the characteristics of the water during its transportation. In this study, ecotoxicity and potential antibiotic resistance were used in a global method to assess the change of UWW quality in a sewage system and determine if sewer pipes can act as a bioreactor spread. Three bioassays were conducted to assess the ecotoxicity of the samples and the concentration and relative abundance of two classes of integrons (as a proxy for antibiotic resistance) were measured. The results of the bioassay battery do not show a pattern, despite the fact that differences were noticeable between upstream and downstream samples. Antibiotic resistance appeared to decrease during transport in the pipe as the concentration and relative abundance of integrons decreased during several campaigns. This result should be confirmed in other sewer networks but already provides useful information for the management of urban sewage system.

Hospital discharges in urban sanitation systems: Long-term monitoring of wastewater resistome and microbiota in relationship to their eco-exposome.

Wastewaters (WW) are important sources for the dissemination of antimicrobial resistance (AMR) into the environment. Hospital WW (HWW) contain higher loads of micro-pollutants and AMR markers than urban WW (UWW). Little is known about the long-term dynamics of H and U WW and the impact of their joined treatment on the general burden of AMR. Here, we characterized the resistome, microbiota and eco-exposome signature of 126 H and U WW samples treated separately for three years, and then mixed, over one year. Multi-variate analysis and machine learning revealed a robust signature for each WW with no significant variation over time before mixing, and once mixed, both WW closely resembled Urban signatures. We demonstrated a significant impact of pharmaceuticals and surfactants on the resistome and microbiota of H and U WW. Our results present considerable targets for AMR related risk assessment of WW.

Transformation of roxarsone in the anoxic-oxic process when treating the livestock wastewater.

In order to evaluate the influence of roxarsone (ROX) on the livestock wastewater treatment, a lab-scale pilot employing an anoxic-oxic (A-O) process was investigated by adding different concentrations of ROX at different periods. The mass balance of arsenic (As) in the A-O system was established through the analysis of As speciation and As migration in the gas, liquid and solid phases. The results showed that around 80% of total ROX (initial concentration was 50mgROXL-1) was eliminated in the anoxic reactor (R1) in which at least about 11% of total ROX was transformed to inorganic Asv (iAsv) due to the direct breaking of the C-As bond of ROX. Inorganic AsIII (iAsIII) and arsine (AsH3) were produced in R1, while the generated iAsIII in the effluent of R1 was almost completely oxidized to iAsV in the aerobic reactor (R2). However, the concentration of ROX in the effluent of R2 was almost the same as that in the effluent of R1. After 85days operation, iAsV and residual ROX as the main forms of As were observed after the A-O process. Furthermore, the mass balance of As at steady state revealed that around 0.08%, 3.91% and 96.01% of total As was transformed into gas (biogas), solid (excess sludge) and liquid (effluent). Additionally, the 16S rRNA analysis demonstrated that the existence of ROX in livestock wastewater may play a crucial role in the diversity of bacterial community in the A-O system.

Ecotoxicity and antibiotic resistance of a mixture of hospital and urban sewage in a wastewater treatment plant.

Hospital and urban effluents are a source of diverse pollutants such as organic compounds, heavy metals, detergents, disinfectants, pharmaceuticals, and microorganisms resistant to antibiotics. Usually, these two types of effluent are mixed in the sewage network, but a pilot site in France now allows studying them separately or mixed to understand more about their characteristics and the phenomena that occur following their mixing. In this study, their ecotoxicity (Daphnia magna mobility, Pseudokirchneriella subcapitata growth, Brachionus calyciflorus reproduction, and SOS Chromotest) and antibiotic resistance (integron quantification) were assessed during mixing and treatment steps. The main results of this study are (i) the ecotoxicity and antibiotic resistance potentials of hospital wastewater are higher than in urban wastewater and (ii) mixing two different effluents does not lead to global synergistic or antagonistic effects on ecotoxicity and antibiotic resistance potential. The global additivity effect observed in this case must be confirmed by other studies on hospital and urban effluents on other sites to improve knowledge relating to this source of pollution and its management.

The SIPIBEL project: treatment of hospital and urban wastewater in a conventional urban wastewater treatment plant.

