Guillaume Daufouy

Nathalie Paniel

Thierry Noguer

Lise Barthelmebs
email: barthelm@univ-perp.fr

Selection of spore-specific aptamers for Geobacillus stearothermophilus, a food spoilage bacterium

Keywords: Aptamers, Spore-SELEX, Geobacillus stearothermophilus, Food spoilage

Due to their ability to form extremely heat resistant spores, anaerobic bacteria are responsible for frequent food spoilage. The development of rapid and specific methods for the detection and quantification of spore contamination is therefore of major interest. In this paper, we describe for the first time the selection of aptamers specific to spores of Geobacillus stearothermophilus (Gbs), which induce flat sour spoilage in vegetable cans. Eighteen Spore-SELEX cycles were performed including 4 counter-selections with 12 bacteria commonly found in cannery. To optimise candidate amplification, PCR in emulsion was performed, and high-throughput sequencing analysis was applied to follow candidate evolution. Sequencing of aptamers from cycle 18 revealed 43 overrepresented sequences whose copy number exceeds 0.15% of the total obtained sequences. Within this group, the A01 aptamer presented a much higher enrichment with a relative abundance of 17.71%. Affinity and specificity for Gbs spores of the 10 most abundant candidates at cycle 18 were confirmed by PCR assay based on aptamerspore complex formation and filtration step. Obtaining these aptamers is the starting point for the future development of biosensors dedicated to the detection of Gbs spores.

Highlights:

-G. stearothermophilus spore-specific aptamers were selected by Spore-SELEX -Next Generation Sequencing data analysis identified 43 candidate sequences -Spore binding affinity was confirmed for top 10 candidates

INTRODUCTION

The development of spore-forming thermophilic bacteria in canned food may result in food spoilage and food poisoning [START_REF] Prevost | PCR Detection of Thermophilic Spore-Forming Bacteria Involved in Canned Food Spoilage[END_REF]. The thermophilic bacteria species usually found in those products are either facultative anaerobes such as Geobacillus stearothermophilus (Gbs) or obligate anaerobes such as Thermoanaerobacterium thermosaccharolyticum and Moorella thermoacetica [START_REF] Prevost | PCR Detection of Thermophilic Spore-Forming Bacteria Involved in Canned Food Spoilage[END_REF][START_REF] Burgess | Insights into the Geobacillus stearothermophilus species based on phylogenomic principles[END_REF]. These species are non-pathogenic Gram-positive bacteria which are able to grow in high temperature environments. Their spores are extremely heat resistant, with a decimal reduction time of a few minutes at 121 °C [START_REF] Prevost | PCR Detection of Thermophilic Spore-Forming Bacteria Involved in Canned Food Spoilage[END_REF][START_REF] Malleck | Temperature impacts the sporulation capacities and spore resistance of Moorella thermoacetica[END_REF]. In food production, growth of Gbs spores results in flat sour spoilage due to acid production associated to little or no gas production [START_REF] Scott | The formation of thermophilic spores during the manufacture of whole milk powder[END_REF][START_REF] Olson | Thermophilic Flat Sour Sporeformers[END_REF] while thermophilic obligate anaerobes usually produce abundant gas, responsible for can swelling [START_REF] Prevost | PCR Detection of Thermophilic Spore-Forming Bacteria Involved in Canned Food Spoilage[END_REF]. These spoilages are mainly observed in low acidic food such as milk, vegetables, meat and marine products under improper storage conditions. The others most frequent spore forming bacteria identified in canned food belong to the Bacillus, Thermoanaerobacter, Caldanaerobius, Anoxybacillus, Paenibacillus and Clostridium genera [START_REF] Postollec | Tracking sporeforming bacteria in food: From natural biodiversity to selection by processes[END_REF][START_REF] André | Thermophilic spore-forming bacteria isolated from spoiled canned food and their heat resistance. Results of a French ten-year survey[END_REF].

Standardised heat treatments are generally applied to prevent canned food spoilage. Their effectiveness can be verified by carrying out stability controls on the finished products. The Codex Alimentarius, a collection of internationally adopted food standards and related texts designed to protect the health of consumers, defines an essential incubation period for international shipments of canned foods. The containers must be incubated for 10-14 days at 37°C or 10 days at 35°C. These controls are also subject to regional or national standards. For example, stability tests for canned products are defined by the French standards NF V08-401 [START_REF] Afnor | Microbiologie des aliments -Contrôle de la stabilité des produits appertisés et assimilés -Méthode de référence[END_REF] as reference method and NF V08-408 [START_REF] Afnor | Microbiologie des aliments -Contrôle de la stabilité des produits appertisés et assimilés -Méthode de routine[END_REF] as routine method, consisting in food product incubation for 21 days or 7 days respectively at 32°C, 37°C or 55°C. Following this incubation step, various observations are performed such as aspect, odour, texture, pH measurement and microscopic examination. Specific detection of spore-forming bacteria species involves classical plate count method. These traditional culture-based assays require multiple steps and are guided by different standards such as the following French standards NF V08-405 [START_REF] Afnor | Microbiologie alimentaire -Conserves -Recherche de {Clostridium} thermophiles[END_REF], NF V08-407 [START_REF] Afnor | Microbiologie alimentaire -Matières premières entrant dans la composition des conserves -Dénombrement des spores thermorésistantes de Bacillus et Clostridium thermophiles -Technique du nombre le plus probable[END_REF], NF V08-250 [START_REF] Afnor | Microbiologie des aliments -Traitements thermiques préalables au dénombrement ou à la recherche de spores bactériennes[END_REF], NF V08-602 [START_REF] Afnor | Microbiologie des aliments -Dénombrement des spores dans les produits alimentaires avant traitement d'appertisation par comptage des colonies[END_REF]. These methods allow the detection of 4 groups of spore-forming bacteria including aerobic mesophilic and thermophilic, and anaerobic mesophilic and thermophilic, using two culture media (either BromoCresol Purple agar (BCP) or Meat Liver Agar (MLA)) and two incubation temperatures (37 °C or 55 °C).

