Pamela Afouda

Grégory Dubourg

Didier Raoult
email: didier.raoult@gmail.com

Dr Didier

Archeomicrobiology applied to environmental samples

The study of ancient microorganisms represents one of the main ways to understand how microbes have evolved to date, especially those associated with humans or ecosystems of interest. However, these studies are always tedious because the viability of the microbes is difficult to maintain and the degradation of their DNA, can make their detection difficult. The explosion in the number of studies on ancient microorganisms in recent years is partly due to improved methods and their availability, ranging from microscopy to next generation sequencing techniques (NGS). In this article, we discuss these methods and their contribution to deciphering the ancient environmental microbial community, with particular emphasis on permafrost, ancient halite, amber and ancient rocks.

INTRODUCTION

While the recent dramatic evolution of methods commonly used in the field of microbiology is mainly used to analyze contemporary specimen, studies on ancient samples have increased considerably in recent years. Notably, these studies clarify our understanding on the changes of microbial community at different periods as well as the associated phenomena [START_REF] Gilbert | Assessing ancient DNA studies[END_REF][START_REF] Adler | Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions[END_REF], including climatic conditions [START_REF] Graham | Microbes in thawing permafrost: the unknown variable in the climate change equation[END_REF][START_REF] Wagner | High-Throughput DNA sequencing of ancient wood[END_REF], infectious diseases or outbreaks [START_REF] Raoult | Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death[END_REF][START_REF] Bianucci | Détection de l'antigène F1 de Yersinia pestis dans les restes humains anciens à l'aide d'un test de diagnostic rapide[END_REF]. These studies are useful for understanding the history of diseases associated with significant human mortality in the past [START_REF] Rivera-Perez | Paleomicrobiology: a Snapshot of Ancient Microbes and Approaches to Forensic Microbiology[END_REF] or discovering the antiquity of certain diseases, long considered to be associated with modern humans [START_REF] Houldcroft | Human biology and ancient DNA: exploring disease, domestication and movement[END_REF]. In addition, studies on ancient samples provide an understanding of how individuals in microbial communities in an entire population or in an ancient environment have evolved [START_REF] Wayne | Full of Sound and Fury: History of Ancient DNA[END_REF][START_REF] Cano | Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman[END_REF][START_REF] Steven | Microbial ecology and biodiversity in permafrost[END_REF]. Among the factors facilitating these studies, the reduction in the cost of high throughput sequencing techniques, as well as the advanced and advent of other OMICS strategies have contributed widely to the field [START_REF] Adler | Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions[END_REF][START_REF] Raoult | Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death[END_REF][START_REF] Gardner | A microbial detection array (MDA) for viral and bacterial detection[END_REF].

In this review, we propose first to detail the methods used to recover ancient microorganisms through several examples and then to highlight the conclusions of environmental studies in terms of ancient microbes' diversity, through examples such as permafrost, ancient halite, amber and ancient rocks that have prompted several investigations.

BIBLIOGRAPHICAL METHODS

A literature was manually performed through Google, PubMed, Web of Science and Google Scholar databases covering the period from September 1928 to August 2019 using the following keywords: "ancient microbes", "ancient microbiome", "ancient bacteria evolution", "paleomicrobiology", "permafrost", "ancient rocks", "ancient halite", "amber", "prehistory of antibiotic resistance", "resistance mechanisms in ancient microorganism", "resistance mechanisms in permafrost", "contaminant in ancient environment sample", "contaminant in permafrost sample", "contaminant in amber sample", "contaminant in ancient rocks sample", "contaminant in ancient halite sample".

In view of the many investigations we found using the keyword "permafrost", we also performed a more specific literature for "Permafrost" by performing the queries specific to each database (Table 1).

Literature allowed us to identify 51512 scientific articles, among which we selected 343 articles based on of their summaries and titles. Finally, 172 articles were retained for this review given their relevance for this work (Figure 1).

TECHNIQUES USED FOR THE DETECTION OF ANCIENT MICROORGANISMS

Techniques for studying ancient microorganisms use multiomics techniques including microscopy techniques, culture and DNA-based approaches.

Microscopy

Microscopy allows an open approach for screening bacteria, fungi, parasites or other microorganisms in ancient samples and highlights their different forms of survival in ancient environments. In ancient human samples, different parasites (such as helminths) and their eggs along with cysts and molds can be easily detected by light microscopy in coprolites and rectum of a mummy dated middle age or even before [START_REF] Bianucci | Trichuris trichiura in a post-Colonial Brazilian mummy[END_REF][START_REF] Jaeger | Evidence of Helminth Infection in Guanche Mummies: Integrating Paleoparasitological and Paleogenetic Investigations[END_REF][START_REF] Appelt | Polyphasic analysis of a middle ages coprolite microbiota, Belgium[END_REF]. Transmission Electron Microscopy (TEM) was also used in ancient samples studies, and its superior resolution to light microscopy makes it possible to identify calcified and non-calcified bacterial species in archaeological studies [START_REF] Preus | Ancient bacterial DNA (aDNA) in dental calculus from archaeological human remains[END_REF]. TEM technique was used to study long-term survival mechanisms of bacteria in permafrost and their physiology [START_REF] Soina | Adaptation of Bacteria to the Terrestrial Permafrost Environment[END_REF][START_REF] Dmitriev | Ultrastructural Characteristics of Natural Forms of Microorganisms Isolated from Permafrost Grounds of Eastern Siberia by the Method of Low-Temperature Fractionation[END_REF]. Indeed, using TEM technique, the shapes of the intact bacterial species found were almost identical regardless of the age of the permafrost. These small cells housed capsular layers, thickened cell walls and a nonhomogenous or/ and dense cytoplasm that would allow bacteria to survive in this extreme environment [START_REF] Soina | Adaptation of Bacteria to the Terrestrial Permafrost Environment[END_REF][START_REF] Dmitriev | Ultrastructural Characteristics of Natural Forms of Microorganisms Isolated from Permafrost Grounds of Eastern Siberia by the Method of Low-Temperature Fractionation[END_REF]. Used as a prelude to Omics techniques, the electron microscopy also makes it possible to identify new bacterial taxa in permafrost [START_REF] Alawi | Cultivation of a novel coldadapted nitrite oxidizing betaproteobacterium from the Siberian Arctic[END_REF]. Also, both bacilli and cocci were revealed in fluid inclusions in ancient halite from different depths by TEM [START_REF] Schubert | Microscopic identification of prokaryotes in modern and ancient halite, Saline Valley and Death Valley, California[END_REF][START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF].

Another microscopy technique, scanning electron microscope (SEM), has been used to detect cribriform and rounded bacteria, but also bacterial molds from ancient samples [START_REF] Arensburg | Ancient dental calculus and diet[END_REF] and revealed the microbial morphology and the formation of biofilm in permafrost samples [START_REF] Soina | Adaptation of Bacteria to the Terrestrial Permafrost Environment[END_REF].

After staining with DAPI, SEM has also been associated with light microscopy to identify and categorize different morphologies of microbial cells from permafrost samples [START_REF] Blanco | Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica)[END_REF]. In addition, the use of epifluorescence microscopy has been reported and has revealed the presence of prokaryotic and eukaryotic microorganisms on the surface of ancient seeds buried for 30,000 years in permanently frozen permafrost [START_REF] Stakhov | Microbial communities of ancient seeds derived from permanently frozen Pleistocene deposits[END_REF]. Fluorescence microscopy coupled with three other techniques: Live / Dead staining, endospore enrichment, and selective depletion of dead cell DNA provide a means of differentiating dormant, dead, or active microbial cells in ancient permafrost samples [START_REF] Burkert | Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence[END_REF]. This differentiation has made it possible to highlight in permafrost samples aged 19,000, 27,000 and 33,000 years, that dormancy is also part of one of the strategies used by microorganisms to survive long-term in permafrost [START_REF] Burkert | Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence[END_REF].

This finding shows that although microbes have the ability to sporulate, some (Clostridia) prioritize survival in their vegetative forms in this extreme environment [START_REF] Burkert | Changes in the Active, Dead, and Dormant Microbial Community Structure across a Pleistocene Permafrost Chronosequence[END_REF].

Culture techniques

Culture techniques have greatly contributed to the understanding of the biodiversity and microbial community associated with ancient samples. These techniques imply that microorganisms are still alive in dormant or active state in the samples before studies [START_REF] Steven | Microbial ecology and biodiversity in permafrost[END_REF][START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF][START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF]. Microorganisms dormant in ancient samples such as permafrost can be activated after samples treatment with chitin [START_REF] Manucharova | Characterization of the Structure of the Prokaryotic Complex of Antarctic Permafrost by Molecular Genetic Techniques[END_REF]. These culture techniques provide microbial isolates, whose identification is generally performed by 16S rRNA gene sequencing [START_REF] Marcolefas | Culture-Dependent Bioprospecting of Bacterial Isolates From the Canadian High Arctic Displaying Antibacterial Activity[END_REF].

