From anaerobes to aerointolerant prokaryotes

Human Microbiome Journal 15 (2020) 100068

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Human Microbiome Journal journal homepage: www.elsevier.com/locate/humic

From anaerobes to aerointolerant prokaryotes a,b

Sokhna Ndongo a b

a,b

, Saber Khelaifia

a,b

, Jean-Christophe Lagier

, Didier Raoult

a,b,⁎

T

Aix-Marseille Univ, IRD, APHM, MEPHI, Marseille, France Institut Hospitalo-Universitaire Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseillle, France

ARTICLE INFO

ABSTRACT

Keywords: Oxygen sensitive bacteria Antioxidant Culture techniques Media Faecalibacterium prausnitzii Akkermansia muciniphila Christensenella spp.

An increasing number of scientists are turning to the microbiota to understand and/or explain the origin of various human metabolic or inflammatory diseases. Oxygen-intolerant bacteria represent the major population of the human intestinal microbiota. Their isolation is often difficult or even fastidious. The number of studies showing their beneficial role in human health is growing exponentially. Faecalibacterium prausnitzii and Akkermansia muciniphila are abundantly represented in healthy intestinal microbiota and their imbalance is positively correlated with inflammatory diseases and metabolic disorders (obesity, diabetes, cancers). Their use as probiotics presents very promising results in restoring the balance of microbial flora but also in the treatment of certain pathological conditions. The Christensenellaceae family has recently emerged as a hereditary taxon and studies have shown that its abundance is positively correlated with leanness and controls obesity in recipient mice. Here, we report the different culture strategies and techniques used for their isolation; the role of antioxidants in the survival of these oxygen-sensitive species in clinical sample and their maintenance in culture isolates.

1. Introduction The human intestinal microbiota is largely dominated by anaerobic microorganisms, and more than 99% of uncultivated species are oxygen intolerant [1]. In addition to their fastidious and difficult culture, the reactive oxygen species (ROS) produced during the metabolism process of anaerobic microorganisms are very toxic [2–4]. Most strict anaerobes have few or no antioxidant defense systems against ROSs, which are powerful oxidative agents [5]. Nowadays, scientists are increasingly turning to the microbiota to understand and/or explain the origin of various human metabolic or inflammatory diseases. There is a particular interest for some bacteria such as Faecalibacterium prausnitzii, an important butyrate producer bacterium [6,7] and Akkermansia muciniphila a mucin degrader bacterium [8] often associated with numerous metabolic or inflammatory disorders in humans such as Crohn’s disease, irritable bowel syndrome, Colorectal cancer diabetes obesity and autism [1,9–15]. The number of works and the reaffirmation of their involvement in human health are constantly increasing (Table 1). A reduction in Akkermansia muciniphila as Faecalibacterium prausnitzii levels has recently been reported in the fecal of children with allergic asthma [16]. In the same context, where the human microbiota has become linked with metabolic diseases and obesity (a major public health problem), metagenomic studies have shown that the host

⁎

genome is associated with the heritability of specific bacterial families such as Christensenellaceae, which forms a network in co-occurrence with other hereditary bacteria and methanogenic archaea, and is associated with leanness and healthy metabolism [17] Here, a review of the literature was done to evidence the role of antioxidants in the evolution of the concept, from strict anaerobic to oxygen intolerance bacteria, through its role in the fight against oxidative stress and its importance in the isolation and conservation of extremely oxygen-sensitive bacteria. Faecalibacterium prausnitzii, Akkermansia muciniphila species and Christensenella genus have beneficial relationships for human health, Hence the need to link the different techniques and culture conditions used for their isolation in order to explore a possible probiotic effect for health promotion 2. The anaerobes microbes 2.1. Historic The discovery of anaerobic micro-organisms dates back to the 19th century, with Louis Pasteur's work on fermentation processes [18]. After their studies on crystallography and molecular asymmetry leading to a correlation between the presence of microorganisms and fermentation processes, Louis Pasteur was called by manufacturers to inspect

Corresponding author at: Institut Hospitalo-Universitaire Méditerranée-Infection, 19-21 Boulevard Jean Moulin, 13385 Marseille cedex 05, France. E-mail address: [email protected] (D. Raoult).

https://doi.org/10.1016/j.humic.2019.100068

Available online 14 December 2019 2452-2317/ © 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

