Cheick O Guindo

Elodie Terrer

Eric Chabriere

Gérard Aboudharam

Michel Drancourt
email: michel.drancourt@univ-amu.fr.

Ghiles Grine

Eric Chabrière

Prof Michel Drancourt

Culture of salivary methanogens assisted by chemically produced hydrogen

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

Introduction

Methanogenic archaea (referred to herein as methanogens) are acknowledged members of the digestive tract microbiota, and they have been detected by PCR-based methods and cultured from the oral cavity and the stools of apparently healthy individuals [START_REF] Sogodogo | Methanogens as emerging pathogens in anaerobic abscesses[END_REF]. More particularly, Methanobrevibacter oralis, Methanobrevibacter smithii and Methanobrevibacter massiliense have been isolated from the oral cavity, whereas M. smithii, Methanosphaera stadtmanae, Methanomassiliicoccus luminiyensis, Methanobrevibacter arboriphilicus, M. oralis, Ca. Methanomethylophilus alvus and Ca. Methanomassiliicoccus intestinalis have been isolated from stools [START_REF] Sogodogo | Methanogens as emerging pathogens in anaerobic abscesses[END_REF]. Accordingly, we previously showed that virtually all apparently healthy individuals would carry methanogens in the digestive tract microbiota [START_REF] Dridi | High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol[END_REF] This observation indeed corroborated the pivotal role of methanogens, which detoxify hydrogen produced by bacterial fermentations into methane [START_REF] Deppenmeier | Novel reactions involved in energy conservation by methanogenic archaea[END_REF][START_REF] Deppenmeier | Redox-driven proton translocation in methanogenic Archaea[END_REF]. Moreover, methanogens are increasingly implicated in diseases; their presence or absence is associated with dysbioses such as those observed in the gut microbiota in cases of chronic constipation [START_REF] Chatterjee | The Degree of breath methane production in IBS correlates with the severity of constipation[END_REF], obesity [START_REF] Zhang | Human gut microbiota in obesity and after gastric bypass[END_REF] and colonic diseases including ulcerative colitis, Crohn's disease and colorectal cancer [START_REF] Piqué | Methane production and colon cancer[END_REF][START_REF] Holma | Colonic Methane Production Modifies Gastrointestinal Toxicity Associated With Adjuvant 5-Fluorouracil Chemotherapy for Colorectal Cancer[END_REF][START_REF] Ghavami | Alterations of the human gut Methanobrevibacter smithii as a biomarker for inflammatory bowel diseases[END_REF], in the vaginal microbiota in cases of vaginosis [START_REF] Belay | Methanogenic bacteria in human vaginal samples[END_REF][START_REF] Grine | Detection of Methanobrevibacter smithii in vaginal samples collected from women diagnosed with bacterial vaginosis[END_REF]. In addition, the presence of methanogens is associated with anaerobic pus abscesses such as brain abscesses [START_REF] Drancourt | Evidence of Archaeal methanogens in brain abscess[END_REF][START_REF] Nkamga | Methanobrevibacter oralis detected along with Aggregatibacter actinomycetemcomitans in a series of community-acquired brain abscesses[END_REF] and muscular abscesses [START_REF] Nkamga | Methanobrevibacter smithii and Bacteroides thetaiotaomicron cultivated from a chronic paravertebral muscle abscess[END_REF].

As is usual in clinical microbiology, isolation and culture of methanogens is the gold standard to assess the detection of living methanogens in microbiota and in pathological clinical specimens collected by puncture or biopsy [START_REF] Sogodogo | Methanogens as emerging pathogens in anaerobic abscesses[END_REF]. The routine application of methanogen culturing is hampered by the fact that methanogens are strictly aero-intolerant microbes and require hydrogen for culturing [START_REF] Garcia | Taxonomic, phylogenetic, and ecological diversity of methanogenic Archaea[END_REF][START_REF] Ferrari | Isolation and characterization of Methanobrevibacter oralis sp[END_REF]. To facilitate the isolation and culture of methanogens from a routine perspective, we previously designed a new process in which methanogens are cultivated in the presence of hydrogen-producing Bacteroides thetaiotaomicron [START_REF] Khelaifia | Aerobic culture of methanogenic archaea without an external source of hydrogen[END_REF][START_REF] Traore | Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates[END_REF].

