Team 1: MILLION Matthieu & LAGIER Jean-Christophe

Human and zoonotic microbiota through culturomics in health and disease

UR D-258, Microbes, Evolution, Phylogénie et Infection (MEPHI)
Aix-Marseille Université (AMU)

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Context and objectives

By 2022, it has become clear that human health and disease cannot be conceived by reducing the scope of study to humans. On the one hand, recent emerging diseases, including recent epidemics and pandemics (SARS-CoV-2 and monkeypox (MPX)), are mostly anthropozoonoses whose emergence depends on environmental factors and proximity to transmitting animals. This has led to the placing of man in the context of a single health, in the image of ‘OneHealth‘. On the other hand, microorganisms hosted by humans, animals and plants (their microbiota) play a critical role in health (symbiosis) but also in diseases and infections (dysbiosis). The recent pandemic of Clostridoides difficile has clearly shown the possibility of deadly epidemics in which the pathogen could spread by silent colonization of human microbiota all around the world (microbiotic pandemic) and cause infection only in the presence of an enabling factor (antibiotic therapy). In addition, some human-associated microbes have beenshown to be associated with host genetic determinants and are heritable (Goodrich, 2014). On the other hand, the microbiota determines the majority of blood metabolites while the host genome would only determine 15% (Diener, 2022). It has also been proposed that symbiosis and the exchange of genetic material between the host and the microorganisms that cohabit with it could be an evolutionary power (Margulis, 1991). All this leads to the consideration of humans, animals and plants as supraorganisms or holobionts (evolutionarily stable symbiotic complexes) (Margulis, 1991). The human microbiota is therefore not a set of microbes passively associated with humans. Microbiota are therefore partners recently rediscovered thanks to new molecular technologies of high throughput sequencing (cloning, pyrosequencing and next generation sequencing). In this context, team 1 has therefore set itself the task of exploring the repertoire and interrelationships between human and animal microbiota and their hosts in health and disease through innovative and original approaches.

The specificity of the team’s approach to the study of microbiota is, for more than 10 years (2009), to have shown the limits of high-throughput sequencing approaches by developing approaches based on its expertise, the unrestricted microbial culture. One of the strengths of the team is to be able to culture microbes from all domains (Hammoudi, 2021) as well as bacteria, archaea, viruses, giant viruses, intracellular, amoebae, yeasts, mycobacteria or CPR (candidate phyla radiation). The team was able to show that molecular and culture approaches are inseparable and complementary. The advantages and limitations of each approach are the strengths of the other. In fact, the limitations and weaknesses of each approach represent the development opportunities for the next quadrennium.

Although culture remains the most valuable expertise, the team has the new, state-of-the-art Illumina NovaSeq, capable of generating up to 10¹² DNA or RNA sequences. This opens the opportunity to perform ultra-deep sequencing. Molecular approaches are not neglected and are exploited to the fullest since the team has been at the center of the development of genome-based description of new species with the emphasis on taxonogenomics (Ramasamy, 2014).

This expertise of international level is what made the reputation of the team and of the institute in which our team is registered. Indeed, the unit became known in the 1980’s for the cultivation of intracellular or fastidious bacteria, such as rickettsiae. From the beginning, the discovery of the unknown has been the main objective. This expertise was then put at the service of other fastidious bacteria such as Coxiella burnetii, Tropheryma whipplei, Bartonella on cell culture with the implementation of a biosafety level 3 laboratory (BSL3). The Tropheryma case (first culture of T. whipplei in 2000 (Raoult, 2000)) shows the complementarity of molecular and culture approaches as the axenic culture medium has been developed thanks to the analysis of the bacterial genome (Renesto, 2003). Finally, the identification and culture of unknown microbes led to the discovery of a new set of living organisms, the giant viruses and their first representative mimivirus (Raoult, 2004), later identified as a member of the human microbiota (Lagier, 2012).

While the team has had world-class microbial culture experience for 30 years, it was only in 2010 that this experience was really applied to the human microbiota. The advent of molecular methods (cloning, pyrosequencing) for the exploration of the digestive microbiota (Eckburg, 2005 – Gill, 2006) and the association of a microbiota profile associated with obesity as early as 2005 (Ley, 2005) were triggering factors for the renewal of the interest of the community in human microbiota. However, while exceptional work on culture had been done in the 1960s by Dubos, Savage and Schaedler (Dubos, 1967), culture had since been relatively neglected.

