- Email: [email protected]
Linking gut redox to human microbiome
Accepted Manuscript Original Article Linking Gut Redox to Human Microbiome Matthieu Million, Didier Raoult PII: DOI: Reference:
S2452-2317(18)30015-0 https://doi.org/10.1016/j.humic.2018.07.002 HUMIC 41
To appear in:
Human Microbiome Journal
Received Date: Revised Date: Accepted Date:
6 May 2018 26 June 2018 12 July 2018
Please cite this article as: M. Million, D. Raoult, Linking Gut Redox to Human Microbiome, Human Microbiome Journal (2018), doi: https://doi.org/10.1016/j.humic.2018.07.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Linking Gut Redox to Human Microbiome Matthieu MILLION1,*, Didier RAOULT1 1
Aix Marseille Univ, APHM, IRD, MEPHI, IHU-Méditerranée Infection, Marseille, France
*To whom the correspondence should be addressed: [email protected] MEPHI, Aix Marseille Université, IRD IHU - Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille Phone number: 04 13 73 24 01 Fax number: 04 13 73 24 02 Abstract word count: 248 Body text word count: 2,944 Keywords: Gut, redox, microbiome, microbiota, oxidative stress, metagenomics
ABSTRACT Uncontrolled oxidative stress has been associated with many diseases and aging. We previously report an increased gut redox and depletion of the anaerobic microbiome in severe acute malnutrition. Here, we extended the analysis to test if this link could be generalized by including individuals with various age and dietary status. Seventy individuals (children and adults, French and African, healthy individuals and teenagers with anorexia nervosa, marasmus and kwashiorkor) were included. Fecal redox potential was measured using a simple redox probe. v3v4 16S gene targeted metagenomics was used to characterize the microbiota. The Metagenomic Aerotolerant Predominance Index (MAPI) was defined as the natural logarithm of the ratio of the relative abundance of aerotolerant on strict anaerobic species. This index is easily understandable (MAPI > 0: aerotolerant predominance, MAPI < 0: anaerobic predominance), can be calculated for any metagenome and follows a normal distribution among our 70 included individuals. Fecal redox potential (mV) and the Metagenomic Aerotolerant Predominance Index were dose-dependently related (linear regression, p < .001). This link, if confirmed, will allow humans to take care of their microbiome and prevent, treat and/or alleviate gut redox associated chronic diseases by (i) controlling the concentration of reactive species in the gut by avoiding behavior associated with uncontrolled oxidative stress (alcoholism,…) in the gut and using reduced water, and (ii) by improving gut anti-oxidant capacities by an adequate diet rich in nutrients allowing the human gut to maintain a very low redox potential in the gut as a key for homeostasis.
INTRODUCTION The role of the microbiota in health and disease has been particularly demonstrated in malnutrition (1, 2) and obesity (3). We have shown that malnutrition leads to an alteration of the digestive microbiota with a disappearance of methanogenic arches that are intolerant to oxygen, a depletion of anaerobic bacteria and a relative proliferation of Proteobacteria, Streptococcus and Staphylococcus aureus which are oxygen tolerant bacterial groups containing many pathogens potentially responsible for infectious diarrhea (1, 4, 5). Anaerobic depletion was previously reported in malnourished children (6) and mice (7). Conversely, ingestion of one of these potential digestive pathogens orally can lead to malnutrition by oxidative disruption of the digestive microbiota (8). This suggests that the microbiota is a key instrumental partner necessary for host homeostasis at the interface with the environment. Therefore, identifying the determinants capable of preserving or altering the microbiota is a major challenge for research and medicine in the new millennium. On the other hand, and completely independently, oxidative stress has been associated with many diseases and aging. Oxidative stress is a type of aggression of the cell constituents due to reactive oxygen species (ROS) and reactive nitrogen species (RNS, N for nitrogen). The three best known ROS are superoxide anion, hydrogen peroxide and hydroxyl radical. The production of ROS and RNS is normal for all aerobic organisms and is not, in itself, an oxidative stress situation. Indeed, eukaryotic cells and bacteria called "aerotolerant" bacteria have a complex detoxification system against ROS including enzymes (superoxide dismutase, catalase, glutathione peroxidase, etc.), chaperone proteins and small molecules (glutathione, uric acid, vitamin E, vitamin C...). Oxidative stress is a pathological cellular state with damage to its constituents resulting from the inadequacy between an excessive concentration of ROS and RNS in the environment and a lack of antioxidant capacities of the cell. The pathogenic role of excessive
oxidative stress has been confirmed in numerous studies for decades, particularly in cardiovascular diseases (9). The role of oxidative stress in malnutrition has been clearly demonstrated by several teams including Golden who proposed that free radicals are critical in pathogenesis of kwashiorkor (10, 11). Moreover, animals deficient in vitamin E have an endurance 40% lower than controls in relation to cellular damage secondary to oxidative stress by insufficient antioxidant capacities (10). At the cellular level, a link between redox potential regulation and NF-kappa B activation (12) has demonstrated that the balance between oxidants and reducing agents in the environment impacts the entire cellular machinery thus leading to inflammation and cell death. However, to our knowledge, no studies have studied the direct link between fecal redox potential and the human digestive microbiome. After showing a link between anaerobic depletion and increased redox potential in a particular situation, severe acute malnutrition (4), we have here extended the analysis to test a universal link between digestive redox and microbiome including very different situations.
METHODS We have included individuals of different ages, geographical origin and nutritional status to obtain the maximum variability of microbiota and intestinal redox potential (1, 4, 13). Participants were 21 healthy adult controls from France, 3 anorexic French adolescents, 9 healthy children from Senegal, 8 children with marasmus (MRS) and 9 with kwashiorkor (KW) from Senegal, as well as 9 children with marasmus (MRS) and 11 with kwashiorkor (KW) from Niger. The French controls were healthy adults who were non-obese and without antibiotics for at least 1 month recruited by the snowball approach. Consent was obtained, and the study was validated by the local ethics committee. Most of these patients, methods for microbiota analysis, aerobic or anaerobic metabolism of each microbe, pH and redox
measurement have been previously described (4). For the redox assessment, 1 g of stool were diluted in 10 mL of distilled water and centrifugated at 8000 rpm for 10 minutes. A pH and redox meter (PCE-228-R, PCE Instruments, Southampton, United Kingdom) was used to measure the redox potential according to the manufacturer’s instructions. This previously published article focusing only on severe acute malnutrition (4) is an open access article distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Compared to this previous article, this new study includes new participants (3 French anorexic adolescents and 21 healthy adult controls) and focuses only on a possible statistical link between digestive redox potential and aerobic/anaerobic balance in stool. To assess the aerobic/anaerobic balance in stools we previously developed a database “List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism” previously published in (4) (v1.1) and updated in (14) (v1.2 – provided as Table S1). To test such a link between fecal redox (mV) and aerotolerant / strict anaerobic balance, different variables were analyzed. Two quantitative variables were obtained by measurement (redox potential, ratio of the metagenomic relative abundance of aerotolerant species on the relative abundance of strict anaerobes). Normal and log-normal distributions were tested by the Kolmogorov-Smirnov test. Automatic distribution fitting was also used to confirmed log-normal distribution. When a log-normal distribution was identified, the natural logarithm of the variable was used instead of the variable itself. Two qualitative variables were also defined: positive redox (redox potential > 0mV) and aerobic predominance (relative abundance of reads corresponding to aerotolerant species > relative abundance of reads corresponding to strict anaerobic species). Qualitative variables were compared using the bilateral khi-square test. Comparison of quantitative variables between groups were analyzed using student t test or Mann-Whitney test according to the
normal distribution test. Finally, a dose-dependent relationship between quantitative variables was tested by correlation and linear regression. Statistical analyses were performed using GraphPad Prism version 8.00 for Windows, (GraphPad Software, La Jolla California USA) and XLSTAT 2018.2 (Addinsoft, Paris, France).