A. muciniphila

Oral administration of F. prausnitzii

Preweaned Dairy Heifers Human

2 Observational study/case control

Human Human Murine Human

Diabetes

Pouchitis

High-fat fed diet

Oral administration of A. muciniphila

Murine

Human murine

Autism Anxiety-induced depression

Observational study/ case control Observational/ control case

Observation study

Human/Murine

Observational study/case control

Skin diseases (atopic dermatitis, eczema) Human health Crohn’s disease, cystic fibrosis, Ulcerative colitis Hepatic desease/Atherosclerosis

Oral treatment of F. prausnitzii

Observational study/case control

Intrarectal administration of F. prausnitzii and extracellular polymeric matrix

Caco-2 cells treated with F. prausnitzii and its extracellular vesicles

Observational study/control case

DSS- induced colitis

Human

Oral administration or Intragastric inoculation of F. prausnitzii/Microbial anti-inflammatory molecule from F. prausnitzii

Murine

DNBS-induced chronic moderate and severe colitis models DSS-Induced Colitis Diarrhea, mortality and weigh gain Ulcerative Colitis Atrial fibrillation: Cystic fibrosis Chronic heart failure Crohn’s disease Intestinal epithelial cells.

Intragastric or oral administration of F. prausnitzii

Murine

TNBS-induced ulcerative colitis

Intervention

F. prausnitzii

Model

Pathology

M

Abundance of A. mucniphila was suggested as biomarkers for a healthy intestinal microbiota Disbiosis and decrease of A. muciniphila Recovery of ethanol-induced A. muciniphila depletion and reduction of hepatic injury, steatosis and neutrophil infiltration. A. muciniphila attenuates the atherosclerotic lesions and the metabolic endotoxemia-induced inflammation and then restores the gut barrier. Low levels of A. muciniphila in cases compared to controls The defeated animals showed decreases in Akkermansia spp. Positive correlation between the relative abundance of Akkermansia spp and behavioral metrics of anxiety and depression

Increasing expression in TNF-α, IL-4, IL-8, and IL-10 expression and significant decreasing in IL-1, IL-2, IL-6, IL-12, IL-17a, IFN-γ in cells treated than control Extracellular vesicles have shown greater efficacy in reducing inflammatory cytokines and producing anti-inflammatory cytokines than F. prausnitzii Anti-inflammatory effects by modulation of IL-12 and IL-10 cytokine production. Reduction in mucosal cytokine expression and improvement in histological score in mice treated with live F. prausnitzii compared to the group DSS‑fed control only. Decrease in the proportions of F. prausnitzii and A. muciniphila in T1DM and T2DM Significant decrease of F. prausnitzii in the total UC compared to the total FAP cohort Decrease in fat tissue inflammation, improves hepatic AST and ALT, lipid profile, and enhances adiponectin signaling and lipid oxidation of the liver of treated mice Dysbiose characterized by an enrichment of F. prausnitzii

Anti-inflammatory effect, protective effect in this colitis model Prevents physiological damage, on macroscopic and histologic criteria Amelioration of the colitis lesions and colonic cytokine secretion profile /counterbalance the disbiosis associated Significant anti-inflammatory effects and decrease in colitis severity in these models by modulation of metabolic profile Production of anti-inflammatory molecules and reduction of proinflammatory markers Reduced the incidence of severe diarrhea and related mortality rate and increases weight gain Significant decrease in F. prausnitzii compared to control patients Temporal changes in the relative abundance of F. prausnitzii inversely correlated to changes in the inflammatory activity in patients with ileal Crohn’s disease-

Findings

Table 1 Summary of the importance of these oxygen sensitive bacteria in health (F. prausnitzii, A. muciniphila and C. minuta). See supplementary material for reference of table.