It is therefore the intersection of a unique experience in microbial culture and the renewed interest in microbiota that led us to develop a pioneering approach to microbial culturomics (Lagier, 2012). The concept was described in the seminal paper on microbial culturomics (Lagier, 2012): « By using different atmospheres, temperatures, pH, nutrients, minerals, antibiotics or phages, ‘microbial culturomics’ provides comprehensive culture conditions simulating, reproducing or mimicking the entirety of selective constraints that have shaped the gut microbiota for millions of years. In fact, each isolated microorganism is one among the possible viable solutions to the evolutionary equation whose constants are the selective constraints of the environment, corresponding here to the human gut. This is why microbial culturomics is the best way to capture the functional and viable gut microbiota biodiversity of each human individual through large-scale isolation, and to capture the deepest informational genetic gut biodiversity by sequencing the complete genomes of the previously isolated microorganism. »

This approach, aiming at capturing the living microbiota, is therefore intrinsically based on a fine and precise understanding of the microenvironment studied (knowledge of the physicochemical and nutritional characteristics of the environment), which is not the case for molecular methods that do not require any knowledge of the microenvironment studied and do not differentiate between living and dead microbiota. The team has therefore pioneered ‘microbial culturomics’, a new ‘omics’ approach that is thus culture applied to the untargeted exploration of microbiota to capture their maximum diversity (Kambouris, 2018). This term, proposed by our team, is now an approach recognized by other teams in the scientific community. Its strong point is to go in search of the unknown, i.e. to explore and illuminate microbial dark matter (Bellali, 2021 – Dickson, 2017 – Dance, 2020). Its other strong point is to distinguish the living from the dead, which is not the case of metagenomics, since it has been shown that a significant part of the uncultured sequences do not correspond to living microbes (Bellali, 2021). The known and unknown but living microbiota is therefore the real object of research of team 1.

Finally, another strong point of the team is to include researchers, clinical microbiologists, infectious diseases specialists, and veterinarians who all work in the same institute and the same building. There are no barriers between humans and animals and the laboratory and interactions are facilitated.

The team’s objective is therefore to explore, observe and describe the living human and zoonotic microbiota in health and disease with a priority focus on the isolation of strains that can be characterized, preserved, and used in in vitro or in vivo models and potentially as constituents of probiotics. The development and use of innovative high technologies is a methodological objective.

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Project

Among the prospects for equipment that could optimize the team’s ability to develop its theme and achieve its objectives, we can mention automated ‘single cell’ culture capabilities (microfluidics, flow cytometry adapted for one microbial cell culturomics) or targeted microscopy (laser microdissection (Chassaing, 2019) with culture module). Some examples are represented by the MilliDrop instrument (https://www.millidrop.com/) or automated single cell culture solutions or using flow cytometry (Afrizal, 2022 – Bellais, 2022). Culture on human mucosa and/or mucosal cells (enterocytes, nasal or bronchial mucosa cells) also represents a critical perspective for bacteria associated with the mucosa (SFB,…). A work in progress with a Parisian team (MICALIS) will allow us to test an in-vitro model of SHIME® digestive microbiota. If this test is conclusive, such equipment could also be part of our perspectives since we wish to develop in vitro and in vivo models (axenic mice).

Situation report on the current microbial culture in the world: approaches of our team and development of new strategies

Establishment of a repertoire of human bacteria and expansion of the strain collection 

In 2021, the culturomics approach added nearly 1100 bacteria to the repertoire of prokaryotes cultured at least once in humans, representing a 26% increase in that same repertoire. This same approach has allowed the discovery of more than 400 bacterial taxa (Diakité, 2021 – Bilen, 2018) and has contributed significantly to the expansion of the catalog of the Strain Collection of the Rickettsia Unit, which to date includes more than 12,000 bacterial isolates for nearly 2,700 species of prokaryotes.

Expertise in the isolation of new human microbes by culture

While the use of rapid identification means (i.e., MALDI-TOF MS) has been an indispensable technological element in the development of this approach, one of the keys to the success of culturomics lies in the development of innovative culture media, the analysis of their performance, as well as the rationalization of their use. Thus, our team initially used nearly 270 culture conditions for the pioneering study, which were later reduced to 70 and then 18 (Lagier, 2016). We recently reported the development of a streamlined standardized approach to capture bacterial diversity comparable to that obtained with the 18 culture conditions using a so-called « fast » culturomic technique using only 4 culture conditions. These last two approaches will also be used in case control studies in which our team is involved (see paragraph 4 & Table 2).

New strategies for microbial culturomics

Despite these advances, a substantial number of bacterial species of human origin remain uncultured to this day, making it essential to pursue efforts dedicated to the development of innovative culture approaches. Among the approaches developed during the development of culturomics, media aiming to mimic the natural environment of bacteria, particularly digestive, have proven to be particularly fertile. Thus, we reported that the addition of rumen to conventional media used in clinical microbiology (i.e., blood cultures) significantly increased the diversity captured by culture approach (Diakité, 2020). This observation echoes several works in environmental microbiology using diffusion chambers to mimic the natural environment of bacteria (Kaeberlein, 2002). To our knowledge, this process has never been adapted to the study of human microbiota. Thus, to go further, we wish to develop a model of diffusion chamber dedicated to the study of human digestive microbiota that can be used as a proof-of-concept in larger scale works, and applied to other microbiota.