RESULTS Seventy individuals were included with a large variability in age (46 children, 3 teenagers and 21 adults), geography (24 from France, 20 from Niger and 26 from Senegal), and nutritional status (30 healthy controls, 3 with anorexia nervosa, 17 with marasmus, and 20 with kwashiorkor) (Table S2). Among these 70 individuals, redox potential variability was high (minimum -137.5 mV, maximum +137.1, normal distribution confirmed by the Kolmorogov-Smirnov test, mean ± standard deviation -15.76 ± 69.07). Comparison of the redox potential between the 7 groups showed that mean was negative in all healthy groups and positive in groups with malnutrition (Table 1 - Figure 1a). Comparisons between groups showed significant differences (Table S3) but this was not the objective of this study limited solely to the link between redox potential and aerotolerant/anaerobic balance in the digestive microbiota. The 95% confidence interval of redox potential of French adult healthy controls was -90mv to 40mV (Table 1 & Table S3). From the metagenomic taxonomic assignment of each metagenome (Table S4), we determined the "aerotolerant", "strict anaerobic" or "oxidative stress resistance unknown" status for each species (Table S1 and S4). This allowed us to calculate the total number of reads that corresponded to strict aerotolerant or anaerobic prokaryotes in each metagenome (Table S5). From this, we calculated the relative abundance of strict anaerobic reads and aerotolerant reads for each metagenome (Table S6 – Figure 1b). We then calculated the ratio
of aerotolerant relative abundance to strict anaerobic relative abundance (Table S6). This ratio was > 1 for aerotolerant predominance and < 1 for strict anaerobic predominance. The variability of the aerotolerant ratio was significant (minimum 0.025, maximum 139.9). The distribution was not normal but log-normal (probability 100%, likelihood ratio 2.07 e-57, kolmogorov-smirnov test), so the natural logarithm of the aerotolerant ratio was calculated for each metagenome for further analysis (Table S6). The log normal distribution was confirmed by automatic distribution fitting using XLSTAT 2018.2 (Table S7). This variable was called the metagenomic aerotolerant predominance index (MAPI) and correspond to the variable “ln(Ae/Ana)”. The MAPI was > 0 for aerotolerant predominance and < 0 for strict anaerobic predominance. The distribution of the MAPI was normal by the Kolmogorov-Smirnov test. The variability was large (minimum -3.67, maximum +4.94, mean ± standard deviation 0.37 ± 2.16). Comparison of the aerotolerant predominance ratio between the 7 groups showed that mean was negative in all healthy individuals (anaerobic predominance) and positive in all children with malnutrition (aerotolerant predominance – Table 2 & Figure 1c). Comparisons between groups showed significant differences (Table S8). The 95% confidence interval of MAPI of French adult healthy controls was -1.88 to -0.44 (Table 2 & Table S8). Positive fecal redox potential was associated with positive MAPI (aerotolerant predominance) (27/41 (65.8%) versus 6/29 (20.7%), bilateral Khi-square test, p = .00019, Conditional maximum likelihood estimates of Odds Ratio (CMLE OR) = 7.16, 95% confidence interval (95%CI) = 2.43 – 23.36). Redox potential was significantly higher in individuals with positive MAPI (aerobic predominance, n = 37, mean ± standard deviation, 9.39 ± 11.82 mV) than in individuals with negative MAPI (anaerobic predominance, n = 33, 44.0 ± 9.4 mV, unpaired bilateral t-test, p = .0009 – Figure 2a). An outlier was detected with anaerobic predominance but positive redox potential (Figure 2a). Strikingly this individual
was also an outlier in his group (KW09_S; children with kwashiorkor from Senegal) because most of the children of this group had aerotolerant predominance. This suggests other factors than redox potential for regulating the gut aero-anaerobic balance or a non-nutritional edema (misclassified child). The MAPI was significantly higher in individuals with positive fecal redox potential than in individuals with negative fecal redox potential (n = 29, 1.60 ± 1.56 versus n = 41, 0.50 ± 2.12, p <.0001 – Figure 2b). Finally, an extremely significant relationship was found between fecal redox potential (mV) and the MAPI by correlation test (Pearson r 0.41, 95% confidence interval 0.19-0.59, R2 = 0.17, p = .0004) and by linear regression (R2 = 0.17, p = .0004. Equation: MAPI = 0.013 * redox potential (mV) + 0.57 (Figure 3)).
DISCUSSION Here we have developed a new index (MAPI: Metagenomic Aerotolerant Predominance Index = ln(relative abudance of aerotolerant species/relative abundance of strict anaerobes)) to evaluate dysbiosis related to oxidative stress. This index is clinically relevant because more and more studies suggest a role of oxidative stress on microbiota alteration (1, 14, 15). This index can be calculated for any metagenome with taxonomic assignment, notably by using our database (List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism available at our website : http://www.mediterraneeinfection.com/article.php?larub=143&titre=base-de-donnees – Table S1). The MAPI is easily understandable (MAPI > 0 corresponds to an aerotolerant predominance, MAPI < 0 corresponds to a strict anaerobic predominance). This index has a normal distribution among a group of human individuals with different ages, geographical origin and nutritional status. This allows it to be used with parametric statistics. For the first time a metagenome is no
longer characterized solely by ecological indices (global diversity) but on microbiological knowledge. This index allowed us to demonstrate a dose-dependent link between redox and the global profile of the microbiota. Why is this important? First, the concentration of reactive oxidative species and antioxidants in the digestive tract depends on the host but also largely on potentially modifiable environmental variables. Measures to control digestive oxidative stress in humans could include (i) avoiding oxidative liquids and nutrients (soda usually have a redox > +300mV, unfiltered tap water has a redox >+200mV due to its antiseptic oxidation by chlorine or ozone, some spring water have a negative redox potential up to -300mV (16)), (ii) favouring nutrients that allow the host to develop its antioxidant capacities (rather than giving only direct antioxidants, as this expose to the risk of inhibiting the host's antioxidant defenses by hormesis). In another context, it has been shown that spring water is able to decrease oxidative stress-related biomarkers (17). Secondly, the control of redox and digestive oxidative stress can allow the preservation of Healthy Human Mature Anaerobic Gut Microbiota (HMAGM (1)) which is the part of the microbiota most early altered in case of uncontrolled oxidative stress. This HMAGM is a key to homeostasis (1, 4, 18) notably with Lachnospiraceae, Ruminococcaceae and Faecalibacterium prausnitzii which produce butyrate (19), one of the key molecules produced by the anaerobic microbiota (HMAGM) with beneficial effects for the host (anti-inflammatory and anti-cancer (18)). Maturation of the gut environment in humans and mammals depend both on age and diet (5) and is associated both with gut redox decrease (20-22) and microbiome changes with anaerobic predominance (13). In few months after birth, human fecal redox decreases by 450mV (From +150 to -300mV) (20) and from positive to negative. In parallel, anaerobes proliferate to become predominant (13, 21, 22). In contrast, geography is a critical factor for microbiome (13) but this has not been reported for gut redox. Experimental data suggest that