Hospital wastewater (HWW) receives increasing attention because of its specific composition and higher concentrations of some micropollutants. Better knowledge of HWW is needed in order to improve management strategies and to ensure the preservation of wastewater treatment efficiency and freshwater ecosystems. This context pushed forward the development of a pilot study site named Site Pilote de Bellecombe (SIPIBEL), which collects and treats HWW separately from urban wastewater, applying the same conventional treatment process. This particular configuration offers the opportunity for various scientific investigations. It enables to compare hospital and urban wastewater, the efficiency of the two parallel treatment lines, and the composition of the resulting hospital and urban treated effluents, as well as the evaluation of their effects on the environment. The study site takes into account environmental, economic, and social issues and promotes scientific and technical multidisciplinary actions. ᅟ.

Flexible biological arsenite oxidation utilizing NOx and O2 as alternative electron acceptors.

The feasibility of flexible microbial arsenite (AsIII) oxidation coupled with the reduction of different electron acceptors was investigated. The results indicated the acclimated microorganisms could oxidize AsIII with oxygen, nitrate and nitrite as the alternative electron acceptors. A series of batch tests were conducted to measure the kinetic parameters of AsIII oxidation and to evaluate the effects of environmental conditions including pH and temperature on the activity of biological AsIII oxidation dependent on different electron acceptors. Kinetic results showed that oxygen-dependent AsIII oxidation had the highest oxidation rate (0.59 mg As g-1 VSS min-1), followed by nitrate- (0.40 mg As g-1 VSS min-1) and nitrite-dependent AsIII oxidation (0.32 mg As g-1 VSS min-1). The kinetic data of aerobic AsIII oxidation were fitted well with the Monod kinetic model, while the Haldane substrate inhibition model was better applicable to describe the inhibition of anoxic AsIII oxidation. Both aerobic and anoxic AsIII oxidation performed the optimal activity at the near neutral pH. Besides, the optimal temperature for oxygen-, nitrate- and nitrite-dependent AsIII oxidation was 30 ± 1 °C, 40 ± 1 °C and 20 ± 1 °C, respectively.

Recovery of phosphorus via harvesting phosphorus-accumulating granular sludge in sequencing batch airlift reactor.

A novel approach was developed for phosphorus recovery from wastewater through thermal treatment of matured phosphorus-accumulating granular sludge cultivated in sequencing batch airlift reactor (SBAR) system. Results showed that SBAR system had stable performances, in which COD, total phosphorus (TP) and total nitrogen (TN) removal efficiencies were stabilized at 80%, 89% and 86%, respectively. The matured granules were gathered from SBAR reactor and heated at relatively low temperature (100°C, 200°C, 300°C). The total P content in thermal treated granular sludge was more than half of total nutrient. Furthermore, the phosphorus release rate for treated granules was negatively correlated with thermal treatment temperature. These results demonstrated that the granules harvested from SBAR system followed with thermal pre-treatment could probably be applied as excellent slow-release phosphorus fertilizer. Hence, low temperature treatment of phosphate-accumulating granules is efficient for phosphorus recovery from wastewater, which is likely to promote the application of granulation technology.

Impact of wastewater treatment plant discharge on the contamination of river biofilms by pharmaceuticals and antibiotic resistance.

Wastewater treatment plants (WWTPs) are one of the main sources of pharmaceutical residue in surface water. Epilithic biofilms were collected downstream from 12 WWTPs of various types and capacities to study the impacts of their discharge through the changes in biofilm composition (compared to a corresponding upstream biofilm) in terms of pharmaceutical concentrations and bacterial community modifications (microbial diversity and resistance integrons). The biofilm is a promising indicator to evaluate the impacts of WWTPs on the surrounding aquatic environment. Indeed, the use of biofilms reveals contamination hot spots. All of the downstream biofilms present significant concentrations (up to 965ng/g) of five to 11 pharmaceuticals (among the 12 analysed). Moreover, the exposition to the discharge point increases the presence of resistance integrons (three to 31 fold for Class 1) and modifies the diversity of the bacterial communities (for example cyanobacteria). The present study confirms that the discharge from WWTPs has an impact on the aquatic environment.