Caroline Le Marechal
email: caroline.lemarechal@anses.fr

Cécile Gateau

T Poëzévara

Jeanne Couturier

Sandra Rouxel

R Syed Zaidi

Emmanuelle Houard

Anne-Marie A M Pourcher

Martine Denis

Félix Barbut

Caroline Le Maréchal

Typhaine Poezevara

Rabab Zyed-Saidi

Frédéric Barbut

Characterization of Clostridioides difficile strains isolated from manure and digestate in five agricultural biogas plants

Keywords: Clostridioides difficile, PCR ribotype 126 and 078, wgMLST, biogas plant, pig, cattle

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacterium considered as an emerging pathogen responsible for diarrhea and colitis in both humans and animals. C. difficile infection (CDI) is the major cause of nosocomial diarrhea in adults, and has been increasingly reported in the community in the past decade or so [START_REF] Barbut | Toxigenic Clostridium difficile carriage in general practice: results of a laboratorybased cohort study[END_REF]. Nowadays, the incidence of community-acquired CDI in some regions accounts for up to a quarter of all cases [START_REF] Khanna | Clostridium difficile infection: new insights into management[END_REF][START_REF] Knight | Diversity and evolution in the genome of Clostridium difficile[END_REF].

Food, animals and the environment are considered potential reservoirs of C. difficile strains [START_REF] Knight | Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance[END_REF]. Indeed, animal and food PCR ribotypes (RT) have been correlated with those found in humans. Although the zoonotic status of C. difficile is still debated [START_REF] Hensgens | Clostridium difficile infection in the community: a zoonotic disease? Clinical microbiology and infection : the official publication of the[END_REF], recent data highlight a potential risk of transmission from animals or environmental reservoirs to humans [START_REF] Knetsch | Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011[END_REF]. CDI should therefore be managed through a One Health approach.

It has recently been demonstrated that C. difficile can be detected in digestate from agricultural biogas plants (BPs) and is able to survive anaerobic digestion [START_REF] Froschle | Fate of Clostridium botulinum and incidence of pathogenic clostridia in biogas processes[END_REF][START_REF] Le Maréchal | Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants[END_REF]. The conversion of livestock manure into biogas through anaerobic digestion in BPs has become increasingly popular in the past few decades in some European countries.

However, the C. difficile strains isolated from agricultural BPs have never been characterized up to now. The PCR ribotypes of these strains and their virulence gene contents are unknown, yet this information is of prime importance to be able to evaluate the risks associated with its detection in digestate, which can be spread over fields after its storage or post-digestion and may constitute a reservoir of C. difficile strains.

The aim of our study was to characterize C. difficile isolates collected from manure and raw digestate in five different agricultural BPs so as to determine which kind of C. difficile strains can survive anaerobic digestion.

Materials and methods

Strain collection

A total of 54 C. difficiIe isolates were collected during a study evaluating the presence of various pathogens in manure and digestate samples from five agricultural BPs [START_REF] Le Maréchal | Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants[END_REF].

Briefly, 1 g of each sample was 10-fold diluted in Brain Heart Infusion (BHI; BioMérieux, Craponne, France) supplemented with 0.1% taurocholate (Sigma Aldrich, Lyon, France), cefoxitin (8 mg/l) and cycloserine (250 mg/l) (Oxoid, Dardilly, France). Tubes were incubated at 37°C in an anaerobic chamber (A35; Don Whitley distributed by BioMérieux, Bruz, France) filled with anaerobic gas (10% H2, 10% CO2, 80% N2). After 7 days of incubation, 10 µl of broth was plated on ChromID C. difficile agar (BioMérieux, Craponne, France). The plates were incubated for 48 hours at 37°C in the anaerobic chamber. C. difficile colonies were suspected by their specific black color and/or shape and two colonies from each positive sample were re-isolated on BHI agar. These plates were incubated for 48 hours at 37°C in the anaerobic chamber. The isolates were then stored at -80°C. The strains were identified using PCR as described below.

For each BP, three replicate samples of manure and three of digestate were collected on the same day. Two different colonies were stored from each positive replicate. Fifty-four strains were collected from the five BPs as depicted in Table 1, either from manure [START_REF] Bauer | Clostridium difficile infection in Europe: a hospital-based survey[END_REF] or from digestate (29).

DNA extraction

DNA for PCR and PCR ribotyping was extracted from colonies grown on Brucella agar (BioMérieux, Craponne, France) after anaerobic incubation for 48 h. After centrifuging a suspension of bacterial colony in sterile water, the supernatant was removed and the bacterial pellet re-suspended with 200 µL of InstaGene™ matrix (Bio-Rad®, Marnes-la-Coquette, France). This suspension was incubated at 56°C for 20 min, then 96°C for 8 min. The supernatant containing the bacterial DNA was recovered after centrifugation and stored at -20°C.

Characterization of C. difficile and detection of toxin genes

A multiplex PCR according to Barbut et al. [START_REF] Barbut | Toxigenic Clostridium difficile carriage in general practice: results of a laboratorybased cohort study[END_REF] was used to detect the PaLoc genes (tcdA, tcdB, tcdC) and CDTLoc genes (cdtA and cdtB). It also detected the 117 bp fragment (lok) present in non-toxigenic strains and the tpi (triose phosphate isomerase) gene fragment used to identify C. difficile. The amplified products were analyzed after a 1/25 dilution by capillary electrophoresis with an ABI 3500 sequencer. Migration profiles were analyzed with the GeneMapper® software (Applied Biosystems®, Foster City, USA) by comparing the size of the fragments (peaks) obtained with the expected sizes for each specific gene fragment.

PCR ribotyping

Capillary gel electrophoresis PCR ribotyping was performed as previously described by Bidet et al. [START_REF] Bidet | Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing[END_REF]. After DNA amplification, 1 µL of a 1/200 dilution of each PCR product was mixed with 10.5 µL formamide and 0.5 µL GeneScan LIZ600 (Applied Biosystems®, Foster City, USA) as an internal marker. After 30 s of denaturation at 90°C, capillary electrophoresis was performed on an 8-capillary 3500 Genetic Analyzer (Applied Biosystems®, Foster City, USA). The GeneMapper software (Thermo Fisher Scientific, Villebon-sur-Yvette, France) was used to analyze the banding patterns. PCR ribotypes (RTs) were identified using the webribo software (https://webribo.ages.at/).

Whole genome sequencing (WGS)

WGS was performed on 18 strains (two isolates from manure and two from digestate for BP1, BP3, BP4 and BP5, and one of each for BP2, based on the PCR-ribotyping and PCR results). DNA was extracted as described in [START_REF] Sim | Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing[END_REF]. Sequencing was performed as per the ICM institute using the NovaSeq 6000 Illumina technology (2x150 paired-end sequencing, Nextera XT DNA Sample Prep Kit, Illumina). Genome sequences generated as part of this study were deposited in SRA (PRJNA599117).

The quality of reads was evaluated using BioNumerics 7.6.3 (Applied Maths NV, Sint-Martens-Latem, Belgium): isolates with an average quality below 30 and estimated coverage below 30 were excluded from subsequent analysis.

Whole genome multilocus sequence typing (wgMLST) analyses

The wgMLST scheme used contains 8,745 coding loci, representing a pan-genome of C. difficile identified from 259 previously-published genomes. WgMLST analyses were performed using BioNumerics 7.6.3 as described in [START_REF] Gateau | Local outbreak of Clostridioides difficile PCR-Ribotype 018 investigated by multi locus variable number tandem repeat analysis, whole genome multi locus sequence typing and core genome single nucleotide polymorphism typing[END_REF]. Briefly, the genetic relationship between two isolates was assessed by calculating the number of different alleles for wgMLST. We defined two isolates as genetically related or belonging to the same clonal complex (CC) when they had an allelic difference ≤ 200 or ≤ 20 respectively.

Antimicrobial susceptibility

Antimicrobial susceptibility to vancomycin (30 µg), metronidazole (4 µg [Sanofi Diagnostics Pasteur, Marnes la Coquette, France]), erythromycin (15 IU), clindamycin(2 IU), moxifloxaxin (5 µg), chloramphenicol (30 µg), imipenem (10 µg) and tetracycline (30 IU) was determined by the disk diffusion method on pre-reduced Brucella agar plates as described elsewhere [START_REF] Barbut | Clinical features of Clostridium difficile-associated infections and molecular characterization of strains: results of a retrospective study, 2000-2004[END_REF]. The results were interpreted according to the French Society of Microbiology's "Comité de l'Antibiogramme" (2013, available at https://resapath.anses.fr/resapath_uploadfiles/files/Documents/2013_CASFM.pdf).