antioxidants and reducing agents are capable of modifying the redox of the medium (23, 24), that redox is capable of modifying bacterial growth (23-25) and that an in vitro microbiota (bioreactor) is capable of lowering redox (26). Accordingly, in order to show that digestive redox is a key variable in the development and maintenance of the anaerobically predominant healthy human microbiota, we deliberately included individuals of different age and nutritional status. Children with severe acute malnutrition were recruited in Africa because this is an endemic area (6% of children from Niger are severely wasted according to the UNICEF (https://data.unicef.org/data/malnutrition/)) whereas kwashiorkor for instance is exceptional in Western countries (0 to 0.1%) with sporadic cases of fad diets, perceived and true milk allergy, but above all nutritional ignorance (not epidemic or endemic poverty) (27, 28). For instance, Liu reported only 12 cases of kwashiorkor in 9 years in the USA (27). As for age, the maximal geographical bias was used to obtain the maximal diversity in factors influencing both the microbiome and the gut redox and this also prevent overmatching bias (1). This is an innovative but preliminary study. Oxygen has already been suspected to drive gut dysbiosis (29) but we extend this link to the more generalizable and universal redox potential. Further studies are needed to confirm this link. Redox measurement should be standardized (great variability between probes, depending on the diluent used (tap water has a redox of +200mV), impact of exposure time to ambient air and stool preservation, possible direct measurement in the rectum (20), etc...). The measurement of MAPI will be improved by culturomics (30) analysis to define an oxidative stress resistance profile for reads not currently assigned to the species. Moreover, this database could be completed at the level of the operational taxonomy units (OTUs) by considering that if all the cultivated species corresponding to a metagenomic cluster have the same oxidative stress resistance profile, all the OTUs of this cluster have this oxidative stress resistance profile (example:
Lachnospiraceae are all strictly anaerobic). This would increase the accuracy of the MAPI measurement and the ability to diagnose oxidative dysbiosis. In conclusion, we present here preliminary but innovative results proposing a link between redox and anaerobic aerotolerant balance in the digestive tract. If this link is confirmed, it will open several promising pathways for human health: (i) the discovery of two markers of oxidative dysbiosis (redox potential, MAPI) allowing progress in the diagnosis of dysbiosis. It will theoretically be possible to define a "normal" value for redox potential (Table 1) and digestive MAPI and thus identify individuals with or without oxidative dysbiosis (whatever the global diversity assessed by ecological but not microbiological indexes), (ii) The identification of a modifiable determinant of at least part of the digestive microbiota opens the way to new therapeutic approaches for diseases associated with uncontrolled oxidative stress in whom the digestive microbiota plays at least an instrumental role. Many diseases are suspected to be associated with an alteration of the microbiota linked to abnormal gut redox. In addition to severe acute malnutrition (4), non-alcoholic steatohepatitis (NASH) is one of the best candidate as it is associated with an oxidative diet (soda), a diet low in fiber and unsaturated fatty acid and plasmatic oxidative stress (31, 32). Other candidate diseases are colitis (33), HIV (14), acute necrotizing enterocolitis of the newborn (34), type I (35) and type II diabetes (36), and rheumatoid arthritis (37). We also proposed a link between uncontrolled oxidative stress and disruption of mucosal immunity through gut microbiota alteration (18). Other teams have also suspected this mechanism, particularly during weaning (37), but without demonstrating any experimental causal role. More generally, this could be applicable to other human microbiota. For example, we have shown very recently that chlorexidine (an oxidizing agent containing chlorine for antiseptic purposes) does not reduce post-operative respiratory infections after thoracic
surgery (38). In this study, there was a trend for an increase in pneumococcus by chlorexidine in the pharyngeal microbiota. This is consistent with the fact that the pneumococcus is very well armed against oxidative stress. Oxidative alteration of the respiratory microbiota could be the missing piece to explain why, in this study, an antiseptic on a mucosa associated with a microbiota has no preventive effect to avoid the occurrence of infection. If the link between redox, oxidative stress and aero-anaerobic dysbiosis is confirmed, this could represent a major step forward and hope to break or slow down the vicious circle including oxidative stress and dysbiosis at play in many human diseases. Possible options to modulate this gut redox include reducing diet and water (39), however in vivo experimental and clinical studies are urgently needed. Overall, our results suggest that redox status and control of oxidative stress could be major determinants of animal and specifically human microbiomes. Beyond smoking and alcohol consumption, redox quality of diet and water could be critical in preventing, treating and/or alleviating chronic diseases associated with oxidative stress which represent the greatest public health burden and the major human threat for the new millennium.
References 1.
Million M, Diallo A, Raoult D. Gut microbiota and malnutrition. Microb Pathog.
2017;106:127-38. 2.
Tidjani Alou M, Million M, Traore SI, Mouelhi D, Khelaifia S, Bachar D, et al. Gut
Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics? Front Microbiol. 2017;8:899. 3.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An
obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444(7122):1027-31. 4.
Million M, Tidjani Alou M, Khelaifia S, Bachar D, Lagier J-C, Dione N, et al.
Increased Gut Redox and Depletion of Anaerobic and Methanogenic Prokaryotes in Severe Acute Malnutrition. Sci Rep. 2016;6:26051. 5.
Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M, Alam MA, et al.
Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature. 2014;510(7505):417-21. 6.
Mata LJ, Jimenez F, Cordon M, Rosales R, Prera E, Schneider RE, et al.
Gastrointestinal flora of children with protein--calorie malnutrition. Am J Clin Nutr. 1972;25(10):118-26. 7.
Allori C, Aguero G, de Ruiz Holgado AP, de Nader OM, Perdigon G. Gut mucosa
morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei. Journal of food protection. 2000;63(1):83-90. 8.
Rivera-Chavez F, Zhang LF, Faber F, Lopez CA, Byndloss MX, Olsan EE, et al.
Depletion of Butyrate-Producing Clostridia from the Gut Microbiota Drives an Aerobic Luminal Expansion of Salmonella. Cell Host Microbe. 2016;19(4):443-54.
9.
Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction,
oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001;104(22):2673-8. 10.
Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage
produced by exercise. Biochem Biophys Res Commun. 1982;107(4):1198-205. 11.
Golden MH, Ramdath D. Free radicals in the pathogenesis of kwashiorkor. Proc Nutr
Soc. 1987;46(1):53-68. 12.
Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L. Redox regulation of NF-
kappa B activation. Free Radic Biol Med. 1997;22(6):1115-26. 13.
Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et
al. Human gut microbiome viewed across age and geography. Nature. 2012;486(7402):222-7. 14.
Dubourg G, Lagier J-C, Hue S, Surenaud M, Bachar D, Robert C, et al. Gut
microbiota associated with HIV infection is significantly enriched in bacteria tolerant to oxygen. BMJ Open Gastroenterol. 2016;3(1):e000080. 15.
Darnaud M, Dos Santos A, Gonzalez P, Augui S, Lacoste C, Desterke C, et al. Enteric
Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation inMice WithColitis. Gastroenterology. 2018;154(4):1009-23.e14. 16.
Ioka S, Muraoka H, Matsuyama K, Tomita K. In situ redox potential measurements as
a monitoring technique for hot spring water quality. Sustain Water Resour Manag. 2016;2:5. 17.
Bajgai J, Fadriquela A, Ara J, Begum R, Ahmed MF, Kim CS, et al. Balneotherapeutic
effects of high mineral spring water on the atopic dermatitis-like inflammation in hairless mice via immunomodulation and redox balance. BMC complementary and alternative medicine. 2017;17(1):481. 18.
Million MT, J.; Wagner, C.; Lelouard, H.; Raoult, D.; Gorvel, J.-P. New insights in
gut microbiota and mucosal immunity of the small intestine. Human Microbiome J. 2018.
19.
Vital M, Howe AC, Tiedje JM. Revealing the bacterial butyrate synthesis pathways by
analyzing (meta)genomic data. MBio. 2014;5(2):e00889. 20.
Grütte F-K, Horn R, Haenel H. Ernährung und biochemisch-mikroökologische
Vorgänge im Enddarm von Säuglingen. Zeitschrift für Kinderheilkunde. 1965;93(1):28-39. 21.
Schulze F, Jacob HE. Redox potential of the gastrointestinal tract of swine of different
ages. Archiv fur experimentelle Veterinarmedizin. 1981;35(3):349-57. 22.
Schulze F, Jacob HE. Diarrhea in young calves. 4. Redox potential of the
gastrointestinal tract of clinically healthy calves, and calves with spontaneous diarrhea, experimental Escherichia coli infection, and cyclophosphamide treatment. Archiv fur experimentelle Veterinarmedizin. 1981;35(3):337-48. 23.
Zehnder AJ, Wuhrmann K. Titanium (III) citrate as a nontoxic oxidation-reduction
buffering system for the culture of obligate anaerobes. Science. 1976;194(4270):1165-6. 24.