[9] [124]

[122,123]

(continued on next page)

[118–12011,63,121]

[115–117]

[114]

[113]

[112]

[110111]

[109]

[105–108]

[104]

[98–103]

[6,15,95–97]

Studies

S. Ndongo, et al.

Human Microbiome Journal 15 (2020) 100068

Human Microbiome Journal 15 (2020) 100068

[84] [17,139]

[137,138]

Positive association with A. muciniphila abundance and weight loss Increase in A. muciniphila abundance after gastric bypass Low rate of A. muciniphila correlated with obesity and ype 2 diabetes, Reduction of inflammatory biomarkers and markers of insulin resistance or dyslipidemia Restores A. muciniphila abundance and improves the gut barrier and metabolic parameters Enhances glucose tolerance and attenuate adipose tissue inflammation Increased abundance of A. muciniphila and reduced fat-mass development and oxidative stress A. muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders Reduces diabetes incidence and controls autoimmunity. Increases the integrity of gut barrier Depletion of F. prausnitzii and A. muciniphila Negative correlation with intestinal inflammatory disorders Reduces adiposity gains in recipient mice/associated with low BMI Heritable family and protects against obesity Association with elevated acetate levels Observational study/dietary intervention or surgical

[61,62,66,126,133–136]

Findings Intervention

[10,64,125,12612714,67,128–132]

the origin of the fermentation of preserved foods previously heated and stored in closed containers. Pasteur rejected Gya-lussac's proposals on the Appert process and discovered that optional anaerobic yeasts were at the origin of the fermentation of milk sugar. In 1861, he demonstrated that the fermentation of butyrate was due to a microorganism that lived in the absence of oxygen, “life without air” called Butyric Vibrion [18]. Pursuing this research, in 1865, he also discovered that these Vibrions were involved in the fermentation of calcium tartrate and calcium lactate. He then refers to them as “anaerobic” microorganisms, which cannot reproduce in the presence of oxygen in the air. Their involvement in medical microbiology was first reported by Pasteur in 1865 following the discovery of a septic vibrio during sepsis in an animal and which was not cultivated until 1877 [18]. In the 1970s, the diversity of the intestinal microbiota and its involvement in human infections was the focus of several research studies. Bacteria exploration was dominated by methods dependent on axenic culture [19]. Different strategies were adopted for the culture of anaerobic microorganisms, including Hungates tubes [20], sealed jars (GasPak jar from BD Diagnostic Systems) and plastics bags with anaerobic generators and anaerobic chambers. The cultivation of anaerobes was a long, difficult and expensive technique. It was abandoned in favors of techniques independent of the culture, such as the latest generation sequencing, realtime PCR and FISH. With the advent of metagenomic techniques in 2005, researchers estimated that more than 80% of the bacterial species sequences evaluated in the digestive tract originated from uncultivated bacteria [1,21]. However, it was considered that only 1% of bacteria could be easily cultured in vitro. More than 90% of the bacteria in the intestinal flora are represented by anaerobic microbes [22]. Thus, many biases and contradictory results have been identified. In the 2010’s, the arrival of Microbial culturomics, an approach multiplying culture conditions, coupled with a rapid identification method using MALDI-TOF mass spectrometry to establish a more exhaustive picture of digestive microbiota diversity, has led to a renewal of culture in clinical microbiology [23–25].

oral gavage Observational study Murine Human

Human Skin disease

Obesity

Human/Murine Diabetic/Obesity Dietary intervention by: High-fat diet or Calorie restriction

Oxygen is a necessary substrate for many enzymes involved in the metabolism of living organisms, with the exception of strict anaerobic microorganisms. Anaerobic bacteria are unable to synthesize a respiratory chain with the O2 molecule as the final electron acceptor. During the oxidation-reduction processes, oxygen generates reactive oxygen species (ROS), in particular hydroxyl radicals (OH), hydrogen peroxide (H2O2) and superoxide anion radicals (O2-). When these reactive oxygen species accumulate in cells, they can be lethal by causing direct damage to DNA, proteins or lipids [4,26,27]. Although oxygen is essential for aerobic organisms, it is paradoxically dangerous for these same life forms [28]. But, these evolutionary changes in the metabolism of organisms that previously lived in anaerobiosis, also caused the development of many defense systems against reactive oxygen species. To face these formations of toxic compounds, aerotolerant organisms possess a highly regulated enzymatic systems of antioxidative defense, such as superoxide dismutase (SOD), superoxide reductase (SOR), peroxidase and catalase [29]. For a long time, strict anaerobes were considered to lack antioxidant defense enzymes, but over the past 30 years, studies have shown that even strict anaerobes have defense enzymes such as catalase, SOD and others enzymatic and non-enzymatic mechanisms against oxygen toxicity [5]. Many obligatory anaerobes can support short time of exposure to atmospheric oxygen and resume their growth after the restoration of anaerobiosis [30,31]. During the period of transient, aerobiosis or the presence of free radical in the environment, defense systems ensure cells viability. This ability to tolerate oxygen resides in the fact that they possess little or almost none of these enzymes and their effectiveness to eliminate the active oxygen species. Thus, the higher the activity of SOD and catalase, the greater the aerotolerance [5]. Among