Compared to anaerobic culture, a technological leap has been made with inoculation in anaerobic blood culture bottles. This is a miniaturization process (anaerobic hood => anaerobic box with gas generator => anaerobic flask with direct inoculation through an impermeable cap). The continuation of this process leads us to think about miniaturized devices. On the other hand, one of the tedious aspects of culturomics is the considerable handling time dedicated to the subculturing of colonies in order to obtain a pure isolate. One possible solution is the use of single cell bacterial delivery systems coupled to a microfluidic system, allowing the direct isolation of bacterial species in pure culture (Afrizal, 2022). If these approaches are often used with conventional or commercial media, the combination of such an approach with innovative or specific culture media that we have already developed would allow to increase the yield of culturomics while considerably reducing the handling time.

We also wish to develop the « directed » culture method. Indeed, operational taxonomic units (OTUs) or Amplicon Sequence Variants (ASVs) of interest can be identified by high-throughput sequencing methods in case-control studies without having a representative in culture, making their isolation extremely difficult. We aim to develop the species-targeted sorting approach, designating monoclonal antibodies to sort species of interest by flow cytometry and thus facilitate their growth (Bellais, 2022). The development of this approach would be facilitated by the prior existence of all the necessary equipment within the different platforms of the IHU Méditerranée Infection (Table 1). This approach could be coupled with the detection of the viability of bacterial species of interest by methods that we already master, such as cytometry or electron microscopy (Bellali, 2019).

Exploration of Candidate Phyla radiation (CPR)

CPR represent an emerging division of the bacterial domain that are grouped in the superphylum Candidatus Patescibacteria (Naud, 2022). These minimicrobes are described as obligate episymbionts, invisible to light microscopy with a size of less than 400 nm and possessing a ribosome as well as a reduced genome (<1 Mb).

Our team initially observed CPR in stools following a study of the microbial composition of stools from healthy volunteers by electron microscopy. Following this, we performed an exhaustive literature review on the subject and studied their prevalence in different human samples using a standard PCR system specific for Candidatus Saccharibacteria. In addition, we designed a real-time PCR system, also specific for Candidatus Saccharibacteria (Ibrahim, 2021 – Ibrahim, 2022). CPR are reported as quasi-ubiquitous in the oral cavity and detected in the gastrointestinal, urogenital and respiratory tracts mainly. They are also associated with pathologies such as gingivitis and Crohn’s disease. Our study aimed at mapping them in the human microbiota by standard PCR, real-time PCR and electron microscopy confirms their presence in the oral, intestinal, urogenital and respiratory microbiota. Unexpectedly, we also detected them in breast milk as well as in the blood of febrile patients and the heart valves of patients suffering from infective endocarditis.

Currently, CPR are only isolated in co-culture. However, these isolation methods are not very reproducible because obtaining stable co-cultures following host infection is rare. We therefore aim to improve the methods of co-culture of Ca. Saccharibacteria in order to obtain isolates and to better understand their phylogenetic diversity, their metabolism in order to obtain axenic cultures.

Our methods for studying CPR are currently based on electron microscopy, specific PCR as well as metagenomics and bioinformatics. However, we aim to develop their detection by fluorescence in situ hybridization (FISH) which would allow a specific identification contrary to electron microscopy which currently allows us only to confirm the results of PCR. Moreover, there is a discrepancy between what we observe by electron microscopy and the results of PCR and metagenomics, which implies an extraction defect. We are currently optimizing the CPR extraction methods in order to obtain easily genomes of Ca. Saccharibacteria.

Impact on microbial taxonomy

The criteria for taxonomic discrimination of new taxa discovered by culturomics have evolved since the creation of the culturomics approach. While sequence homology with the 16S rRNA gene was used for several years, species definition is now achieved through genome comparison by digital DNA-DNA hybridization (dDDH) coupled with increasingly exhaustive phenotypic criteria. These increasingly complete descriptions of new taxa have allowed us to reclassify many species of Ruminococcus (Togo, 2018) or Faecalibacterium spp.

Complementarity with high-throughput sequencing approaches

Finally, it is essential to complement our culturomic work with an optimized high-throughput sequencing approach. At present, our 16S metagenomic data are analyzed by a commercially licensed pipeline that does not include the latest advances in bioinformatics analysis. We would like to develop our own analysis pipeline that would allow us to be in line with the latest standards, to analyze taxa that are differentially abundant between several groups in an automated way and for which we could autonomously integrate our own databases (i.e., 16S sequence libraries, and complete genomes).

In a circular way, the optimization of our high-throughput sequencing approach and their bioinformatics analysis would allow us to measure more precisely the impact of culturomics on the decrease of the so-called « dark matter » (i.e., bacterial species considered to date as non-cultivated) and thus to valorize a decade of work.