Strains were considered susceptible if the inhibition diameters for vancomycin (VA), metronidazole (MZ), erythromycin (ERY), clindamycin (CM), moxifloxacin (MXF), chloramphenicol (C), imipenem (IMI) and tetracycline (TE) were greater than or equal to 17mm, 21 mm, 22 mm, 15 mm, 23 mm, 23 mm, 24 mm and 23 mm respectively.

Results

All the results are fully presented in Table 1.

Identification of C. difficile and detection of toxin genes

All the strains were positive for tpi, thus confirming the identification of C. difficile. All also harbored the tcdA and tcdB genes. Forty-five (83.3%) contained cdtA and cdtB genes coding for the binary toxin and had a -39 bp deletion in regulator gene tcdC.

Nine strains (16.7%) harbored only the cdtA gene and had no deletion in regulator gene tcdC.

PCR ribotyping

Among the five BPs investigated, only six different RTs (Fig. 1A) were identified, with a predominance of RT 078 (37%) and 126 (46.3%) in manure and digestate. Other RTs included 005 (7.4%), 003 (3.7%), 014 (3.7%) and 106 (1.8%) (Fig. 1A). Isolates from BP2 that contained bovine and poultry manure had the widest RT diversity (Fig. 1B). On the contrary, there was little variability among the RTs: two RTs in BP1 and BP5 (see Fig. 1B) and only one in BP3 and BP4 (see Fig. 1B).

wgMLST

The minimum spanning tree of wgMLST typing is shown in Fig. 2. wgMLST analysis revealed that isolates clustered by BP, with fewer than five alleles of difference within a BP except for strains from RT 005 isolated from BP2. wgMLST results confirm results from PCR ribotyping, with little variability within the same BP except for BP2.

Antimicrobial susceptibility

The antimicrobial susceptibility of C. difficile strains was evaluated for nine antibiotics.

All the strains were susceptible to vancomycin, metronidazole, imipenem and chloramphenicol. Resistance rates to erythromycin, clindamycin, moxifloxacin, and tetracycline were 85.2%, 98.2%, 24.1%, and 83.3% respectively. Strains from BP1, BP2 and BP3 were susceptible to moxifloxacin, whereas all the isolates from BP4 and two out of the ten isolates from BP5 were resistant. Strains from BP3, BP4 and BP5 were resistant to erythromycin and 85.3% were resistant to tetracycline. All but one of the isolates from BP2 were susceptible to erythromycin and all were susceptible to tetracycline.

Discussion

As previously reported [START_REF] Froschle | Fate of Clostridium botulinum and incidence of pathogenic clostridia in biogas processes[END_REF], C. difficile strains were easily detected and collected from the five agricultural BPs investigated during this study. Manure samples collected from BP2 had a lower positive rate (only one positive sample out of the three analyzed [START_REF] Le Maréchal | Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants[END_REF]) than the others, but it was still possible to isolate eight strains from this BP (two from manure and six from digestate). Ten isolates were collected from BP5 instead of 12 because one isolate from a manure sample and one from a digestate sample were tpi-negative by PCR so were not included in this study. To our knowledge, this study is the first to characterize C. difficile strains isolated from agricultural BPs.

As already stated in previous studies [START_REF] Koene | Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates[END_REF], there is no standard method for the detection and isolation of C. difficile in animal samples. Our study collected isolates using supplemented BHI followed by isolation on ChromID. Two isolates from the same sample were characterized to evaluate the diversity of strains and profiles that could be encountered within a single sample. Considering together the results presented here from PCR ribotyping, multiplex PCR and antibiotic susceptibility assays, it appears that isolates were very similar when pig manure was present, and indeed were similar both in manure and digestate samples. Considering this result, two isolates from manure and two from digestate for BP1, BP3, BP4 and BP5 were analyzed using wgMLST. Isolates from BP3, BP4 and BP5 analyzed by wgMLST had fewer than one allelic difference, and those from BP1 fewer than five allelic differences, confirming the very close proximity of strains. This seems to show the low impact of anaerobic digestion on a potential selection of strains during the process. Greater diversity in isolated strains was observed in BP2 however, where both cattle and poultry manure was used as input, even for isolates from the same RT analyzed using wgMLST and showing 41 allelic differences (Fig. 2). This difference in strain variability between pigs, cattle or poultry has already been described [START_REF] Diaz | Non-human C. difficile Reservoirs and Sources: Animals, Food, Environment[END_REF]. According to available studies, a single RT is expected on pig farms, while greater variability is reported on cattle farms and the greatest on poultry and rabbit farms [START_REF] Diaz | Non-human C. difficile Reservoirs and Sources: Animals, Food, Environment[END_REF]. This variability could have been missed if only one isolate had been characterized per matrix or per BP.

Organic co-substrates are used to supply the BPs, [START_REF] Le Maréchal | Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants[END_REF] but it was not possible to analyze them during the study mainly because of their diversity from one plant to the next and their variability within the same BP, depending what matrices are available.

As mentioned above, the characteristics of strains isolated from BPs supplied with pig manure are very similar, suggesting that C. difficile strains isolated from digestate originated from manure. Conclusions are more difficult to draw for BP2 considering that the PCR ribotypes from the manure and digestate are not the same, and that the two RT 005 strains isolated from manure or digestate differ by 41 alleles. It is necessary to detect and characterize C. difficile strains in co-substrates or monitor C. difficile profiles in manure and digestate over a period of time in order to determine the origin of C. difficile strains recovered from digestate.

RT 078 and RT 126 were the most common ribotypes identified in manure and digestate from the BPs investigated. This finding is in accordance with studies conducted on pigs, as RT 078 is the most reported PCR ribotype in pigs worldwide [START_REF] Koene | Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates[END_REF]. Except in BP2, all the manure in this study contained at least pig manure, which may explain this result. RT 126 is part of the ribotype 078 lineage that includes RT 078, 045, 066, 126 and 127 [START_REF] Knetsch | Genetic markers for Clostridium difficile lineages linked to hypervirulence[END_REF][START_REF] Stabler | Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations[END_REF] and is a fluoroquinolone-resistant descendant of RT 078 [START_REF] Dingle | A Role for Tetracycline Selection in Recent Evolution of Agriculture-Associated Clostridium difficile PCR Ribotype 078[END_REF]. It has also been isolated from pigs in previous studies worldwide [START_REF] Wu | Potentially hypervirulent Clostridium difficile PCR ribotype 078 lineage isolates in pigs and possible implications for humans in Taiwan[END_REF][START_REF] Álvarez-Pérez | Distribution and tracking of Clostridium difficile and Clostridium perfringens in a free-range pig abattoir and processing plant[END_REF][START_REF] Schneeberg | Clostridium difficile Genotypes in Piglet Populations in Germany[END_REF][START_REF] Janezic | International Clostridium difficile animal strain collection and large diversity of animal associated strains[END_REF].

RT 078 is considered to be an emerging cause of community-acquired CDI in various countries or regions around the world (the Netherlands, England, Scotland, North America and Europe [START_REF] Goorhuis | Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078[END_REF][START_REF] Barbut | Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates[END_REF][START_REF] Fawley | Enhanced surveillance of Clostridium difficile infection occurring outside hospital, England, 2011 to 2013[END_REF][START_REF] Bauer | Clostridium difficile infection in Europe: a hospital-based survey[END_REF]). The zoonotic potential of RT 078 from Sequence Type 11 has been recently demonstrated by genomic analyses, [START_REF] Knight | Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance[END_REF] and it is hypothesized that the food chain and/or the environment are its reservoirs [START_REF] Hensgens | Clostridium difficile infection in the community: a zoonotic disease? Clinical microbiology and infection : the official publication of the[END_REF]. By detecting this RT in manure and digestate, our study appears to confirm this hypothesis.

RT 014, identified in BP2, has been previously detected in wastewater effluents and influents, in human patients, shellfish, and sewage sludge [START_REF] Romano | Clostridioides difficile in the environment, food, animals and humans in southern Italy: Occurrence and genetic relatedness[END_REF]. RT 014 is the third most prevalent ribotype associated with CDI in Europe [START_REF] Davies | Diversity of Clostridium difficile PCR ribotypes in Europe: results from the European, multicentre, prospective, biannual, point-prevalence study of Clostridium difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013[END_REF] and the USA [START_REF] Tickler | (Clostridium) difficile strains in the United States between 2011-2017[END_REF]. RT 005 has also been detected in wastewater effluents and RT 106 from shellfish [START_REF] Romano | Clostridioides difficile in the environment, food, animals and humans in southern Italy: Occurrence and genetic relatedness[END_REF]. They have been associated with CDI in France but to a lesser extent than RT 014 or RT 078/126 [29]. However, an increase in the prevalence of RT 106 in cases of CDI was observed in the USA between 2011 and 2017 [START_REF] Tickler | (Clostridium) difficile strains in the United States between 2011-2017[END_REF].