Culture media reported in the literature are varied, from oligotrophic media to nutrient-rich media. However, in most cases, these are oligotrophic culture media that can isolate the most microorganisms from ancient environmental samples after long incubation period (up to 15 weeks or more) [START_REF] Pulschen | Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media[END_REF]. Usually, the culture media reported for the culture of the ancient permafrost samples are nutrient-poor media, such as the R2A (Reasoner's 2A) [START_REF] Marcolefas | Culture-Dependent Bioprospecting of Bacterial Isolates From the Canadian High Arctic Displaying Antibacterial Activity[END_REF][START_REF] Cheptsov | 100 kGy gamma-affected microbial communities within the ancient Arctic permafrost under simulated Martian conditions[END_REF] or PYG (Peptone, Yeast Extract, Glucose) [START_REF] Cheptsov | 100 kGy gamma-affected microbial communities within the ancient Arctic permafrost under simulated Martian conditions[END_REF] medium. Yet, nutrient media, such as LB (Luria Bertani), TSA (trypticase soy agar) have been used for the culture of Siberian and Canadian permafrost microorganisms [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF]. These media can be enriched with vitamins or made selective by the addition of antibiotics [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF] or by the addition of nitrite [START_REF] Alawi | Cultivation of a novel coldadapted nitrite oxidizing betaproteobacterium from the Siberian Arctic[END_REF] to optimize the culture of microorganisms as reported for the culture of amber samples [START_REF] Lambert | Staphylococcus succinus sp. nov., isolated from Dominican amber[END_REF][START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Greenblatt | Micrococcus luteus --survival in amber[END_REF] as well as the culture of permafrost bacteria oxidizing nitrite [START_REF] Alawi | Cultivation of a novel coldadapted nitrite oxidizing betaproteobacterium from the Siberian Arctic[END_REF]. Oriented media, such as halophilic media, with different salt concentrations have also been reported, particularly in permafrost [START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF], but also in halite samples [START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF]. These culture media included Casamino Acids Binghamton (CAB) media, Pyruvate Glycerol Binghamton media [START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF].

Molecular diagnostic techniques

The first sequencing of an ancient DNA dates back to 1984 and focused on the study of DNA from extinct ancient species, including the sequencing of mitochondrial DNA from a museum specimen of the zebra species quagga (Equus quagga) [START_REF] Higuchi | DNA sequences from the quagga, an extinct member of the horse family[END_REF]. Since then, curiosity for the molecular evolution, behavior and ecology of ancient DNA has increased and the techniques being used now combine PCR, cloning and standard sequencing [START_REF] Rizzi | Ancient DNA studies: new perspectives on old samples[END_REF] on the one hand, but also next generation sequencing techniques (NGS) [START_REF] Adler | Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions[END_REF][START_REF] Rizzi | Ancient DNA studies: new perspectives on old samples[END_REF][START_REF] Gorgé | Analysis of Ancient DNA in Microbial Ecology[END_REF], metagenomics [START_REF] Warinner | Ancient human microbiomes[END_REF][START_REF] Lugli | Ancient bacteria of the Ötzi's microbiome: a genomic tale from the Copper Age[END_REF][START_REF] Fornaciari | Environmental Microbial Forensics and Archaeology of Past Pandemics[END_REF] and microbial detection arrays [START_REF] Devault | Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array[END_REF] on the other. DNA-based approaches are nowadays the most widely used in the study of ancient samples because they provide a better understanding of the origin and evolution of ancient species than culture techniques [START_REF] Drancourt | Palaeomicrobiology: current issues and perspectives[END_REF]. The use of NGS methods for the study of ancient DNA microorganisms is expanding [START_REF] Warinner | Ancient human microbiomes[END_REF][START_REF] Fornaciari | Environmental Microbial Forensics and Archaeology of Past Pandemics[END_REF][START_REF] Duan | The microbial community characteristics of ancient painted sculptures in Maijishan Grottoes, China[END_REF], but other techniques, such as mass spectrometry techniques [START_REF] Warinner | Ancient human microbiomes[END_REF][START_REF] Huynh | Bacteria and archaea paleomicrobiology of the dental calculus: a review[END_REF][START_REF] Asara | Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry[END_REF][START_REF] Hollemeyer | Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests[END_REF][START_REF] Tran | Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling[END_REF], fluorescent in situ hybridization techniques [START_REF] Kobabe | Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridisation[END_REF] and immunoassay techniques [START_REF] Allison | Further studies on fecal parasites in antiquity[END_REF][START_REF] Gonçalves | Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay[END_REF][START_REF] Goncalves | Amoebiasis distribution in the past: first steps using an immunoassay technique[END_REF][START_REF] Appelt | Human Coprolites as a Source for Paleomicrobiology[END_REF], are also widely associated with ancient microorganism studies.

DNA extraction

DNA extraction is one of the most delicate parts for all studies based on ancient DNAs. In fact, not only the ancient DNA is confronted with multiple contaminants including inhibitors of PCR, but especially with numerous damages thus generating molecules of small sizes [START_REF] Pääbo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF].

Ancient DNA faces oxidative damage, which significantly affects cytosine and thymine bases [START_REF] Pääbo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF]. Authors suggest treatments of its ancient DNA extracts with alkali and various enzymes, such as endonuclease IV; endonuclease III; uracil-DNA glycosylase + endonuclease IV [START_REF] Pääbo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF].

More specifically with regard to DNA extracts from ancient sediments, some authors described procedures for the appropriate extraction of environmental DNAs (contained in frozen sediment cores) with appropriate solutions to remove PCR inhibitors [START_REF] Epp | Sampling and Extraction of Ancient DNA from Sediments[END_REF]. This protocol includes pre-treatment of the sample under rigorous aseptic conditions; and essential stages of total sediment dissolution allowing optimization of the amount of DNA [START_REF] Epp | Sampling and Extraction of Ancient DNA from Sediments[END_REF].

The extraction of genomic DNA from permafrost samples for example, use commercial kits suitable for extracting DNAs present in the environment, water or soil among which the highest gDNA concentrations noted come from the FastDNA SPIN Kit for Soil (MP Biomedicals, Irvine, CA) kit [START_REF] Vishnivetskaya | Commercial DNA extraction kits impact observed microbial community composition in permafrost samples[END_REF].

Polymerase chain reaction (PCR) and its variants

The objective of the PCR technique is to obtain several copies of a targeted DNA, initially in small quantities in the samples to be analyzed, so it was included very early in the study of ancient DNA. This has greatly facilitated the diversification and remarkable perspective that we now have on ancient DNAs [START_REF] Pääbo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF][START_REF] Pääbo | Molecular cloning of Ancient Egyptian mummy DNA[END_REF]. Different types of PCR are performed on ancient DNAs. These PCRs include standard PCR, quantitative PCR, multiplex PCR and nested PCR, suicide PCR and immuno-PCR.

Standard PCR

Since there are several degradation conditions specific to ancient samples, including damaged bases, weak DNA fragments that can be degraded and the presence of PCR inhibitors associated with ancient DNA, modifications have been suggested in the commonly used standard PCR protocol [START_REF] Hänni | Isopropanol precipitation removes PCR inhibitors from ancient bone extracts[END_REF][START_REF] Fulton | PCR amplification, cloning, and sequencing of ancient DNA[END_REF]. Therefore, it is recommended to carry out several PCRs on different extracts of the same sample to compare the results of the PCRs [START_REF] Fulton | PCR amplification, cloning, and sequencing of ancient DNA[END_REF][START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF]. Also, these modifications include pretreatment with bovine serum albumin (BSA) [START_REF] Rohland | Comparison and optimization of ancient DNA extraction[END_REF], with uracil DNA glycosylase (UNG or UDG) [START_REF] Willerslev | Ancient DNA[END_REF], or N -phenacylthiazolium bromide (PTB) [START_REF] Poinar | Molecular coproscopy: dung and diet of the extinct ground sloth Nothrotheriops shastensis[END_REF], but also isopropanol precipitation [START_REF] Hänni | Isopropanol precipitation removes PCR inhibitors from ancient bone extracts[END_REF] to remove PCR inhibitors co-extracted with DNAs. Standard PCR systems used to amplify ancient microbial DNAs sometimes have different targets. The 16S RNA Gene Region is usually the most targeted in the study of haloarchaeal diversity [START_REF] Park | Haloarchaeal diversity in 23, 121 and 419 MYA salts[END_REF][START_REF] Santiago-Rodriguez | Microbial communities in precolumbian coprolites[END_REF], but also bacterial diversity [START_REF] Appelt | Human Coprolites as a Source for Paleomicrobiology[END_REF]. The ITS region is often the one targeted in the fungal community study [START_REF] Santiago-Rodriguez | Microbial communities in precolumbian coprolites[END_REF], but also the algal community [START_REF] Sankaranarayanan | Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction[END_REF]. As for the study of eukaryotes, 18S rRNA gene, cytochromes or NADH dehydrogenase can been targeted [START_REF] Appelt | Human Coprolites as a Source for Paleomicrobiology[END_REF].