Here, we tested the conditions to replace the biological production of hydrogen with the chemical production of hydrogen and applied it to isolate methanogens from saliva as a proof-of-concept.

Materials and methods

Chemical production of hydrogen

The immediate chemical production of hydrogen resulting from the oxidation of iron by a weak acid has long been known [START_REF] Boddien | Ironcatalyzed hydrogen production from formic acid[END_REF]. We used acetic acid in our experiment because of its ability to oxidize iron, resulting in sufficient production of hydrogen [START_REF] Crouzet | Hydrogen production by hydrothermal oxidation of FeO under acidic conditions[END_REF]. We used two solutions for our experiments, namely, SAB culture broth [START_REF] Khelaifia | A Versatile Medium for Cultivating Methanogenic Archaea[END_REF] and distilled water (Fresenius, Bad Homburg voor Hoehe, Germany) with pH values of 7.3 and 7, respectively. We used 5 mL of each solution in our experiments. The experiments were performed in 10 Hungate tubes (Dominique Dutscher, Brumath, France) for each solution. Iron filings (Amazon, Clichy, France) were used at increasing quantities: 0.5 g for tube n°1, 1 g for tube n°2, and a 0.25 g increase from tube n°3 to tube n°10. The amount of acetic acid (VWR International, Pennsylvania, USA) used was 100 μL for tube n°1 to tube n°7 and 50 μL for tube n°8 to tube n°10. We first placed the iron filings in the tubes, followed by the solution (SAB broth or water) and then the acetic acid. We used three negative control Hungate tubes, one tube with iron filings in SAB culture medium, another tube with iron filings in distilled water and finally a third tube containing iron filings alone. Gas chromatography was then performed using a Clarus 580 FID chromatograph (PerkinElmer, Villebon-sur-Yvette, France) to measure hydrogen production in the different tubes, and the pH was monitored (Fisher Scientific, Illkirch, France).

Culture of methanogen strains using the chemical method for hydrogen production.

M. oralis CSUR P9633, a human saliva isolate [START_REF] Grine | Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota[END_REF], was used for the development of the chemical method. We used the mini-double-chamber flask technique, which was derived from the previously described technique [START_REF] Khelaifia | A Versatile Medium for Cultivating Methanogenic Archaea[END_REF]. SAB agar plates (5-cm plates, Fisher Scientific) inoculated with 200 μL of a M. oralis suspension at 10 7 colony-forming units (CFUs) were placed in the upper compartment of a mini-double-chamber flask, which was sealed with parafilm to ensure an anaerobic atmosphere. We then placed 1 g of iron filings and 100 μL of acetic acid in 10 mL of distilled water in the lower compartment of the minidouble-chamber flask in a microbiological safety station. We used an SAB agar plate inoculated with 200 μL of sterile phosphate buffered saline (PBS) (Fisher Scientific) as a negative control. We then incubated the plates at 37°C for nine days with visual inspection on day 3, day 6 and day 9 postinoculation.

Then, the growth of M. smithii CSUR P9632, a human stool isolate, using the chemical method was compared with that using the reference biological method using a doublechamber system as previously described for the aerobic culture of methanogens [START_REF] Khelaifia | Aerobic culture of methanogenic archaea without an external source of hydrogen[END_REF]. For the chemical method, we put 1.5 g of iron filings and 150 μL of acetic acid in 200 mL of distilled water in the lower compartment instead of Bacteroides thetaiotaomicron, which was used in the biological method. SAB agar plates inoculated with 200 μL of a M. smithii suspension at 10 6 CFU or 200 µL of PBS for the negative controls were placed in the upper compartment.