C. minuta

Model

Control case

2.2. Sensitivity of anaerobes to oxygen

Pathology M

Table 1 (continued)

Studies

S. Ndongo, et al.

3

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obligates anaerobes, different levels of oxygen sensitivity can also be distinguished up to those commonly referred to as “extremely oxygen sensitive” (EOS) [32,33]. They differ considerably in their ability to grow at defined oxygen concentrations. Classically, obligate anaerobes can be divided into 2 types according to their degree of tolerance to oxygen: strictly obligate anaerobes, that will not survive at oxygen tension levels (P02) greater than 0.5% and moderate obligate anaerobes, able to grow in the presence of oxygen levels up to 2% to 8% in the environment [2,5,34]. Oxygen is harmful for anaerobic organisms [26], however, some researchers theorize that, true respiration of certain strict obligate anaerobes requires an extremely low partial pressure of oxygen, while others consider it unnecessary [5].

the medium is supplemented with cysteine, riboflavin and the cryoprotectant inulin [38]. 3.3. Antioxidant and the isolation of oxygen sensitive microbes in the presence of oxygen Previously, La Scola et al. demonstrated the efficacy of antioxidants, such as ascorbic acid and glutathione, in the culture of strictly anaerobic bacterial species in aerobic atmosphere [48]. Recently, a new culture medium named “R-medium” has been developed by Dione et al. [46] for the culture of strict anaerobes in aerobic atmosphere. This culture medium is based on Schaedler agar medium modified by adding an antioxidant solution composed of glutathione, ascorbic acid, uric acid and L-cysteine [49]. This R-medium allowed the culture of 82 strictly anaerobic species in aerobic atmosphere, as well as Francisella tularensis, Bartonella henselae and Mycobacterium smegmatis, which are fastidious bacteria [46].

3. The role of antioxidant in the evolution of strict anaerobes to oxygen intolerant 3.1. Role of antioxidants in the neutralization of free radicals and fighting oxidative stress

4. Culture techniques of extremely oxygen-sensitive bacteria (Eos): through the example of Faecalibacterium prausnitzii Akkermansia muciniphila and Christensenella genus

Through its small size, oxygen freely penetrates the bacterial cell and interacts with flavoenzymes by enzymatic autoxidation, thereby inducing the formation of superoxide (O2−) by electron transfer [3,27]. The presence of superoxide (O2−) then leads to an oxidation phenomenon leading to the progressive formation of hydrogen peroxide (H2O2), and hydroxyl radical (HO−) [35]. To fight against the formation and accumulation of these ROS and oxidative stress, these organisms use enzymatic antioxidant defense systems, the best known of which are SOD, SOR, peroxidase and catalase, and/or non-enzymatic defense systems. SORs catalyze the reduction of an electron of O2− to give H2O2 and SODs catalyze the disproportionation of superoxide into dioxide (O2) and hydrogen peroxide (H2O2) [29,35]. In order to avoid accumulation of hydrogen peroxide (H2O2) at a low level not toxic for the cell, it is transformed into H2O and O2 by the catalase and the peroxidase ensures the conversion of 1NADH + H2O2 into 2NAD and O2 [36]. There are several types of non-enzymatic antioxidants, including ascorbic acid, glutathione, vitamin E [37], riboflavin and cysteine [38]. These non-enzymatic antioxidants play a major role in the fight against oxidative stress. They are often supplied by the environment of anaerobic microorganisms via the host's dietary sources (in vivo) or supplemented in culture media (in vitro). In vivo, the redox status of intestinal light is mainly determined by the redox pair Cys/CySS [39–41] and regulated by the shuttle Cys/CySS [42] and dietary sources where the most part is sequestered by the small intestine [43] and luminal hydrolysis GSH [44]. Oxygen delivered into the intestinal lumen by the chyme is diffused into the lumen via the underlying tissues of the mucosa. In addition of regulation system of the oxidative stress by the antioxidants brought by the dietary sources, the bacterial metabolism of facultative anaerobes in the colon contributes to lowering the redox potential and promoting the proliferation of aerointolerant species [45]. In vitro, the addition of antioxidants to the culture medium can help reduce the level of free radicals generated in contact with oxygen in the air and ensure the growth or viability of aerointolerant bacteria [38,46].