Multiplex PCR and Quantitative PCR (qPCR)

The advantage of multiplexed PCR in the analysis of ancient samples lies in its ability to have several amplified fragments simultaneously in a single PCR [START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. This would allow, even from very small quantities of ancient samples, to have enough easily analysable DNA fragments [START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. Based on what already existed, Stiller and Fulton proposed a multiplex PCR protocol better suited to target the ancient highly degraded microbial DNAs [START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. This protocol includes two steps: a first step that consists of a normal multiplex PCR and a second step that uses this first multiplex PCR product as matrix, along with adding nested primers that increase the specificity and selectivity of the PCR reaction [START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. As for the qPCR, it has also been reported in the study of ancient samples. Used on permafrost, it has recovered up to 37.5 ± 1.9 % total genomic DNAs [START_REF] Vishnivetskaya | Commercial DNA extraction kits impact observed microbial community composition in permafrost samples[END_REF] Nested PCR Much more sensitive than conventional PCR, nested PCR has the advantage of a high level of amplification of the target DNA. It consists in amplifying the PCR products resulting from a first reaction of the conventional PCR [START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. The involvement of nested PCR in determining the microbial community of the ancient samples has been much reported [START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Stiller | Multiplex PCR amplification of ancient DNA[END_REF]. Associated with culture, this technique has proven effective in comparing the archaic community at different depths of ancient halite samples dated to the Pliocene (5.3 to 1.8 million years) [START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF].

Suicide PCR

"The suicide PCR" has been designed to overcome the problems of false positives in PCR techniques usually caused by cross-contamination. Indeed, the principle of the "suicide PCR" relies in a unique utilization of the same PCR primers without a positive control in the same laboratory [START_REF] Raoult | Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death[END_REF]. This protocol confirmed the presence of Yersinia pestis DNA in dental pulp from skeletons of Black Death victims [START_REF] Raoult | Molecular identification by "suicide PCR" of Yersinia pestis as the agent of medieval black death[END_REF][START_REF] Drancourt | Molecular insights into the history of plague[END_REF]. Moreover, this technique allowed the identification of Bartonella quintana in 4,000-year-old human remains [START_REF] Fournier | Paleomicrobiology of Bartonella infections[END_REF]. Preceded by a rapid strip test, it also proved effective for the detection of Yersinia pestis antigen F1 in human remains, corresponding to the victims of the plague of the 16 th , 17 th and 18 th centuries in the south-east of France [START_REF] Bianucci | Détection de l'antigène F1 de Yersinia pestis dans les restes humains anciens à l'aide d'un test de diagnostic rapide[END_REF].

Immuno-PCR

Immuno-PCR is a technique that has been developed in 1992, which combines the advantages of immunodetection and the strong amplification and specificity of PCR, thus forming a very strong antibody-DNA complex capable of detecting very low amount of microbial DNAs [START_REF] Sano | Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates[END_REF]. This technique allows the amplification of DNA sequences from ancient samples, which are sometimes difficult to obtain with a simple standard PCR amplification [START_REF] Sano | Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates[END_REF]. So far, its use has only been reported once in the microbial detection of ancient samples [START_REF] Malou | Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm[END_REF]. It exhibited a higher sensitivity compared to conventional ELISA and PCR techniques for the detection of plague agent Yersinia pestis in the dental pulp samples taken from Black Death victims [START_REF] Malou | Immuno-PCR--a new tool for paleomicrobiology: the plague paradigm[END_REF].

Molecular cloning techniques

Ancients DNAs often undergo damage by oxidative processes [START_REF] Pääbo | Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification[END_REF], by hydrolysis processes, by DNA crosslinks or also degradation by microorganisms' nucleases in the post mortem cell [START_REF] Rizzi | Ancient DNA studies: new perspectives on old samples[END_REF] that can affect their matrix [START_REF] Hofreiter | DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA[END_REF], and this does not guarantee the authenticity of results from sequencing. For this, the cloning of independent amplicons is privileged by some researchers as one of the major precautions before sequencing in order to minimize the determination of incorrect sequences of ancients DNAs [START_REF] Hofreiter | DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA[END_REF][START_REF] Winters | To clone or not to clone: method analysis for retrieving consensus sequences in ancient DNA samples[END_REF].

Next generation sequencing (NGS) techniques

Next-generation sequencing provides an easy way to study the microbial community in ancient samples [START_REF] Adler | Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions[END_REF] by its rapidity to sequence the microbial genomes. The sequencing strategies for ancient samples studies included Illumina MiSeq sequencing strategy [START_REF] Duan | The microbial community characteristics of ancient painted sculptures in Maijishan Grottoes, China[END_REF],

Roche 454 GS FLX Titanium pyrosequencer [START_REF] Tito | Insights from Characterizing Extinct Human Gut Microbiomes[END_REF]. Their use has facilitated advances in microbial genomic studies, particularly on the history of certain infectious diseases, but also on the bacterial ecology [START_REF] Andam | Microbial Genomics of Ancient Plagues and Outbreaks[END_REF]. For instance, the genomic reconstruction of Clostridium perfringens found in the soft tissue of frozen mummified sample (Tyrolean Iceman) revealed virulence genes, which would specialized very early in these bacteria, while in the Pseudomonas veronii, these virulence genes would be acquired for its adaptation to its ecological niche [START_REF] Lugli | Ancient bacteria of the Ötzi's microbiome: a genomic tale from the Copper Age[END_REF]. Applied to old calcified plaque, it reveals that the presence of chronic diseases in the subjects of the Industrial Revolution is due to a low diversification of microorganisms compared to the Neolithic oral microbiota [START_REF] Adler | Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions[END_REF]. However, when applied to excrement found in archaeological contexts, a change in its microbial community was observed, compared to the modern community [START_REF] Tito | Insights from Characterizing Extinct Human Gut Microbiomes[END_REF].

Shotgun sequencing (metagenomics)

The advent of metagenomics makes it possible to characterize the microbial community of the 16S rRNA gene in a sample much more quickly, making it possible to characterize complex communities in the environment [START_REF] Yergeau | The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses[END_REF][START_REF] Liang | Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost[END_REF]. In contrast to culture, metagenomics provides a much faster overview of the microbial community, and allows comparative genomic studies [START_REF] Bos | Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis[END_REF]. Metagenomics contributes to the identification and taxonomy of ancient bacterial species [START_REF] Yergeau | The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses[END_REF] and provides a better understanding of the microbial life [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF], the microbial diversity and the metabolic functions [START_REF] Liang | Predominance of Anaerobic, Spore-Forming Bacteria in Metabolically Active Microbial Communities from Ancient Siberian Permafrost[END_REF] under extreme conditions and the impact of age on the microbial community in permafrost samples several million years old.

Fluorescent in situ hybridization (FISH)

Fluorescence in situ Hybridization (FISH) technique reveals the different morphologies of cultivable and non-cultivable colonies contained in a sample. This molecular technique uses labeled fluorescent probes to detect nucleic acid sequences contained in the cell itself. These targets include RNA or DNA at different chromosomal phases of the cell [START_REF] Tsuchiya | Fluorescence In Situ Hybridization[END_REF], thus the metabolic activities of cells greatly influence FISH results [START_REF] Poulsen | Use of rRNA fluorescence in situ hybridization for measuring the activity of single cells in young and established biofilms[END_REF]. Its high efficiency for the rapid detection of pathogenic microorganisms has been widely reported, to such an extent that it is recommended as one of the routine diagnostic tools [START_REF] Kempf | Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures[END_REF][START_REF] Peters | Faster Identification of Pathogens in Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice[END_REF][START_REF] Machado | Diagnosis of bacterial vaginosis by a new multiplex peptide nucleic acid fluorescence in situ hybridization method[END_REF]. This technique is also very effective for reliable quantification of various microbial taxa involved in anammox processes into complex environments such as the black sea [START_REF] Amann | Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques[END_REF]. Its effectiveness is real in assessing the microbial community and its use on the soil of the Siberian tundra has shown that a decrease in the microbial cell community is more significant in the deep layers [START_REF] Kobabe | Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridisation[END_REF]. FISH was also effective in evaluating the number of bacterial cell and archaea metabolically active in permafrost samples after exposure to a 100 kGy irradiation dose, thus revealing that microorganisms in this environment are radiation-resistant [START_REF] Cheptsov | 100 kGy gamma-affected microbial communities within the ancient Arctic permafrost under simulated Martian conditions[END_REF].

Microbial Detection Array

Given the degradations often observed on ancient samples, studies focusing on microbial pathogens based on ancient DNA sequence analysis are sometimes very expensive [START_REF] Devault | Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array[END_REF].