Status report on the knowledge of human microbiota associated with health and disease

Knowledge of the human and zoonotic microbiota cannot be limited to knowledge of the species that constitute it, even if establishing its repertoire is a key step. Understanding the role of the human and zoonotic microbiota in health and disease is a complementary objective. In fact, describing the repertoire and understanding the mechanisms involved in health and disease are again complementary. For example, the study of the microbiota associated with malnutrition led the team to discover a depletion of aerointolerant microbes and to propose a link between oxidative stress, antioxidants, and maintenance of the healthy, aerointolerant-dominant microbiota (Million, 2016 – Million, 2018). This ‘anaerobic’ predominance was known but had been overlooked in molecular studies. The identification of this link had a direct impact on the description of the repertoire by the development of media enriched with the 3 main human antioxidants: vitamin C, glutathione and uric acid (La Scola, 2014) and the filing of a patent (WO 2014/064359 A1 ‘Use of an antioxidant compound for the cultivation of oxygen tension sensitive bacteria’). Once again, this evidence that association in the same team of clinician, microbiologists collaborating for the description of the repertoire and its variations in health and disease is a strength of the team.

There are currently no recognized or accepted criteria to characterize a normal microbiota. The very large diversity of human microbiota makes this a challenge. It remains a challenge for the coming years to determine whether it is possible and which markers can be used to determine a healthy or an ‘abnormal’ microbiota. The approach that seems most appropriate to characterize a healthy microbiota is ultimately to characterize its variations according to all potentially confounding factors (age, gender, and geography) and diseases (Lagier, 2012b). This is why many teams (Ley, 2005) have conducted case-control studies. In particular, our team has contributed to this effort to compare microbiota in several case-control studies of obesity, anorexia, malnutrition, nonalcoholic steatohepatitis, necrotizing enterocolitis and Clostridoides difficile colitis (Table 4). This allowed the identification of some of the specific characteristics of the microbiota associated with health.

Microbiota and nutritional diseases

In nutritional diseases, the study of obesity, severe acute malnutrition (SAM), anorexia and non-alcoholic steatohepatitis (NASH) has revealed common and specific profiles. For example, enrichment in Proteobacteria is found in several diseases (severe acute malnutrition, non-alcoholic steatohepatitis) and particularly in Klebsiella pneumoniae. This is consistent with the results of other teams that have associated K. pneumoniae with NASH and endogenous ethanol production in these patients (Mbaye, 2022). This led us to identify yeast as another potential candidate for endogenous ethanol production in these patients (Mbaye, 2022). This illustrates the team’s approach of not falling victim to barriers between different microbe domains (trans-kingdom microbiota studies (Hammoudi, 2021)). Moreover, on nutritional studies, the conjunction of medical expertise and understanding lead to reconsider the nutrients and co-factors necessary for cultivation and the metabolites produced:

• Links between oxidative stress, anti-oxidants, and aerointolerant microbiota (Million, 2016)
• Link between salinity, halophilic microbiota, obesity (Seck, 2019)
• Link between fructose, Klebsiella pneumoniae and yeast, ethanol production (Mbaye, 2022). Beyond the medical importance, this led us to develop alcohol media and to describe an ‘ethanophilic’ strain (Mbaye, 2022)

Nutritional diseases are an opportunity for the study of dysbiosis. But the discoveries made can extend beyond nutritional diseases, including infectious diseases (aerointolerant depletion found also in HIV (Dubourg, 2016). It is therefore a new paradigm where the boundary between nutritional disease and infectious diseases becomes blurred. Indeed, the study of the microbiota by our team has shown that malnutrition could lead to dysbiosis with enrichment in invasive pathogen (malnutrition => infection) and it has already been clearly shown that infectious diarrhea could lead to malnutrition (infection => malnutrition).

This interrelationship between nutrition and infection is particularly critical in the first 1000 days of life, a period described as a window of opportunity for the development of the child, especially the immune system. In this context, the team showed the importance of the breast milk microbiota. Unexpectedly, the team discovered the colonization of mother’s milk in Africa (Mali & Senegal) by the pathogenic bacterium Listeria monocytogenes (Togo, 2020 – Sarr, 2021). It was shown that this was the same clonal complex (Sarr, 2021) and that it was also found in the vagina of women with spontaneous abortions (Fall, 2020). This is a good representation of the team’s dynamics, capable of linking high technology, culture, malnutrition, and the North-South link. To go even further, preliminary work by our team suggests that nutrition may have an effect on cancer risk through the epigenetic role of microbiota, and in particular butyrate production (Million, 2020).

Perspectives for the next quadrennium

Perspectives for studying the links between microbiota and nutritional and metabolic diseases include:

• Development of the GDRI DySAM (International Research Grouping – Dysbiosis associated with Severe Acute Malnutrition) with Mali, Niger and Senegal – Funding over 4 years
• New South partnerships on Malnutrition (Republic of Congo, Pr Muyembe, two students planned in Master 2 for 2023)
• Valorization and publication of the HEPATGUT study on liver diseases and microbiota (collaboration with Pr GEROLAMI)
• ANR (pre-proposal filed 2022) on the role of yeasts in NASH with new collaboration with the TAAM center (CNRS UAR 44 TAAM: Typing and archiving of animal models – Director Cécile FREMOND) for the capacity of in vivo axenic models to test the hypothesis that antifungal agents could correct steatohepatitis when associated with ethanol hyperproducing yeasts
• Investigation of dysbiosis associated with NOMA (disease associating facial destruction with malnutrition whose microbial etiology remains to be determined)