Jones GA, Pickard MD. Effect of titanium (III) citrate as reducing agent on growth of
rumen bacteria. Applied and environmental microbiology. 1980;39(6):1144-7. 25.
Lund BM, Knox MR, Sims AP. The effect of oxygen and redox potential on growth of
Clostridium botulinum type E from a spore inoculum. Food Microbiology. 1984;1:10. 26.
Zapletalova M, Kasparovska J, Krizova L, Kasparovsky T, Sery O, Lochman J.
Bacterial community dynamics in a rumen fluid bioreactor during in-vitro cultivation. Journal of biotechnology. 2016;234:43-9. 27.
Liu T, Howard RM, Mancini AJ, Weston WL, Paller AS, Drolet BA, et al.
Kwashiorkor in the United States: fad diets, perceived and true milk allergy, and nutritional ignorance. Arch Dermatol. 2001;137(5):630-6. 28.
Mori F, Serranti D, Barni S, Pucci N, Rossi ME, de Martino M, et al. A kwashiorkor
case due to the use of an exclusive rice milk diet to treat atopic dermatitis. Nutr J. 2015;14:83.
29.
Rivera-Chavez F, Lopez CA, Baumler AJ. Oxygen as a driver of gut dysbiosis. Free
Radic Biol Med. 2017;105:93-101. 30.
Lagier J-C, Khelaifia S, Alou MT, Ndongo S, Dione N, Hugon P, et al. Culture of
previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol. 2016;1:16203. 31.
Machado MV, Ravasco P, Jesus L, Marques-Vidal P, Oliveira CR, Proenca T, et al.
Blood oxidative stress markers in non-alcoholic steatohepatitis and how it correlates with diet. Scandinavian journal of gastroenterology. 2008;43(1):95-102. 32.
Banini BA, Sanyal AJ. Current and future pharmacologic treatment of nonalcoholic
steatohepatitis. Curr Opin Gastroenterol. 2017;33(3):134-41. 33.
Li X, Li X, Shang Q, Gao Z, Hao F, Guo H, et al. Fecal microbiota transplantation
(FMT) could reverse the severity of experimental necrotizing enterocolitis (NEC) via oxidative stress modulation. Free Radic Biol Med. 2017;108:32-43. 34.
Cassir N, Benamar S, Khalil JB, Croce O, Saint-Faust M, Jacquot A, et al. Clostridium
butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates. Clin Infect Dis. 2015;61(7):1107-15. 35.
Soyucen E, Gulcan A, Aktuglu-Zeybek AC, Onal H, Kiykim E, Aydin A. Differences
in the gut microbiota of healthy children and those with type 1 diabetes. Pediatrics international : official journal of the Japan Pediatric Society. 2014;56(3):336-43. 36.
Singh S, Sharma RK, Malhotra S, Pothuraju R, Shandilya UK. Lactobacillus
rhamnosus NCDC17 ameliorates type-2 diabetes by improving gut function, oxidative stress and inflammation in high-fat-diet fed and streptozotocintreated rats. Benef Microbes. 2017;8(2):243-55.
37.
Zhang X, Zhang D, Jia H, Feng Q, Wang D, Liang D, et al. The oral and gut
microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med. 2015;21(8):895-905. 38.
D’Journo XB, Falcoz P-E, Alifano M, Le Rochais J-P, D’Annoville T, Massard G, et
al. Oropharyngeal and nasopharyngeal decontamination with chlorhexidine gluconate in lung cancer surgery: a randomized clinical trial. Intensive Care Medicine. 2018. 39.
Vorobjeva NV. Selective stimulation of the growth of anaerobic microflora in the
human intestinal tract by electrolyzed reducing water. Medical hypotheses. 2005;64(3):543-6.
Acknowledgments We thank Eric ADEHOSSI, Dipankar BACHAR, Caroline BLANC-TAILLEUR, Souleymane BRAH, Aldiouma DIALLO, Alexandre FABRE, Saber KHELAIFIA, JeanChristophe LAGIER, Catherine ROBERT, Cheikh SOKHNA, and Maryam TIDJANI-ALOU for providing samples, measurements and technical help.
Conflict of interest statement Authors have no conflict of interest to declare.
Funding This work has received financial support from the French Government through the Agence Nationale pour la Recherche (ANR), including the “Programme d’Investissement d’Avenir” under the reference Méditerranée Infection 10-IAHU-03. This work was supported by Région Provence Alpes Côte d’Azur and European funding FEDER PRIMMI (Fonds Européen de Développement Régional - Plateformes de Recherche et d'Innovation Mutualisées Méditerranée Infection).
Table 1. Fecal redox potential in Humans with various Age and Dietary Status Fecal redox (mV)
Mean
Lower 95%
Upper 95%
CI of mean
CI of mean
French adult controls (n = 21)
-64.9
-89.6
-40.1
Senegalese healthy children (n = 9)
-42.9
-73.9
-11.9
French teenagers with anorexia nervosa (n = 3)
-104.9
-194.3
-15.5
Senegalese children with marasmus (n = 8)
46.2
14.9
77.4
Senegalese children with kwashiorkor (n = 9)
25.9
-14.5
66.3
Nigerien children with marasumus (n = 9)
17.8
-19.4
55.0
Nigerien children with kwashiorkor (n = 11)
18.0
-31.4
67.3
Values for all groups followed a normal distribution according to Kolmogorov-Smirnov test except the group “French teenagers with anorexia nervosa” for which the sample size was too small.
Table 2. Metagenomic Aerotolerant Predominance Index in Humans with various Age and Dietary Status MAPI
Mean
(Metagenomic Aerotolerant Predominance Index)
Lower 95%
Upper 95%
CI of mean
CI of mean
French adult controls (n = 21)
-1.16
-1.88
-0.44
Senegalese healthy children (n = 9)
-0.92
-2.76
0.92
French teenagers with anorexia nervosa (n = 3)
-0.40
-2.72
1.91
Senegalese children with marasmus (n = 8)
0.92
-0.46
2.3
Senegalese children with kwashiorkor (n = 9)
0.88
-0.83
2.59
Nigerien children with marasumus (n = 9)
1.72
0.61
2.83
Nigerien children with kwashiorkor (n = 11)
2.64
2.14
3.14
Values for all groups followed a normal distribution according to Kolmogorov-Smirnov test except the group “French teenagers with anorexia nervosa” for which the sample size was too small.
Figure legends Figure 1. Fecal redox potential and Metagenomic Aerotolerant Predominance Index (MAPI) a. Fecal redox potential according to each group (mV, mean ± standard deviation (SD)). Results of statistical tests are provided in Table S3. b. Metagenomic Aerotolerant Predominance Index (MAPI) according to each group (mean ± SD). Results of statistical tests are provided in Table S8. c. Relative abundance of anaerobic reads (green), aerotolerant reads (red) and reads for which no oxidative stress tolerance could be defined (not assigned at the species level and/or uncultured species). Note that anaerobic reads and reads without known oxidative stress resistance are higher in healthy controls (French adults: “témoin” and Healthy children from Senegal “S01 to S010”) and teenagers with anorexia nervosa (“Ano”). Conversely, aerotolerant reads predominated in children with malnutrition (MR; marasmus, KW; kwashiorkor).
Figure 2. Dose-dependent association between fecal redox potential and aerotolerant predominance a. Fecal redox potential was significantly higher in individuals with aerotolerant predominance (violin plots, median and quartiles are shown), b. MAPI (Metagenomic Aerotolerant Predominance Index) was significantly higher in individuals with positive fecal redox potential (violin plot, median and quartiles are shown). C. Linear regression evidence a dose-dependent relationship between fecal redox potential and aerotolerant predominance (MAPI = ln(Ae/Ana)).