C. difficile strains are known to be resistant to certain antibiotics, such as quinolones, erythromycin and clindamycin [START_REF] Diaz | Non-human C. difficile Reservoirs and Sources: Animals, Food, Environment[END_REF]. Resistance to erythromycin has in particular been reported among isolates of PCR ribotype 078 in pigs and cattle [START_REF] Keessen | Antimicrobial susceptibility profiles of human and piglet Clostridium difficile PCR-ribotype 078[END_REF]. RT 126/078 has been reported to be highly resistant to tetracycline, moxifloxacin, clindamycin, and erythromycin [START_REF] Knight | Evolutionary and Genomic Insights into Clostridioides difficile Sequence Type 11: a Diverse Zoonotic and Antimicrobial-Resistant Lineage of Global One Health Importance[END_REF]. Our results are thus in accordance with previous studies. Only strains from BP4 and one isolate from BP5 were resistant to moxifloxacin. Fluoroquinolones represented 0.24% of the tonnage of active ingredients sold in the veterinarian field in 2017 in France [START_REF]Suivi des ventes de médicaments vétérinaires contenant des antibiotiques en France[END_REF]. It was not possible to obtain information regarding the antimicrobial treatments of animals whose manure was input into the BPs investigated in this study, and no link between rearing practices and antimicrobial resistance could therefore be evaluated.

Tetracycline is the most widely-used antimicrobial for the treatment, control, and prevention of infections in animals [START_REF]Sales of veterinary antimicrobial agents in 29 European countries in[END_REF]. Tetracycline represented around 38% of veterinary antimicrobial sales in France in 2017 and is the main antimicrobial used in pig and bovine production [START_REF]Suivi des ventes de médicaments vétérinaires contenant des antibiotiques en France[END_REF]. The high level of decreased-susceptibility strains to this antimicrobial (83.3% with an inhibition diameter below 23) in our study is not therefore surprising. The resistance of RT078 and RT126 to tetracycline may also play a role in its high prevalence in pigs by providing these strains with a selective advantage in comparison with susceptible strains.

The resistances to antimicrobials reported here are similar to those observed in strains involved in human CDI [29]. Fortunately, no resistance to antibiotics such as vancomycin or metronidazole, used to treat human C. difficile infections, was detected in the strains isolated from the BPs studied.

Conclusion

This study shows that toxigenic C. difficile strains resistant to a number of antimicrobials can be isolated from manure and digestate. Such matrices represent a reservoir of C. difficile strains whose RTs are similar to those found in patients with CDI. This illustrates the relevance of the One Health approach to this issue and our results show that, in addition to monitoring the strains involved in CDI, C. difficile strains from animal and environmental reservoirs should be monitored to identify circulating strains so as to be able to detect at an early stage any emergence that could impact human health.

Figures:Fig. 1 :Fig. 2 :

 12 Figures:

Figure

  Figure 1

Table 1 :

 1 1 PCR ribotypes, toxin gene profiles and antimicrobial susceptibility of Clostridioides difficile isolated from manure and digestate samples collected from French agricultural biogas plants (BPs). LOK: gene encoding for the lok gene, characteristic of non-toxic C. difficile; tpi: gene fragment used to identify C. difficile; Binary Tox: cdtA and cdtB genes encoding for the binary toxin; del. tcdC: détection of deletions in tcdC gene; +: detected; -:

	413

not detected Strain reference Sample Type BP Production Antimicrobial Strain gene content ERY CN MOXI MZ VA TE IMP C Tox A

		D17MD20 D17MD132	17MD01-01-5 Digestate BP1 17MD01-04-21 Manure BP4 Pig, Bovine, Poultry 6 6 Pig 6 6	26 34 31 20 31 31 + 6 35 31 14 26 30 +	+ +	--	+ +	+ +	-39 -39
		D17MD21 D17MD134	17MD01-01-5 Digestate BP1 17MD01-04-22 Digestate BP4 Pig, Bovine, Poultry 6 6 Pig 6 6	26 32 31 16 29 30 + 6 40 34 14 30 36 +	+ +	--	+ +	+ +	-39 -39
		D17MD23 D17MD135	17MD01-01-6 Digestate BP1 17MD01-04-22 Digestate BP4 Pig, Bovine, Poultry 6 10 Pig 6 7	26 35 32 14 30 30 + 6 40 35 17 32 36 +	+ +	--	+ +	+ +	-39 -39
		D17MD25 D17MD137	17MD01-01-6 Digestate BP1 17MD01-04-23 Digestate BP4 Pig, Bovine, Poultry 6 6 Pig 6 7	28 34 31 15 31 32 + 6 36 32 16 28 31 +	+ +	--	+ +	+ +	-39 -39
		D17MD62 D17MD138	17MD01-02-9 Manure BP2 Bovine, Poultry 17MD01-04-23 Digestate BP4 Pig, Bovine, Poultry 6 8 28 8	24 32 34 40 30 36 + 6 37 31 16 29 31 +	+ +	--	+ Truncated 0 + + -39
		D17MD64 D17MD140	17MD01-02-9 Manure BP2 Bovine, Poultry 17MD01-04-24 Digestate BP4 Pig, Bovine, Poultry 6 7 28 6	25 34 32 44 27 33 + 6 37 34 17 33 35 +	+ +	--	+ Truncated 0 + + -39
		D17MD65 D17MD141	17MD01-02-10 Digestate BP2 Bovine, Poultry 17MD01-04-24 Digestate BP4 Pig, Bovine, Poultry 6 6 36 6	23 36 31 50 28 34 + 6 37 31 11 27 27 +	+ +	--	+ Truncated 0 + + -39
		D17MD66 D17MD162	17MD01-02-10 Digestate BP2 Bovine, Poultry 17MD01-05-25 Manure BP5 Pig, Bovine	6 9 6 6	26 40 34 25 31 35 + 28 31 29 17 26 27 +	+ +	--	+ Truncated 0 + + -39
		D17MD69 D17MD165	17MD01-02-11 Digestate BP2 Bovine, Poultry 17MD01-05-26 Manure BP5 Pig, Bovine	26 6 6 6	23 34 31 44 24 28 + 27 30 30 17 27 27 +	+ +	--	+ Truncated 0 + + -39
		D17MD70 D17MD166	17MD01-02-11 Digestate BP2 Bovine, Poultry 17MD01-05-26 Manure BP5 Pig, Bovine	36 6 6 6	27 41 35 56 30 38 + 28 34 31 17 28 31 +	+ +	--	+ Truncated 0 + + -39
		D17MD71 D17MD168	17MD01-02-12 Digestate BP2 Bovine, Poultry 17MD01-05-27 Manure BP5 Pig, Bovine	24 6 6 6	24 36 32 40 27 23 + 28 33 31 16 28 31 +	+ +	--	+ Truncated 0 + + -39
		D17MD73 D17MD169	17MD01-02-12 Digestate BP2 Bovine, Poultry 17MD01-05-27 Manure BP5 Pig, Bovine	30 10 30 40 40 60 50 38 + 6 6 26 32 30 18 26 30 +	+ +	--	+ Truncated 0 + + -39
		D17MD99 D17MD171	17MD01-03-13 Manure BP3 17MD01-05-28 Digestate BP5	Pig Pig, Bovine	6 11 29 40 34 20 30 36 + 6 6 26 29 31 19 26 29 +	+ +	--	+ +	+ +	-39 -39
		D17MD100 D17MD174	17MD01-03-13 Manure BP3 17MD01-05-29 Digestate BP5	Pig Pig, Bovine	6 7 6 6	28 38 35 16 33 34 + 26 31 31 18 26 28 +	+ +	--	+ +	+ +	-39 -39
		D17MD102 D17MD175	17MD01-03-14 Manure BP3 17MD01-05-29 Digestate BP5	Pig Pig, Bovine	6 7 6 6	29 34 35 15 30 37 + 6 39 31 14 28 28 +	+ +	--	+ +	+ +	-39 -39
		D17MD103 D17MD177	17MD01-03-14 Manure BP3 17MD01-05-30 Digestate BP5	Pig Pig, Bovine	6 8 6 6	31 35 36 15 32 36 + 26 30 28 16 26 27 +	+ +	--	+ +	+ +	-39 -39
		D17MD105 D17MD178	17MD01-03-15 Manure BP3 17MD01-05-30 Digestate BP5	Pig Pig, Bovine	6 8 6 6	29 39 34 16 30 35 + 27 32 30 19 27 29 +	+ +	--	+ +	+ +	-39 -39
	438	D17MD106	17MD01-03-15 Manure BP3	Pig	6 6	30 38 35 15 32 35 +	+	-	+	+	-39
		D17MD108	17MD01-03-16 Digestate BP3	Pig	6 8	30 39 34 13 30 36 +	+	-	+	+	-39
		D17MD109	17MD01-03-16 Digestate BP3	Pig	6 12 30 44 36 19 33 37 +	+	-	+	+	-39
		D17MD111 D17MD112	17MD01-03-17 Digestate BP3 17MD01-03-17 Digestate BP3	Pig Pig	6 6 6 7	28 36 34 15 31 33 + 30 34 34 10 34 34 +	Tox B + +	LOK tpi -+ -+	Binary Tox + +	del. tcdC -39 -39	Ribotype
		D17MD08 D17MD114	17MD01-01-1 Manure BP1 17MD01-03-18 Digestate BP3	Pig Pig	6 6 6 7	26 36 30 16 32 27 + 30 38 35 15 29 36 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD09 D17MD115	17MD01-01-1 Manure BP1 17MD01-03-18 Digestate BP3	Pig Pig	6 6 6 6	26 36 31 16 31 28 + 28 42 34 17 33 35 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD11 D17MD125	17MD01-01-2 Manure BP1 17MD01-04-19 Manure BP4 Pig, Bovine, Poultry 6 6 Pig 6 6	26 40 32 16 28 30 + 6 44 33 18 33 32 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD12 D17MD126	17MD01-01-2 Manure BP1 17MD01-04-19 Manure BP4 Pig, Bovine, Poultry 6 6 Pig 32 16 31 46 34 50 29 36 + 6 42 32 19 30 34 +	+ +	--	+ Truncated 0 + + -39	005
		D17MD14 D17MD128	17MD01-01-3 Manure BP1 17MD01-04-20 Manure BP4 Pig, Bovine, Poultry 6 6 Pig 6 7	26 32 32 15 27 32 + 6 42 33 15 30 34 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD15 D17MD129	17MD01-01-3 Manure BP1 17MD01-04-20 Manure BP4 Pig, Bovine, Poultry 6 8 Pig 6 6	26 33 28 15 30 27 + 6 43 35 16 31 35 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD17 D17MD131	17MD01-01-4 Digestate BP1 17MD01-04-21 Manure BP4 Pig, Bovine, Poultry 6 6 Pig 6 7	29 38 33 16 30 33 + 6 41 31 14 34 34 +	+ +	--	+ +	+ +	-39 -39	078
		D17MD18	17MD01-01-4 Digestate BP1	Pig	6 8	28 34 31 16 32 31 +	+	-	+	+	-39	078