Microarray technologies, have recently been shown to be effective in detecting microorganisms in ancient samples [START_REF] Devault | Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array[END_REF], as they are very fast and less expensive than sequencing techniques [START_REF] Thissen | Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray[END_REF]. Several types of microarray systems have been reported for the study of ancient samples. The most reported microarray system is based on the use of long oligonucleotide probes (50-65 bp) and constitutes a database named Lawrence Livermore Microbial Detection Array (LLMDA) containing at least a total of 388,000 oligonucleotide of microorganisms [START_REF] Gardner | A microbial detection array (MDA) for viral and bacterial detection[END_REF][START_REF] Thissen | Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray[END_REF]. This enables to identify simultaneously and very quickly the genomic content of several microorganisms contained in a sample [START_REF] Thissen | Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray[END_REF]. For example, the LLMDA technique has allowed the rapid identification of Vibrio cholerae and Yersinia pestis strains in tooth samples from 19th century and a medieval period, while their sequences recovered only weakly with high-throughput sequencing techniques [START_REF] Devault | Ancient pathogen DNA in archaeological samples detected with a Microbial Detection Array[END_REF]. Otherwise, Yergeau et al. have extensively investigated the use of much more specific microarray for the screening of 16S rRNA genes of polar soil microorganisms [START_REF] Yergeau | The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses[END_REF][START_REF] Yergeau | Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments[END_REF][START_REF] Yergeau | Metagenomic Analysis of the Bioremediation of Diesel-Contaminated Canadian High Arctic Soils[END_REF]. The most widely used microarray database for cold environments such as permafrost [START_REF] Yergeau | The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses[END_REF] consists of 525 25-mer oligonucleotides targeting several bacterial and archaeal taxa [START_REF] Yergeau | The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses[END_REF][START_REF] Yergeau | Microarray and Real-Time PCR Analyses of the Responses of High-Arctic Soil Bacteria to Hydrocarbon Pollution and Bioremediation Treatments[END_REF].

Immunoassay techniques

Numerous studies have demonstrated the sensitivity and specificity of immunoassay techniques for the detection of parasitic coproantigens in contemporary samples [START_REF] Sharp | Evaluation of the Triage Micro Parasite Panel for Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in Patient Stool Specimens[END_REF][START_REF] Pillai | Entamoeba histolytica and Entamoeba dispar: Epidemiology and Comparison of Diagnostic Methods in a Setting of Nonendemicity[END_REF][START_REF] Garcia | Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay[END_REF][START_REF] Haque | Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits[END_REF][START_REF] Haque | Comparison of PCR, Isoenzyme Analysis, and Antigen Detection for Diagnosis of Entamoeba histolytica Infection[END_REF].

Although the elements contained in the ancient samples may be damaged by time due to adverse conditions, immunoassay techniques have, in several cases, supplemented microscopic analysis by allowing the detection of parasites in ancient samples [START_REF] Gonçalves | Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay[END_REF]. ELISA is an immunoassay technique, easy to perform, which consists of a monoclonal antibody assay kit. An antibody-antigen complex will therefore be formed with the antigen of the microorganisms and the reaction is revealed by a color indicator due to the enzyme fixed on the antibody [START_REF] Gonçalves | Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay[END_REF]. For example, the detection of ancient intestinal parasites' coproantigen, such as Giardia duodenalis coproantigen [START_REF] Gonçalves | Detection of Giardia duodenalis antigen in coprolites using a commercially available enzyme-linked immunosorbent assay[END_REF] and Entamoeba histolytica coproantigen [START_REF] Goncalves | Amoebiasis distribution in the past: first steps using an immunoassay technique[END_REF] has been easily achieved through the use of enzyme-linked immunosorbent assay (ELISA).

In addition, the detection of microbial antigens permafrost was made possible by an immunosensor named LDChip300 (Life Detector Chip) with hundreds of antibodies. This technique allowed the determination of several bacterial clusters (Firmicutes, Proteobacteria, Bacteroides, Actinobacteria) and archaeal clusters (mainly Methanobacterium spp) [START_REF] Blanco | Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica)[END_REF]

Mass spectrometry

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF MS)

Matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF MS), based on the analysis of the proteins, has proven in recent years to be a fast and efficient tool for the detection and classification of proteins from ancient samples [START_REF] Asara | Protein sequences from mastodon and Tyrannosaurus rex revealed by mass spectrometry[END_REF][START_REF] Hollemeyer | Species identification of Oetzi's clothing with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based on peptide pattern similarities of hair digests[END_REF][START_REF] Tran | Classification of Ancient Mammal Individuals Using Dental Pulp MALDI-TOF MS Peptide Profiling[END_REF][START_REF] Ostrom | New strategies for characterizing ancient proteins using matrix-assisted laser desorption ionization mass spectrometry[END_REF][START_REF] Nielsen-Marsh | Sequence preservation of osteocalcin protein and mitochondrial DNA in bison bones older than 55 ka[END_REF]. As for the determination of microorganisms, its use in the analysis and identification of 1400-year-old mycolic acids, a tuberculosis biomarkers [START_REF] Mark | High-throughput mass spectrometric analysis of 1400-year-old mycolic acids as biomarkers for ancient tuberculosis infection[END_REF], has opened a way on for its possible efficacy in the study of ancient microorganisms. Recently used as screening for bacterial identification in a culturomic study, it allowed us to obtain a unique protein profile of a new bacterial species of the genus Bacillus isolated in Siberian permafrost [START_REF] Afouda | Bacillus massiliglaciei', a new bacterial species isolated from Siberian permafrost[END_REF]; whose species-level identification and complete characterization required the sequencing of the 16S rRNA gene [START_REF] Cadoret | Noncontiguous finished genome sequences and description ofBacillus massiliglaciei, Bacillus mediterraneensis, Bacillus massilinigeriensis, Bacillus phocaeensisandBacillus tuaregi,five new species identified by culturomics[END_REF].

Metaproteomic

Metaproteomic is a method of analysis of non-nucleoside biomolecules. Associated with metagenomics for microorganisms study, it provides all the proteomic information contained in a complex sample [START_REF] Huynh | Bacteria and archaea paleomicrobiology of the dental calculus: a review[END_REF]. This technique uses tandem mass spectrometry (MS/MS) to generate protein profile, peptide signals, whose correspondences is determined by the available bioinformatics resources such as the Byonic™ database [START_REF] Hill | Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen[END_REF]. Using the metaproteomic approach on 120,000-year-old bone samples from the Ziegler Reservoir fossil site in Snowmass Village, Colorado, the authors reported the presence of "Hydroxylysine glucosylgalactosylation'', a post-translational modification of collagen, which would be strongly involved in the conservation of organic matter, as well as new peptide and protein sequences, whether or not derived from the extracellular matrix [START_REF] Hill | Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen[END_REF]. For instance, the study of microorganisms in medieval dental calculus by metaproteomic revealed well-preserved proteins with abundant and clinically important proportions, such as proteins of periodontal pathogens: Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola [START_REF] Warinner | Pathogens and host immunity in the ancient human oral cavity[END_REF].

Raman spectroscopy

Raman spectroscopy, also known as Raman spectrometry, is involved in the study of ancient microorganisms as a non-destructive method of searching for microbial biomarkers [START_REF] Vítek | Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy[END_REF]. Its sole use in paleomicrobiology has been reported in the study of ancient halite microorganisms and consists in identifying, in a record time, biomolecules belonging to microorganisms integrated into fluid halite inclusions. The entrapped amino acids detected included glycine, L-alanine, β-alanine, L-serine and γ-aminobutyric acid [START_REF] Osterrothová | Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy[END_REF]. Recognition of the microorganism is made possible by the interpretation of spectra generated by biomarkers [START_REF] Vítek | Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy[END_REF][START_REF] Osterrothová | Investigation of biomolecules trapped in fluid inclusions inside halite crystals by Raman spectroscopy[END_REF][START_REF] Conner | Acidophilic halophilic microorganisms in fluid inclusions in halite from Lake Magic, Western Australia[END_REF].

Contaminants

Microbial diversity is important in environmental samples, but one of the major problems that often raises doubts about their authenticity is the contamination of ancient samples with external material [START_REF] Yang | Hydrocarbon degraders establish at the costs of microbial richness, abundance and keystone taxa after crude oil contamination in permafrost environments[END_REF][START_REF] Willerslev | Isolation of nucleic acids and cultures from fossil ice and permafrost[END_REF]. The contaminants involved may be other exogenous microorganisms or viruses contracted due to carelessness in sampling or laboratory handling [START_REF] Willerslev | Isolation of nucleic acids and cultures from fossil ice and permafrost[END_REF] or reagents used in handling [START_REF] Boessenkool | Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA[END_REF]. Moreover, it is sometimes difficult to avoid contamination despite precautions. For example, in constantly cold environments, ice tissue cracks or thawing are also a constant source of contamination [START_REF] Rike | Response of cold-adapted microbial populations in a permafrost profile to hydrocarbon contaminants[END_REF][START_REF] Dyke | Contaminant migration through the permafrost active layer, Mackenzie Delta area, Northwest Territories, Canada[END_REF] because they increase hydraulic conductivity and allow solute movement that promotes the migration of dissolved or insoluble contaminants [START_REF] Dyke | Contaminant migration through the permafrost active layer, Mackenzie Delta area, Northwest Territories, Canada[END_REF]. Consequently, a heterotrophic microbial population emerges greatly at contaminated thawed sites compared to uncontaminated sites [START_REF] Rike | Response of cold-adapted microbial populations in a permafrost profile to hydrocarbon contaminants[END_REF].