The incubation times are comprised between 2 and 5 days depending on the analysed group. These methods require specific equipment, are labour intensive, time consuming and are not always successful if spores fail to germinate [START_REF] Nakano | Detection and quantification of thermophilic spore-forming Moorella thermoacetica in canned beverages using real-time PCR[END_REF].

Faster and more sensitive detection methods have been recently developed based on molecular techniques. Among these methods, Polymerase Chain Reaction (PCR) has been the subject of considerable attention. Real-time quantitative PCR (qPCR) is now regarded as a robust method for the detection and quantification of microorganisms. Its main advantages over conventional culture methods are related to the rapidity, sensitivity, and specificity of the method, and the possibility of simultaneous detection of different species [START_REF] Postollec | Recent advances in quantitative PCR (qPCR) applications in food microbiology[END_REF]. However, PCR methods are not really specific for bacterial spores detection as they are based on the recognition of genetic material, which does not differ between spores and vegetative forms [START_REF] Nakano | Detection and quantification of thermophilic spore-forming Moorella thermoacetica in canned beverages using real-time PCR[END_REF]. Only the detection of non-spore forming foodborne pathogens by PCR is governed by ISO standards (ISO 22174:2005 [START_REF] Iso | Microbiologie des aliments -Réaction de polymérisation en chaîne (PCR) pour la recherche de micro-organismes pathogènes dans les aliments -Exigences générales et définitions[END_REF], ISO 20836:2021 [START_REF] Iso | Microbiologie de la chaîne alimentaire -Réaction de polymérisation en chaîne (PCR) pour la recherche de micro-organismes -Essais de performance thermique des thermocycleurs[END_REF], ISO 20837:2006 [START_REF] Iso | Microbiologie des aliments -Réaction de polymérisation en chaine (PCR) pour la détection des micro-organismes pathogènes dans les aliments -Exigences relatives à la préparation des échantillons pour la détection qualitative[END_REF], ISO 20838:2006 [START_REF] Iso | Microbiologie des aliments -Réaction de polymérisation en chaine (PCR) pour la recherche des micro-organismes pathogènes dans les aliments -Exigences relatives à l'amplification et à la détection pour les méthodes qualitatives[END_REF]).

Consequently, the development of reliable alternative methods for the detection and quantification of microbiological risk linked to spore contamination has become of major interest. For such purposes, biosensors appear as promising tools as they allow rapid and efficient diagnosis, they are easy to operate, and they can be miniaturised [START_REF] Vidic | Advanced biosensors for detection of pathogens related to livestock and poultry[END_REF]21]. These analytical devices are made of the tight association of a biorecognition molecule, often called bioreceptor, and a physical transducer allowing the conversion of the recognition event into a measurable signal. The bioreceptor is the key element since its properties condition the sensitivity and the selectivity of the biosensor [START_REF] Surribas | Monoclonal Antibody-Based Immunosensor for the Electrochemical Detection of Chlortoluron Herbicide in Groundwaters[END_REF][START_REF] Rocaboy-Faquet | A novel amperometric biosensor for ß-triketone herbicides based on hydroxyphenylpyruvate dioxygenase inhibition: A case study for sulcotrione[END_REF]. Biosensors dedicated to bacteria detection very often involve antibodies able to specifically recognise determinants present on the bacterial cell surface. However, it is often difficult to design antibodies that are able to discriminate between vegetative and spore forms of the same bacteria. In some cases, the knowledge of the composition of cell surfaces can facilitate the development of specific antibodies [START_REF] Da-Silva | Biosensing platforms for Vibrio bacteria detection based on whole cell and nucleic acid analysis: A review[END_REF][START_REF] Leva-Bueno | A review on impedimetric immunosensors for pathogen and biomarker detection[END_REF]. For instance, antibodies have been produced for the spores of B. anthracis and B. cereus, for which a specific oligosaccharide named anthrose, present in the exosporium, has been identified [START_REF] Mehta | Synthesis and antigenic analysis of the BclA glycoprotein oligosaccharide from the Bacillus anthracis exosporium[END_REF]. However, external structures are not so well deciphered for all the spore forming bacteria, thus hampering the development of appropriate antibodies.

One way to tackle selectivity problems could consist in using aptamers as bioreceptors. Aptamers are short synthetic single-stranded oligonucleotides (20-100 nucleotides) that are able to bind to a target with high affinity and specificity thanks to their three-dimensional structures. Aptamer selection occurs in vitro through an iterative process termed Systematic Evolution of Ligands by EXponential enrichment (SELEX), which can be adapted to the needs of the intended application. This protocol has been used for the selection of aptamers against a wide range of targets, from small molecules [START_REF] Mckeague | Screening and Initial Binding Assessment of Fumonisin B1 Aptamers[END_REF][START_REF] Stojanovic | Aptamer-Based Folding Fluorescent Sensor for Cocaine[END_REF][START_REF] Paniel | Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor[END_REF] to whole cells [START_REF] Morris | High affinity ligands from in vitro selection: Complex targets[END_REF], protozoan parasites [START_REF] Homann | Combinatorial selection of high affinity RNA ligands to live African trypanosomes[END_REF] or pathogen bacteria [START_REF] Liu | Aptamers against Pathogenic Bacteria: Selection Strategies and Apta-assay/Aptasensor Application for Food Safety[END_REF][START_REF] Wang | QCM-based aptamer selection and detection of Salmonella typhimurium[END_REF][START_REF] Yu | Whole-bacterium SELEX of DNA aptamers for rapid detection of E.coli O157:H7 using a QCM sensor[END_REF]. To date, specific aptamers against bacterial spores have been selected for B. anthracis [START_REF] Bruno | In vitro selection of DNA aptamers to anthrax spores with electrochemiluminescence detection[END_REF][START_REF] Bruno | Development of aptamer beacons for rapid presumptive detection of Bacillus spores[END_REF], B. thuringiensis [START_REF] Fan | Aptamer selection express: a novel method for rapid single-step selection and sensing of aptamers[END_REF], Alicyclobacillus acidoterrestris [START_REF] Hünniger | Food Sensing: Selection and Characterization of DNA Aptamers to Alicyclobacillus Spores for Trapping and Detection from Orange Juice[END_REF], and B. cereus [START_REF] Fischer | Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk[END_REF].