Acknowledgments

The authors are grateful to all the participating farmers.

We are also very grateful to Edouard Hirchaud and Aurélie Leroux of ANSES's Ploufragan Laboratory, GVB Unit, and Amandine Thépault, of ANSES's Ploufragan Laboratory, HQPAP Unit for their technical support.

Funding

This research received financial support from the French Environment and Energy Management Agency (ADEME) (agreement number: 1606C0022 and 1806C0020), the departmental Council of Côtes d'Armor (Conseil Départemental des Côtes d'Armor).


Diana Soukarié

Vincent Ecochard

Laurence Salomé
email: laurence.salome@ipbs.fr

DNA-based nanobiosensors for monitoring of water quality

Keywords: Biosensor, Environmental contaminant, Nanotechnologies, Aptamer, DNAzyme, Single molecule detection

Introduction

The demand for novel monitoring methods for the control of water status was pointed out by the EU parliament and council in the founding Directive of 2013 regarding priority substances in the field of water policy [START_REF] Eudirective | DIRECTIVE 2006/7/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 15 February 2006 concerning the management of bathing water quality and[END_REF]. This directive announced the establishment of the first watch list of substances and was followed by vast Union-wide monitoring programs. Among contaminants posing threat to aquatic environment and human health are i) the microorganisms (bacteria, viruses and parasites) and ii) micropollutants with negative effects at low concentrations, ranging from µg/L to ng/L. The latter include an immense variety of inorganic and organic chemicals: metal ions, in particular heavy metal ions, hydrocarbons, plasticizers, pesticides, pharmaceuticals, personal care products, detergents and sanitizer products... Massive analytic, ecotoxicological and toxicological data are collected and reviewed on the basis of what risks are assessed for determining new priority substances and environmental quality standards (EQS). The discharge of micropollutants in surface waters comes for a large part from the wastewater treatment plants which are rarely designed to eliminate micropollutants. In addition, an important diffuse pollution exists also with contamination of ground and surface waters from rainfall runoff draining pollutants from agriculture and livestock. Pesticides, for example, are among the contaminants for which the EQS are the most frequently exceeded [START_REF] Petit | The status of surface water and groundwater[END_REF]. Climate change and, since micropollutants are mainly of anthropogenic origin, demographic growth will increase water stress, the ratio between water withdrawals and the available renewable water supplies. Preserving the quality of water has hence become a major worldwide concern and monitoring devices will help to set regulatory limits for micropollutants and enable their continuous control with the most extensive coverage.

The disclosure of micropollutants occurrence in surface water owes much to the progress of analytical methods, in particular liquid chromatography coupled to mass spectrometry (LC-MS) which has become a gold standard of water micropollutant analyses (EUdirective 2013). Thanks to the sensitivity of the technique and its capacity of identification of a large multiplicity of compounds in a complex matrix [START_REF] Cotton | Development and validation of a multiresidue method for the analysis of more than 500 pesticides and drugs in water based on on-line and liquid chromatography coupled to high resolution mass spectrometry[END_REF], massive campaigns of measurements revealed the extent of the contamination and also of the variety of the compounds [START_REF] Petrie | A review on emerging contaminants in wastewaters and the environment: Current knowledge, understudied areas and recommendations for future monitoring[END_REF].

While LC-MS is extremely useful to unveil this environmental concern, such a technique is not adapted for a regular and systematic control of catchment stations, urban and industrial WWTP effluents, … To meet the future challenges, in addition to high sensitivity, the pre-requisites for devices adapted to such a mass use are: portability, automation, rapidity, versatility, reduced cost, ease of use and, ideally, sustainability.

The use of antibodies has led to the development of a panel of fast and cost-effective alternatives to this analytic approach relies on the detection of analytes through their specific recognition. The well-known ELISA tests (for Enzyme Linked Immuno Sorbent Assay) allow to reach detection limits comparable to LC-MS/MS in some cases and sometimes even below the European Union limit for drinking water and. Antibody-based commercial kits are widely used in biology and biomedical laboratories, where conditions are met to perform those assays routinely but their format is not usually suitable for field applications.

Nanoscale or nanotechnology-enabled sensors offer the potential of integration in miniaturized and automated devices. Based on a nanomaterial, a recognition element and a mechanism for the transduction of the detection event efficient at the molecular level, they promise high performance with extremely low detection limit and rapid analysis [START_REF] Vikesland | Nanosensors for water quality monitoring[END_REF]. Over the past decades, DNA, apart from its obvious interest for species identification, has emerged as a highly potent recognition element, with enormous advantages: versatility, robustness and cost-effective mass synthesis [START_REF] Palchetti | Nucleic acid biosensors for environmental pollution monitoring[END_REF]. DNA has also made a breakthrough in the domain of nanotechnology as a building block for the construction of refined nanostructures [START_REF] Abu-Salah | DNA-Based Applications in Nanobiotechnology[END_REF].

Devices embarking DNA-based nanobiosensors are most promising candidates. This mini-review depicts the essential features of recent achievements in the field of DNA-based nanobiosensors, focusing on functional solutions for the monitoring of ions, pesticides and pathogens and discusses the future developments in progress.

Detection principles of DNA-based nanosensors

Biosensors can be classified according to the different components employed for their design: the recognition element used, the signal transduction method, or the analyte of interest (IUPAC definition) [START_REF] Bhalla | Introduction to biosensors[END_REF]. The selectivity of a sensor is provided by the recognition element chosen. While it is commonly thought that the biorecognition element is the most important part of a biosensor, we will also discuss how choosing the transducer affects the device's performance. Accordingly, biosensors can be designed as electrochemical, optical, or piezoelectric devices. Here we present another classification, showing how different assay configurations can be designed to exploit the polymorphism of DNA.

Conformational change

DNA molecules, contrary to protein recognition elements, present a potential for malleability often exploited in the conception of bioassays. Conformational change tests are based on the measurement of a distance shift consecutive to the recognition event. To the polymorphism of DNA is added the versatility of surfaces used in sensors: modified electrodes, noble metal nanoparticles, quantum dots, carbon nanotubes, graphene sheets,… offering a wide range of experimental designs [START_REF] Dolatabadi | Optical and electrochemical DNA nanobiosensors[END_REF].

We distinguish two categories of sensors: one using (de)hybridization, and the other nucleic acid probes (aptamers and DNAzymes). In the first case, the capacity of nucleic acids chains to shift from single-stranded molecules to double-stranded helices in the presence of a complementary strand is exploited. Alternatively, a single-stranded oligonucleotide can be functionalized with a fluorophore and its complementary sequence carries the quencher or vice versa.