Contaminants management

To avoid the contaminants caused by a lack of standard precautions, the authors suggested strict asepsis and antiseptic conditions consisting mainly of the use of sterile disposable equipment (wearing gloves, masks, caps, etc.), the use of a clean laboratory dedicated solely to the handling of ancient samples, systematic cleaning of all PCR equipment before handling [START_REF] Willerslev | Isolation of nucleic acids and cultures from fossil ice and permafrost[END_REF]. Then, decontamination techniques on ancient samples such as permafrost , amber, ancient halite and ancient rocks samples have been proposed [START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Sankaranarayanan | Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction[END_REF][START_REF] Jaakkola | Buried Alive: Microbes from Ancient Halite[END_REF][START_REF] William | Development of a Protocol to Retrieve Microorganisms from Ancient Salt Crystals[END_REF][START_REF] Radax | Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt[END_REF] to avoid any amplification of DNA from contaminants. The various decontamination protocols included: the use of ethanol and/or a Bunsen lamp; use of acid; use of bleach wash; use of combination of 6% (w/v) sodium hypochlorite saturated NaCl, NaOH, saturated NaCl, HCl, saturated Na2CO3/NaCl; use of saturated NaCl and 70% (v/v) ethanol [START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Radax | Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt[END_REF]; use of peroxide, ethanol and heat [START_REF] Lipman | THE DISCOVERY OF LIVING MICRORGANISMS IN ANCIENT ROCKS[END_REF][START_REF] Lipman | Living Microörganisms in Ancient Rocks[END_REF]; use of 2% Glutaraldehyde [START_REF] Cano | Revival and identification of bacterial spores in 25-to 40million-year-old Dominican amber[END_REF].

APPLICATIONS TO DIFFERENT ENVIRONMENTAL MICROBIOMES

The environmental microbiome is filled with all types of microorganisms and certainly the oldest known microorganisms on earth [START_REF] Willerslev | Isolation of nucleic acids and cultures from fossil ice and permafrost[END_REF]. Regarding environmental microbiome studies, the case of permafrost has been the subject of extensive studies in recent decades [START_REF] Steven | Microbial ecology and biodiversity in permafrost[END_REF][START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF] because this environment is constantly at a low temperature and capable of containing and preserving ancient DNAs for thousands of years [START_REF] Willerslev | Isolation of nucleic acids and cultures from fossil ice and permafrost[END_REF]. Its microorganisms adapt to their environmental conditions and, as a result, their survival modes vary according to the environment in which they are found. In samples collected in cold environments for example, the GC content and sporulation of the isolates can be a mean of survival [START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF]. Indeed, about 30% of the isolates found in permafrost are sporulated [START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF]. In general, the GC contents of 16S rRNA gene sequences from permafrost are related to their optimum growth temperature. In fact, a high GC content is related to a high growth temperature and vice versa.

In permafrost, the GC content can reach 72-80% with an optimal growth temperature that can also reach 55-80°C [START_REF] Bakermans | Molecular characterization of bacteria from permafrost of the Taylor Valley, Antarctica[END_REF]. GC high content isolates are linked to samples several million years old, while low-content GC isolates are linked to many recent samples only a few thousand years old [START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF].

Permafrost

Permafrost is defined as a soil that has been frozen for at least two years. It currently accounts for almost 25% of the Earth's surface and is mainly found in Alaska [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF], Siberia [START_REF] Dmitriev | Ultrastructural Characteristics of Natural Forms of Microorganisms Isolated from Permafrost Grounds of Eastern Siberia by the Method of Low-Temperature Fractionation[END_REF][START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Hinsa-Leasure | Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample[END_REF], the Antarctic [START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF] and Canada [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF][START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF], but also in ice-free regions, such as the alpine mountains of Europe [START_REF] Frey | Microbial diversity in European alpine permafrost and active layers[END_REF], America and China [START_REF] Hu | Diversity and community structure of fungi through a permafrost core profile from the Qinghai-Tibet Plateau of China[END_REF]. Despite extreme conditions, many endogenous culturable microorganisms have been reported, and the mesophilic, psychotropic, halotolerant, but also acidotolerant species are represented [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF]. In general, culture-independent methods, such as sequencing identification of the 16S rRNA gene, are combined with culture-dependent methods to determine microbial diversity of permafrost.

Microbial diversity

The cultivable bacteria in permafrost soil are represented by the 4 major phyla : Firmicutes, Actinobacteria (with a predominance of order Actinomycetales), Proteobacteria, and Bacteroidetes [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF][START_REF] Hinsa-Leasure | Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample[END_REF][START_REF] Bai | Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China[END_REF][START_REF] Hansen | Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway[END_REF]. Bacteroides are predominant in high pH soils, whereas Proteobacteria are associated with low pH and high nitrogen and carbon levels [START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF]. In addition, the implication of genes belonging to Actinobacteria and Bacteroidetes on the degradation processes of the Earth's organic carbon stored in Arctic permafrost peatlands has recently been reported [START_REF] Tveit | Organic carbon transformations in high-Arctic peat soils: key functions and microorganisms[END_REF]. The advance of molecular high throughput sequencing techniques revealed a different picture as Acidobacteria, Gemmatimonadetes, Actinobacteria and Proteobacteria [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF][START_REF] Seok | Microbial Community of the Arctic Soil from the Glacier Foreland of Midtre Lovenbreen in Svalbard by Metagenome Analysis[END_REF] are found in high proportion from both in the active layer and in the permafrost. The cultivable bacteria genera found in the permafrost soils are diversified.

The genus Arthrobacter, Planococcus and Bacillus are the most commonly isolated in all types of permafrost, probably because they are easier to grow and require less nutrients growth than others [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF][START_REF] Cadoret | Noncontiguous finished genome sequences and description ofBacillus massiliglaciei, Bacillus mediterraneensis, Bacillus massilinigeriensis, Bacillus phocaeensisandBacillus tuaregi,five new species identified by culturomics[END_REF][START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF][START_REF] Hinsa-Leasure | Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample[END_REF][START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF][START_REF] Bai | Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China[END_REF][START_REF] Zhang | Isolation and characterization of bacteria from ancient siberian permafrost sediment[END_REF] (Table 2). Table 2 summarizes the most isolated bacterial genera by culture in different types of permafrost. Some of these bacterial species isolated from permafrost samples may exhibit antimicrobial activities against pathogenic microorganisms. For instance, Bacillus tequilensis and Pseudomonas spp isolated from Canadian permafrost, have demonstrated inhibitory activities against foodborne pathogens such as S. aureus, L. monocytogenes, S. enterica and E. coli O157:H7 [START_REF] Marcolefas | Culture-Dependent Bioprospecting of Bacterial Isolates From the Canadian High Arctic Displaying Antibacterial Activity[END_REF].

Otherwise, the probiotic activity of Bacillus cereus isolated from permafrost on Salmonella has also been reported [START_REF] Fursova | Probiotic Activity of a Bacterial Strain Isolated from Ancient Permafrost Against Salmonella Infection in Mice[END_REF].

The 16S sequences of the majority of these bacterial isolates are extremely close, sometimes identical to those reported in various environmental or human samples [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF]. More precisely, apart from a few genera (Table 2), all others are associated with humans [START_REF] Bilen | The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species[END_REF].

In addition, the microbial diversities obtained in the permafrost soil and in the active layer depend on the techniques used and the origin of the permafrost [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF][START_REF] Frey | Microbial diversity in European alpine permafrost and active layers[END_REF][START_REF] Gittel | Distinct microbial communities associated with buried soils in the Siberian tundra[END_REF][START_REF] Gittel | Site-and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland[END_REF]. For example, the most abundant bacterial diversity was identified in the Canadian permafrost soil, even if the number of viable aerobic cells found in this soil can be up to 100 times lower than that of the active layer [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF]. However, other studies have shown that the greatest microbial diversity of permafrost (Siberian, Greenland ) can be found in the active layer [START_REF] Gittel | Distinct microbial communities associated with buried soils in the Siberian tundra[END_REF][START_REF] Gittel | Site-and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland[END_REF].