However, only a few aptamers against bacterial spores have been used for analytical purposes. In 2007, an assay was developed for the detection of B. thuringiensis spores based on the fluorescence observed upon binding of an aptamer-quantum dot conjugate to spores [START_REF] Ikanovic | Fluorescence Assay Based on Aptamer-Quantum Dot Binding to Bacillus thuringiensis Spores[END_REF]. In 2017, Fischer et al. integrated an aptamer recognising A. acidoterrestris spores into a Lateral Flow Assay (LFA) [START_REF] Fischer | Aptamers: Universal capture units for lateral flow applications[END_REF]. More recently, a colorimetric paper chip was developed in 2020 for the detection of B. thuringiensis spores [START_REF] Zhou | Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores[END_REF]. These analytical methods were all tested only under laboratory conditions and in buffered solutions.

The aim of this work was to select aptamers able to bind with spores of Gbs, which is with M. thermoacetica one of the two strains most frequently found in spoiled canned food [START_REF] André | Thermophilic spore-forming bacteria isolated from spoiled canned food and their heat resistance. Results of a French ten-year survey[END_REF]. For this purpose, a SELEX protocol was carried out using spores produced from a Gbs strain isolated from green beans sample, and including numerous counter-selection cycles either with vegetative Gbs cells, or spore suspensions from 12 others spore forming bacteria. PCR was performed in emulsion (emPCR) for a better amplification of DNA from random libraries. Next Generation Sequencing (NGS) was then applied to allow a deeper candidate analysis and to monitor DNA aptamer evolution over cycles. The interaction of spores with the most frequently selected aptamers was then confirmed by aptamer-target complex formation confirmation by PCR.

MATERIALS AND METHODS

Reagents, buffers and culture media

All reagents and chemicals used were of either analytical grade or HPLC grade. All oligonucleotides, including single stranded DNAs (ssDNAs) library, aptamers and primers, were purchased from Microsynth AG (Balgach, Switzerland). The ssDNA library contained a central randomised sequence of 40 nucleotides flanked by two 18 nucleotides primer-binding regions for SELEX PCR amplification: 5'-CATCCGTCACACCTGCTC-40N-GGTGTTGGCTCCCGTATC-3'. Two different primers were used for SELEX PCR amplification: Gbs-F 5'-CATCCGTCACACCTGCTC-3', and Gbs-R 5'-biotin-GATACGGGAGCCAACACC-3'. Two other primers were used for NGS analysis: NGS-F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCATCCGTC ACACCTGCTC-3' and NGS-R 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATACGGG AGCCAACACC-3'. The aptamer used as negative control for spore-aptamer binding assays was designed as follow: 5'-CATCCGTCACACCTGCTCGTCAGCGATCAGTTAGCTAGAAGCGGTGTTGGCTCCCGTATC-3'. The working solution of ssDNA library was prepared at 1 nM in the selection buffer (100 mM NaCl, 20 mM Tris HCl pH 7.6, 5 mM KCl, 1 mM CaCl2, 5 mM MgCl2). All PCR amplifications were carried out with the Go Taq ® G2 Flexi PCR kits from Promega (Charbonnières les Bains, France).

Bacterial strain and spore production

All strains used in this study were isolated from canned food and identified using 16S sequencing by the French Technical Centre for the Preservation of Agricultural Products (CTCPA). A primary revivification culture was incubated during 48 h at 55 °C in Brain-Heart Infusion (BHI) broth. This primary culture was used as an inoculum for the scale-up multiplication culture that was incubated for 48 h at 55 °C in BHI broth. This suspension was designed as vegetative cell suspension. To produce spores, 1 mL of vegetative cell suspension was inoculated on Gbs sporulation medium agar plates (GSM), composed of meat extract (10 g/L), yeast extract (2 g/L), type E agar (15 g/L), and MnSO4 monohydrate (0.04 g/L). The plates were incubated at 55 °C for at least 3 days. Sporulation, following by phase-contrast microscopy counting (Olympus BX50), was stopped when free spores represented more than 90% of cells. Spores were then harvested by pouring distilled sterile water into the plates and gently scraping their surface, and the obtained suspensions were centrifuged at 6 000 g for 20 min at 4 °C. The resulting pellet was resuspended in 5 mL of sterile water, heated 10 min at 100 °C to kill vegetative cells, cooled and stored at 4 °C until use. Spore production from other bacterial strains used for counter-selections were performed similarly but using their corresponding medium (Table 1). All strains were isolated from spoiled canned food and identified by the CTCPA.

Enumeration method

To estimate bacterial concentration, ten-fold dilution series were prepared from vegetative cell suspensions in tryptone salt broth, and 1 mL of each dilution was spread onto agar plates containing various media following incubation at the appropriate temperature (Table 1). Colonies Forming Units (CFU) were then enumerated.

Counting of spores was carried out using the same protocol with a preheating step of 10 min at 100 °C, performed prior to enumeration, to eliminate vegetative cells and induce germination.

Purification of Gbs spore suspension

To optimise the purity of Gbs spore suspension, an additional step was added. The remaining vegetative cells and cellular debris were eliminated by three washing steps followed by centrifugation at 200 g for 5 min at 4 °C. The obtained pellet was resuspended in cold sterile water. Spore purity was estimated after each washing step by phasecontrast microscopy. After the three washing steps, spore concentration was enumerated as described before.

In vitro selection of Gbs spore aptamer

The SELEX protocol used in this work was adapted from the one described by Hünniger et al. [START_REF] Hünniger | Food Sensing: Selection and Characterization of DNA Aptamers to Alicyclobacillus Spores for Trapping and Detection from Orange Juice[END_REF] with some modifications (Fig. 1).

