In activated sludge, the antibiotic resistance genes (ARGs) can be present either in the intracellular (iDNA) or extracellular DNA fraction (exDNA). Recent advances in the exDNA extraction methodology allow a better profiling of the pool of ARGs. However, little is known about how stress conditions modify the distribution of ARGs between both DNA fractions. Here, we performed two batch tests for analyzing the effects of two different stress conditions, namely nutrient starvation and high concentrations of sulfamethoxazole (1, 10 and 150 mg L⁻¹) in activated sludge. We tracked by qPCR the resulting relative abundances of four target genes, namely the universal 16S rRNA gene, the class 1 integron-integrase gene intI1, and the sulfonamide resistance genes sul1 and sul2 in both the iDNA and exDNA fractions. In the exDNA pool, unlike starvation, which provoked a decrease of 1-2 log₁₀ [copies] per ng DNA in the concentration of sul1 and intI1, the presence of sulfamethoxazole did not influence the abundances of sul1 and sul2. However, high concentrations of sulfamethoxazole (150 mg L⁻¹) selected for microorganisms harboring sul1 and, more remarkably, sul2 genes in their iDNA during their exponential growth phase. The abundances of intI1 and sul1 were positively correlated in the exDNA fraction (r > 0.7), whereas no significant correlation (p < 0.05) between the abundance of these two genes was found in the iDNA fraction of the sludge. High SMX concentrations influenced the abundance of ARGs in the iDNA; their abundance in the exDNA was influenced by nutrient limitations. Further studies should consider the profiling of exDNA fractions because of the relationship between ARGs and mobile genetic elements. Besides, the surveillance of antimicrobial resistance is encouraged in wastewater treatment plants facing high antibiotic concentrations.

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Antibiotic resistance is one of the biggest threats to global health, food security, and development today, leading to a growing number of difficult-to-treat infections and an economic burden. It can affect anyone of any age and in any country. It is mainly accelerated by the misuse and abuse of antibiotics, poor hygiene, and a lack of sanitation infrastructure. From the One Health concept, water is the main link connecting all the compartments where antibiotic resistance has primarily developed (human, animal, and natural environments). It carries microorganisms, pharmaceuticals such as antibiotics, floating genetic information in the form of mobile genetic elements (MGEs), and genes conferring antibiotic resistance. It is thought that bacteria found in anthropogenic barriers such as wastewater and drinking water treatment plants could play a role in transferring and disseminating resistant bacteria into the natural environment. However, the mechanisms by which bacteria can exchange via horizontal gene transfer (HGT) to further disseminate antibiotic resistance genes (ARGs) in such compartments are unknown. Natural transformation is one of the main HGT phenomena by which competent bacteria pull extracellular DNA into their cytoplasm. Still, it remains widely unknown which bacteria can use such a mechanism and under which circumstances. Unraveling the composition of such free-floating extracellular DNA (exDNA) fraction in complex systems such as wastewater is crucial to identify the environmental conditions promoting gene transfer. This thesis aims to understand further the role of exDNA in the transfer and development of antibiotic-resistant bacteria (ARBs) from complex systems. The status of released DNA from different model microorganisms after different sterilization procedures was evaluated in Chapter 2. The results showed that current sterilization methods are effective in microorganism inactivation. However, stable DNA is released from microbial cultures and ends up in sewage streams with genetic information from microorganisms originating from human and animal discharges. In Chapter 3, a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 μg exDNA was obtained out of 1 L), activated sludge (5.6 μg out of 1 L), and treated effluent (4.3 μg out of 1 L) was developed. Thus, this was necessary to profile its genetic composition. Surprisingly, results highlighted that exDNA is mainly comprised of MGEs (65%), posing a risk as the prevalence of MGEs in the extracellular fraction can indirectly promote antibiotic resistance development mainly via natural transformation. In the two field investigation chapters (Chapters 4 and 5), the transfer of ARGs and MGEs and their removal capacity in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge and in chlorine-free drinking water treatment plants were evaluated. These two chapters summarize the journey that antibiotic-resistant bacteria follow toward water sanitation. Resistance determinants decreased their load reaching effluents from wastewater (1.1 log gene copies mL−1) and drinking water treatment plants (2.5 log gene copies mL−1), at least when inside active bacterial cells. It is less clear regarding exDNA since the treatment process involves cell decay and lysis that releases exDNA into the environment. After profiling the exDNA both in lab-scale and full-scale experiments, the effect of environmental factors such as increasing antibiotic concentrations was evaluated on exDNA transformation in an activated sludge enrichment in Chapter 6. We showed the feasibility of distantly-related microorganisms for DNA uptake when strong environmental pressures (≥50 mg L−1) were applied. Thus, it shows that natural transformation under environmental antibiotic concentrations may not be the driving force by which bacteria take up exDNA in complex systems. However, the focus should be on other compartments such as research facilities and pharmaceutical industrial discharges. Finally, strategies to remediate ARGs (intracellular and extracellular) and ARBs from wastewater effluents were evaluated in Chapter 7. We showed how byproducts from wastewater and drinking water treatment plants, such as sewage-sludge biochar and iron-oxide coated sands, were effective at removing ARBs and exDNA from effluent waters. Collectively, this thesis shows that the exDNA fraction from water matrices is an overlooked pool of genetic fragments containing MGEs and ARGs. Thus, these could be used as genetic material to transform competent bacteria and develop ARBs. However, exDNA transformation under environmental antibiotic concentrations is not the main mechanism by which bacteria evolve and adapt in mixed cultures. It is important to highlight that anthropogenic barriers are effective at remediating ARBs, which should redirect the focus from wastewater treatment plants and tackle the antibiotic resistance issue from multiple compartments simultaneously. @en