The pH of the SAB agar plates was measured at day 3, day 6 and day 9 postinoculation using pH indicator strips (Macherey-Nagel SARL, Hoerdt, France). We placed the pH indicator strip directly on the SAB agar plate for two minutes. We then took the value corresponding to the color obtained on the strip from the values indicated by the supplier. The pH and redox potential of the broth in the lower chamber were measured at day 3, day 6 and day 9 postinoculation using the Accumet™ AE150 apparatus (Fisher Scientific). The probe was rinsed thoroughly with distilled water and then immersed in the lower chamber until the device displayed the value on the reading screen. The rinsing step was performed after each use to avoid any error in reading the device. We used the pH and redox potential of distilled water alone and the SAB culture broth alone as controls. We also measured the redox potential of the upper compartment. For the two methods, we used one double-chamber system, in which we placed one Petri plate with 10 mL of distilled water and one Petri plate with 10 mL of the SAB culture broth. These double-chamber systems were then incubated at 37°C, and the redox potential was measured in each of the Petri dishes at day 3, day 6 and day 9 postinoculation. The effectiveness of the two methods for the growth of methanogens was compared by observing the appearance of the first colonies as well as the average number of colonies at day 3, day 6 and day 9 postinoculation. Colonies were confirmed by autofluorescence optical microscopy as follows. Briefly, the colony deposited on a microscopy slide was observed at 63X using an epifluorescence microscope (Leica DMI 3000, Wetzlar, Germany) and at 100X magnification using another epifluorescence microscope (Leica DMI 6000). Colonies were enumerated at day 3, day 6 and day 9 postinoculation by focusing the image of the entire 5-cm plate on a black background using a portable camera without flash (Lenovo L38011, Beijing, China). Numbered images were analyzed and counted using ImageJ version 8 (https://imagej.nih.gov/ij/download.html) (Wayne Rasband, Java, National Institutes of Health, Bethesda, USA) as follows: each image was imported into ImageJ software and analyzed in a first step using the blue, diamidino phenylindole filter to normalized the image. In a second step, the normalized image was analyzed using the green fluorescein isothiocyanate filter and saved in the Joint Photographic Experts Group format. We standardized the images using the elliptical selection tabulation of the software. We then proceeded to manually count the enlarged colonies using the magnifying glass and the multipoint tabulation of the software.

Comparison of the two methods on saliva samples

The study was previously approved by the Ethics Committee of the IHU Méditerranée infection under n°2016-011. After having informed and collected the consent of the participants, we collected saliva samples from 50 people, including 25 tobacco smokers (15 men, 10 women, median age of 30) and 25 nonsmokers (12 men, 13 women, median age of 28). The collection and processing of samples were performed as previously described [START_REF] Grine | Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota[END_REF].

We used SAB agar plates inoculated with 200 μL of PBS as negative controls and then proceeded in the same manner as described above for the M. smithii strain.

Molecular analysis

PCR was performed to confirm the identity of the colonies. Colonies were suspended in 200 μL of ultrapure water (Fisher Scientific), and a sonication step was performed for 30 minutes. DNA was then extracted with the EZ1 Advanced XL Extraction Kit (QIAGEN, Hilden, Germany) using 200 μL as the sample volume and 200 μL as the elution volume.

Gene amplification and PCR sequencing were performed as previously described [START_REF] Grine | Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota[END_REF][START_REF] Grine | Methanobrevibacter smithii, a methanogen consistently colonising the newborn stomach[END_REF][START_REF] Nkamga | Diversity of human-associated Methanobrevibacter smithii isolates revealed by multispacer sequence typing[END_REF].

Real-time PCR analyses were performed as previously described [START_REF] Bringuier | Real-time PCR quantification of Methanobrevibacter oralis in periodontitis[END_REF].

Results and discussion

We report on the use of chemically produced hydrogen for the isolation and culture of methanogens as an alternative to the currently used biological method [START_REF] Khelaifia | Aerobic culture of methanogenic archaea without an external source of hydrogen[END_REF]. The method we report is based on the production of hydrogen resulting from the chemical reaction between iron filings and a weak acid such as acetic acid. All reported results were validated by negative controls that remained negative, and the colonies were identified by autofluorescence and PCR-based analyses.