4.1. Faecalibacterium prausnitzii Today, it is clear that Faecalibacterium prausnitzii is the most common bacterial species found in healthy human intestinal microbiota with a rate greater than 5% from the total bacterial flora up to 15% in some subjects [12]. Given its involvement in promoting human health, it may possibly serve as an indicator of intestinal health status in adults. It is an important butyrate producer and acetate consumer in the colon [6,7], hence its major role in several inflammatory and metabolic diseases in humans, such as Crohn's disease, irritable bowel syndrome, Colorectal cancer and obesity [12]. A depletion of F. prausnitzii level has been reported in children with severe acute malnutrition [50]. A recent study showed that F. prausnitzii has an antidepressant effect in rats and can be considered as potential psychobiotic [51]. It is an extremely oxygen-sensitive bacterium, but it is capable of colonizing the intestinal mucosa despite the low level of oxygen that diffuses from the epithelial cells by an extracellular transfer of electrons to oxygen provided by flavine and antioxidants such as cysteine or glutathione [47]. 4.2. The isolation medium of Faecalibacterium prausnitzii Culture media such as YCFAG and M2GSC have often been used for the cultivation and isolation of F. prausnitzii strains. These media were modified by adding antioxidant to better optimize the culture of this fastidious and extreme oxygen sensitive bacteria [32,38,52]. M2GSC medium contains yeast extract, casitone, soluble starch, glucose, cellibiose, NaHCO3, cysteine, KH2PO4, K2HPO4, NaCl, CaCl2, (NH4)2 SO4, MgSO4 and can be also supplemented by 30% micro-filtered rumen fluid (20–21). YCFAG medium, on the other hand, is a basic medium composed of nutrients, such as casitone, yeast extract, glucose and mineral salts; a solution of short chain fatty acids (acetate, propionate, isobutyrate, isovalerate, and valerate); and vitamin solution (biotin, cobalamin, p-aminobenzoic acid, folic acid, pyridoxamine, thiamine and riboflavin). Vitamins and short chain fatty acids solutions were filtered and added in the medium after autoclaving and distribution in Hungate tubes. Resazurin is added to the solution as controls for the redox potential. For the subculture on solid media, the agar plates are previously incubated under anaerobic conditions for 24 h [54]. In 2014, F. prausnitsii strains were isolated from the fecal samples of Calves and Piglets using the anaerobic media Versa TREK REDOX 2 (Trek Diagnostic Systems, Cleveland-OH) enriched with rumen fluid (VTR2RF) [55]. Recently, Brain–heart infusion medium supplemented with 0.5% yeast extract (YBHI) and 20% of filtered rumen fluid was used for the isolation of several strains of F. prausnitzii from human stool [33].

3.2. Utilization of antioxidants in the transport and preservation of oxygen sensitive microbes Antioxidants are increasingly used in transport medium for the conservation of strict anaerobes. AnaeroGRO™ Anaerobic Transport Media Systems is the Cary-Blair transport medium modified by addition of L-cysteine and resazurin. In 2012, Khan et al. had shown in a study that F. prausnitzii had the ability to use oxidized flavines and thiols as oxidation-reduction mediators, thus ensuring the transfer of electrons to oxygen [47]. In a recent study, they have showed that F. prausnitzii, an extreme oxygen sensitive, can be kept alive in the air for 24 h when 4

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Trypticase soy agar medium was also used [56]. Growth studies revealed that F. prausnitzii can degrade fructose, fructo-oligosaccharides and glucose [32]. YCFAG supplemented with maltose, cellobiose and starch allowed the isolation of others strains of this bacterium [54]. Lopez-Siles et al. showed that most strains of F. prausnitzii can grow on apple pectin [52]. Acetate strongly stimulate the growth of F. prausnitzii [32]. The growth of this bacterium is observed at pH between 5.5 and 6.7 [55] with an optimum at pH 6.7 [52]. F. prausnitzii strains are sensitive to bile salts and only few stains resist to the presence of 0.1% of bile salts [52,55]. The growth of the majority of F. prausnitzii spots was inhibited by the addition of 0.25% or 0.5% bile salts to the culture medium on average 95% and 97% respectively [52]. Several studies mentioned that F. prausnitzii stains were found to be resistant to antibiotics such as ciprofloxacin, sulfamethoxazole-trimethoprim and streptomycin [33,55].