The transformation of environmental microorganisms by extracellular DNA is an overlooked mechanism of horizontal gene transfer and evolution. It initiates the acquisition of exogenous genes and propagates antimicrobial resistance alongside vertical and conjugative transfers. We combined mixed-culture biotechnology and Hi-C sequencing to elucidate the transformation of wastewater microorganisms with a synthetic plasmid encoding GFP and kanamycin resistance genes, in the mixed culture of chemostats exposed to kanamycin at concentrations representing wastewater, gut and polluted environments (0.01–2.5–50–100 mg L⁻¹). We found that the phylogenetically distant Gram-negative Runella (102 Hi-C links), Bosea (35), Gemmobacter (33) and Zoogloea (24) spp., and Gram-positive Microbacterium sp. (90) were transformed by the foreign plasmid, under high antibiotic exposure (50 mg L⁻¹). In addition, the antibiotic pressure shifted the origin of aminoglycoside resistance genes from genomic DNA to mobile genetic elements on plasmids accumulating in microorganisms. These results reveal the power of Hi-C sequencing to catch and surveil the transfer of xenogenetic elements inside microbiomes.@en

Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL−1, despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs.@en

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3′’)-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.@en

This study investigates the effects, conversions, and resistance induction, following the addition of 150 μg·L⁻¹ of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), in a laboratory-scale micro-aerated anaerobic membrane bioreactor (MA-AnMBR). TMP and SMX were removed at 97 and 86%, indicating that micro-aeration did not hamper their removal. These antibiotics only affected the pH and biogas composition of the process, with a significant change in pH from 7.8 to 7.5, and a decrease in biogas methane content from 84 to 78%. TMP was rapidly adsorbed onto the sludge and subsequently degraded during the long solids retention time of 27 days. SMX adsorption was minimal, but the applied hydraulic retention time of 2.6 days was sufficiently long to biodegrade SMX. The levels of three antibiotic-resistant genes (ARGs) (sul1, sul2, and dfrA1) and one mobile genetic element biomarker (intI1) were analyzed by qPCR. Additions of the antibiotics increased the relative abundances of all ARGs and intI1 in the MA-AnMBR sludge, with the sul2 gene folding 15 times after 310 days of operation. The MA-AnMBR was able to reduce the concentration of antibiotic-resistant bacteria (ARB) in the permeate by 3 log.@en

The success of antibiotics as a therapeutic agent has led to their ineffectiveness. The continuous use and misuse in clinical and non-clinical areas have led to the emergence and spread of antibiotic-resistant bacteria and its genetic determinants. This is a multi-dimensional problem that has now become a global health crisis. Antibiotic resistance research has primarily focused on the clinical healthcare sectors while overlooking the non-clinical sectors. The increasing antibiotic usage in the environment – including animals, plants, soil, and water – are drivers of antibiotic resistance and function as a transmission route for antibiotic resistant pathogens and is a source for resistance genes. These natural compartments are interconnected with each other and humans, allowing the spread of antibiotic resistance via horizontal gene transfer between commensal and pathogenic bacteria. Identifying and understanding genetic exchange within and between natural compartments can provide insight into the transmission, dissemination, and emergence mechanisms. The development of high-throughput DNA sequencing technologies has made antibiotic resistance research more accessible and feasible. In particular, the combination of metagenomics and powerful bioinformatic tools and platforms have facilitated the identification of microbial communities and has allowed access to genomic data by bypassing the need for isolating and culturing microorganisms. This review aimed to reflect on the different sequencing techniques, metagenomic approaches, and bioinformatics tools and pipelines with their respective advantages and limitations for antibiotic resistance research. These approaches can provide insight into resistance mechanisms, the microbial population, emerging pathogens, resistance genes, and their dissemination. This information can influence policies, develop preventative measures and alleviate the burden caused by antibiotic resistance.@en