Sediments of permafrost are also often rich in methanotrophic bacteria [START_REF] Trotsenko | Aerobic methanotrophic bacteria of cold ecosystems[END_REF], highlighting that methane can be used by bacteria living there as a source of carbon and energy for their survival in such an extreme environment. Furthermore, the microbial diversification of permafrost also widens to molds [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF][START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF] and yeasts [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF][START_REF] Xin | Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil[END_REF], whose determination is based on the analysis of internal transcribed spacer (ITS2) sequences in total DNA of sample [START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF], but also 18S rDNA sequences analysis [START_REF] Xin | Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil[END_REF]. The yeasts isolated in the permafrost are mainly represented by the genera Mrakia and Leucosporidium [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF], known to adapt to cold environments [START_REF] Xin | Mrakia psychrophila sp. nov., a new species isolated from Antarctic soil[END_REF][START_REF] Laich | Leucosporidium escuderoi f.a., sp. nov., a basidiomycetous yeast associated with an Antarctic marine sponge[END_REF][START_REF] Tsuji | Mrakia arctica sp. nov., a new psychrophilic yeast isolated from an ice island in the Canadian High Arctic[END_REF].

With regard to molds, a wide variety of cosmopolitan fungal species have been found in different types of permafrost [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF][START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF][START_REF] Hu | Diversity and community structure of fungi through a permafrost core profile from the Qinghai-Tibet Plateau of China[END_REF], of which more than 26 are viable in the Antarctic permafrost [START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF]. Among them, a predominance of Ascomycota in Chinese and Antarctic permafrost [START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF][START_REF] Hu | Diversity and community structure of fungi through a permafrost core profile from the Qinghai-Tibet Plateau of China[END_REF] and Geomyces in Alaskan permafrost [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF] has been reported. Besides, genera Penicillium and Cladosporium, known for their spores, adaptations and resistance to climate change, have been isolated in the Antarctic permafrost [START_REF] Kochkina | Ancient fungi in Antarctic permafrost environments[END_REF].

In addition to bacteria, archaea are also present in permafrost through DNA-based approaches [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF][START_REF] Shcherbakova | Archaeal communities of Arctic methane-containing permafrost[END_REF]. Archaea can reach 0.1% of the total 16S rRNA gene copy number of permafrost [START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF]. Analysis of DNA fragments from 5 permafrost samples collected from a borehole in the Kolyma River plain (northeastern Russia) revealed a high diversity of archaea at a depth of more than 20 meters [START_REF] Shcherbakova | Archaeal communities of Arctic methane-containing permafrost[END_REF]. The Euryarchaeota (especially methanogenic archaea) were found abundant in both Russian and Canadian permafrost [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Shcherbakova | Archaeal communities of Arctic methane-containing permafrost[END_REF]. The amount of methane released in permafrost can be significant, especially just after a period of thawing permafrost [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]; this gas can be a source of energy and carbon for the anaerobic oxidation of microbial metabolism, which also explains the important quantity of OTU related to methanogens and methanogenesis genes found in different types of permafrost [START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF][START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]. The abundance functional metabolic genes present in the permafrost vary with the age and freezing state of the permafrost, which influences the variation of its microbial community [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]. To survive, the microbial community develops adaptive traits to acquire new genes and beneficial pathways depending on permafrost conditions [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]. Therefore, compared to younger permafrost sample, older permafrost sample is associated with enrichment of certain Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]. A thaw of permafrost can lead to a decrease in nitrogen fixation genes and an increase in those of denitrification [START_REF] Mackelprang | Metagenomic analysis of a permafrost microbial community reveals a rapid response to thaw[END_REF].

Besides, the age of the permafrost is correlated with the abundance of aromatic hydrocarbon degradation genes [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF]. The acquisition of adaptive traits for survival in hostile environments of permafrost is facilitated by several elements, including horizontal gene transfers [START_REF] Niehus | Migration and horizontal gene transfer divide microbial genomes into multiple niches[END_REF], chemotaxis, the synthesis of stress-related genes, including those involved in the synthesis of polyamines [START_REF] Mackelprang | Microbial survival strategies in ancient permafrost: insights from metagenomics[END_REF], but also metabolic activity of some sporulated bacterial species of permafrost [START_REF] Hultman | Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes[END_REF]. The increase in other KEGG pathways involved in cell envelope components, amino-acid and peptide metabolism, carbohydrate metabolism, environmental sensing and response, membrane transport and the degradation of recalcitrant biomass have also been reported in ancient permafrost [START_REF] Hultman | Multi-omics of permafrost, active layer and thermokarst bog soil microbiomes[END_REF].

Other environmental microbiomes

Ancient rocks

Several studies have already reported the presence of microorganisms in rocks dating from the Precambrian period (ie the period between 4.55 billion years and 540 million years).

Nonetheless, very little information is available regarding the culture of microorganisms from ancient rocks. The scientist Chas. B. Lipman, first reported the presence of living bacteria living in ancient rocks, and especially in a Pre-Cambrian rock from the Algonkian in Canada [START_REF] Lipman | Living Microörganisms in Ancient Rocks[END_REF]. In particular, rod shaped and sporulated bacteria represented the groups of bacteria found there [START_REF] Lipman | THE DISCOVERY OF LIVING MICRORGANISMS IN ANCIENT ROCKS[END_REF]. In addition, using scanning microscopy, various microorganisms (Cyanobacteria and other bacteria) have been identified in rocks of Karelia dating back to the archaean period. These microorganisms are mostly cocci-shaped, sometimes in division. Some of these, arose in the form of Archean fossils (suspicion of Melanocyrillium) and others in enthalic fossils [START_REF] Astafieva | Fossil microorganisms in Archaean[END_REF]. Later, analysis of the microbial composition of ancient rocks collected in the Yungang Grottoes by combining PCR techniques with denaturing gradient gel electrophoresis (DGGE) and cloning sequencing showed the predominance of phyla of Gammaproteobacteria, Firmicutes and Alphaproteobacteria , but also Sphingobacteria, and Actinobacteria by phylogenetic analysis [START_REF] Yan | Analysis of microbial community on the surface of the historic stone and nearby rock samples in Yungang Grottoes[END_REF].

Ancient halite

Halite or rock salt (NaCl) is a crystalline formed of salts resulting from dried saline environments [START_REF] Fish | Recovery of 16S ribosomal RNA gene fragments from ancient halite[END_REF]. Despite its adverse conditions, many microbiological and geological studies have revealed the survival of microorganisms in fluid inclusions of ancient halite. The techniques used included microscopy, Raman spectroscopy, culture and also metagenomics studies [START_REF] Schubert | Microscopic identification of prokaryotes in modern and ancient halite, Saline Valley and Death Valley, California[END_REF][START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF][START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Sankaranarayanan | Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction[END_REF][START_REF] Vítek | Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy[END_REF][START_REF] Fredrickson | Potential for preservation of halobacteria and their macromolecular constituents in brine inclusions from bedded salt deposits[END_REF][START_REF] Mormile | Isolation of Halobacterium salinarum retrieved directly from halite brine inclusions[END_REF][START_REF] Vreeland | Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal[END_REF]. Although halite may contain ancient microorganisms, the community diversity remains low. There are very little cultivable bacteria, and cultured archaea were mostly halophilic. Several ribosomal 16S sequences found in the halite are attributed to known microbial species but also to new species [START_REF] Jaakkola | Buried Alive: Microbes from Ancient Halite[END_REF]. In the halite, the species of long-term survival are mainly halophilic species and more specifically Halobacterium species. Indeed, halophilic and halotolerant oriented culture media have made it possible to obtain in halite dating back thousands and millions of years, an archaeal community predominated by the isolates Haloarcula, Halobacterium, Halococcus, Halolamina, Halorubrum, Haloterrigena, Natronobacterium, Natronomonas [START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF][START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Jaakkola | Buried Alive: Microbes from Ancient Halite[END_REF]. Besides, the bacterial community includes the genus Oceanobacillus and Virgibacillus isolated from 34-49 million years old halite (Yunying mine, China ) [START_REF] Meng | A newly isolated Haloalkaliphilic bacterium from middle-late Eocene halite formed in salt lakes in China[END_REF] and from 250 million years of age halite (Salado formation, USA) [START_REF] Vreeland | Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal[END_REF], respectively. The ribosomal 16S sequences in halite samples represented in abundance the genera Acinetobacter, Burkholderia and Pseudomonas in halite dating 65-96 million years from Khorat plateau, Thailand, while Stenotrophomonas genus was found in halite dating 11-16 million years ago from Wieliczka mine, Poland [START_REF] Fish | Recovery of 16S ribosomal RNA gene fragments from ancient halite[END_REF].

The archaeal 16S rRNA sequences were composed of Haloarcula, Halobacterium, Halobiforma, Halomicrobium, Halonotius, Halorhabdus, Halorubrum, Halosimplex, Natronomonas from halite dated of thousands or millions of years [START_REF] Gramain | Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)[END_REF][START_REF] Park | Haloarchaeal diversity in 23, 121 and 419 MYA salts[END_REF][START_REF] Jaakkola | Buried Alive: Microbes from Ancient Halite[END_REF][START_REF] Radax | Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt[END_REF][START_REF] Fish | Recovery of 16S ribosomal RNA gene fragments from ancient halite[END_REF][START_REF] Sankaranarayanan | Characterization of ancient DNA supports long-term survival of Haloarchaea[END_REF][START_REF] Jaakkola | The complete genome of a viable archaeum isolated from 123-million-year-old rock salt[END_REF]. The long-term survival of halobacteria would be enhanced by their trapping in salt crystals over time [START_REF] Norton | Survival of Halobacteria Within Fluid Inclusions in Salt Crystals[END_REF].