Microbiota and non-nutritional diseases

Microbiota, cancer and immunotherapy

From 2017 to 2021, the IHU Méditerranée Infection was involved in the RHU LUMIERE, a European consortium led by the Institut Gustave Roussy (IGR) (Pr Laurence Zitvogel) whose objective was to identify « oncomicrobiotics », ie. bacterial species/strains associated with the response to immunotherapy in various cancers (lung cancer, kidney cancer). Our team contributed significantly to this project by leading the culturomic studies, by providing strains from our collection (i.e., Akkermansia muciniphila, Enterococcus hirae) for in vitro experiments, or by designing culture media specifically dedicated to the research of strains of interest. On this last point, we have patented a culture medium allowing the specific isolation of Enterococus hirae isolate (patent number 1H53316 case 24 FR1755310).

Susceptibility to HIV-1 infection and disease progression

Team 1 is an investigator since 2021 in the Workpackage 1 of the European Project MISTRAL led by Pr Roger Paredes (IrwiCaixa, Barcelona) for which a funding of 730,470 euros has been obtained. Recent observations suggest that the composition of the digestive microbiota may predispose to infection and we hypothesize the existence of a microbiota that may conversely induce resistance to the acquisition of HIV-1 infection and, by extension, to disease progression. The project proposes to study the digestive microbiota of 3 cohorts of MSM patients (men who have sex with men) by culturomics and high throughput sequencing in order to identify potential candidate fecal bacteria and to measure their in vitro impact on HIV-1 virus replication in a descriptive and observational population-based study. Two collaborating centers (i.e., Institut Irsi Caixa and BCN Checkpoint, Barcelona) are also included in this project.

Microbiota and neonatal infections

The work of the team on nutritional diseases has led to a precise interest in the milk microbiota. This has interested our pediatric colleagues and has led Aurélie MORAND (MCU-PH, head of the pediatric emergency department of the APHM) to join team 1 for the next quadrennium. A preliminary collaborative work with the supervision of a pediatric intern has shown that the milk of mothers whose children had a neonatal infection did not contain the pathogen responsible for the infection. However, a dysbiotic profile with an enrichment of Corynebacterium kroppenstedtii, a pathogen usually associated with granulomatous mastitis was found.

Microbiota and necrotizing enterocolitis

We have demonstrated an association between the presence of Clostridium butyricum in stool and the occurrence of necrotizing enterocolitis (NEC) in preterm neonates whether by pyrosequencing and culture methods or by quantitative real-time PCR specific for C. butyricum (Cassir, 2015). We also showed that the digestive microbiota of neonates with NEC were less diverse, more acidic and oxidized than those of healthy controls. Furthermore, we identified, after whole genome sequencing of all our C. butyricum strains, the presence of a β-hemolysin gene homolog. Cytotoxic activity of culture supernatants of C. butyricum strains could also be demonstrated. Once again, microbial culturomics was a determining factor. Indeed, this species had not been identified initially by metagenomics. The reanalysis of the sequencing data after the results of the microbial culturomics led to correct the taxonomic assignment. Subsequently, we showed the association between clonal strains of C. butyricum and the epidemic occurrence of NEC (Hosny, 2019 – Benamar, 2017). We obtained a 39,811 € funding in the framework of the AORC 2018 call for projects in order to study the colonization kinetics by C. butyricum and C. neonatale in preterm neonates. Indeed, the identification of the source of C. butyricum and C. neonatale involved in the occurrence of NEC epidemics remains a major issue in the understanding and control of these epidemics in the future.

Finally, in collaboration with Prof. Harry SOKOL of the research team of the MICALIS Unit (Microbiology of food for health) at the Saint-Antoine Hospital, we plan to study the impact of the presence of C. butyricum, C. neonatale, but also other species of Clostridium spp. such as C. difficile, C. perfringens, C. paraputrificum on the composition of the digestive microbiota from an in-vitro model of digestive microbiota that could be adapted to the study of the digestive microbiota of premature newborns. Thus, the in-vitro model of digestive microbiota SHIME® consists of six reactors connected and controlled by computer. The model is specifically designed and validated to simulate the conditions found in the stomach, duodenum/jejunum, ileum, ascending, transverse and descending colon (Van den Abbeele, 2010). In addition to being highly representative of the intestinal microbiota encountered in vivo, this model has the advantage of being less ethically restrictive, opening up prospects for analyzing the effectiveness of bacteriotherapy in the treatment of NEC. In the long term, we plan to import this technique to Marseille in order to apply the analysis of the digestive microbiota to different applications within the framework of different collaborations such as fecal microbiota transplantation (donor selection for example).