Environmental microorganisms evolve constantly under various stressors using different adaptive mechanisms, including horizontal gene transfer. Microorganisms benefit from transferring genetic information that code for antibiotic resistance via mobile genetic elements (plasmids). Due to the complexity of natural microbial ecosystems, quantitative data on the transfer of genetic information in microbial communities remain unclear. Two 1-L chemostats (one control and one test) were inoculated with activated sludge, fed with synthetic wastewater, and operated for 45 days at a hydraulic retention time of 1 day to study the transformation capacity of a rolling-circle plasmid encoding GFP and kanamycin resistance genes, at increasing concentrations of kanamycin (0.01-2.5-50-100 mg L ⁻¹ ) representing environmental, wastewater, lab-selection, and gut or untreated pharmaceutical wastewater discharge environments. The plasmid DNA was spiked daily at 5 µg L ⁻¹ in the test chemostat. The evolution of the microbial community composition was analyzed by 16S rRNA gene amplicon sequencing and metagenomics, and the presence of the plasmid by quantitative PCR. We used Hi-C sequencing to identify natural transformant microorganisms under steady-state conditions with low (2.5 mg L ⁻¹ ) and high (50 mg L ⁻¹ ) concentrations of kanamycin. Both chemostats selected for the same 6 predominant families of Spirosomaceae, Comamonadaceae, Rhodocyclaceae, Rhizobiaceae, Microbacteriaceae , and Chitinophagaceae , while biomass formation in the presence of kanamycin was higher with the plasmid. Hence, the antibiotic exerted the main pressure on microbial selection, while the plasmid helped these populations better resist the antibiotic treatment and grow. The kanamycin resistance gene increased in both reactors (log 7 gene copies g VSS ⁻¹ ). When higher antibiotic concentrations were applied, the GFP/16S ratio was increased, highlighting plasmids accumulation in the test reactor over time. The plasmid transformed mainly inside populations of Bosea sp ., Runella spp ., and Microbacterium sp .. This study made one significant step forward by demonstrating that microorganisms in enrichments from activated sludge biomasses can acquire exogenous synthetic plasmids by transformation. Graphical abstract@en

In the One Health context, wastewater treatment plants (WWTPs) are central to safeguarding water resources. Nonetheless, many questions remain about their effectiveness in preventing antimicrobial resistance (AMR) dissemination. Most surveillance studies monitor the levels and removal of selected antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in intracellular DNA (iDNA) extracted from WWTP influents and effluents. The role of extracellular free DNA (exDNA) in wastewater is mostly overlooked. This study analyzed the transfer of ARGs and MGEs in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge. We tracked the composition and fate of the iDNA and exDNA pools of influent, sludge, and effluent samples. Metagenomics was used to profile the microbiome, resistome, and mobilome signatures of iDNA and exDNA extracts. Selected ARGs and MGEs were analyzed by qPCR. From 2,840 ARGs identified, the genes arr-3 (2%), tetC (1.6%), sul1 (1.5%), oqxB (1.2%), and aph(3")-Ib (1.2%) were the most abundant among all sampling points and bioaggregates. Pseudomonas, Acinetobacter, Aeromonas, Acidovorax, Rhodoferax, and Streptomyces populations were the main potential hosts of ARGs in the sludge. In the effluent, 478 resistance determinants were detected, of which 89% were from exDNA potentially released by cell lysis during aeration in the reactor. MGEs and multiple ARGs were co-localized on the same extracellular genetic contigs. Total intracellular ARGs decreased 3-42% due to wastewater treatment. However, the ermB and sul1 genes increased by 2 and 1 log gene copies mL−1, respectively, in exDNA from influent to effluent. The exDNA fractions need to be considered in AMR surveillance, risk assessment, and mitigation strategies.@en