[33]. An optimization scheme for the isolation of F. prausnitzii strains was represented in Fig. 1. The resistance of F. prausnitzii isolates to bile salts was also evaluated, and most of the tested strains were sensitive. Only few stains resist in the presence of 0.1% of bile salts [52,55]. Antibiotics susceptibility of F. prausnitzii to ciprofloxacin, sulfamethoxazole-trimethoprim and streptomycin was also reported and the tested isolates were resistant [33,55]. 4.4. Bacterial co-culture model promoted the growth of F. prausnitzii It is important to understand the physiological interactions within the intestinal microbiota environment to better implement efficient culture strategies of this beneficial commensal bacteria. In this sense, a study has showed by co-cultivation, that Bacteroides adolescensis was able to promote the growth of F. prausnitzii. Indeed, species of the genus Bifidobacteria are known to be the main producers of acetate in the colon and studies have reported that low levels of Bifidobacteria have been associated with several diseases suggesting the use of prebiotics or probiotics for human health promotion. A study showed that there was a cross-feed between B. adolescensis that provided acetate and F. prausnitzii that consumed it for butyrate production [59]. A very significant stimulation of the growth of F. prausnitzii has been reported in co-culture with B. adolescentis, with a higher butyrate level than in monoculture after 8–24 h, in the presence of fructo-oligosaccharides as a carbon source. Growth factors of bacterial origin, such as the quinone, secreted by certain strains of E. coli (a universal helper growth) favored the growth of F. prausnitzii in co-culture [60].

4.3. Culture conditions and isolation techniques Fusobacterium prausnitzii, reclassified as Faecalibacterium prausnitzii by Duncan et al. [32] was first described by Cato et al., in 1974 [57]. Belonging to the Firmicutes phylum, F. prausnitzii is a Gram-negative, no motile and non-spore-forming bacterium. On agar medium, colonies are translucent, circular or irregular, with borders slightly erose, convex, flat or umbonate [54,57]. In 2002, a new strain of this bacterium was isolated from human stools by Duncan et al. [32]. The stool specimen was serially diluted in the anaerobic Hungate tubes [58] containing the M2GSC medium [53] and cultured as the protocol of Bryant [20]. Several strains were isolated from human fresh stools using the same protocol [47,52]. As the bacterium is very sensitive to short exposure to oxygen [32], immediate culture of freshly emptied stools (18), storage in anaerobic transport medium [55] and rapid transport to the laboratory in an anaerobic container [54] are necessary to optimize their isolation. In the study published by Lopez-Siles et al. [52], 1 µL of the stool specimen freshly sampled was directly seeded on YCFAG medium and incubated at 37 °C for 12–16 h in anaerobic chamber (80% N2, 12% CO2, and 8% H2). Fecal samples can also be diluted in MG2SC broth or YCFAG broth and then seeded on YCFAG agar plates [54]. The choice of the subcultured colonies was based on morphological criteria [52,54] and then into Gram staining [52]. Generally, the growth in liquid medium was observed after 16–18 h of incubation at 37 °C [32,47] and colonies appear on agar plates after 2–4 days of incubation [33,54,55]. However, according to Lopez-Siles et al, the growth of colonies was obtained earlier, after 12–16 h of incubation at 37 °C [52]. F. prausnitzii has a high proportion in the total bacterial population of a stool sample. Thus, serial dilutions are often performed [12] in commercialized liquid media (Anaerobic Dilution Blank (Anaerobe Systems, USA) or prepared (M2GSC, YCFAG or VTR2RF broth) [32,54,55] in Hungate culture tubes using the Bryant method [20]. This pre-dilution step makes it possible to reduce the presence of other minority or fast-growing bacteria such as Enterococcaceae (Enterococcus faecalis, Enterococcus faecium) in the culture samples. These different steps are performed in anaerobic chambers (BacBasic chamber, Sheldon Manufacturing, Inc., Cornellius, OR). A recent study used a new protocol based on the negative screening. This protocol was elaborated to isolate extremely oxygen sensitive bacteria including several strains of F. prausnitzii from the human feces on YBHI agar (Brain–heart infusion medium supplemented with 0.5% yeast extract) enriched with 20% filtered rumen fluid. Briefly, after serials dilutions, the last dilutions (10-8 and 10-9) were seeded on agar plates and incubated at 37 °C for 4 days. The isolated colonies were picked and subcultured in duplicate on YBHI. A batch of plates was maintained under anaerobic chamber and, in parallel, another batch was exposed under aerobic atmosphere after 1 h to eliminate the EOS. After 2 to 4 days of incubation, a negative screening was carried out, showing a percentage up 67,7% of EOS in the fecal samples of subjects