Another, more often used type of DNA probe, is the aptamer, first introduced in 1990 by the laboratories of Szostak and Gold [START_REF] Ellington | In vitro selection of RNA molecules that bind specific ligands[END_REF][START_REF] Tuerk | Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[END_REF]. Aptamers are single-stranded oligonucleotides ranging from 20 to 100 nucleotides in length, which can bind specifically to the target of interest (Figure 1). They are obtained by a process called Systematic Evolution of Ligands by EXponential enrichment (SELEX), developed by the two aforementioned teams. A randomized DNA (or RNA) library of around 10 14 sequences, flanked by specific primers is incubated with the immobilized target to allow binding. The unbound sequenced are washed, the rest is eluted and amplified by Polymerase Chain Reaction (PCR), dehybridized before going through another round of incubation with the target. The cycle is repeated between 8 to 30 times with increasing stringency, and the enrichment of bound sequences is followed by sequencing after each round. Some of the aptamers are consequently tested to single out the best ones. Since the 90s, several researchers have been trying to improve SELEX and adapt it to a wide variety of applications [START_REF] Sharma | ABCs of DNA aptamer and related assay development[END_REF][START_REF] Wang | Three decades of nucleic acid aptamer technologies: Lessons learned, progress and opportunities on aptamer development[END_REF]. Indeed, aptamers can recognize targets such as ions, small molecules, proteins, pathogens and eukaryotic cells. Aptamers are therefore considered as the nucleic acid analogs of antibodies, and their affinity and selectivity can sometime rival those of the more commonly known recognition molecules. Aptasensors can be found in as many different assay configurations as immonusensors based on antibodies, and benefit from the advantages inherent to the molecular nature of aptamers, including versatility in target recognition (even nonimmunogenic molecules), cost-effective development, long shelf-life and possible chemical modifications. The latter allows to graft aptamers on various nanomaterials [START_REF] Nguyen | Aptamer-based environmental biosensors for small molecule contaminants[END_REF][START_REF] Liu | Aptasensors for pesticide detection[END_REF].Aptamers adopt a three-dimensional structure when they recognize their target, a conformational change that is taken advantage of for sensing methods. It is interesting to note that, while many RNA aptamers have been generated by SELEX, very few can be found in the water quality field as they are more fragile than DNA, despite the various chemical modifications made to improve their stability [START_REF] Meek | Enhancing aptamer function and stability via in vitro selection using modified nucleic acids[END_REF]. . Alongside the development of aptamers, it is also possible to carry out the in vitro selection of oligonucleotides called DNAzymes , which possess catalytic properties that only work in the presence of metal ions as co-factors for which they have high specificity [START_REF] Mcghee | DNAzyme sensors for detection of metal ions in the environment and imaging them in living cells[END_REF]) (Figure 1).

When functionalized with a fluorophore-quencher pair on each end of the DNA strand, these DNAzymes allow on-site and real-time detection of picomolar concentrations of metal ions including UO2 2+ , Pb 2+ , Hg 2+ , Cu 2+ , Ag + , Cr 3+ , and Ce 3+ , Ni 2+ , Zn 2+ in different aqueous media. This list of the different types of nucleic acid-based sensors is far from being exhaustive. Starting from the same type of biomolecule, a wide variety of assays can be designed. Reminding us of building blocks, researchers can combine nucleic acids with different nanomaterials and different signal transducers. This is how, and why, hundreds of different biosensors have been developed; some more fit than others for the specific detection of a target of interest. The complexity of this field is then easily understood when we realize that it comes from its wealth in sensors architecture.

When we take an interest into what makes a biosensor fit for commercialization and used by the public, we find that optical based devices are the most successful. Based on plasmonic resonance, colorimetry or fluorescence [START_REF] Damborský | Optical biosensors[END_REF], their coupling to nanomaterials such as nanoparticles strongly enhances signal transduction (Table 1). Among noble metals, gold nanoparticles (AuNPs) are by far the most popular [START_REF] Aldewachi | Gold nanoparticle-based colorimetric biosensors[END_REF]. Smaller than 100 nm, they offer an ease of preparation and biocompatibility, making them perfect candidates for bioassay designs, all the more so since their unique optical, chemical, and electric properties can be explored for signal amplification. Their most common utilization is as platforms for colorimetric sensors: a color shift is observed according to their shape, size and aggregation state [START_REF] Jain | Calculated Absorption and Scattering Properties of Gold Nanoparticles of Different Size, Shape, and Composition: Applications in Biological Imaging and Biomedicine[END_REF]). The amino function groups found in the bases of nucleic acids non-covalently bind to the negative charges of AuNPS, stabilizing the particles' suspension. When a molecule of interest is added to the suspension, the DNA will preferentially bind to its target, leaving the AuNPS bare and susceptible to aggregation due to the presence of high salt concentration. The extent of the resulting absorbance shift being target concentration dependent [START_REF] Li | Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles[END_REF]. While simple visual observation can be used to detect this phenomenon, the resonance behavior of AuNPs provides an alternative transduction method. As AuNPs present unique Localized Surface Plasmon Resonance (LSPR) properties, sensitive to the mean interparticle distance, transduction is often coupled to Surface Enhanced Raman Spectroscopy (SERS). SERS enhances Raman scattering by a 10 10 factor when molecules are adsorbed on metal surfaces, allowing us to reach single molecule sensitivity, a perfect example of how nanotechnology can be used to improve detection limits [START_REF] Blackie | Single-Molecule Surface-Enhanced Raman Spectroscopy of Nonresonant Molecules[END_REF][START_REF] Wei | Plasmonic colorimetric and SERS sensors for environmental analysis[END_REF].

Despite their numerous advantages, sensors based on particle aggregation are yet to be used for in situ studies as further development is still needed. Since their properties are shape and sizedependent, a better control of these parameters is required during the particles' preparation. On the other hand, progresses made for point-of-care (POC) devices using such colorimetric assays could be transposed to environmental applications.

The other frequently developed type of sensor is based on electrochemistry. Ease-of-use and facile miniaturization are the main advantages of this sensing method [START_REF] Willner | Nanomaterial enabled sensors for environmental contaminants[END_REF].

The interaction of an analyte of interest with an electrode is measured, using one of four main modes: amperometric (change in current), potentiometric (change in potential), conductometry or impedance, and finally field-effect methods [START_REF] Thévenot | Electrochemical biosensors: recommended definitions and classification1International Union of Pure and Applied Chemistry: Physical Chemistry Division, Commission I.7[END_REF]) (Table 1). The last one is based on transistor technology and measures current as a result of a potentiometric effect at a gate electrode. The surface architecture of the electrodes has benefited from the development of nanotechnology, improving detection limits and lowering signal-to-noise ratios [START_REF] Hammond | Electrochemical biosensors and nanobiosensors[END_REF]. Overall, electrochemical sensing presents the most advantageous detection limits, and the possibility of reuse as regeneration of the surfaces is possible. Portability in most cases is also feasible making them interesting candidates for their use in environmental monitoring.

However, they do require instrumentation and complex, expensive fabrication steps when nanotechnology is involved.

Other transduction methods are not discussed in this review: piezoelectric effect, sensitive to mass change, and thermal-based sensors that rely on the energy released by biochemical reactions.

Since both are too sensitive to environmental conditions and not adapted for onsite use, we will not discuss them further here.

Nucleic acid amplification

Stemming from a growing need in the biomedical field, gene amplification techniques have been developed alongside biosensing strategies. The most popular, Polymerase Chain Reaction (PCR), was created by K. Mullis in 1983[START_REF] Mullis | Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction[END_REF]): a specific region of a DNA strand is amplified by a polymerase enzyme after repeated and cyclic steps of denaturation, specific primers hybridization to the target DNA strand (annealing) and elongation (Figure 1). PCR-based conventional methods rely on nucleic acid amplification of a specific target sequence, and are used for detection of pathogens at low concentration [START_REF] Ahmed | Biosensors for whole-cell bacterial detection[END_REF]. A limit of this strategy is the potential amplification of a contaminant sequence due to non-specific or mismatched hybridization of PCR primers. Therefore, a precise knowledge of the genetic information of interest is vital. Other downsides to its use on the field are the length and complexity of the sample preparation steps [START_REF] Lazcka | Pathogen detection: A perspective of traditional methods and biosensors[END_REF]) and the potential amplification of genetic material from non-viable cells [START_REF] Velusamy | An overview of foodborne pathogen detection: In the perspective of biosensors[END_REF]. The limitations of conventional PCR should be overcome by lab-onchip platforms integrating solid-phase PCR and recently proven to work for the detection of pathogens [START_REF] Hung | A novel lab-on-chip platform with integrated solid phase PCR and Supercritical Angle Fluorescence (SAF) microlens array for highly sensitive and multiplexed pathogen detection[END_REF].