The first four cycles started by a counter-selection step against Gbs vegetative cells suspension. Before use, the ssDNA library was heated at 95 °C for 5 min, cooled on ice for 5 min and kept at room temperature for 20 min to allow the formation of folded DNA structures. Denaturation step was performed at the beginning of each SELEX cycle. For the first cycle, 100 µL of 1 nM denatured ssDNA library were incubated for 60 min with Gbs vegetative cells suspension at a final concentration of 10 5 CFU/mL. After the binding reaction, the mixture was centrifuged during 12 min at 10 500 g. The pellet containing vegetative cells-bound oligonucleotides was discarded, and the supernatant was used for the next SELEX step. After a new denaturation step, the collected oligonucleotides were incubated for 60 min with purified Gbs spores at a final concentration of 10 5 spores/mL. A centrifugation was then performed during 12 min at 10 500 g, the supernatant was discarded, and the pellet was washed several times with 200 µL of binding-and-washing buffer (BW: 1 mM EDTA, 2 M NaCl, 10 mM Tris-HCl, pH 7.5), and resuspended in 35 µL of sterile nuclease-free water. A heating step of 5 min at 95 °C was performed to separate the spores from the oligonucleotides, followed by a quick centrifugation (10 s) at 10 500 g. The aptamer candidates were finally collected in the supernatant. Amplification of ssDNAs was achieved by emPCR, carried out using the Micellula DNA Emulsion and Purification Kit (EURX, Gdańsk, Poland). The PCR mixture for amplifying ssDNA candidates contained 0.025 U of DNA Polymerase, 1X of buffer, 2 mM of MgCL2, 0.8 mM of dNTPs and 4 µM of each primer Gbs-F and Gbs-R. Positive and negative emPCR controls were performed using ssDNAs from the starting library and ultrapure water, respectively.

Oil-phase was prepared separately according to Micellula protocol and kept on ice. PCR mixture was added in the cold oil-phase and mixed vigorously for 5 min at room temperature to create emulsion. The mixture was divided into several 50 µL aliquots and emPCR was carried out under the following conditions: preheat at 95 °C for 5 min, 30 cycles of 95 °C for 30 s, 61.5 °C for 30 s, 72 °C for 30 s and final elongation at 72 °C for 5 min. Finally, the emulsion was broken using methanol, the oil-phase was discarded and emPCR product was purified using Micellula kit.

A strand separation step was mandatory to retrieve only ssDNA aptamers. For this purpose, 100 µL of streptavidin magnetic beads (Dynabeads M-270 Streptavidin, Fisher Scientifics S.A.S.) were incubated under agitation with biotinylated emPCR products in BW buffer. After 15 min, the supernatant was discarded, the beads were resuspended in 80 µL of sterile distilled water and heated during 10 min at 95 °C for emPCR products denaturation.

The tube was immediately centrifuged for 10 s at 10 500 g and placed on a magnetic rack. The hot supernatant containing the desired aptamers was collected and kept at 4 °C until the following cycle.

The monitoring of the SELEX process was assessed by 7% denaturing PolyAcrylamide Gel Electrophoresis (PAGE). Thus, the emPCR products after purification and after strand separation were deposited as well as the emPCR positive and negative controls. The bands were revealed by post staining using 3X GelRed ® Nucleic Acid Stain.

Subsequent selection cycles were performed with decreasing the amount of input target (from 20 µL to 15 µL spore solution) and incubation times (from 60 to 10 min) and increasing the number of washing steps (from 1 to 3). All conditions are summarised in Fig. 1.

Elimination of non-specific binding sequences was performed by carrying out from cycle 15 several counterselection cycles with mixtures of non-target spores, as performed with vegetative Gbs cells. Each bacterial strain was used at 10 6 spores/mL and exposed to the aptamer candidate pool for 60 min. Mixture specie compositions used in counter-selection cycles are described in Table 1. 4 cycles with counter-selection were carried out following the previous 14 classical cycles, bringing the total number of SELEX cycles to 18.

Aptamer identification

Aptamer candidate diversity was monitored using a high throughput sequencing of emPCR products after strand separation obtained upon cycles 8, 10, 12, 14, 16 and 18. A two-step procedure was used to amplify the emPCR products after the strand separation step of the six selected SELEX cycles. In the first step, a PCR was done using the NGS-F and NGS-R primers under the following conditions: 1 U of DNA Polymerase, 1X of buffer, 2 mM of MgCl2, 0.2 mM of dNTPs and 0.2 µM of each primer NGS-F and NGS-R in a total volume of 50 µL. After a preheat step at 95 °C for 5 min; 10 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s were carried out followed by a final elongation step at 72 °C for 5 min. The second barcoding step, size amplicons verification and normalization were performed by the Bio-Environment platform (University of Perpignan). Diversity of the aptamer candidates from the different cycles was assessed by Illumina 2x250 base-pair MiSeq sequencing run.

Sequencing data analysis

The sequencing data were first analysed with FastQC software and confirmed using QIIME 2. The frequency of occurrence of sequences, as well as the sequence categories were expressed as a percentage of the total number of reads in each SELEX run (% of total reads). Over-represented sequences, considered to represent more than 0.1% of all aptamers sequenced in each run, were listed for each sequenced run. Multiple sequence alignment was carried out using ClustalW. Alignment and phylogenetic reconstructions were performed using the "build" function of ETE3 3.1.2 [START_REF] Huerta-Cepas | ETE 3: Reconstruction, Analysis, and Visualization of Phylogenomic Data[END_REF] as implemented on the GenomeNet (https://www.genome.jp/tools/ete/). The tree was constructed using FastTree v2.1.8 with default parameters [START_REF] Price | FastTree 2 -Approximately Maximum-Likelihood Trees for Large Alignments[END_REF]. Secondary structure and free energy estimation of the aptamers were predicted using the NUPACK web application [START_REF] Zadeh | NUPACK: Analysis and design of nucleic acid systems[END_REF].