S2452-2317(18)30015-0 https://doi.org/10.1016/j.humic.2018.07.002 HUMIC 41
To appear in:
Human Microbiome Journal
Received Date: Revised Date: Accepted Date:
6 May 2018 26 June 2018 12 July 2018
Please cite this article as: M. Million, D. Raoult, Linking Gut Redox to Human Microbiome, Human Microbiome Journal (2018), doi: https://doi.org/10.1016/j.humic.2018.07.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Linking Gut Redox to Human Microbiome Matthieu MILLION1,*, Didier RAOULT1 1
Aix Marseille Univ, APHM, IRD, MEPHI, IHU-Méditerranée Infection, Marseille, France
*To whom the correspondence should be addressed: [email protected] MEPHI, Aix Marseille Université, IRD IHU - Méditerranée Infection 19-21 Boulevard Jean Moulin 13005 Marseille Phone number: 04 13 73 24 01 Fax number: 04 13 73 24 02 Abstract word count: 248 Body text word count: 2,944 Keywords: Gut, redox, microbiome, microbiota, oxidative stress, metagenomics
ABSTRACT Uncontrolled oxidative stress has been associated with many diseases and aging. We previously report an increased gut redox and depletion of the anaerobic microbiome in severe acute malnutrition. Here, we extended the analysis to test if this link could be generalized by including individuals with various age and dietary status. Seventy individuals (children and adults, French and African, healthy individuals and teenagers with anorexia nervosa, marasmus and kwashiorkor) were included. Fecal redox potential was measured using a simple redox probe. v3v4 16S gene targeted metagenomics was used to characterize the microbiota. The Metagenomic Aerotolerant Predominance Index (MAPI) was defined as the natural logarithm of the ratio of the relative abundance of aerotolerant on strict anaerobic species. This index is easily understandable (MAPI > 0: aerotolerant predominance, MAPI < 0: anaerobic predominance), can be calculated for any metagenome and follows a normal distribution among our 70 included individuals. Fecal redox potential (mV) and the Metagenomic Aerotolerant Predominance Index were dose-dependently related (linear regression, p < .001). This link, if confirmed, will allow humans to take care of their microbiome and prevent, treat and/or alleviate gut redox associated chronic diseases by (i) controlling the concentration of reactive species in the gut by avoiding behavior associated with uncontrolled oxidative stress (alcoholism,…) in the gut and using reduced water, and (ii) by improving gut anti-oxidant capacities by an adequate diet rich in nutrients allowing the human gut to maintain a very low redox potential in the gut as a key for homeostasis.
INTRODUCTION The role of the microbiota in health and disease has been particularly demonstrated in malnutrition (1, 2) and obesity (3). We have shown that malnutrition leads to an alteration of the digestive microbiota with a disappearance of methanogenic arches that are intolerant to oxygen, a depletion of anaerobic bacteria and a relative proliferation of Proteobacteria, Streptococcus and Staphylococcus aureus which are oxygen tolerant bacterial groups containing many pathogens potentially responsible for infectious diarrhea (1, 4, 5). Anaerobic depletion was previously reported in malnourished children (6) and mice (7). Conversely, ingestion of one of these potential digestive pathogens orally can lead to malnutrition by oxidative disruption of the digestive microbiota (8). This suggests that the microbiota is a key instrumental partner necessary for host homeostasis at the interface with the environment. Therefore, identifying the determinants capable of preserving or altering the microbiota is a major challenge for research and medicine in the new millennium. On the other hand, and completely independently, oxidative stress has been associated with many diseases and aging. Oxidative stress is a type of aggression of the cell constituents due to reactive oxygen species (ROS) and reactive nitrogen species (RNS, N for nitrogen). The three best known ROS are superoxide anion, hydrogen peroxide and hydroxyl radical. The production of ROS and RNS is normal for all aerobic organisms and is not, in itself, an oxidative stress situation. Indeed, eukaryotic cells and bacteria called "aerotolerant" bacteria have a complex detoxification system against ROS including enzymes (superoxide dismutase, catalase, glutathione peroxidase, etc.), chaperone proteins and small molecules (glutathione, uric acid, vitamin E, vitamin C...). Oxidative stress is a pathological cellular state with damage to its constituents resulting from the inadequacy between an excessive concentration of ROS and RNS in the environment and a lack of antioxidant capacities of the cell. The pathogenic role of excessive
oxidative stress has been confirmed in numerous studies for decades, particularly in cardiovascular diseases (9). The role of oxidative stress in malnutrition has been clearly demonstrated by several teams including Golden who proposed that free radicals are critical in pathogenesis of kwashiorkor (10, 11). Moreover, animals deficient in vitamin E have an endurance 40% lower than controls in relation to cellular damage secondary to oxidative stress by insufficient antioxidant capacities (10). At the cellular level, a link between redox potential regulation and NF-kappa B activation (12) has demonstrated that the balance between oxidants and reducing agents in the environment impacts the entire cellular machinery thus leading to inflammation and cell death. However, to our knowledge, no studies have studied the direct link between fecal redox potential and the human digestive microbiome. After showing a link between anaerobic depletion and increased redox potential in a particular situation, severe acute malnutrition (4), we have here extended the analysis to test a universal link between digestive redox and microbiome including very different situations.
METHODS We have included individuals of different ages, geographical origin and nutritional status to obtain the maximum variability of microbiota and intestinal redox potential (1, 4, 13). Participants were 21 healthy adult controls from France, 3 anorexic French adolescents, 9 healthy children from Senegal, 8 children with marasmus (MRS) and 9 with kwashiorkor (KW) from Senegal, as well as 9 children with marasmus (MRS) and 11 with kwashiorkor (KW) from Niger. The French controls were healthy adults who were non-obese and without antibiotics for at least 1 month recruited by the snowball approach. Consent was obtained, and the study was validated by the local ethics committee. Most of these patients, methods for microbiota analysis, aerobic or anaerobic metabolism of each microbe, pH and redox
measurement have been previously described (4). For the redox assessment, 1 g of stool were diluted in 10 mL of distilled water and centrifugated at 8000 rpm for 10 minutes. A pH and redox meter (PCE-228-R, PCE Instruments, Southampton, United Kingdom) was used to measure the redox potential according to the manufacturer’s instructions. This previously published article focusing only on severe acute malnutrition (4) is an open access article distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Compared to this previous article, this new study includes new participants (3 French anorexic adolescents and 21 healthy adult controls) and focuses only on a possible statistical link between digestive redox potential and aerobic/anaerobic balance in stool. To assess the aerobic/anaerobic balance in stools we previously developed a database “List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism” previously published in (4) (v1.1) and updated in (14) (v1.2 – provided as Table S1). To test such a link between fecal redox (mV) and aerotolerant / strict anaerobic balance, different variables were analyzed. Two quantitative variables were obtained by measurement (redox potential, ratio of the metagenomic relative abundance of aerotolerant species on the relative abundance of strict anaerobes). Normal and log-normal distributions were tested by the Kolmogorov-Smirnov test. Automatic distribution fitting was also used to confirmed log-normal distribution. When a log-normal distribution was identified, the natural logarithm of the variable was used instead of the variable itself. Two qualitative variables were also defined: positive redox (redox potential > 0mV) and aerobic predominance (relative abundance of reads corresponding to aerotolerant species > relative abundance of reads corresponding to strict anaerobic species). Qualitative variables were compared using the bilateral khi-square test. Comparison of quantitative variables between groups were analyzed using student t test or Mann-Whitney test according to the
normal distribution test. Finally, a dose-dependent relationship between quantitative variables was tested by correlation and linear regression. Statistical analyses were performed using GraphPad Prism version 8.00 for Windows, (GraphPad Software, La Jolla California USA) and XLSTAT 2018.2 (Addinsoft, Paris, France).