Skin microbiota

Recent studies have identified associations between altered skin microbiota composition and physiological changes such as skin stigmata of aging and various dermatological diseases (Boxberger, 2021). We established a collaboration between the IHU-Mediterranean Infection (IHU-MI) and the L’Occitane Group. The aim of this collaboration was to study the composition of the skin microbiota and its variations according to age using combined metagenomic and culturomic approaches. It is thanks to the expertise of our MEPHI (Microbe, Evolution and Phylogeny) team within the IHU-MI, that the financing of this project was obtained. The research and development laboratories of the L’Occitane Group (skin biology laboratory) allowed us to analyze the interactions between microbiota and 3D skin models. The use of commensal bacteria of interest as an active ingredient in cosmetic formulations is invaestigated. For this, the use of 3D skin models will provide information on the behavior of the skin in reaction to the co-culture of these commensal bacteria of interest. It will thus be possible to assess the probiotic potential of each of these strains. Finally, the analysis of compounds secreted by the bacterial strains of interest that can be isolated, produced and used as prebiotic active ingredients such as carotenoids, constitutes a major issue in cosmetology but also in dermatological pathology.

Perspectives for the next quadrennium for microbiota and non-nutritional diseases

For the next quadrennium, the team wishes to develop its expertise on the role of the microbiota in non-nutritional diseases:

• Cancer: renewal of the collaboration with Laurence Zitvogel’s team (RHU Immunolife). New collaboration since 2022 with an African-European consortium MICAFRICA on microbiota and cancer (Leila KESKES, Sfax, Tunisia). Prospects of collaboration with the Institut Paoli Calmette.
• Neonatal infections: recruitment of a pediatrician student in Master II to continue the collaboration on milk microbiota and neonatal infections (Call for clinical research, AORC MINEOS in progress).
• Necrotizing enterocolitis: new partnership with Nice University Hospital (AORC) and MICALIS Institute (H. Sokol)
• Skin microbiota: development of probiotic potential – industrial partnership (L’Occitane)

Interactions of human and animal microbiota in a OneHealth perspective

Veterinary laboratory of the IHU Méditerranée Infection

Sixty percent of pathogens affecting humans are zoonotic and originate from domestic or wild animals. The « One Health » concept was born from the recognition of the need to control pathogens at the level of animal populations at the human/animal/environment interface. This approach is implemented within the IHU, mainly by a veterinary field epidemiologist and his assistant veterinary microbiologist. They are responsible for the IHU veterinary research center, which is organized as a reactive structure, light in its operation, mobile and adapted to the care and study of animals (domestic or wild, living or dead) suspected of zoonotic or unknown infectious diseases, or likely to be sentinel animals and/or reservoirs of pathogens for humans. An adapted room is dedicated to this activity in the IHU.  The veterinarians intervene in the south-eastern Mediterranean but also wherever transmissible, potentially zoonotic, endemic or emerging diseases are spreading. By collaborating with the world of veterinarians and other professionals in the animal world, it is possible to bring to the IHU, in the shortest possible time, samples likely to provide the microbiological or parasitological data necessary for the realization of analyses of epidemiosurveillance of zoonotic risks with the objective: « the right sample, at the right time, well preserved ». Thus, a biobank has been set up containing nearly 40,000 samples (blood, organs, feces, swabs, ectoparasites, etc.) collected from dozens of animal species from the five continents. Thousands of analyses (PCR, serologies, cultures, coproscopies…) are carried out to detect nearly a hundred infections, including coronaviroses, arboviroses, plague, Q fever, bartonellosis, anaplasmosis, leptospirosis, tuberculosis, leishmaniasis, toxoplasmosis, babesiosis, trypanosomiasis, filariasis… The analyses are mainly performed at the IHU or in collaboration with about fifteen reference laboratories. Recently, these investigations, from the field to the laboratory, have allowed the identification of the ovine source of an outbreak of human Q fever in the Hérault, the epidemiological role of the dog as a sentinel of arboviroses in the southeast (West Nile, Usutu and unidentified flavivirosis), the characterization of colistin-resistant enterobacteria in the feces of wild boars in the southeast. In addition, in partnership with the city of Marseille, an epidemiological monitoring of the carriage of zoonotic agents by commensal rats allows to compare by genotyping the leptospires detected in the surmulot and in the patients diagnosed at the IHU. These murine investigations also include Bartonella and Pasteurella isolated in culture. These microorganisms have the potential to cross the species barrier.

Our « One Health » project is to show that a new « systemic » approach centered on man (sick or exposed to potential risks) is possible. It starts from man, but, from this positioning, it must aggregate, beyond the strictly medical vision, other knowledge that makes infectiology a coherent whole and integrated with the environment (animal world, environment, ecosystems, climate, …). Since 2019, Bernard Davoust and Younes Laidoudi are co-authors of 81 articles referenced on PubMed, all of them carrying this « One Health » vision.