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.@en

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemia has been one of the most difficult challenges humankind has recently faced. Wastewater-based epidemiology has emerged as a tool for surveillance and mitigation of potential viral outbreaks, circumventing biases introduced by clinical patient testing. Due to the situation urgency, protocols followed for isolating viral RNA from sewage were not adapted for such sample matrices. In parallel to their implementation for fast collection of data to sustain surveillance and mitigation decisions, molecular protocols need to be harmonized to deliver accurate, reproducible, and comparable analytical outputs. Here we studied analytical variabilities linked to viral RNA isolation methods from sewage. Three different influent wastewater volumes were used to assess the effects of filtered volumes (50, 100 or 500 mL) for capturing viral particles. Three different concentration strategies were tested: electronegative membranes, polyethersulfone membranes, and anion-exchange diethylaminoethyl cellulose columns. To compare the number of viral particles, different RNA isolation methods (column-based vs. magnetic beads) were compared. The effect of extra RNA purification steps and different RT-qPCR strategies (one step vs. two-step) were also evaluated. Results showed that the combination of 500 mL filtration volume through electronegative membranes and without multiple RNA purification steps (using column-based RNA purification) using two-step RT-qPCR avoided false negatives when basal viral load in sewage are present and yielded more consistent results during the surveillance done during the second-wave in Delft (The Hague area, The Netherlands). By paving the way for standardization of methods for the sampling, concentration and molecular detection of SARS-CoV-2 viruses from sewage, these findings can help water and health surveillance authorities to use and trust results coming from wastewater based epidemiology studies in order to anticipate SARS-CoV-2 outbreaks.@en

Wastewater treatment is challenged by the continuous emergence of chemical and biological contaminants. Disinfection, advanced oxidation, and activated carbon technologies are accessible in high-income countries to suppress them. Low-cost, easily implementable, and scalable solutions are needed for sanitation across regions. We studied the properties of low-cost absorbents recycled from drinking water and wastewater treatment plant residues to remove environmental DNA and xenogenetic elements from used water. Materials characteristics and DNA adsorption properties of used iron-oxide-coated sands and of sewage-sludge biochar obtained by pyrolysis of surplus activated sludge were examined in bench-scale batch and up-flow column systems. Adsorption profiles followed Freundlich isotherms, suggesting a multilayer adsorption of nucleic acids on these materials. Sewage-sludge biochar exhibited high DNA adsorption capacity (1 mg g−1) and long saturation breakthrough times compared to iron-oxide-coated sand (0.2 mg g−1). Selected antibiotic resistance genes and mobile genetic elements present on the free-floating extracellular DNA fraction and on the total environmental DNA (i.e., both extra/intracellular) were removed at 85% and 97% by sewage-sludge biochar and at 54% and 66% by iron-oxide-coated sand, respectively. Sewage-sludge biochar is attractive as low-cost adsorbent to minimize the spread of antimicrobial resistances to the aquatic environment while strengthening the role of sewage treatment plants as resource recovery factories.
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The free-floating extracellular DNA (exDNA) fraction of microbial ecosystems harbors antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Natural transformation of these xenogenetic elements can generate microbial cells resistant to one or more antibiotics. Isolating and obtaining high yield exDNA is challenging due to its low concentration in wastewater environments. Profiling exDNA is crucial to unravel the ecology of free-floating ARGs and MGEs and their contribution to horizontal transfer. We developed a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 µg exDNA out of 1 L), activated sludge (5.6 µg), and treated effluent (4.3 µg). ARGs and MGEs were metagenomically profiled for both the exDNA and intracellular DNA (iDNA) of activated sludge, and quantified by qPCR in effluent water. qPCR revealed that ARGs and MGEs are more abundant in the iDNA fraction while still significant on exDNA (100-1000 gene copies mL−1) in effluent water. The metagenome highlighted that exDNA is mainly composed of MGEs (65%). According to their relatively low abundance in the resistome of exDNA, ARGs uptake by natural transformation is likely not the main transfer mechanism. Although ARGs are not highly abundant in exDNA, the prevalence of MGEs in the exDNA fraction can indirectly promote antibiotic resistance development. The combination of this method with functional metagenomics can help to elucidate the transfer and development of resistances in microbial communities. A systematic profiling of the different DNA fractions will foster microbial risk assessments across water systems, supporting water authorities to delineate measures to safeguard environmental and public health. @en

The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.@en