Isothermal Amplification of nucleic acids have been developed as a simpler alternative to the former technique [START_REF] Zhao | Isothermal Amplification of Nucleic Acids[END_REF], allowing the detection of a wide spectrum of targets with higher sensitivities. They are commonly divided into three categories: linear amplification, cascade amplification and exponential amplification. Hybridization Chain Reaction (HCR), is an example of enzyme-free amplification process where an initiator sequence triggers a cascade of hybridization between two populations of DNA hairpins. The latter can be directly functionalized with labels or signaling molecules or incorporated to nanostructured platforms such as NPs for the detection of a broad range of targets [START_REF] Bi | Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine[END_REF]. Another method, called Exponential Amplification Reaction (EXPAR) has been thoroughly explored using a simple template, a polymerase and a nicking endonuclease achieving amplification up to a 10 8 fold in less than 30 min [START_REF] Reid | Exponential Isothermal Amplification of Nucleic Acids and Assays for Proteins, Cells, Small Molecules, and Enzyme Activities: An EXPAR Example[END_REF].

Although the detection of nucleic acids (miRNA, genomic DNA, ...) is the most straightforward application of isothermal amplification techniques, research has been led to incorporate them in assays for a broader range of targets by linking the oligos with recognition molecules and DNA probes, increasing the sensitivity of the detection of molecules that cannot be chemically amplified such as proteins or metal ions. To this versatility is the added bonus of a wide choice in the mode of detection of the amplification products, from simple colorimetry (AuNPs), to fluorescence and electrochemical transducers, displaying the important potential in developing amplification techniques to carry out on-site tests.

Proven sensing solutions

Among the abundant literature reporting a multiplicity of detection sensing solutions, it is worth emphasizing the following few examples of advanced or very successful developments commercially available (see also Table 2).

Metal ions

The real-time and on-site detection of metal ions is of primary importance as they have strong negative impact on human health. Lead, mercury, cadmium, chromium, copper, zinc, arsenic… produced by industrial activity or released by various equipment can compromise the quality of water resources. These elements are easily and efficiently identified by standard laboratory techniques, but those are not adapted to field measurements. Although aptamers have been successfully selected against a variety of metal ions (Abu-Ali, Nabok et al. 2019), DNAzymes sensors indisputably took the lead in the field of metal ion sensing due to their extremely high selectivity.

Several methods with limits of detection (LOD) in the pM range have been developed using the ease of DNA functionalization, to label it with fluorophores, electrochemical agents, or attach it to nanomaterials followed by various detection modes: fluorescence, SERS, colorimetry, electrochemistry or electroluminescence.

DNAzymes are synthetic single-stranded DNA sequences consisting of a substrate strand, with a ribonucleoside adenosine at the cleavage site for the RNA-cleaving DNAzymes, and an enzyme strand with catalytic activity in the presence of the analyte (Figure 1). The group of Yi Lu [START_REF] Li | A Highly Sensitive and Selective Catalytic DNA Biosensor for Lead Ions[END_REF] was the first to report the utilization of DNAzymes as metal ion biosensors, starting with Pb 2+ , which could be detected down to 10 nM with a high selectivity over other metal ions. After this breakthrough obtained using a fluorophore-quencher pair as a signaling method, alternative methods for the detection were investigated, aiming at reducing the required instrumentation and cost and/or enhancing the sensitivity, such as colorimetry with gold nanomaterials [START_REF] Liu | Accelerated Color Change of Gold Nanoparticles Assembled by DNAzymes for Simple and Fast Colorimetric Pb2+ Detection[END_REF] and SERS spectroscopy with Ag nanoparticles-on-a-film [START_REF] Fu | DNAzyme-Based Plasmonic Nanomachine for Ultrasensitive Selective Surface-Enhanced Raman Scattering Detection of Lead Ions via a Particle-on-a-Film Hot Spot Construction[END_REF]. Nowadays fluorescent DNAzyme-based sensors have reached LOD in the pM range. This is well below the guideline value in drinking water of 50 nM for lead for example which is one of the more stringent prescription with arsenic and selenium (WHO 2017). Commercially available in a portable format (ANDalyze) they are successfully applied to an extended list of metal ions in a large range of water types.

Chemicals and drugs

Synthetic chemicals are introduced in the water cycle by industrial and urban WWTP effluents and agriculture. While advanced processes can be used to improve the rate of chemicals' abatement at a WWTP output, and thus reduce the contamination of surface waters by chemicals of domestic and industrial origin, the application of crop protection products and fertilizers in agriculture is followed by their penetration in groundwater and their runoff in rivers in case of heavy rains. Pesticide contamination is one of the major problem of public health worldwide and will remain so for a long time. Indeed, pesticides can have long break down times and products banned more than ten years ago are still found in rivers and groundwater [START_REF] Cotton | Development and validation of a multiresidue method for the analysis of more than 500 pesticides and drugs in water based on on-line and liquid chromatography coupled to high resolution mass spectrometry[END_REF]. Excessive concentrations of pesticides and fertilizers is the major cause of abandonment of drinking water catchments. The demand for frequent and rapid control of pesticide content is strong and devices to meet this great challenge are eagerly expected. This context has motivated massive research efforts for the development of aptasensors. The first and essential step in this direction is the selection of fully potent aptamers. A dozen of aptamers are presently available for a variety of pesticides with good (Kd around 1 µM) to very good affinities (Kd between 10 to 100 nM), the best candidate being the aptamer against atrazine with a Kd of 0.6 nM [START_REF] Liu | Aptasensors for pesticide detection[END_REF]).

Colorimetric platforms using aptamers and gold nanoparticles allowing simple visual detection (see section 2) proved to be functional. In one version of these assays, DNA aptamers are used as stabilizers of a gold nanoparticle suspension and desorb from the particles surface in the presence of the analyte. Such assays are susceptible to be biased by the ion composition of matrices.

Here again sensors based on fluorescence detection have been designed following various strategies. Typically, a fluorophore is attached at one end of the aptamer and the other end is bound to a quencher, or to a surface acting as a quencher, leading to a loss of fluorescence as the analyte binds to the aptamer and changes its conformation. More complex designs, involving a complementary DNA sequence functionalized with the quencher that is released upon analyte binding, result in an increase of fluorescence signal upon aptamer conformational change. Validated in a large variety of aqueous matrices, the main limiting step of such aptasensors is the optimization of the design which becomes all the more difficult the longer the aptamer is.

Finally, an original impedimetric biosensor was proposed that is highly selective and has the lowest LOD of electrochemical sensors for atrazine equal to 10 pM [START_REF] Madianos | A highly sensitive impedimetric aptasensor for the selective detection of acetamiprid and atrazine based on microwires formed by platinum nanoparticles[END_REF].

Conventional Ti interdigitated electrodes are bridged by Pt nanoparticles microwires deposited along patterns designed by electron beam lithography. The sensor surface is then silanized to immobilize the thiol modified aptamers that hinder the charge transfer between electrodes upon binding of the analyte. This method which has been tested with two pesticides in tap and bottle mineral waters can be easily integrated in portable devices and could be exploited for the control of drinking water.

Pathogens

According to the World Health Organization, a plethora of bacteria, viruses, protozoa and parasites are recognized as harmful and are classified by health significance in their water quality guidelines. Conventional methodologies such as colony counting do not allow the continuous, realtime monitoring of the presence of pathogens in the environment, and while their use is still widespread researchers all over the world work to develop portable, rapid and sensitive biosensing platforms to overcome their limitations [START_REF] Sutarlie | Nanomaterials-based biosensors for detection of microorganisms and microbial toxins[END_REF][START_REF] Rajapaksha | A review of methods for the detection of pathogenic microorganisms[END_REF].

Waterborne pathogen sensing relies on either the detection of whole cells, a representative epitope (a membrane protein for example), genetic material or pathogen product such as toxins.

In terms of specificity, detecting the genetic material of a microorganism is by far the most secure way to determine its presence in a sample. As was discussed above, nucleic acid amplification techniques, particularly isothermal and enzyme-free methods, have taken over this particular field.

Paradoxically, however, most of the research published using these techniques is for the detection of cancer biomarkers, tumor cells, and pathogens for diagnostics and point-of-care purposes [START_REF] Mayboroda | Multiplexed isothermal nucleic acid amplification[END_REF]). The problem with genetic material, however, is that it does not provide information on the viability of a pathogen detected. Combining this type of assay with the detection of toxins released or RNA expression would offer better and more informative alternatives for water quality analysis. Here we present a few examples.