We first determined an optimal balance between the concentration of iron filings and acetic acid to maintain the production of hydrogen over several days at a relative concentration compatible with the isolation and culture of methanogens. These preliminary experiments enabled the development of a safe and controlled production of hydrogen that was shown to be efficient in the culture of M. oralis and M. smithii reference strains.

In the first step, we measured the average redox potential and pH values of the various media investigated here. The pH and redox potential of the controls consisting of distilled water and SAB broth culture medium were 7 and + 81.3 mV and 7.3 and -11.9 mV, respectively. After incubation at 37°C for 3, 6 and 9 days, these values were 6.98, 6.92 and 6.87 units and +64.6 mV, +67.3 mV and +79.9 mV for distilled water; and 6.83, 6.78 and 6.27 units and +11.1 mV, +12.9 mV and +14.8 mV for the SAB broth culture medium, respectively (Table 1). These results agree with previous observations that most types of water, including tap water and bottled water, are oxidizing agents, and the value of their redox potential is positive, ≥ 20 mV [START_REF] Adamberg | Degradation of fructans and production of propionic acid by Bacteroides thetaiotaomicron are enhanced by the shortage of amino acids[END_REF]. The addition of antioxidants in the SAB medium makes this culture medium reductive. Then, the pH and redox potential of distilled water enriched with iron filings and acetic acid (chemical method) were measured at 5.23, 5.16 and 5.08 units and +109.77 mV, +108.6 mV and +112.7 mV, respectively (Table 2). These values were 5.8, 5.25, and 5.24 units and +113.8 mV, +113.8 mV and +114.5 mV at day 3, day 6 and day 9 for the SAB culture medium incubated with B. thetaiotaomicron, respectively (Table 2). These data indicated that the cultivation of B. thetaiotaomicron in the SAB medium releases fermenting compounds such as CO 2 , hydrogen and acetate, which could oxidize the SAB medium [START_REF] Adamberg | Degradation of fructans and production of propionic acid by Bacteroides thetaiotaomicron are enhanced by the shortage of amino acids[END_REF]. Then, we measured these values in the Petri dishes placed in the upper compartment of the double-chamber system, which hosted the methanogen culture (Fig. 1). The redox potential values were -28.1 mV, -81.2 mV and -108.9 mV at day 3, day 6 and day 9 for distilled water, respectively, and +13.5 mV, -116.5 mV and -77.5 mV at day 3, day 6 and day 9 for the SAB culture broth in the chemical method, respectively. For the biological method, the redox potential values were -4.5 mV, -66 mV and -91.6 mV at day 3, day 6 and day 9 for distilled water, respectively, and +19.1 mV, -7.1 mV and -34.9 mV at day 3, day 6 and day 9 for the SAB culture broth in the biological method, respectively (Table 1). These data indicated that the hydrogen, being a reducer released into the bottom compartment, reaches the upper compartment and reduces the SAB medium or the water placed in the Petri dishes in the upper compartment [START_REF] Thauer | Methanogenic archaea: ecologically relevant differences in energy conservation[END_REF].

In a second step, by culturing the reference methanogen strains in the presence of the negative and positive controls, we obtained hydrogen production in all tubes and observed a correlation in the hydrogen production, the amount of iron filings used and the acid acetic concentration. The hydrogen production using the chemical method was maintained until day 5, and the average pH values of the mixture were 4.09 ± 0.15 and 4.35 ± 0.23 in the SAB culture broth and distilled water, respectively. M. oralis colonies that were confirmed by PCR sequencing were visible on day 3 postinoculation using the chemical method (Supplementary Fig. 1). The negative control consisting of PBS inoculation instead of M. oralis remained negative.