Spore-aptamer binding assays

The recognition capacity for the target was evaluated using the 10 most abundant aptamers of cycle 18. Briefly, 0.1 µM of denatured A01 to A10 aptamers was incubated for 60 min with 10 7 Gbs spores in 1 mL of selection buffer under agitation. Then, the volume was adjusted to 10 mL with selection buffer and the suspension was immediately filtered through a Nuclepore Track-Etch Membrane (PC MB 47 mm 0.2 µm, Whatman) to eliminate unbound aptamers. The filter was then washed with 10 mL of selection buffer, transferred in a new tube with 1 mL physiological water and strongly agitated for 2 h to transfer the spore-aptamer complex into the liquid phase.

The resulting retentate was amplified by PCR to reveal the presence of bound aptamers. The PCR mixture contained 0.025 U of DNA Polymerase, 1X of buffer, 0.1 mM of MgCl2, 0.2 mM of dNTPs and 0.1 µM of each primer in a total volume of 50 µL. 20 µL of retentate was added to this mix and PCR was carried out under the following conditions: preheat at 95 °C for 5 min; 30 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 15 s and final elongation at 72 °C for 5 min. The PCR products were then analysed by 7% PAGE.

Several negative controls were performed in technical triplicates to ensure the validity of the results. Firstly, to verify that aptamers are not retained by the filters, 0.1 µM of each aptamer were incubated without spore suspension, then filtered under the same conditions as described before, and the retentate was submitted to PCR amplification.

Then, the specificity of the 10 most abundant aptamers was controlled by carrying out the same experiments using Paenibacillus sp. spores instead of Gbs spores.

Finally, to verify that the aptamers were not retained during the filtration process due to spore obstruction on the filter membrane surface, an aptamer having the two nucleotides primer-binding sequences and a random internal sequence of 24 nucleotides was designed. 0.1 µM of this aptamer was incubated with Gbs spores suspension as described above, and the retentate was submitted to PCR amplification.

RESULTS

Preparation of bacterial spore suspensions

In a first time, spore suspensions of Gbs and of the different strains used in the counter-selection cycles were produced on agar plates under appropriate conditions and temperature. Spores concentrations were then determined using a culturable enumeration method (Table 1). The concentrations obtained from the spore suspensions intended for the counter-selection cycles allowed their use at a final concentration of 10 6 spores/mL. A specific protocol of purification was applied to Gbs spore suspension to increase the specificity of candidate aptamers for spore binding rather than to vegetative forms. The resulting spore concentration after purification was 5.4 10 6 spores/mL, which allowed this suspension to be used during the SELEX at a final concentration of 10 5 spores/mL.

Spore-SELEX

The SELEX protocol for the selection of aptamers specific to Gbs spores was conducted over a total of 18 cycles.

The first cycle consisted in a counter-selection with Gbs vegetative, allowing elimination of DNA sequences specific to these forms. Similarly, to increase aptamer specificity, the last 4 cycles (cycles 15 to 18) were preceded by counter-selection phases performed with a mixture of various bacterial spores frequently found in the food environment (Table 1). In addition, working conditions were modified throughout the SELEX process to increase the stringency and improve selection pressure and screening efficiency, and finally retain the aptamers presenting the highest affinity (Fig. 1). The monitoring of the SELEX process was done by electrophoresis at the end of each cycle to confirm the size and abundance of the selected DNAs. emPCR products were deposited on PAGE gels before and after the strand separation step as well as the emPCR positive and negative controls. As a reminder, 76 nucleotides-long DNA were used in the starting library. Migration patterns obtained before and after strand separation were identical, and were also similar to this obtained for the positive control, as shown in Fig. S1 for cycles 8 and 9. Nevertheless, some differences in length of emPCR products compared to the positive control appeared since cycle 11, with additional shorter fragments that became more and more visible until they became the majority at cycle 14 (data not shown). In any case, no band has ever been observed for the negative controls.

Those observations were constant all over the SELEX cycles, validating each cycle before proceeding to the next.

To allow a complete analysis of the aptamers and to follow their evolution during the SELEX, DNA candidates obtained at the end of cycles 8, 10, 12, 14, 16 and 18 were sequenced by NGS. This method generates between 912,496 and 1,585,742 sequences per cycle for a total of 8,001,215 sequences.

Nucleotide sequence analysis aimed to characterise the most widely represented sequences that should correspond to the best candidates in terms of target recognition capacity. Indeed, a large number of copies of a single sequence reflects its better conservation during the selection process and thus its possible good affinity for the target. It is therefore important to classify the data obtained during sequencing in order to highlight the most frequently encountered nucleotide sequences. Thus, the sequences were grouped into five frequency categories according to their number of reads: from 1 to 9, from 10 to 99, from 100 to 999, from 1,000 to 9,999 and more than 10,000 times. This data processing was adapted from Kolm et al. [START_REF] Kolm | DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing[END_REF]. Analysis of the NGS data showed that sequence enrichment was already observable in cycle 8, where sequences comprised between 1,000 to 9,999 reads were already present (1.65%) (Fig. 2A). For the cycles 8 and 10, the sequences counted 9 times or less are in a majority with 60.7% and 53.4%, respectively. The proportion of this category of sequences decreased over the SELEX cycles, reaching 17.5% in cycle 18. Similarly, the sequences with a number of reads comprised between 10 and 99 were shown to decrease from 27.7% in cycle 8 to 9.4% in cycle 18. Sequences with a number of reads between 100 and 999 were observed at a maximum frequency of 14%, after a minimum of 10.0% observed in cycle 8. Conversely, the frequencies of the sequences with a number of reads between 1,000 and 9,999 increased during the SELEX, from 1.7% in cycle 8 to 16.4% in cycle 18. Finally, the most significant increase was observed for the category of sequences having more than 10,000 reads. Although it was not present in cycle 8, this category appeared in cycle 10 with a frequency of 1.9% and increased rapidly to become a large majority in cycle 18 (43.6%).