Several aptasensors have been developed for the detection of whole bacteria, viruses or spores [START_REF] Hong | Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications[END_REF][START_REF] Davydova | Aptamers against pathogenic microorganisms[END_REF]. Based on aptamers hybridized with complementary DNA, grafted on AuNPs and Upconversion Nano Particles respectively, an aptasensor reached a detection limit of 3 cfu/mL for the ATCC 8739 strain of E. coli in tap and pond water [START_REF] Jin | Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection[END_REF]). This sensitivity does not meet the requirements for drinking water [START_REF] Who | Guidelines for drinking-water quality[END_REF] but is suitable to evaluate the quality of recreational waters [START_REF] Eudirective | DIRECTIVE 2006/7/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 15 February 2006 concerning the management of bathing water quality and[END_REF][START_REF] Khang | Chemiluminescent aptasensor capable of rapidly quantifying Escherichia Coli O157:H7[END_REF].

Detection of toxins produced by pathogens is also useful. Of special concern are the toxins released by cyanobacteria because of their potential lethal effect. One of them, the microcystin-LR is hepatotoxic and can be detected using an aptamer developed against the toxin in 2012 [START_REF] Ng | Selection, Characterization, and Biosensing Application of High Affinity Congener-Specific Microcystin-Targeting Aptamers[END_REF]) leading to many different nanomaterial-based aptasensors [START_REF] Bostan | Determination of microcystin-LR, employing aptasensors[END_REF] reaching detection limits in the ng/L range, well below the guideline value set by WHO (WHO 2017) for drinking water.

Ozkan-Ariksoyal et al designed an electrochemical sensor targeting the conserved 16s-rDNA region of E. coli using DNA wrapped multi-walled carbon nanotubes from PCR amplified real samples, in less than 20 minutes and with a detection limit as low as 0.5 pmol (Ozkan-Ariksoysal, Kayran et al.

2017). Melaine et al successfully and simultaneously detected the RNA of Pseudomonas aeruginosa,

Legionella pneumophila and Salmonella typhimurium in a multiplex SPR imaging platform based on DNA probes coupled to AuNPS, applicable to a wider range of waterborne bacteria, with a LOD of 10 pg.mL -1 [START_REF] Melaine | Selective and High Dynamic Range Assay Format for Multiplex Detection of Pathogenic Pseudomonas aeruginosa, Salmonella typhimurium, and Legionella pneumophila RNAs Using Surface Plasmon Resonance Imaging[END_REF]. These methods require additional preparation steps, for which many integrative sensors are being tested.

Several methodologies were developed for diagnostics, assessment of water and food contaminations, using paper, microfluidics or lateral flow [START_REF] Kumar | Point-of-Care Strategies for Detection of Waterborne Pathogens[END_REF], showing promising future breakthroughs for rapid in situ microorganisms detection. For example, Feng Xu's lab created a paper-based device combining all the steps necessary for genetic material detection and analysis, including extraction and amplification [START_REF] Tang | A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection[END_REF].

Ongoing developments

DNA origami

As was stated previously, the polymorphism of DNA allows us to use it not only as a recognition element (probes) or a signal transducer (amplification), but also as the building block of a sensor architecture. In this section, we present DNA as a nanoscale platform upon which it is possible to graft recognition elements. The interactions between the recognition element and the target can be observed directly on the nanostructure, or indirectly by the modification of the shape of the platform. This last concept counts as an emerging tool for the development of bioassays and biosensors.

The in vitro constructs of DNA nanostructures have evolved during the last forty years [START_REF] Seeman | Nucleic acid junctions and lattices[END_REF][START_REF] Seeman | DNA in a material world[END_REF][START_REF] Goodman | Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication[END_REF]) to be used as sophisticated large scaffolds called origami with broad potentials. Using bioinformatics and the large single stranded DNA of the M13 phage, combined to short ssDNA called "staples", it is now possible to create a diverse catalog of DNA structures [START_REF] Rothemund | Folding DNA to create nanoscale shapes and patterns[END_REF]. Many studies demonstrated the potential use of origami for target drug delivery and diagnostics (for a complete review concerning recent progress in origami studies see Ijäs [START_REF] Ijäs | Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems[END_REF]) and Sakai [START_REF] Sakai | DNA Aptamers for the Functionalisation of DNA Origami Nanostructures[END_REF]). We focus here on the evolution of DNA origami for biosensing applications. Ke and al. designed tiles as arrays (built like DNA chips) with a 2D origami on which DNA probes were grafted to detect (RNA) target in solution and observed by Atomic Force Microscopy (AFM) to quantify hybridization events [START_REF] Ke | Self-Assembled Water-Soluble Nucleic Acid Probe Tiles for Label-Free RNA Hybridization Assays[END_REF]. This paved the way to use origami in sensing devices, demonstrated in 2016 by grafting the aptamer targeting the malaria biomarker PfLDH on origami tiles [START_REF] Godonoga | A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly[END_REF]. Beyond the simple use of origami as a support for recognition molecules, Andersen showed that it was possible to have a conformational transition of a 3D origami (a box) in response to stimuli to detect DNA (or RNA) in solution, observable by AFM, TEM or FRET [START_REF] Andersen | Self-assembly of a nanoscale DNA box with a controllable lid[END_REF]. Recently the same origami box was designed to detect plasmodium using the malaria biomarker aptamer [START_REF] Tang | An aptamer-enabled DNA nanobox for protein sensing[END_REF].

Other designs of DNA origami that fold differently in response to various stimuli were published by Kuzuya [START_REF] Kuzuya | Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy[END_REF]. In this article authors constructed origami pliers that could detect metal ions by zipping, small molecules (miRNA and ATP) by unzipping and proteins by pinching mechanisms, at the single molecule resolution. In a similar manner, Ke and al. [START_REF] Ke | Regulation at a distance of biomolecular interactions using a DNA origami nanoactuator[END_REF] designed an elegant tunable DNA origami device named "nanoactutor" responding to different stimuli ranging from small ions, to large molecules like miRNA and proteins.

These studies demonstrated the potential of origami as a signal transducer. However reaching single molecule detection still has to be improved, and the most important problem is of course the signal readout, as AFM is not transposable to carry out in situ studies and demands expertise.

Another limitation is the growing cost of some of these complex structures compared to the more basic sensors discussed in this review. It is too early to have an idea about the reproducibility and the selectivity of biosensors based on origami. In terms of sensitivity that could be reached, Selnihhin et al. [START_REF] Selnihhin | Multifluorophore DNA Origami Beacon as a Biosensing Platform[END_REF]) recently developed a high-throughput, optical DNA origami nanobiosensor based on strand displacement by the target DNA sequence at concentrations down to 100 pM, with future multiplexing development (Figure 2). We believe DNA origami is still in the early stages of its development for application purposes, but presents an important potential that still needs to be explored. One family of these methods where the motion of a particle linked via a DNA tether to a surface is observed by video-microscopy can be diverted for a use as a biosensor for the analysis of water pollutants. Indeed, the efforts devoted to the development of these new tools have led to progress in nanoscale engineering leading to reliable, accurate and robust techniques. Pulling on the particle, while measuring the extension of the DNA molecule determined by tracking the particle's position in real time, informs about DNA conformation and elasticity. It is referred to as single molecule force spectroscopy measurements [START_REF] Bustamante | Ten years of tension: single-molecule DNA mechanics[END_REF].

Single DNA sensing

Halvorsen et al designed a clever approach using DNA origami to construct a DNA tether with two distant functional sites [START_REF] Halvorsen | Nanoengineering a single-molecule mechanical switch using DNA self-assembly[END_REF]) and optical tweezers to exert a force on the DNA tether. This simple method gives in principle access to measurements of force-dependent bond rupture and bond formation of pairs of interacting molecules which can be performed repeatedly for the analysis of molecular interactions (Figure 3 A). A simpler design of DNA tethered particle pulling assay is proposed by Silver and Neuman [START_REF] Silver | Tethered-bead, immune sandwich assay[END_REF] as sandwich immunoassay. The sensor uses DNA tethers attached by one end to a flow cell surface, particles bind the other end of the DNAs via an immune sandwich formed in case of capture of the analyte (Figure 3 B). The number of tethered particles determined by the particles displacement upon application of a flow allows to quantify the concentration of analyte with a detection limit down to pM, at the prototype stage.

Even more simple but effective is the so-called particle mobility sensing by Visser et al [START_REF] Visser | Continuous biomarker monitoring by particle mobility sensing with single molecule resolution[END_REF] wherein no force is applied on the DNA tethered particle. In this sandwich assay, the particle and the surface are functionalized with affinity molecules (Figure 3 C). The particle experiences successive reversible binding events, leading to immobilization periods upon recognition of the analyte, the duration of which is related to the analyte concentration. Demonstrated on model biomarkers, single stranded DNA and a protein in complex media, the technology has a picomolar range sensitivity and the potential for a broad range of applications.