To compare the chemical method with the biological method, M. smithii was inoculated in SAB agar plates using both methods. Using the chemical method, inoculation of SAB agar plates with M. smithii yielded colonies as early as day 3 postinoculation, whereas they appeared on day 9 using the biological method (Supplementary Fig. 1, Supplementary Fig. 2). All negative controls remained negative. The colonies were confirmed using PCR sequencing and real-time PCR. The average number of colonies of M. smithii was significantly higher with the chemical method than with the biological method, regardless of the day of follow-up (Student's test, p <0.0001, p <0.001 and p = 0.0001 for day 3, day 6 and day 9 postinoculation, respectively) (Table 1). Moreover, cultures of these two strains were obtained in three days using the chemical method rather than in nine days using the biological method [START_REF] Khelaifia | Aerobic culture of methanogenic archaea without an external source of hydrogen[END_REF]. In addition, the chemical method was easier to set up than the biological method because it incorporated distilled water instead of SAB culture broth, in contrast to the biological method in which only SAB culture broth can be used (Supplementary Table 1).

Additionally, the biological method requires maintaining a B. thetaiotaomicron culture in the exponential growth phase to release efficient hydrogen production, which is not the case for the chemical method.

Finally, we compared the ability of the chemical method and the biological method to isolate methanogens from 50 saliva samples collected from 50 individuals after 9 days of incubation. The number of positive samples was 6/50 (12%) at day 3 post inoculation using both methods, and one additional saliva sample was positive at day 6, so that a total of 7/50 (14%) were positive using both methods. No additional sample was positive at day 9 postinoculation, regardless of the culture method. In these 7 culture-positive saliva samples, the average number of colonies at day 6 and day 9 postinoculation was significantly higher using the biological method than using the chemical method (Student's test, p = 0.005 and p = 0.04, respectively), whereas there was no significant difference in the average number of colonies on day 3 (Student's test, p = 0.09) (Table 1). Cultures from 6/25 (24%) of the samples from tobacco smokers yielded colonies as early as day 3 in both methods versus 1/25 (4%) of the samples from nonsmokers at day 6 in both methods. Colonies yielded autofluorescence in both methods (Fig. 2 and Fig. 3), whereas no colony or autofluorescence was observed in the negative controls. PCR sequencing and real-time PCR identified three M. oralis-positive and three M. smithii-positive samples in the tobacco smoker samples and one M. oralis-positive sample in one nonsmoker sample (Supplementary Figure 2). Applying the chemical method for the isolation of M. oralis from saliva samples yielded significantly more colonies more rapidly than the biological method used in parallel. We hypothesized that the chemical method allowed the kinetics of hydrogen production to be mastered in such a way to optimize the atmosphere in the double-chamber system used in our experiments. These results show that the chemical method is more effective than the biological method for the rapid culture of methanogens. Accordingly, the observation that almost a quarter of the saliva samples contained M. oralis and M. smithii is in agreement with a previously published report [START_REF] Grine | Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota[END_REF]. Interestingly, we confirmed that methanogens are more prevalent in the saliva from tobacco smokers than in the saliva collected from nonsmokers. These results are consistent with those found previously [START_REF] Grine | Tobacco-smoking-related prevalence of methanogens in the oral fluid microbiota[END_REF], in which there was also a predominance of M. oralis over M. smithii in saliva samples. These observations also confirm that M. oralis is the most prevalent methanogen in the oral cavity. We measured the redox potential in the culture using the chemical method to monitor whether this chemical reaction led to anaerobiosis. Our results indicate that this chemical reaction is reducing and confirm that acetic acid is an efficient oxidizer of iron filings [START_REF] Crouzet | Hydrogen production by hydrothermal oxidation of FeO under acidic conditions[END_REF]. The pH of the agar plates remained neutral throughout the inoculation period both in the chemical method and the biological method; this observation suggests that the acidity produced in the lower compartment of the doublechamber device in the chemical method does not reach the upper compartment.