4.5. Akkermansia muciniphila Akkermansia muciniphila is an important mucin-degrading among bacteria colonizing the intestinal mucosa. It uses the mucin as sole carbon and nitrogen source [8]. A. muciniphila is involved in the protection of the mucosal barrier by stimulating the production of mucin by the immune response of the intestinal mucosa [61,62]. It is abundant in the microbiota of healthy subjects and can be used as a potential biomarker [63]. Several studies showed that low proportion of A. muciniphila was correlated with several inflammatory diseases [63,11,13], obesity in mice and human [10,14,64–67] and with autism in children [9]. 4.6. Culture medium and bacterial isolation techniques A. muciniphila was first isolated from the human feces in 2004 by Derrien et al. [8]. The stool sample was mixed and serially diluted in anaerobic Ringer’s solution containing antioxidant (0.5 g of cysteine), then inoculated in liquid medium autoclaved and containing 0.53 g Na2HPO4, 0.4 g KH2PO4, 0.3 g NH4Cl, 0.3 g NaCl, 0.11 g CaCl2, 0.1 g MgCl2·6H2O, 0.25 g Na2S, 4 g NaHCO3, 0.5 mg resazurin, 1 mL acid trace component solution, 1 mL alkaline trace mineral solution, 1 mL filtered vitamin solution, Stams et al. [68], rumen fluid 0.7% (v/v) and gastric mucin 0.25% (v/v). Pure colonies were obtained after subculturing on the same culture medium containing 0.75% of agar. A. muciniphila is a Gram-negative, non-motile and non-spore forming bacterium. Growth was observed at temperatures between 20 and 40 °C and pH between 5.5 and 8.0, with an optimum at 37 °C and a pH of 6.5 [8]. A. muciniphila was very sensitive to low pH hence its abundance in the distal colon its strong regression in the proximal colon [69,70]. This same culture medium and protocol was used to isolate 22 strains of A. muciniphila from stools sample after bacterial quantification by real-time PCR in China [71]. Several others strains have been isolated using several strategies [72]. Recently, the blood culture vial was also used to isolate a new strain from human stool after a Columbia agar subculture supplemented with 5% sheep blood (Biomerieux, Craponne, France) [73]. 5

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Fig. 1. Strategy to maintain the viability of oxygen-sensitive.

A. muciniphila is considered sensitive to oxygen bacterium [8] even if it adheres well to the mucus of intestinal epithelium despite the minimal presence of oxygen. An interesting fact is that it can use oxygen at nanomolar concentrations through the genes encoding the cythochrome bd complex acting as a terminal oxidase and surprisingly favoring its growth [74]. Indeed, its growth rate and yield by culture under anaerobic conditions in the presence of small amounts of oxygen was greater than under strict anaerobic conditions, thus suggesting its addition in the culture of some anaerobic organisms [74]. Growth of A. muciniphila was inhibited by the addition of 0.5% of purified bile salts but not 0.1% [75]. In another previous study conducted by Ark et al, the addition of porcine bile extract up to 1% in the medium showed an increase in the growth of A. muciniphila [76]. Several studies found that A. muciniphila was resistant to vancomycin [77,73]. The type strain of A muciniphila MucT was also found to be resistant to metronidazole and penicillin G, but susceptible to doxycycline, imipenem, and piperacillin/tazobactam [77]. The strain from the Dubourg et al. study was also resistant to ofloxacin, but was sensitive to penicillin, imipenem, ceftriaxone and amoxicillin [73].