The microbiota of non-human primates as reservoirs of zoonotic diseases

Most emerging diseases are zoonoses. Since the AIDS pandemic and the indisputable demonstration that it originated in the accidental transmission of simian retroviruses to humans, viruses as deadly as rabies, herpes B virus, Marburg and Ebola hemorrhagic fever viruses have been transferred from apes to humans. Since apes are our closest genetic relatives, the pathogens that colonize them are probably the best adapted to be transferred to humans in case of accidental exposure. We plan to continue using non-invasive methods to explore the microbiota of simians in Africa. For our studies we would like to use monkey feces. Non-invasive sampling (stool collection) is very important for endangered animals (e.g. all great apes). We would like to search for microorganisms (especially pathogens) by molecular biology (specifically PCR, Sanger sequencing and NGS), by classical microbiology tools (culture, etc.) and by serology. We consider this approach to have prospects in the solution of still existing enigmas in the epidemiology of zoonotic diseases such as COVID-19, Ebola fever etc. For this project we have already obtained a financing within the framework of PREZODE/AFRICAM in Senegal; the project for European financing (EDCTP3) has been submitted in collaboration with our partners in Senegal, Gabon, Congo-Brazzaville and DRC.

The unexplored microbiota of chiropterans, reservoirs of zoonotic diseases

Despite the importance of the role of bats in the functioning of the biosphere, their ecology and their role as reservoirs of zoonotic diseases remain poorly known. Several microorganisms have been isolated or detected in bats, including Rhabdoviridae of the genus Lyssavirus, Paramyxoviridae such as Nipah and Hendra viruses, Filoviridae (Ebola and Marburg viruses) or Coronaviridae (SARS and MERS). Some bat species appear to be reservoirs or effective disseminators of these infectious agents, or even transmitters. Our team has recently documented a serious new disease with high mortality in hunters and meat consumers of large fruit bats (dogfish) in New Caledonia. These bats of the genus Pteropus were confirmed as the source and reservoir of the new pathogen responsible for this disease, Candidatus Mycoplasma haemohominis (Descloux, 2021). 

We plan to continue research on the patho-microbiota of bats especially in Africa. Among the goals are the determination of the presence of haemotropic mycoplasmas in bats in Africa, the isolation of Bartonella, the detection of antibodies against Rhabdoviridae, Paramyxoviridae, Filoviridae and Coronaviridae in order to determine the hypothetical role of chiropterans in the circulation of these pathogens and, probably, in the origin of zoonotic epidemics. We have already obtained a permit to capture and collect bats in Senegal (Direction Des Eaux Et Forêts, Chasses Et Conservation Des Sols du Sénégal).

Termites that cause antibiotic resistance

Termites are important ecosystem engineers in tropical habitats. So-called mushroom termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialized fungus wheel structures, with no apparent problems of infectious disease. We have performed in vitro testing and found that chemical extracts of mushroom cap material can inhibit the growth of bacteria and fungi. Chemical analyses of the mushroom wheel material indicate a very complex metabolome, including compounds that may play a role in inhibiting the growth of bacteria. This confirms our previous data on the presence of multidrug-resistant bacteria (Klebsiella pneumoniae) in termites and chimpanzees – termite consumers. We plan to continue our work on the following hypothesis: termite fungus wheels (made of residues of organic matter harvested by termites in the environment) contain several antibiotic and antifungal molecules; the passage of environmental bacteria through this wheel constitutes a natural filter for the selection of antibiotic-resistant microorganisms. This hypothetical mechanism could possibly explain the abundance of multidrug-resistant bacteria in tropical environments, taking into account the moderate consumption of antibiotics in these environments.

Vectorized microorganisms

Deadly lice-borne diseases such as typhus, relapsing fever, and trench fever affect susceptible populations and flare up easily during socioeconomic disasters. We are targeting two lines of research in the project:

a) Human lice are among the oldest parasites of humans, making them an excellent marker of the evolution and migration of the Homo species over time. Pediculosis of the scalp and pediculosis of the body are highly contagious external parasites. Human lice are also vectors of human diseases of primary importance. We plan to compare the proteomics of head lice versus body lice. This research may give us keys to explain the specialization and different abilities to transmit pathogens between two ecotypes. We also target to continue studies on the resistance of lice to ivermectin and the effects of naturally occurring products on lice and its endosymbiotic bacteria in the context of researching new insecticidal tools.

b)In order to decipher the microbiota of human infectious disease vectors, we will continue our work on the isolation of fastidious microorganisms from different arthropods. Several examples demonstrate that microorganisms initially isolated from arthropods are pathogenic to humans (Rickettsia africae, Neoerhlichia mikurensis etc.) or may play an important role in the vector capabilities of arthropods (Wolbachia spp.). We will continue to use such approaches as cell culture and the development of new axenic media to isolate difficult microorganisms. Microbial culturomics applied to arthropods, including methods for intracellular, could for example allow to better characterize a Coxiella burnetii-like endosymbiont very frequently detected by molecular biology in some arthropods (Angelakis, 2016).

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Ethics / GMOs / Parity

All of the team’s work respects the ethical principles of the Helsinki declaration revised in 2013 in Fortaleza (World Medical Association, 2013). The team within the UMR plans to streamline the circuit of research projects for validation by the ethics committee and a research support cell before starting any process of participant inclusion or sample collection. If requested by the ethics committee, projects will be submitted to the personal protection committee. As the team is particularly oriented towards the South, particular attention will be paid to the respect of the NAGOYA protocol. One of the principles of Team 1 is to explore natural human and zoonotic microbial strains, to study them and eventually to propose them as therapeutics (probiotics). As such, no genetically modified organisms are produced by Team 1.