The present study highlighted some advantages of the chemical method over the biological method (Supplementary Table 1). The chemical method is simple to set up, the hydrogen production is controlled so that the first colonies appear quickly due to the immediate hydrogen production, and this chemical method can be performed with both distilled water and SAB culture broth. Given the simplicity, speed and efficiency of this chemical method of hydrogen production, it could replace the biological method incorporating B. thetaiotaomicron in the isolation and culture of methanogens. Using the chemical method will improve the routine isolation and culture of methanogens in microbiology laboratories to ease and speed the isolation and culture of these opportunistic pathogens [START_REF] Sogodogo | Methanogens as emerging pathogens in anaerobic abscesses[END_REF] and rarely encountered Methanomassiliicoccales, of medical interest as potential archaeobiotics [START_REF] Dridi | Methanomassiliicoccus luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces[END_REF][START_REF] Gorlas | Complete genome sequence of Methanomassiliicoccus luminyensis, the largest genome of a humanassociated Archaea species[END_REF][30].

[30] J.F. Brugère, G. Borrel, N. Gaci, W. Tottey, P.W. O'Toole, C. Malpuech-Brugère.

Archaebiotics: proposed therapeutic use of archaea to prevent trimethylaminuria and cardiovascular disease. Gut Microbes. 5 (2014) 5-10.

TABLES

FIGURES

Figure 1. Aerobic culture of methanogens in a double-chamber system on agar plates using either a microbiological method for hydrogen production (red arrow) or a chemical method for hydrogen production investigated here (blue arrow). Methane released by methanogens.

7-day incubation at 37°C

Hydrogen released by chemical reaction and consumed by methanogens.

A B

A B

Figure 2 .

 2 Figure 2. (A) Fluorescence emitted by M. smithii by applying the blue filter set. (B)

Figure 3 .Supplementary figure 1 :Supplementary Figure 2 .

 312 Figure 3. (A) Fluorescence emitted by M. oralis by applying the blue filter set. (B)

  Distilled water: 200 mL +Iron filings: 1.5 g +Acetic acid: 150 μL SAB medium : 200 mL + B. thetaiotaomicron

Table 1 .

 1 Controls of pH and redox potential.

		Redox potential of the bottom compartment
	Controls		Redox potential (mV)			pH
		D0	D3	D6	D9	D0	D3	D6	D9
	Distilled water	+81.3 +64.6	+67.3	+79.9	7	6.98	6.92	6.87
	SAB medium	-8.7	+11.1	+12.9	+14.8	7.3	6.83	6.78	6.27
		Redox potential of the upper compartment
	Distilled water		-28.1	-81.2	-108.9		-4.5	-66	-91.6
	SAB medium		+13.5	-116.5	-77.5		+19.1	-7.1	-34.9
	mV= millivolts; D: day.

Table 2 .

 2 pH and redox potential of the lower compartment.

	Methods	Redox potential (mV)		pH
		D3	D6	D9	D3	D6	D9
	Chemical method	+109.77	+108.6	+112.7	5.23	5.16	5.08
	Biological method	+113.8	+113.8	+114.5	5.8	5.25	5.24
	mV= millivolts; D: day.

Table 3 .

 3 Comparison of the two methods according to the average of the colonies counted byImageJ.

	Methods Chemical method		Biological method		P-value
		D3	D6	D9	D3	D6	D9	D3	D6	D9
	M. smithii 377±72 659±168 1116±193 0	0	436±74	<0.0001 <0.001 0.0001
	Saliva	190±90 509±133 700 ± 232 305±139 722±96 936±136 0.09	0.005	0.04
	samples
	Negative	0	0	0	0	0	0	NA	NA	NA
	controls
	NA : Not adapted; D: Day.

ACKNOWLEDGEMENTS

COG and GG benefit from Ph.D. grants from the Fondation Méditerranée Infection, Marseille, France. This work was supported by the French Government under the «Investissements d'avenir» (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), (reference: Méditerranée Infection 10-IAHU-03). This work was supported by Région Sud (Provence Alpes Côte d'Azur) and European funding FEDER PRIMMI.