An improvement of these single molecule sensing methods is their controlled parallelization by self-assembly of the DNA tethered particles on regular nanoarrays. Using a standard microcontact printing technique, Plénat, Tardin et al [START_REF] Plénat | High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion[END_REF] successfully developed a single DNA biochip for the simultaneous acquisition of data from more than 500 DNA molecules, strongly increasing the yield and quality of the output (Figure 3 D). Together with the possible miniaturization of the readout system using mobile phone-based wide field fluorescence or dark-field imaging [START_REF] Mcleod | Democratization of Nanoscale Imaging and Sensing Tools Using Photonics[END_REF], these capabilities make single DNA tethered particle methods promising platforms for the development of systems for water monitoring in either laboratory or field conditions.

Concluding remarks

Biosensor development for environmental contamination is growing rapidly, benefiting from the advances made in new nanotechnologies and single molecule assays. As demonstrated by a first successful industrialization based on DNAzymes, DNA nanobiosensors are excellent candidates for the implementation of highly sensitive, specific and rapid methods into portable and cost-effective devices. We soon expect to see more to arrive on the market, combining DNA with nanomaterials in creative ways, continuously enriching the field.

The ideal assay would be a fully integrated device, something already conceptualized with lab-onchip and paper-based sensors, overcoming the tedious preparation steps necessary in conventional methods [START_REF] Mahadeva | Paper as a Platform for Sensing Applications and Other Devices: A Review[END_REF]. Another benefit would be the accessibility of these cheaper, one-shot devices for low income countries.

We foresee that new solutions, transposable to environmental monitoring, inspired from biomedical devices will emerge in the future. Indeed, sensors developed for point-of-care diagnostics and personalized medicine share similar technical specifications with water quality monitoring systems. (1,6x10 3 -100x10 3 ) a 2 (2-50) Fluorimetric DNAzyme sensor ANDalyze.com Atrazine 100 0,1 2,5x10 -6 (10 -3 -0,064) Aptamer grafted on graphene in a photoelectrochemical sensor. [START_REF] Sun | Immobilization-free photoelectrochemical aptasensor for environmental pollutants: Design, fabrication and mechanism[END_REF] 0-10 b 0,002 (0,02-200) Aptamer and platinum NPs grafted on microwires for impedance spectroscopy. [START_REF] Madianos | A highly sensitive impedimetric aptasensor for the selective detection of acetamiprid and atrazine based on microwires formed by platinum nanoparticles[END_REF] Microcystin-LR 1 1 0,03 Steric hindrance initiated signal amplification effect [START_REF] Du | Building a Three-Dimensional Nano-Bio Interface for Aptasensing: An Analytical Methodology Based on Steric Hindrance Initiated Signal Amplification Effect[END_REF]) 0,005 (0,03-1) c 0,002 (0,004-0,512)

Interaction with DNA-modified electrodes in impedimetric sensor [START_REF] Zhang | Electrochemical detection of microcystin-LR based on its deleterious effect on DNA[END_REF] E. coli 0/100mL 0/100mL 3 cfu/mL (5-10 6 cfu/mL) AuNPs and Upconverstion particles FRET aptasensor [START_REF] Jin | Upconversion nanoparticles based FRET aptasensor for rapid and ultrasenstive bacteria detection[END_REF]) <10 3 cfu/mL (6×10 3 -4×10 5 cfu/ml) d 10 cfu/mL (10-10 6 cfu/mL) Graphene functionalized impedimetric aptasensor [START_REF] Kaur | Bridged Rebar Graphene functionalized aptasensor for pathogenic E. coli O78:K80:H11 detection[END_REF])

Figure captions

  In contrast to the complex DNA origami structures, single molecule approaches emerge that propose simple designs of detection assays. Single molecule sensing is an exciting and extremely active field bearing the promise of very high sensitivity. After a few decades of instrumental developments in optics, scanning probe microscopy and nanofabrication, measurements at the single molecule level are now part of the scientist's toolbox. DNA has been one of the favorite object of interest for the pioneers of single molecule methods. With a view to elucidate biological behaviors by unmasking the effects of averaging inherent to conventional methods, a panel of techniques have been developed to visualize and manipulate DNA leading to important contributions in the detailed understanding of the cellular processes involving DNA (Kapanidis and Strick 2009). Remarkable technological achievements are the third generation sequencing methods, single molecule long-read methods, which took only about 10 years to go successfully from the bench to the market (van Dijk, Jaszczyszyn et al. 2018).

Figure 1 :

 1 Figure 1: Main features of DNA nanobiosensors for water monitoring. The various water contaminants (metal ions M x+ , chemicals and pathogens) can be detected through different processes via DNA-based recognition elements: target induced conformational changes for DNAzymes and aptamers and, nucleic acid amplification for oligos (primers).

Figure 2 :

 2 Figure 2: DNA origami sensing platform. Two arrays made of DNA origami harbor multiple donor and acceptor fluorophore pairs for enhanced FRET signal. The device immobilized on a glass slide switches from closed to opened state upon binding of the target to the recognition elements (sensor modules) on the free edges of the arrays. Reproduced and adapted from Selnihhin et al ACS Nano 2018 (Selnihhin, Sparvath et al. 2018) with permission of the American Chemical Society and from Xavier and Chadrasekaran Nanotechnology 2018 (Xavier and Chandrasekaran 2018) with permission of IOP Publishing Ltd.

Figure 3 :

 3 Figure 3: Single DNA tethered particle mobility sensing. The displacement of the particle under an applied force using either magnetic or optical tweezers (A) or flow (B) allows, with ad hoc functionalizations of the DNA tether, to analyze interactions between receptor and ligand grafted to distant locations on the DNA (A, adapted from Halvorsen et al Nanotechnology 2011 (Halvorsen, Schaak et al. 2011) with permission of IOP Publishing Ltd or to identify the presence of an analyte (B, adapted from Silver et al Biosensors and Bioelectronics 2015 (Silver, Li et al. 2015) with permission of Elsevier). In the absence of force, target binding events between the recognition elements on the particle and the substrate are detected through immobilization periods of the particle (C, reproduced from Visser et al Nature Communications 2018 (Visser, Yan et al. 2018)). The parallelization of the Tethered particle Motion technique by the controlled positioning of the tethered particles on regular nanoarrays drastically improves the throughput capacity (D, reproduced from Plénat, Tardin et al Nucleic Acids Research 2012 (Plénat, Tardin et al. 2012) with permission of Oxford University Press). Dark field microscopy images of single DNA tethered particles scale bars equal to 50 µm and 20 µm for upper and lower frames, respectively.

Table 1 : Overview of nanomaterial-based biosensors transduction methods.

 1 For further information, see[START_REF] Sutarlie | Nanomaterials-based biosensors for detection of microorganisms and microbial toxins[END_REF]).

	Transduction	Methods	Nanomaterials associated
	Optical	Colorimetry
		Raman spectroscopy (SERS) and infrared spectroscopy (FTIR)	Noble metal nanoparticles
		Surface Plasmon resonnace (SPR)
		Fluorescence Chemiluminescence	Quantum dots, carbon nanotubes, graphene
		Single-molecule microscopy	Polystyrene or magnetic nanoparticles
	Electrochemical	Amperometry
		Conductimetry
			Modification of the electrode itself
		Impedance spectroscopy	(gold, silver…) Carbon nanotubes,
			graphene…
		Potentiometry
		Field-effect transistors

Table 2 : Performances of DNA-based nanobiosensors for the detection of micropollutants and microorganisms in water, with regard to EU and WHO guideline values for drinking water and commercial ELISA tests.

 2 Examples of targets were selected to represent the following types of contaminants: metal ions, chemicals, microorganisms. ( a ) Abcam, ( b ) Creative Diagnostics, ( c,d ) MyBioSource. N.A. not available.

	Micropollutant/ microorganism	WHO Guideline values (WHO 2017)	EU quality standards (EUdirective 2015)		DNA-based nanobiosensors tested in natural water	ELISA commercial test
		µg/L (in drinking water)	LOD (range) µg/L	Method	Reference	LOD (range) µg/L
				0,002 (0,01-20)	Chronocoulometric DNAzyme sensor using AuNPs.	(Zhang, Lai et al. 2016)
	Lead	10	10	0,1 (0,5-20)	Portable DNAzyme sensor with magnetic beads and Platinum NPs.	(Huang, Lin et al. 2019)	N.A.
	Mercury	6	1	0,3 (0,5-20)	Colorimetric aptasensor using Rolling Cycle Amplification	(Wu, Yu et al. 2020)	1,6 x10 3

Acknowledgments

We thank the colleagues of our research teams of IPBS "Membrane and DNA dynamics" and "Cellular Biophysics". D. Soukarié is the recipient of a joint Agence Innovation Defense/University of Toulouse PhD fellowship.