Amber

Amber is a fossil resin that has the natural property of preserving microbial cells and their DNAs, making this sample a good candidate for evolutionary microbiological studies [START_REF] Veiga-Crespo | Putative ancient microorganisms from amber nuggets[END_REF].

Several studies have already reported the presence of microorganisms in amber [START_REF] Lambert | Staphylococcus succinus sp. nov., isolated from Dominican amber[END_REF][START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Greenblatt | Micrococcus luteus --survival in amber[END_REF][START_REF] Veiga-Crespo | Putative ancient microorganisms from amber nuggets[END_REF][START_REF] Santiago-Rodriguez | luxS in bacteria isolated from 25-to 40-millionyear-old amber[END_REF]. The methods for identifying the microorganisms reported include comparison of FAMES, enzymatic profiles, culture and 16S rRNA sequencing [START_REF] Lambert | Staphylococcus succinus sp. nov., isolated from Dominican amber[END_REF][START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Cano | Revival and identification of bacterial spores in 25-to 40million-year-old Dominican amber[END_REF]. Bacillus (sporulated species) and Staphylococcus genera are predominant in the majority of ancient ambers of all ages [START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Santiago-Rodriguez | Microbial communities in precolumbian coprolites[END_REF][START_REF] Cano | Revival and identification of bacterial spores in 25-to 40million-year-old Dominican amber[END_REF]. Moreover, Dominican amber preserved 25-to 40-millionyear-old was characterized by Curtobacterium, Paenibacillus, and Brevundimonas genera isolates [START_REF] Lambert | Staphylococcus succinus sp. nov., isolated from Dominican amber[END_REF][START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Cano | Revival and identification of bacterial spores in 25-to 40million-year-old Dominican amber[END_REF][START_REF] Santiago-Rodriguez | luxS in bacteria isolated from 25-to 40-millionyear-old amber[END_REF]. The genera Micrococcus, Rathayibacter, Nocardioides, Caryophanon, Arthrobacter, Streptomyces, Amycolatopsis, Nocardioides, Micromonospora were also predominated in Dominican amber and Israeli amber preserved about 120 millionyear-old [START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Greenblatt | Micrococcus luteus --survival in amber[END_REF]. These bacteria are mostly bacterial genera in the soil and environment, but some are also the main producers of antimicrobials [START_REF] Zhao | Actinobacteria-Derived peptide antibiotics since[END_REF][START_REF] Gong | Tetrocarcin Q, a New Spirotetronate with a Unique Glycosyl Group from a Marine-Derived Actinomycete Micromonospora carbonacea LS276[END_REF][START_REF] Gui | Antimicrobial Spirotetronate Metabolites from Marine-Derived Micromonospora harpali SCSIO GJ089[END_REF]. The long-term survival of the bacteria in ancient amber would have been modulated both by spores, but also by bacterial communications, notably via luxS. Indeed, the discovery of the LuxS gene, a promoter of quorum sensing detection in ancient bacteria isolated from amber stored between 25 and 40 million years ago, suggests that there would be horizontal transfers over millions of years between these ancient and contemporaries bacteria [START_REF] Santiago-Rodriguez | luxS in bacteria isolated from 25-to 40-millionyear-old amber[END_REF]. Some authors doubt these studies considering that DNA could not survive more than 1.5 million years in an environment [START_REF] Weyrich | Was luxS really isolated from 25-to 40-million-year-old bacteria?[END_REF], while these studies exclude any possibility of contamination given the precautions taken during sampling and laboratory manipulations. However, although some of these studies on amber have been the subject of much criticism [START_REF] Weyrich | Was luxS really isolated from 25-to 40-million-year-old bacteria?[END_REF][START_REF] Eisenhofer | proper authentication of ancient DNA is essential[END_REF], there is no tangible scientific evidence to assert or not the actual survival of these microorganisms during all these millions of years in amber.

THE ANTIQUITY OF ANTIBIOTIC RESISTANCE

Studies on ancient samples have shown that these samples represent an important source of discovery of the antiquity of antibiotic resistance. The most relevant samples are permafrost samples thousands and millions of years old, wherein many resistance genes have been reported. The first discovery of multiresistant strain in permafrost sediments dates back to 2008 in a study conducted by Mindlin et al. revealing bacterial strains resistant to aminoglycosides, chloramphenicol, tetracycline and harboring strA-strB genes encoding aminoglycoside phosphotransferases and aadA genes encoding aminoglycoside adenylyltransferases [START_REF] Mindlin | Isolation of antibiotic resistance bacterial strains from Eastern Siberia permafrost sediments[END_REF]. However, other studies have confirmed that some bacteria isolated from permafrost were resistant to the majority of classes of antibiotics produced naturally in microorganisms, but also modern synthetic antibiotics such as amikacin [START_REF] Perron | Functional Characterization of Bacteria Isolated from Ancient Arctic Soil Exposes Diverse Resistance Mechanisms to Modern Antibiotics[END_REF]. Their genomic analyzes confirm the presence of genes (most of which are localized on plasmids), encoding resistance to streptomycin, tetracycline [START_REF] Petrova | Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain[END_REF][START_REF] Mindlin | Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis[END_REF], Beta-lactam, Fosfomycin, Fusidic acid, MLS, Macrolide, Phenicol [START_REF] Kashuba | Ancient permafrost staphylococci carry antibiotic resistance genes[END_REF], bacitracin [START_REF] Zhang | Distribution of ARGs and MGEs among glacial soil, permafrost, and sediment using metagenomic analysis[END_REF] and to heavy metals, such as mercury, cobalt, zinc, cadmium, copper, chromium and arsenic compounds [START_REF] Mindlin | Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis[END_REF].

These antibiotic resistance genes found in permafrost are sometimes followed by mobile genetic elements, such as integrons, plasmids and insertion sequences [START_REF] Zhang | Distribution of ARGs and MGEs among glacial soil, permafrost, and sediment using metagenomic analysis[END_REF], the majority of which are already known, but some may also been found that have never been identified in studies of contemporary microorganisms. This is the case of a new insertion sequence named "ISPpy1" discovered in Psychrobacter psychrophilus, a bacterium isolated from permafrost aged 35,000 years [START_REF] Petrova | Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain[END_REF]. In addition, a variety of resistance genes coding for β-lactam, tetracycline and glycopeptide has also been reported in metagenome data of 30,000 year old permafrost sediment [START_REF] D'costa | Antibiotic resistance is ancient[END_REF].

Comparative studies of resistance profiles confirm that these resistance genes are similar to their modern counterparts and variants [START_REF] Petrova | Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain[END_REF][START_REF] Mindlin | Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis[END_REF][START_REF] Kashuba | Ancient permafrost staphylococci carry antibiotic resistance genes[END_REF][START_REF] D'costa | Antibiotic resistance is ancient[END_REF], thus stipulating a reduced evolution of these resistance genes over several thousands of years.

CONCLUSIONS

A wide array of methods is currently available to decipher microbial diversity in ancient samples (Table 3). Currently, next generation sequencing (NGS) methods provide exciting perspectives enabling both compositional and functional analyses. For a more comprehensive overview of the microbial community of the ancient samples, association of these methods could be required as a polyphasic approach. Studies on ancient microorganisms have revealed that certain metabolic pathways have emerged or increased over the years as witnesses of the evolution or adaptation to changing environments and that many antibiotic resistance genes have niches in ancient environments. The fact that many of the ancient bacterial genera are reported at least once in humans (Table 2) [START_REF] Bilen | The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species[END_REF] underlines the importance of such studies.

PERSPECTIVES ON MICROBIAL EVOLUTION

Studies on microorganisms from ancient environments are crucial as they are currently the single ones that can confirm or disprove the various theories of evolution, as microorganisms in these environments can survive for millions of years, whereas such studies on human beings could not exist. In this sense, the survival of microorganisms in ancient samples can perpetuate for a considerable time, as bacterial strains that can survive in permafrost up to 3.5 million years [START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF][START_REF] Zhang | Isolation and characterization of bacteria from ancient siberian permafrost sediment[END_REF][START_REF] Gilichinsky | Long-term preservation of microbial ecosystems in permafrost[END_REF], in amber dated about 120 million years old, [START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Greenblatt | Micrococcus luteus --survival in amber[END_REF] or in halite dated 250 million years old [START_REF] Vreeland | Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal[END_REF].

Thus, even when trapped by cold and salinity, microorganisms can be preserved in ancient environments under conditions that are unfavorable to their survival. Moreover, the melting of permafrost due to global warming is causing the reappearance of these longtrapped and preserved bacterial populations [START_REF] Woodcroft | Genome-centric view of carbon processing in thawing permafrost[END_REF].The first conclusions that can be drawn from the comparison made between ancient and contemporary strains suggest that Darwinian Theory regarding the evolution is not relevant when applied to bacterial populations.