RESULTS Seventy individuals were included with a large variability in age (46 children, 3 teenagers and 21 adults), geography (24 from France, 20 from Niger and 26 from Senegal), and nutritional status (30 healthy controls, 3 with anorexia nervosa, 17 with marasmus, and 20 with kwashiorkor) (Table S2). Among these 70 individuals, redox potential variability was high (minimum -137.5 mV, maximum +137.1, normal distribution confirmed by the Kolmorogov-Smirnov test, mean ± standard deviation -15.76 ± 69.07). Comparison of the redox potential between the 7 groups showed that mean was negative in all healthy groups and positive in groups with malnutrition (Table 1 - Figure 1a). Comparisons between groups showed significant differences (Table S3) but this was not the objective of this study limited solely to the link between redox potential and aerotolerant/anaerobic balance in the digestive microbiota. The 95% confidence interval of redox potential of French adult healthy controls was -90mv to 40mV (Table 1 & Table S3). From the metagenomic taxonomic assignment of each metagenome (Table S4), we determined the "aerotolerant", "strict anaerobic" or "oxidative stress resistance unknown" status for each species (Table S1 and S4). This allowed us to calculate the total number of reads that corresponded to strict aerotolerant or anaerobic prokaryotes in each metagenome (Table S5). From this, we calculated the relative abundance of strict anaerobic reads and aerotolerant reads for each metagenome (Table S6 – Figure 1b). We then calculated the ratio
of aerotolerant relative abundance to strict anaerobic relative abundance (Table S6). This ratio was > 1 for aerotolerant predominance and < 1 for strict anaerobic predominance. The variability of the aerotolerant ratio was significant (minimum 0.025, maximum 139.9). The distribution was not normal but log-normal (probability 100%, likelihood ratio 2.07 e-57, kolmogorov-smirnov test), so the natural logarithm of the aerotolerant ratio was calculated for each metagenome for further analysis (Table S6). The log normal distribution was confirmed by automatic distribution fitting using XLSTAT 2018.2 (Table S7). This variable was called the metagenomic aerotolerant predominance index (MAPI) and correspond to the variable “ln(Ae/Ana)”. The MAPI was > 0 for aerotolerant predominance and < 0 for strict anaerobic predominance. The distribution of the MAPI was normal by the Kolmogorov-Smirnov test. The variability was large (minimum -3.67, maximum +4.94, mean ± standard deviation 0.37 ± 2.16). Comparison of the aerotolerant predominance ratio between the 7 groups showed that mean was negative in all healthy individuals (anaerobic predominance) and positive in all children with malnutrition (aerotolerant predominance – Table 2 & Figure 1c). Comparisons between groups showed significant differences (Table S8). The 95% confidence interval of MAPI of French adult healthy controls was -1.88 to -0.44 (Table 2 & Table S8). Positive fecal redox potential was associated with positive MAPI (aerotolerant predominance) (27/41 (65.8%) versus 6/29 (20.7%), bilateral Khi-square test, p = .00019, Conditional maximum likelihood estimates of Odds Ratio (CMLE OR) = 7.16, 95% confidence interval (95%CI) = 2.43 – 23.36). Redox potential was significantly higher in individuals with positive MAPI (aerobic predominance, n = 37, mean ± standard deviation, 9.39 ± 11.82 mV) than in individuals with negative MAPI (anaerobic predominance, n = 33, 44.0 ± 9.4 mV, unpaired bilateral t-test, p = .0009 – Figure 2a). An outlier was detected with anaerobic predominance but positive redox potential (Figure 2a). Strikingly this individual
was also an outlier in his group (KW09_S; children with kwashiorkor from Senegal) because most of the children of this group had aerotolerant predominance. This suggests other factors than redox potential for regulating the gut aero-anaerobic balance or a non-nutritional edema (misclassified child). The MAPI was significantly higher in individuals with positive fecal redox potential than in individuals with negative fecal redox potential (n = 29, 1.60 ± 1.56 versus n = 41, 0.50 ± 2.12, p <.0001 – Figure 2b). Finally, an extremely significant relationship was found between fecal redox potential (mV) and the MAPI by correlation test (Pearson r 0.41, 95% confidence interval 0.19-0.59, R2 = 0.17, p = .0004) and by linear regression (R2 = 0.17, p = .0004. Equation: MAPI = 0.013 * redox potential (mV) + 0.57 (Figure 3)).
DISCUSSION Here we have developed a new index (MAPI: Metagenomic Aerotolerant Predominance Index = ln(relative abudance of aerotolerant species/relative abundance of strict anaerobes)) to evaluate dysbiosis related to oxidative stress. This index is clinically relevant because more and more studies suggest a role of oxidative stress on microbiota alteration (1, 14, 15). This index can be calculated for any metagenome with taxonomic assignment, notably by using our database (List of Prokaryotes according to their Aerotolerant or Obligate Anaerobic Metabolism available at our website : http://www.mediterraneeinfection.com/article.php?larub=143&titre=base-de-donnees – Table S1). The MAPI is easily understandable (MAPI > 0 corresponds to an aerotolerant predominance, MAPI < 0 corresponds to a strict anaerobic predominance). This index has a normal distribution among a group of human individuals with different ages, geographical origin and nutritional status. This allows it to be used with parametric statistics. For the first time a metagenome is no
longer characterized solely by ecological indices (global diversity) but on microbiological knowledge. This index allowed us to demonstrate a dose-dependent link between redox and the global profile of the microbiota. Why is this important? First, the concentration of reactive oxidative species and antioxidants in the digestive tract depends on the host but also largely on potentially modifiable environmental variables. Measures to control digestive oxidative stress in humans could include (i) avoiding oxidative liquids and nutrients (soda usually have a redox > +300mV, unfiltered tap water has a redox >+200mV due to its antiseptic oxidation by chlorine or ozone, some spring water have a negative redox potential up to -300mV (16)), (ii) favouring nutrients that allow the host to develop its antioxidant capacities (rather than giving only direct antioxidants, as this expose to the risk of inhibiting the host's antioxidant defenses by hormesis). In another context, it has been shown that spring water is able to decrease oxidative stress-related biomarkers (17). Secondly, the control of redox and digestive oxidative stress can allow the preservation of Healthy Human Mature Anaerobic Gut Microbiota (HMAGM (1)) which is the part of the microbiota most early altered in case of uncontrolled oxidative stress. This HMAGM is a key to homeostasis (1, 4, 18) notably with Lachnospiraceae, Ruminococcaceae and Faecalibacterium prausnitzii which produce butyrate (19), one of the key molecules produced by the anaerobic microbiota (HMAGM) with beneficial effects for the host (anti-inflammatory and anti-cancer (18)). Maturation of the gut environment in humans and mammals depend both on age and diet (5) and is associated both with gut redox decrease (20-22) and microbiome changes with anaerobic predominance (13). In few months after birth, human fecal redox decreases by 450mV (From +150 to -300mV) (20) and from positive to negative. In parallel, anaerobes proliferate to become predominant (13, 21, 22). In contrast, geography is a critical factor for microbiome (13) but this has not been reported for gut redox. Experimental data suggest that