The analysis of the sequencing data also permits monitoring the evolution of the selected DNAs length along the SELEX process. These data confirmed the results of emPCR product size observed with PAGE analysis. Fig. 2B shows the distribution of sequenced DNAs grouped into length categories at the end of cycles 8, 10, 12, 14, 16 and 18. Similarly to the previous analysis, obtained values were normalised to the total number of reads in each SELEX run. Up to cycle 10, fragments between 75 and 79 nucleotides were by far the most representative. In subsequent cycles, shorter fragments emerged at the expense of longer ones, and the 75-79 category almost disappeared in cycle 18, with an abundance of only 1.98%. Interesting to note that the category that emerged most rapidly from cycle 12 consisted of fragments of 55 to 59 nucleotides, followed by those with sizes between 60 and 64 nucleotides. In cycle 18, those two categories represented respectively 53.43% and 39.10% of the total sequences. Surprisingly, the intermediate categories with a length between 65 and 74 nucleotides were globally not very abundant, with a maximum of 17.17% in cycle 16.

A sequence table was then generated including sequences with a copy number exceeding 0.15% of the total sequences obtained in each cycle. Due to the enrichment of DNAs during SELEX, the cycle with the most of these sequences is cycle 18 with 43 candidates. Those sequences were ranked according to their frequency, and their evolution was tracked from the first sequenced cycle to the last one. The abundances throughout the SELEX cycles of the 10 most represented aptamers in cycle 18 are presented in Fig. 3. These data show that the percentage of abundance of those 10 aptamers globally increased during SELEX. The A01 aptamer was by far the most abundant from cycles 14 to 18, accounting for 17.71% of all the sequenced ssDNAs in cycle 18 and thus being more than 5 times more abundant than A02 aptamer in cycle 18. Furthermore, it is important to note that the abundance of A01 aptamer did not decrease during the counter-selection cycles (cycles 15 to 18), suggesting that this sequence is specific to Gbs spores and is not likely to bind to other spores used in counter-selections.

In contrast, a drop in abundance was observed for the A10 aptamer between cycles 16 and 18, suggesting that this sequence could be specific to at least one of the spores used in counter-selections. This aptamer was also included in the list of candidate sequences for further testing as it showed high abundance in all other cycles, being even the most abundant up to cycle 12.

Alignment of sequences obtained in cycle 18

In order to study the similarities between the over-represented sequences highlighted by the NGS data, an alignment of the 43 selected aptamers was performed and represented as a phylogenetic tree (Fig. 4).

As can be seen in Fig. 4, the 43 candidates are divided into 5 heterogeneous main branches. The 10 most represented aptamers in cycle 18 are fairly evenly distributed among these branches. Thus, the aptamers A01 and A02 belong to the same family, as for A05 and A08, A03 and A09 and A04, A06 and A07. Only the sequence A10 is separated in a branch of only two candidates.

In the first family, 13 sequences are included in a phylogenetic group with closely related individuals. This set of sequences, including the A01 aptamer, is referred to as the A01 cluster, and those 13 candidates are present among the 43 most represented sequences in cycle 18. Alignment of these sequences (Fig. 5) shows that they differ from each other by only one nucleotide, placed at different positions. Together, these aptamers represent 21.8% of the sequences obtained in cycle 18.

The A01 sequence, used here as reference, displays a high cytosine ratio of 42%. No deletions or additions are observed among the A01 cluster but only replacements. 6 of the 12 replacements involve cytosines that are systematically substituted by thymines. Although they have very similar sequences, the aptamers of the A01 cluster show very different percentages of abundance at cycle 18. For instance, the second highest ranked aptamer in this cluster after A01 is A11, showing an abundance of only 0.59%, being 30 times lower than A01. A prediction of their most likely secondary structures by NUPACK allowed visualising the opening of a loop structure due to the replacement of a guanine by a cytosine at position 36 (Fig. 6).

The 2D structure analysis of the cluster A01 aptamers also showed that certain patterns are highly conserved. This is the case for the stem-loop structure composed of bases 4 to 29, which is identical in 12 of the 13 sequences, as A17 aptamer displays a very similar structure whose stem is shortened by 1 base.

Alignment and comparison of the secondary structures of the first 10 aptamers obtained in cycle 18 were also performed (Fig. S2 andS3). The analysis of conserved patterns among these sequences was made difficult by the variable size of the aptamers.

Affinity tests

To access the recognition capacity of the 10 most abundant aptamers of cycle 18, each of them was incubated with the same amount of Gbs spores for 1 hour, following by a 0.2 µm filtration step of the resulting retentate in order to separate the spores-aptamer complex from the unbound aptamers. Bound aptamers were then revealed by PCR amplification.

The designed protocol was first validated by several assays. It was first checked that the used membrane did not retain the aptamers in the absence of spores. As shown in Fig. 7a, lane C1, no PCR product was observed in samples when filtration assays were performed only with aptamers. Migration pattern revealed only the presence of the primers at the bottom of the gel. This condition was tested three times in independent trials.

The risk of membrane saturation by spores, resulting in an unspecific retention of aptamers, was also assessed in three independent trials using a 60 nucleotides ssDNA whose sequence was not found among the 8,001,215 sequences obtained by NGS. Absence of PCR amplification from the resulting retentate obtained after incubation of this aptamer with Gbs spores validated our protocol, as shown in Fig. 7a, lane C2.

The developed protocol was then applied to the 10 most abundant aptamers. The results of the assays on aptamers A01 to A10 are shown in Fig. 7b. The presence of PCR products in all cases demonstrated that these aptamers are able to bind to Gbs spores during incubation time. Differences in migration height were assigned to differences in aptamer sizes.

In order to test the specificity of the selected aptamers, similar assays were carried out with Paenibacillus sp. spores. As shown in Fig. 7c, no amplification of the aptamers was observed, confirming that the tested aptamers were not able to bind to Paenibacillus sp. as they do to Gbs spores.