their gut [66,78,79]. In the presence of mucin, A. muciniphila is also able to degrade certain monosaccharides, such as galactose, fucose, glucose and N-acetylhexosamines such as N-acetylglucosamine and Nacetylgalactosamine [80]. Recently, a study on the genome analysis of the bacterium showed the deficiency of an enzyme (GlmS) involved in the synthesis of glucosamine 6-phosphate by conversion of fructose-6phosphate, essential in the process of peptidoglucan formation [81,82]. Supported by in vitro culture experiments, they have demonstrated that the presence of N-acetylglucosamine is essential for the culture of A. muciniphila, and which is thus present in mucin, encouraging its importance in isolation media [83]. The biliary resistance study of A. muciniphila howed that 1% pork bile acid added to the culture medium increased its growth [76] but on the other hand it was inhibited with 0.5% purified bile salts or ox bile extract [75]. 4.8. The Christensenella genus The host genetics was associated with the transmission of the bacterial community and consequently, the gut microbiota can also be associated with the host metabolism. Recently, Goodrich et al. reported that Christensenellaceae is the most hereditary bacterial family and that it is strongly correlated with leanness and obesity control in mice [17,84]. This new family was firstly isolated from the human feces with the only species Christensenella minuta, a strict anaerobic bacteria, Gram-negative, non-motile and no-spore-forming. After collection, the stool sample was immediately placed in anaerobic glovebox (Coy Laboratory Products, USA, Grass Lake). Christensenella minuta was isolated on modified Gifu anaerobic medium (Nissui Pharmaceutical) supplemented with 1.5% agar (w/v) and 8% Bacto oxgall (Difco™ Oxgall), equivalent to 80% of fresh bile solution. The isolate resists to 20% bile and optimum growth was observed at 37 °C with pH 7.5. In the light of

4.7. Factors stimulating the growth of Akkermansia muciniphila In several studies, the major role of mucin in A. muciniphila growth has been demonstrated [8]. The presence of mucin in culture media stimulates strongly in-vitro A. muciniphila abundance [69] . Preliminary results from a study on carbon sources of A. muciniphila showed that the fructo-oligosaccharides added to the culture medium strongly promoted its growth [72], while it was previously demonstrated that only mucin can be used as the sole source of carbon [8]. Previous studies had already shown in animal models that oral administration of fructo-oligosaccharides as probiotics increased the abundance of A. muciniphila in 6

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these recent discoveries, the Christensenellaceae family plays an important role in leanness [17,84,85]. Thus, the search for effective strategies to optimize the isolation of microorganisms that can prevent obesity is an important objective for the human health. The involvement of gut microbiota in obesity [86–90] has already been demonstrated, knowing that its risk factor is strongly linked to most cardiovascular and metabolic diseases [91], the search for effective strategies to optimize the isolation of microorganisms that can be used to prevent obesity, is an important application to human health. In 2016, through culturomics investigations, two news species of the genus Christensenella were isolated from the human feces, and named Christensenella massiliensis and Christensenella timonensis [92,93]. The isolated colonies were obtained after a pre-incubation of the stool sample for 7 days in an anaerobic blood bottle culture supplemented with 5 mL of rumen fluid, and then subcultured on 5% sheep's blood agar (bioMérieux, Marcy l'Etoile, France) under anaerobic condition using AnaeroGen (bioMérieux).

[8] [9]

[10] [11] [12] [13] [14]

5. Conclusion and perspectives

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We started from a Pasteurian anaerobic model that describes anaerobic bacteria as growing only in the absence of oxygen based on the fermentation model, to aero-intolerance bacteria based on their ability to grow even under atmospheres composed of 20% O2 through protective components such as antioxidants. This new approach, puts an end to the concept of strict anaerobic organisms. The use of antioxidants would be a key element in the culture and conservation of bacteria designated as having probiotic potential or in fecal transplants where highly oxygen-sensitive species are significantly reduced [94]. A diet rich in antioxidants or supplemented with antioxidants could also be a therapeutic approach for some diseases related to the restriction of extreme oxygen-sensitive bacteria beneficial for human health and the fight against oxidative stress. The potential probiotic role of bacteria such as F. prausnitzii and A. muciniphila has become a current topic for several research projects, hence the interest in reporting the different strategies used for their isolation in order to identify new strains, to better understand their relationship with human health and diseases.

[16] [17] [18] [19] [20] [21] [22] [23]

Declaration of Competing Interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[25]

Acknowledgements

[26]

Funding sources: This research was supported by the National Research AgencyNational Research Agency under the program "Investissements d'avenir", reference ANR-10-IAHU-03.

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