antioxidants and reducing agents are capable of modifying the redox of the medium (23, 24), that redox is capable of modifying bacterial growth (23-25) and that an in vitro microbiota (bioreactor) is capable of lowering redox (26). Accordingly, in order to show that digestive redox is a key variable in the development and maintenance of the anaerobically predominant healthy human microbiota, we deliberately included individuals of different age and nutritional status. Children with severe acute malnutrition were recruited in Africa because this is an endemic area (6% of children from Niger are severely wasted according to the UNICEF (https://data.unicef.org/data/malnutrition/)) whereas kwashiorkor for instance is exceptional in Western countries (0 to 0.1%) with sporadic cases of fad diets, perceived and true milk allergy, but above all nutritional ignorance (not epidemic or endemic poverty) (27, 28). For instance, Liu reported only 12 cases of kwashiorkor in 9 years in the USA (27). As for age, the maximal geographical bias was used to obtain the maximal diversity in factors influencing both the microbiome and the gut redox and this also prevent overmatching bias (1). This is an innovative but preliminary study. Oxygen has already been suspected to drive gut dysbiosis (29) but we extend this link to the more generalizable and universal redox potential. Further studies are needed to confirm this link. Redox measurement should be standardized (great variability between probes, depending on the diluent used (tap water has a redox of +200mV), impact of exposure time to ambient air and stool preservation, possible direct measurement in the rectum (20), etc...). The measurement of MAPI will be improved by culturomics (30) analysis to define an oxidative stress resistance profile for reads not currently assigned to the species. Moreover, this database could be completed at the level of the operational taxonomy units (OTUs) by considering that if all the cultivated species corresponding to a metagenomic cluster have the same oxidative stress resistance profile, all the OTUs of this cluster have this oxidative stress resistance profile (example:
Lachnospiraceae are all strictly anaerobic). This would increase the accuracy of the MAPI measurement and the ability to diagnose oxidative dysbiosis. In conclusion, we present here preliminary but innovative results proposing a link between redox and anaerobic aerotolerant balance in the digestive tract. If this link is confirmed, it will open several promising pathways for human health: (i) the discovery of two markers of oxidative dysbiosis (redox potential, MAPI) allowing progress in the diagnosis of dysbiosis. It will theoretically be possible to define a "normal" value for redox potential (Table 1) and digestive MAPI and thus identify individuals with or without oxidative dysbiosis (whatever the global diversity assessed by ecological but not microbiological indexes), (ii) The identification of a modifiable determinant of at least part of the digestive microbiota opens the way to new therapeutic approaches for diseases associated with uncontrolled oxidative stress in whom the digestive microbiota plays at least an instrumental role. Many diseases are suspected to be associated with an alteration of the microbiota linked to abnormal gut redox. In addition to severe acute malnutrition (4), non-alcoholic steatohepatitis (NASH) is one of the best candidate as it is associated with an oxidative diet (soda), a diet low in fiber and unsaturated fatty acid and plasmatic oxidative stress (31, 32). Other candidate diseases are colitis (33), HIV (14), acute necrotizing enterocolitis of the newborn (34), type I (35) and type II diabetes (36), and rheumatoid arthritis (37). We also proposed a link between uncontrolled oxidative stress and disruption of mucosal immunity through gut microbiota alteration (18). Other teams have also suspected this mechanism, particularly during weaning (37), but without demonstrating any experimental causal role. More generally, this could be applicable to other human microbiota. For example, we have shown very recently that chlorexidine (an oxidizing agent containing chlorine for antiseptic purposes) does not reduce post-operative respiratory infections after thoracic
surgery (38). In this study, there was a trend for an increase in pneumococcus by chlorexidine in the pharyngeal microbiota. This is consistent with the fact that the pneumococcus is very well armed against oxidative stress. Oxidative alteration of the respiratory microbiota could be the missing piece to explain why, in this study, an antiseptic on a mucosa associated with a microbiota has no preventive effect to avoid the occurrence of infection. If the link between redox, oxidative stress and aero-anaerobic dysbiosis is confirmed, this could represent a major step forward and hope to break or slow down the vicious circle including oxidative stress and dysbiosis at play in many human diseases. Possible options to modulate this gut redox include reducing diet and water (39), however in vivo experimental and clinical studies are urgently needed. Overall, our results suggest that redox status and control of oxidative stress could be major determinants of animal and specifically human microbiomes. Beyond smoking and alcohol consumption, redox quality of diet and water could be critical in preventing, treating and/or alleviating chronic diseases associated with oxidative stress which represent the greatest public health burden and the major human threat for the new millennium.
References 1.
Million M, Diallo A, Raoult D. Gut microbiota and malnutrition. Microb Pathog.
2017;106:127-38. 2.
Tidjani Alou M, Million M, Traore SI, Mouelhi D, Khelaifia S, Bachar D, et al. Gut
Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics? Front Microbiol. 2017;8:899. 3.
Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An
obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444(7122):1027-31. 4.
Million M, Tidjani Alou M, Khelaifia S, Bachar D, Lagier J-C, Dione N, et al.
Increased Gut Redox and Depletion of Anaerobic and Methanogenic Prokaryotes in Severe Acute Malnutrition. Sci Rep. 2016;6:26051. 5.
Subramanian S, Huq S, Yatsunenko T, Haque R, Mahfuz M, Alam MA, et al.
Persistent gut microbiota immaturity in malnourished Bangladeshi children. Nature. 2014;510(7505):417-21. 6.
Mata LJ, Jimenez F, Cordon M, Rosales R, Prera E, Schneider RE, et al.
Gastrointestinal flora of children with protein--calorie malnutrition. Am J Clin Nutr. 1972;25(10):118-26. 7.
Allori C, Aguero G, de Ruiz Holgado AP, de Nader OM, Perdigon G. Gut mucosa
morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei. Journal of food protection. 2000;63(1):83-90. 8.
Rivera-Chavez F, Zhang LF, Faber F, Lopez CA, Byndloss MX, Olsan EE, et al.
Depletion of Butyrate-Producing Clostridia from the Gut Microbiota Drives an Aerobic Luminal Expansion of Salmonella. Cell Host Microbe. 2016;19(4):443-54.
9.
Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T. Endothelial dysfunction,
oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation. 2001;104(22):2673-8. 10.
Davies KJ, Quintanilha AT, Brooks GA, Packer L. Free radicals and tissue damage
produced by exercise. Biochem Biophys Res Commun. 1982;107(4):1198-205. 11.
Golden MH, Ramdath D. Free radicals in the pathogenesis of kwashiorkor. Proc Nutr
Soc. 1987;46(1):53-68. 12.
Flohe L, Brigelius-Flohe R, Saliou C, Traber MG, Packer L. Redox regulation of NF-
kappa B activation. Free Radic Biol Med. 1997;22(6):1115-26. 13.
Yatsunenko T, Rey FE, Manary MJ, Trehan I, Dominguez-Bello MG, Contreras M, et
al. Human gut microbiome viewed across age and geography. Nature. 2012;486(7402):222-7. 14.
Dubourg G, Lagier J-C, Hue S, Surenaud M, Bachar D, Robert C, et al. Gut
microbiota associated with HIV infection is significantly enriched in bacteria tolerant to oxygen. BMJ Open Gastroenterol. 2016;3(1):e000080. 15.
Darnaud M, Dos Santos A, Gonzalez P, Augui S, Lacoste C, Desterke C, et al. Enteric
Delivery of Regenerating Family Member 3 alpha Alters the Intestinal Microbiota and Controls Inflammation inMice WithColitis. Gastroenterology. 2018;154(4):1009-23.e14. 16.
Ioka S, Muraoka H, Matsuyama K, Tomita K. In situ redox potential measurements as
a monitoring technique for hot spring water quality. Sustain Water Resour Manag. 2016;2:5. 17.
Bajgai J, Fadriquela A, Ara J, Begum R, Ahmed MF, Kim CS, et al. Balneotherapeutic
effects of high mineral spring water on the atopic dermatitis-like inflammation in hairless mice via immunomodulation and redox balance. BMC complementary and alternative medicine. 2017;17(1):481. 18.
Million MT, J.; Wagner, C.; Lelouard, H.; Raoult, D.; Gorvel, J.-P. New insights in
gut microbiota and mucosal immunity of the small intestine. Human Microbiome J. 2018.
19.
Vital M, Howe AC, Tiedje JM. Revealing the bacterial butyrate synthesis pathways by
analyzing (meta)genomic data. MBio. 2014;5(2):e00889. 20.
Grütte F-K, Horn R, Haenel H. Ernährung und biochemisch-mikroökologische
Vorgänge im Enddarm von Säuglingen. Zeitschrift für Kinderheilkunde. 1965;93(1):28-39. 21.
Schulze F, Jacob HE. Redox potential of the gastrointestinal tract of swine of different
ages. Archiv fur experimentelle Veterinarmedizin. 1981;35(3):349-57. 22.
Schulze F, Jacob HE. Diarrhea in young calves. 4. Redox potential of the
gastrointestinal tract of clinically healthy calves, and calves with spontaneous diarrhea, experimental Escherichia coli infection, and cyclophosphamide treatment. Archiv fur experimentelle Veterinarmedizin. 1981;35(3):337-48. 23.