DISCUSSION

In order to be able to develop specific detection tools for Gbs spores, recognition elements must be identified. To our knowledge, no antibody or other specific element has been designed to date against these spores. This study therefore focused on the development of aptamers specific to Gbs spores. Different variations of SELEX protocol have been described in literature, some of them using whole cells as target, as proposed for the spore-SELEX [START_REF] Hünniger | Food Sensing: Selection and Characterization of DNA Aptamers to Alicyclobacillus Spores for Trapping and Detection from Orange Juice[END_REF].

One of the strengths of this protocol is the use of emulsion PCR for the amplification step of the candidates selected during the cycles. Indeed, the formation of by-products during the PCR amplification step inhibits product generation and therefore limits the application of conventional PCR in aptamer selection [START_REF] Musheev | Selection of aptamers by systematic evolution of ligands by exponential enrichment: Addressing the polymerase chain reaction issue[END_REF][START_REF] Yufa | Emulsion PCR Significantly Improves Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures-Based Aptamer Selection: Allowing for Efficient and Rapid Selection of Aptamer to Unmodified ABH2 Protein[END_REF]. Emulsion PCR, as a single-molecule PCR, allows the amplification of complex DNA libraries whilst avoiding the appearance of chimeric products [START_REF] Williams | Amplification of complex gene libraries by emulsion PCR[END_REF][START_REF] Kanagal-Shamanna | Emulsion PCR: Techniques and Applications[END_REF].

Due to the considerable progress in NGS technologies over the last decade, Sanger sequencing of aptamers obtained after cloning is being gradually replaced by high-throughput sequencing [START_REF] Schütze | Probing the SELEX Process with Next-Generation Sequencing[END_REF][START_REF] Civit | Systematic evaluation of cell-SELEX enriched aptamers binding to breast cancer cells[END_REF][START_REF] Hassan | In vitro selections of mammaglobin A and mammaglobin B aptamers for the recognition of circulating breast tumor cells[END_REF]. Indeed, this technology allows for a very large amount of data to be obtained from different cycles. This allows monitoring of the SELEX protocol as it provides maximum information on the evolution of the pool and is very useful for understanding the process and progression of SELEX [START_REF] Komarova | Inside the Black Box: What Makes SELEX Better?[END_REF]. The data collected during the various sequencing cycles show that the level of overexpression of the candidate sequences evolves more significantly after a dozen SELEX cycles. This confirms that the choice of the number of cycles carried out in this study is relevant.

In our study, the secondary structure predictions did not identify any conserved structures or motifs among the 10 most abundant sequences in cycle 18. Similar results have been recently observed for the aptamers most frequently identified, following NGS analysis against Enterococcus faecalis [START_REF] Kolm | DNA aptamers against bacterial cells can be efficiently selected by a SELEX process using state-of-the art qPCR and ultra-deep sequencing[END_REF] and against Escherichia coli and Listeria monocytogenes [START_REF] Yilmaz | SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers[END_REF]. Nevertheless, our sequences show great structural complexity with hairpin loops, internal loops and bulges. It is recognised that the key molecular interactions that confer the specificity of aptamers to their targets include precise stacking of flat moieties, electrostatic and van der Waals interactions, and hydrogen bonding [START_REF] Hermann | Adaptive recognition by nucleic acid aptamers[END_REF][START_REF] Zhang | Structural Biology for the Molecular Insight between Aptamers and Target Proteins[END_REF]. The high structural diversity observed among the selected aptamers increases the probability of the presence of such molecular interactions. In particular, it is well known that stem regions play an important role in binding the target [START_REF] Katilius | Exploring the sequence space of a DNA aptamer using microarrays[END_REF]. Moreover, this high diversity of structural features may suggest that the obtained aptamers presumably bind to various spore surface areas and not a specific spore determinant.

The results obtained also showed the presence of a cluster of candidate sequences differing from each other by only one base. This suggests that all these sequences could be able to recognise the same target. However, it is probable that the mutations observed among these sequences impact their recognition efficiency, which could explain the overexpression level variability between A01 cluster candidates. In contrast, the locations without mutations, such as the first 25 bases, could reveal the regions essential for target recognition. Analysis of the 2D structures of the A01 cluster shows that the stem-loop structure generated by the first 25 bases is conserved in 12 sequences out of 13 (Fig. 6).

NGS data showed that the level of overexpression of certain sequences, such as A10, decreases during counterselection cycles. It can be expected that the A10 aptamer would be able to bind to an element shared by Gbs spores and spores of one or more of the species exposed in cycles 17 and/or 18, i.e., M. thermoacetica, C. sporogenes, Anoxybacillus sp., Paenibacillus sp., Paenibacillus polymyxa. However, affinity tests showed that aptamer A10 did not seem to be able to bind to Paenibacillus sp. spores. It would therefore be interesting to carry out the same affinity tests with the other spores mentioned. Indeed, an aptamer that is not only specific to Gbs but able to target a wider range of spores could be suitable for the development of an analytical tool that recognises a variety of bacterial spores. There are already aptamers capable of recognising groups of bacteria, presumably based on the presence of common structural compounds on their surfaces. This is the case of aptamers published by Shin et al in 2019 being able to target several Gram-negative bacteria [START_REF] Shin | Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers[END_REF].

Affinity tests were carried out for the 10 most abundant aptamers in cycle 18 with a method adapted from Tuerk and Gold [START_REF] Tuerk | Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase[END_REF]. Due to the nature of these tests, they provided qualitative but not quantitative results. These tests were performed to evaluate the ability of the aptamers to recognise or not Gbs spores under the conditions tested. However, it is not possible to quantitatively compare the aptamers with each other or to determine an association/dissociation constant by this method. In our study, the results showed that all 10 aptamers tested are Color should be used Color should be used Fig. 3 Color should be used Fig. 4 Color should be used Fig. 5 Color should be used Fig. 6 Color should be used Fig. 7 Color should be used

SUPPLEMENTARY MATERIAL

Figure captions Fig. 1 Fig. 2 18 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7

 captions121834567 Figure captions

Fig. S1

 S1 Fig. S1 Denaturant polyacrylamide gel electrophoresis of emPCR products obtained after the SELEX cycles N°8 (A) and N°9 (B). Lane 1: amplification product of resulting DNAs at the end of SELEX cycle. Line 2: amplification product of resulting DNAs at the end of SELEX cycle after strand separation. Lines C+ and C-: positive and negative emPCR control, respectively