For gender, the team is putting all its energy to support the candidacy of Maryam TIDJANI ALOU (microbiota malnutrition theme) to the IRD 2023 CR competition. Moreover, one of the team leaders (MM) is a « representative of equality » in the IRD sectorial scientific commission 2 (CSS2).

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NETWORK

Local :

• Biochemistry Department, Assistance Publique Hôpitaux de Marseille (Pr Régis Guieu) – Active
• Architectures and Function of Biological Macromolecules Laboratory (AFMB) – Active
• Marseille Luminy Immunology Center (CIML) – Active
• Paoli-Calmette Institute (IPC) – To develop

Regional

• Hepatology Department, Nice University Hospital (Rodolphe ANTY – NASH and microbiota) – Active

Nationals :

• Institut Gustave-Roussy (L Zitvogel) – Active
• MICALIS Unit (Microbiology of food for health) at the Saint-Antoine Hospital (H. Sokol) – Currently being set up.
• Neonatology department, CHU Nimes (Microbiota and necrotizing enterocolitis) – Active.
• UMR QUALISUD Institute of Research for Development – Active.
• CNRS UAR44 TAAM: Typing and archiving of animal models (Cécile FREMOND – Orléans) – Perspective.

International :

• Senegal, IRD Cheikh Sokhna Unit – Active.
• European project MISTRAL H2020 (https://cordis.europa.eu/project/id/847943) – Active.
• MICAFRICA project (JC, MM, MTA) – H2020 project – Coordinator Leila KESKES (https://micafrica.eu/) – Active
• GDRI DySAM – Active
  • Mali (Malaria Research Training Center – Pr Thera, Pr Djimde)
  • Niger (CHU Niamey – Pr BRAH)
  • Senegal (IRD VITROME – Cheikh SOKHNA)

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Conclusions

Human and zoonotic microbiota represent microscopic living partners with an exceptional diversity that are keys for the understanding of evolution, health and disease, for precision medicine (personalized medicine) and the OneHealth perspective of tomorrow. The original approach proposed by Team 1 has been from the beginning to focus on the culture of the living microbe. The precise knowledge and identification of new pathogens and probiotics, and the domestication (Dance, 2020) of unknown microbes is a challenge for the future that represents an unparalleled opportunity to develop new preventive and therapeutic options using natural human-derived non-GMO strains.

Specifically, the team wishes to invest maximum energy in developing natural (not artificial, not genetically manipulated or engineered strains) and local solutions. For example, the team is looking for probiotics for malnutrition in Senegal in the microbiota of healthy people in Senegal, or probiotics for necrotizing enterocolitis in France in the microbiota of healthy controls in France. The geographical specificity of the strains remains to be clarified.

The team’s objective is clearly the exploration of the microbial dark matter to discover new microbes, in order to illuminate the entire map of the human microbial world, but also the understanding of the mechanisms of symbiosis, dysbiosis and pathogenicity. After 10 years of microbial culturomics, the transition to the next quadrennium represents a turning point and an opportunity for renewal both conceptually (microbes clinging to the skin and mucous membranes inaccessible by analysis of fluids and excreta (stool), ultra rare microbes, CPR) and technologically (laser microdissection on surgical tissue, ultra deep sequencing, single cell culturomics, microfluidics, diffusion chamber inspired by environmentalists, in vitro models in bioreactor, axenic models)

The better knowledge of the repertoire of microbes associated with humans and zoonotic microbes of interest for humans feeds the clinical perspectives of the team in the investigation of the role of microbiota in health and in nutritional diseases (obesity, anorexia, severe acute malnutrition, non-alcoholic steatohepatitis, noma) and non-nutritional diseases (cancer, ulcerative colitis, neonatal infections,…) This will allow us to find prebiotic and probiotic solutions for these scourges impacting human development (WHO Sustainable Development Goals). Echoing Metchnikov’s book on the potential role of digestive microbiota and probiotics for health (Ilya Ilitch Metchnikov. The prolongation of life: optimistics studies, 1906), knowledge of the microbiota could also play a role in ageing, particularly skin ageing (industrial partnership with L’Occitane), without neglecting aesthetics and cosmetics (skin probiotics). Beyond the study of each bacterial or fungal world taken in isolation, the new studies will associate the different domains (trans-kingdom approach: bacterial, fungal, eukaryotic) allowing to decipher interactions that were neglected until now.

Finally, the study of animal microbiota will contribute to a ‘OneHealth‘ approach. The inclusion of the study of zoonotic microbiota in the team’s objectives demonstrates that human health, which remains at the core of the team’s work, cannot be conceived without the awareness and knowledge of its micro (holobiont) and macroscopic (animal and vector) environment and ecosystem. In summary, the team’s perspectives for the next quadrennium extend to the entire microbial ecosystem in which man is embedded. This knowledge will allow optimism by providing new solutions to the challenges of human fatality.

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Publications

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