Zehnder AJ, Wuhrmann K. Titanium (III) citrate as a nontoxic oxidation-reduction
buffering system for the culture of obligate anaerobes. Science. 1976;194(4270):1165-6. 24.
Jones GA, Pickard MD. Effect of titanium (III) citrate as reducing agent on growth of
rumen bacteria. Applied and environmental microbiology. 1980;39(6):1144-7. 25.
Lund BM, Knox MR, Sims AP. The effect of oxygen and redox potential on growth of
Clostridium botulinum type E from a spore inoculum. Food Microbiology. 1984;1:10. 26.
Zapletalova M, Kasparovska J, Krizova L, Kasparovsky T, Sery O, Lochman J.
Bacterial community dynamics in a rumen fluid bioreactor during in-vitro cultivation. Journal of biotechnology. 2016;234:43-9. 27.
Liu T, Howard RM, Mancini AJ, Weston WL, Paller AS, Drolet BA, et al.
Kwashiorkor in the United States: fad diets, perceived and true milk allergy, and nutritional ignorance. Arch Dermatol. 2001;137(5):630-6. 28.
Mori F, Serranti D, Barni S, Pucci N, Rossi ME, de Martino M, et al. A kwashiorkor
case due to the use of an exclusive rice milk diet to treat atopic dermatitis. Nutr J. 2015;14:83.
29.
Rivera-Chavez F, Lopez CA, Baumler AJ. Oxygen as a driver of gut dysbiosis. Free
Radic Biol Med. 2017;105:93-101. 30.
Lagier J-C, Khelaifia S, Alou MT, Ndongo S, Dione N, Hugon P, et al. Culture of
previously uncultured members of the human gut microbiota by culturomics. Nat Microbiol. 2016;1:16203. 31.
Machado MV, Ravasco P, Jesus L, Marques-Vidal P, Oliveira CR, Proenca T, et al.
Blood oxidative stress markers in non-alcoholic steatohepatitis and how it correlates with diet. Scandinavian journal of gastroenterology. 2008;43(1):95-102. 32.
Banini BA, Sanyal AJ. Current and future pharmacologic treatment of nonalcoholic
steatohepatitis. Curr Opin Gastroenterol. 2017;33(3):134-41. 33.
Li X, Li X, Shang Q, Gao Z, Hao F, Guo H, et al. Fecal microbiota transplantation
(FMT) could reverse the severity of experimental necrotizing enterocolitis (NEC) via oxidative stress modulation. Free Radic Biol Med. 2017;108:32-43. 34.
Cassir N, Benamar S, Khalil JB, Croce O, Saint-Faust M, Jacquot A, et al. Clostridium
butyricum Strains and Dysbiosis Linked to Necrotizing Enterocolitis in Preterm Neonates. Clin Infect Dis. 2015;61(7):1107-15. 35.
Soyucen E, Gulcan A, Aktuglu-Zeybek AC, Onal H, Kiykim E, Aydin A. Differences
in the gut microbiota of healthy children and those with type 1 diabetes. Pediatrics international : official journal of the Japan Pediatric Society. 2014;56(3):336-43. 36.
Singh S, Sharma RK, Malhotra S, Pothuraju R, Shandilya UK. Lactobacillus
rhamnosus NCDC17 ameliorates type-2 diabetes by improving gut function, oxidative stress and inflammation in high-fat-diet fed and streptozotocintreated rats. Benef Microbes. 2017;8(2):243-55.
37.
Zhang X, Zhang D, Jia H, Feng Q, Wang D, Liang D, et al. The oral and gut
microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med. 2015;21(8):895-905. 38.
D’Journo XB, Falcoz P-E, Alifano M, Le Rochais J-P, D’Annoville T, Massard G, et
al. Oropharyngeal and nasopharyngeal decontamination with chlorhexidine gluconate in lung cancer surgery: a randomized clinical trial. Intensive Care Medicine. 2018. 39.
Vorobjeva NV. Selective stimulation of the growth of anaerobic microflora in the
human intestinal tract by electrolyzed reducing water. Medical hypotheses. 2005;64(3):543-6.
Acknowledgments We thank Eric ADEHOSSI, Dipankar BACHAR, Caroline BLANC-TAILLEUR, Souleymane BRAH, Aldiouma DIALLO, Alexandre FABRE, Saber KHELAIFIA, JeanChristophe LAGIER, Catherine ROBERT, Cheikh SOKHNA, and Maryam TIDJANI-ALOU for providing samples, measurements and technical help.
Conflict of interest statement Authors have no conflict of interest to declare.
Funding This work has received financial support from the French Government through the Agence Nationale pour la Recherche (ANR), including the “Programme d’Investissement d’Avenir” under the reference Méditerranée Infection 10-IAHU-03. This work was supported by Région Provence Alpes Côte d’Azur and European funding FEDER PRIMMI (Fonds Européen de Développement Régional - Plateformes de Recherche et d'Innovation Mutualisées Méditerranée Infection).
Table 1. Fecal redox potential in Humans with various Age and Dietary Status Fecal redox (mV)
Mean
Lower 95%
Upper 95%
CI of mean
CI of mean
French adult controls (n = 21)
-64.9
-89.6
-40.1
Senegalese healthy children (n = 9)
-42.9
-73.9
-11.9
French teenagers with anorexia nervosa (n = 3)
-104.9
-194.3
-15.5
Senegalese children with marasmus (n = 8)
46.2
14.9
77.4
Senegalese children with kwashiorkor (n = 9)
25.9
-14.5
66.3
Nigerien children with marasumus (n = 9)
17.8
-19.4
55.0
Nigerien children with kwashiorkor (n = 11)
18.0
-31.4
67.3
Values for all groups followed a normal distribution according to Kolmogorov-Smirnov test except the group “French teenagers with anorexia nervosa” for which the sample size was too small.
Table 2. Metagenomic Aerotolerant Predominance Index in Humans with various Age and Dietary Status MAPI
Mean
(Metagenomic Aerotolerant Predominance Index)
Lower 95%
Upper 95%
CI of mean
CI of mean
French adult controls (n = 21)
-1.16
-1.88
-0.44
Senegalese healthy children (n = 9)
-0.92
-2.76
0.92
French teenagers with anorexia nervosa (n = 3)
-0.40
-2.72
1.91
Senegalese children with marasmus (n = 8)
0.92
-0.46
2.3
Senegalese children with kwashiorkor (n = 9)
0.88
-0.83
2.59
Nigerien children with marasumus (n = 9)
1.72
0.61
2.83
Nigerien children with kwashiorkor (n = 11)
2.64
2.14
3.14
Values for all groups followed a normal distribution according to Kolmogorov-Smirnov test except the group “French teenagers with anorexia nervosa” for which the sample size was too small.
Figure legends Figure 1. Fecal redox potential and Metagenomic Aerotolerant Predominance Index (MAPI) a. Fecal redox potential according to each group (mV, mean ± standard deviation (SD)). Results of statistical tests are provided in Table S3. b. Metagenomic Aerotolerant Predominance Index (MAPI) according to each group (mean ± SD). Results of statistical tests are provided in Table S8. c. Relative abundance of anaerobic reads (green), aerotolerant reads (red) and reads for which no oxidative stress tolerance could be defined (not assigned at the species level and/or uncultured species). Note that anaerobic reads and reads without known oxidative stress resistance are higher in healthy controls (French adults: “témoin” and Healthy children from Senegal “S01 to S010”) and teenagers with anorexia nervosa (“Ano”). Conversely, aerotolerant reads predominated in children with malnutrition (MR; marasmus, KW; kwashiorkor).
Figure 2. Dose-dependent association between fecal redox potential and aerotolerant predominance a. Fecal redox potential was significantly higher in individuals with aerotolerant predominance (violin plots, median and quartiles are shown), b. MAPI (Metagenomic Aerotolerant Predominance Index) was significantly higher in individuals with positive fecal redox potential (violin plot, median and quartiles are shown). C. Linear regression evidence a dose-dependent relationship between fecal redox potential and aerotolerant predominance (MAPI = ln(Ae/Ana)).