Fig. S3

 S3 Fig. S3 2D structures of aptamers A01 to A10 modelled by NUPACK web application (parameters: 25 °C, 100 mM Na + , 5 mM Mg 2+ )

Figure

  Figure Click here to access/download;Figure;Figures Daufouy.pptx

Fig. 2

 2 Fig. 2

Table Table 1

 Table Bacteria species used during selection and counter-selection cycles. Medium, incubation temperatures and concentrations of initial spore suspensions are indicated GSM: Gbs Sporulation Medium, see detailed composition in section "Bacterial strain and spore production" b MLA: Meat Liver Agar c BCP agar: Bromocresol purple lactose agar

	Bacillus subtilis	16	BCP agar c	37 °C	7,40E+09
	Bacillus licheniformis	16	BCP agar c	37 °C	7,00E+10
	Bacillus smithii	16	BCP agar c	55 °C	3,60E+09
	Moorella thermoacetica	17	MLA b	55 °C	1,90E+08
	Clostridium sporogenes	17	MLA b	37 °C	2,70E+08
	Anoxybacillus sp.	18	BCP agar c	55 °C	2,90E+09
	Paenibacillus sp.	18	BCP agar c	37 °C	8,40E+09
	Paenibacillus polymyxa	18	BCP agar c	37 °C	5,60E+08
				Incubation	Spore concentration
	Bacteria species	SELEX cycle	Medium	temperature	(spores/mL)
	Geobacillus stearothermophilus	1-18	GSM a	55 °C	9,90E+08
	Thermoanaerobacterium thermosaccharolyticum/bryantii	15	MLA b	55 °C	3,00E+08
	Thermoanaerobacterium sp.	15	MLA b	55 °C	5,50E+07
	Bacillus coagulans	16	BCP agar c	37 °C	2,40E+08
	Bacillus cereus	16	BCP agar c	37 °C	1,60E+09

a

Acknowledgements: This work was supported by Association Nationale de la Recherche et de la Technologie (ANRT) in the frame of a CIFRE PhD fellowship, under the convention 2019/0206. It was funded through "Spores-Quantum" project of Institut Carnot Qualiment 2018-2021 program. Special thanks to Benoit Budzeszewski from the microbiology team of the CTCPA for carrying out the cultures and the sporulations of the spore-forming bacteria used during this study and to Clara Chamontin for working on the development of affinity tests. Authors: Guillaume DAUFOUY (GD), Nathalie PANIEL (NP), Thierry NOGUER (TN), Lise BARTHELMEBS (LB)

Funding: This study was funded by Association Nationale de la Recherche et de la Technologie (grant number 2019/0206), and "Spores-Quantum" project of Institut Carnot Qualiment 2018-2021 program.

Data availability: All data generated or analysed during this study are included in this published article and its supplementary information files. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

able to bind to Gbs spores. Furthermore, cross-reactivity tests showed no interaction with Peanibacillus sp. spores under the same conditions. More advanced affinity tests could allow the quantitative characterisation of the recognition capacity of the aptamers selected in this study before applying them to further research. Several biophysical instruments or techniques could be investigated to measure aptamer-target binding affinity [START_REF] Zhuo | Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine[END_REF][START_REF] Thevendran | Assays to Estimate the Binding Affinity of Aptamers[END_REF]. For instance, Microscale Thermophoresis (MST) [START_REF] Breitsprecher | Aptamer Binding Studies Using MicroScale Thermophoresis[END_REF][START_REF] El Deeb | Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters[END_REF] and Quartz Crystal Microbalance (QCM) [START_REF] Challier | Multianalytical Study of the Binding between a Small Chiral Molecule and a DNA Aptamer: Evidence for Asymmetric Steric Effect upon 3′-versus 5′-End Sequence Modification[END_REF][START_REF] Hu | Selection of acrylamide-specific aptamers by a quartz crystal microbalance combined SELEX method and their application in rapid and specific detection of acrylamide[END_REF] are two methods that could allow us to measure dissociation constants (Kd) for our aptamers towards Gbs spores.

CONCLUSION

This work reported for the first time the possibility of isolating aptamers specific to Gbs spores using spore-SELEX protocol. An aptamer cluster of 13 over-represented sequences was identified containing the aptamer A01, alone representing 18% of the aptamers present in cycle 18. Qualitative binding experiments confirmed that the isolated aptamers were able to specifically bind to Gbs spores. These aptamers are thus promising candidates for their integration as biorecognition molecules in analytical devices. Several types of biosensors can be considered, based on different transduction methods. Paper chip systems will be first explored due to their simple setup and compatibility to field use. A previous work has demonstrated the feasibility of capturing bacterial spores of Bacillus thuringiensis, on the surface of polyvinylidene fluoride (PVDF) paper strips [START_REF] Zhou | Aptamer-Conjugated Polydiacetylene Colorimetric Paper Chip for the Detection of Bacillus thuringiensis Spores[END_REF]. The possibility of electrochemical transduction methods will be also explored based on the works of Mazzaracchio et al. [START_REF] Mazzaracchio | A label-free impedimetric aptasensor for the detection of Bacillus anthracis spore simulant[END_REF], who described the impedimetric detection of Bacillus cereus spores with a detection limit of only 3.10 3 CFU/mL. Thus, the implementation of the selected Gbs aptamers in a biosensor system will offer a new analytical tool, available for food quality analysis.

Compliance with Ethical Standards

Conflict of Interest: Guillaume Daufouy declares that he has no conflict of interest. Nathalie Paniel declares that she has no conflict of interest. Thierry Noguer declares that he has no conflict of interest. Lise Barthelmebs declares that she has no conflict of interest.

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors. We, as authors, declared that there is no financial and personal interest that could affect our objectivity. We also declared that there is no potential competing interests.