Our anthropocentric vision suggests that the human being evolves rapidly [START_REF] Nongkynrih | Biological clocks: mechanisms and developments[END_REF], but this evolutionary speed seems to be rather applicable to vertebrates than to bacteria. This is therefore a proof of the human vanity to define and delineate bacterial species which, in our view, belong to another side world. The variety of species in the visible and the invisible have in fact few in common.

Considering the significant number of millions of years under different conditions in which these ancient bacterial environmental species can survive, we believe that several parameters (mastered or not) are potentially relevant to understand what has kept them all these years and how they have evolved. It is not possible to define their molecular evolution relying on a substitution rate that would be supposed to be constant over the years [START_REF] Kuo | Inferring clocks when lacking rocks: the variable rates of molecular evolution in bacteria[END_REF]. In conclusion, studies on ancient microorganisms make it possible to build a real molecular clock and gene network, which is neither speculative nor universal since it has long been 570 considered a research tool [START_REF] Ochman | Evolution in bacteria: Evidence for a universal substitution rate in cellular genomes[END_REF]. "bacteria" AND "permafrost" AND " Metagenomics " OR " Molecular Diagnostic Technique" OR "NGS" OR "PCR" OR " Genomics" OR " RNA Sequence Analysis" "Permafrost" AND "metagenomic*"AND "bacter*"AND "microorganism*" ; "Permafrost" AND "PCR*"AND "bacter*"AND "microorganism*"; "Permafrost" AND "genomic*"AND "bacter*"AND "microorganism*"; "Permafrost" AND "molecular*"AND "bacter*"AND "microorganism*"; "Permafrost" AND "RNA"AND "bacter*"AND "microorganism*" Microscopy Permafrost ("permafrost "[Tiab] AND (" Microscopy "[Tiab] OR " Electron, Microscopy "[Tiab] OR " Scanning Probe "[Tiab] OR " Fluorescence, Microscopy "[Tiab])

Queries

"permafrost " AND " Microscopy " OR " Electron, Microscopy " OR " Scanning Probe " OR " Fluorescence, Microscopy " "Permafrost" AND "microscopy" AND "bacter*"AND "microorganism*" Mass Spectrometry Permafrost ("permafrost "[Tiab] AND (" Mass Spectrometry "[Tiab] OR " Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization "[Tiab] OR " Spectrum Analysis mass "[Tiab] OR " Mass Spectroscopy "[Tiab])

"permafrost " AND " Mass Spectrometry " OR " Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization " OR " Spectrum Analysis mass " OR " Mass Spectroscopy " "Permafrost" AND " Mass Spectrometry" AND "bacter*"AND "microorganism*" "permafrost " AND "Antibiotic resistance " OR " Drug Resistance " OR " Drug Resistance, Microbial " OR " Drug Resistance, Microbial " OR " Drug Resistance, Bacterial" OR " Resistance genes" OR " Genes, MDR " "permafrost " AND "Antibiotic resistance "; "permafrost " AND "resistance genes";

  DNA "[Tiab] AND ("culture "[Tiab] OR " metagenomics "[Tiab] OR " microscopy "[Tiab] OR " PCR "[Tiab] OR " Genomics"[Tiab]) ("ancient microorganisms "[Tiab] AND ("culture "[Tiab] OR " metagenomics "[Tiab] OR " microscopy "[Tiab] OR " PCR "[Tiab] OR " Genomics"[Tiab])"ancient microorganisms " AND ("culture " OR " metagenomics "OR " microscopy " OR " PCR " OR " Genomics" Ancient microorganisms Culture Bacteria Permafrost ("bacteria"[Tiab] AND ("permafrost "[Tiab] AND ("isolation "[Tiab] OR "Culture Media"[Tiab] AND "Culture Techniques"[Tiab] OR media[Tiab] OR medium[Tiab] OR cultiv*[Tiab] OR cultur*[Tiab]) "bacteria" AND "permafrost " AND isolation OR Culture Media AND "Culture Techniques" OR media OR medium OR cultiv* OR cultur* "Permafrost" AND "cult*"AND "bacter*"AND "microorganism*" Molecular techniques Permafrost ("bacteria"[Tiab] AND ("permafrost "[Tiab] AND (" Metagenomics "[Tiab] OR " Molecular Diagnostic Technique"[Tiab] OR "NGS"[Tiab] OR "PCR"[Tiab] OR " Genomics"[Tiab]) OR " RNA Sequence Analysis"[Tiab])

Fluorescent

  "[Tiab] AND (" fluorescent in situ hybridization "[Tiab] OR " Staining and Labeling"[Tiab] OR " FISH "[Tiab] OR " In Situ Hybridization, Fluorescence "[Tiab]) "permafrost " AND " fluorescent in situ hybridization " OR " Staining and Labeling" OR " FISH " OR " In Situ Hybridization, Fluorescence "Permafrost" AND " FISH" AND "bacter*"AND "microorganism*" Multidrug Resistance Gene /Permafrost ("permafrost "[Tiab] AND ("Antibiotic resistance "[Tiab] OR " Drug Resistance "[Tiab] OR " Drug Resistance, Microbial "[Tiab] OR " Drug Resistance, Microbial "[Tiab] OR " Drug Resistance, Bacterial"[Tiab] OR " Resistance genes "[Tiab] OR " Genes, MDR "[Tiab])

* Bacterial genera not found in humans. All others are reported at least once in human being[START_REF] Bilen | The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species[END_REF]

FUNDING

This study was supported by the "Méditerranée-Infection" Foundation; program "Investissements d'Avenir", managed by the Agence Nationale de la Recherche (reference Mediterranee Infection ANR-10-IAHU-03); Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI.

CONFLICT OF INTEREST

The authors declare no conflict of interest

Genera of bacterial isolates Site References

Arthrobacter Planococcus Planomicrobium*

Siberia

Canada China [START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Hinsa-Leasure | Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample[END_REF][START_REF] Bai | Phylogenetic diversity of culturable bacteria from alpine permafrost in the Tianshan Mountains, northwestern China[END_REF][START_REF] Reddy | Planococcus antarcticus and Planococcus psychrophilus spp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica[END_REF] Arthrobacter Bacillus Paenibacillus Pseudomonas Sphingomonas

Northeast Greenland Siberia Canada [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF][START_REF] Cadoret | Noncontiguous finished genome sequences and description ofBacillus massiliglaciei, Bacillus mediterraneensis, Bacillus massilinigeriensis, Bacillus phocaeensisandBacillus tuaregi,five new species identified by culturomics[END_REF][START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF][START_REF] Hinsa-Leasure | Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample[END_REF][START_REF] Wilhelm | Microbial diversity of active layer and permafrost in an acidic wetland from the Canadian High Arctic[END_REF][START_REF] Zhang | Isolation and characterization of bacteria from ancient siberian permafrost sediment[END_REF] Flavobacterium Northeast Greenland Siberia [START_REF] Vishnivetskaya | Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods[END_REF][START_REF] Ganzert | Bacterial community composition and diversity of five different permafrost-affected soils of Northeast Greenland[END_REF] Rhodococcus -Determination of bacilli and cocci in ancient halite samples [START_REF] Schubert | Microscopic identification of prokaryotes in modern and ancient halite, Saline Valley and Death Valley, California[END_REF][START_REF] Schubert | Halophilic Archaea cultured from ancient halite, Death Valley, California[END_REF] -Determination of bacteria in ancient teeth samples [START_REF] Linossier | Paleomicrobiological study in dental calculus: Streptococcus mutans[END_REF] -Determination of prokaryotic associated to permafrost samples [START_REF] Stakhov | Microbial communities of ancient seeds derived from permanently frozen Pleistocene deposits[END_REF] -Determination of the microbial morphology and biofilm formation in permafrost samples [START_REF] Soina | Adaptation of Bacteria to the Terrestrial Permafrost Environment[END_REF]. For microorganisms culture in Siberian and Canadian permafrost: -Nutrient-poor media (R2A; PYGV) [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF] -Nutrient nutrients media (LB , TSA) enriched or not with vitamin/antibiotics [START_REF] Steven | Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods[END_REF][START_REF] Shi | Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing[END_REF] -Halophilic media [START_REF] Niederberger | Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic[END_REF] For microorganisms culture in Alaskan permafrost [START_REF] Panikov | Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C[END_REF] -Solid media Frozen For microorganisms culture in amber samples [START_REF] Lambert | Staphylococcus succinus sp. nov., isolated from Dominican amber[END_REF][START_REF] Greenblatt | Diversity of Microorganisms Isolated from Amber[END_REF][START_REF] Greenblatt | Micrococcus luteus --survival in amber[END_REF].

-Nutrient nutrients media (LB, TSA) enriched or not with vitamin/antibiotics For microorganisms culture in halite samples [