Accepted Manuscript
Repertoire of human gut microbes
Perrine Hugon, Jean-Christophe Lagier, Philippe Colson, Fadi Bittar, Didier Raoult
PII: S0882-4010(15)30231-X DOI: 10.1016/j.micpath.2016.06.020 Reference: YMPAT 1868
To appear in: Microbial Pathogenesis
Received Date: 14 December 2015
Accepted Date: 14 June 2016
Please cite this article as: Hugon P, Lagier J-C, Colson P, Bittar F, Raoult D, Repertoire of human gut microbes, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.06.020.
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1 Repertoire of Human Gut Microbes
2
3 Perrine HUGON 1, Jean-Christophe LAGIER 2, Philippe COLSON 2, Fadi BITTAR 2, and
4 Didier RAOULT 2,3
5
6 1. Institut Pasteur, Unité de Biologie des Spirochètes, Paris, France
7 2. Aix-Marseille Université URMITE, UM63, CNRS 7278, IRD 198, INSERM 1095
8 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France
9 3. Special Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz
10 University, Jeddah, 21589, Saudi Arabia.
11
12 * To whom correspondence should be addressed 13 Prof. Didier RAOULT MANUSCRIPT 14 Aix-Marseille Université, URMITE – UMR 63, CNRS 7278, IRD 198, INSERM 1095 15 Faculté de Médecine, 27 Bd Jean Moulin, 13005, Marseille, France
16 Email: [email protected]
17 Phone: (33) 491 38 55 17
18 Fax: (33) 491 83 03 90
19
20
21 22 ACCEPTED 23 Running title: gut microbiota repertoire
24 Words count: 4,292
25 Abstract word count: 206
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26 Abstract
27 In 1675, Antoni Van Leeuwenhoeck was the first to observe several forms using an
28 optical microscope that he named “animalcules”, realizing later that these were
29 microorganisms. The first classification of living organisms proposed by Ehrenberg in 1833
30 was based on what we could visualize. The failure of this kind of classification arises from
31 viral culture, which preceded direct observations that were finally achieved during the 20 th
32 century by electron microscopy.
33 The number of prokaryotic species is estimated at approximately 10 million, although
34 only 1,800 were known in 1980, and 14,000 to date, thanks to the advent of 16S rRNA
35 amplification and sequencing. This highlights our inability to access the entire diversity.
36 Indeed, a large number of bacteria are only,known as Operational Taxonomic Units (OTUs)
37 and detected as a result of metagenomics studies, revealing an unexplored world known as
38 the “dark matter”. Recently, the rebirth of bacterial culture through the example of 39 culturomics has dramatically increased the human MANUSCRIPTgut repertoire as well as the 18SrRNA 40 sequencing allowed to largely extend the repertoire of Eukaryotes. Finally, filtration and co-
41 culture on free-living protists associated with high-throughput culture elucidated a part of the
42 megavirome.
43 While the majority of studies currently performed on the human gut microbiota focus on
44 bacterial diversity, it appears that several other prokaryotes (including archaea) and
45 eukaryotic populations also inhabit this ecosystem; their detection depending exclusively on
46 the tools used. Rational and comprehensive establishment of this ecosystem will allow the 47 understandingACCEPTED of human health associated with gut microbiota and the potential to change 48 this.
49 Key words : gut microbiota; culturomics; taxonogenomics; prokaryotes; eukaryotes; giant
50 viruses; Megavirales; 16SrRNA
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51 Highlights: 52 53 - In addition with bacterial communities, archaea, virus (including giant virus), fungi
54 and parasites inhabits the human gut repertoire.
55
56 - In complement with metagenomics, we introduce microbial culturomics based on the
57 multiplication of culture conditions coupled with a rapid identification
58 method by MALDI-TOF allowed to extend the known gut microbiota repertoire.
59
60 - We consider bacterial taxonomy as inadequate with the exponential number of new
61 bacteria described and must include genome sequencing as proposed by
62 taxonogenomics
63
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64 1. Introduction
65 The exploration of the human gut microbiota has exploded during the last decade. With the
66 tremendous changes in molecular technologies and the new “omics” strategies developed, this
67 ecosystem is now considered for its role in metabolism, immune system and human health
68 [1]. Moreover, numerous metagenomic studies performed during the last years have
69 suggested an association between the microbial composition of the human gut and various
70 diseases including for instance obesity [2], Crohn’s disease [3], or irritable bowel syndrome
71 [4]. The gut microbiota harbours at least 10 11 to 10 12 bacteria per gram of faeces [5], and its
72 composition varies with physiological factors [6] such as geographic provenance, age, dietary
73 habits, malnutrition, and external factors can also imbalance the microbiota as probiotics or
74 antimicrobial agents uses [7]. The relationship between the host and this complex ecosystem
75 composed by prokaryotes, viruses, fungi and parasites is extremely complex. Recent
76 significant efforts have been deployed to characterize the gut repertoire; however there is still 77 a need to provide an efficient repertoire even for allMANUSCRIPT microorganisms isolated or detected in 78 the human gut [8]. Regarding viruses, giant ones have been recently showed being genuine
79 members of the tree of life [9, 10]. Thus, their tremendous gene repertoires contain genes with
80 homologs in cellular organisms, among which those encoding DNA-dependent RNA
81 polymerase. This represents a change of paradigm. Indeed, the predominant use of ribosomal
82 genes to classify organisms that was introduced in the 1970s by C. Woese, who defined three
83 domains of life, namely Bacteria , Archea and Eukarya , led to exclude viruses because they
84 are devoid of such genes [11]. Apart from lacking ribosomes, giant viruses share many 85 features with ACCEPTEDother intracellular microorganisms and can be considered as microbes. This led 86 to propose in 2013 a new classification of microbes in four 'TRUC', an acronym for Things
87 Resisting Uncompleted Classifications, that does not rely on ribosomal genes but takes into
88 account giant viruses alongside with bacteria, archaea and eukaryotic microbes and should
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89 allow more comprehensive description of human gut microbiota [10]. In this review we are
90 focusing on human gut components of the bacterial, fungal, parasites and archaeal diversity,
91 as well as on the gut virome discovery.
92 2. The Prokaryotes
93 2.1. Culture-based methods as the pioneer strategy for human gut microbiota
94 research
95 The first discrepancy arose from initial culture studies [5]. At that time, the 1970s, gram-
96 staining and microscopic examination performed directly on stool samples were the
97 techniques used to study gut microbiota composition [12-14]. While such techniques revealed
98 the predominance of gram-negative bacteria in stool samples [12], culture counts identified a
99 majority of gram-positive bacteria [14] and anaerobes dominated the community. The second
100 discrepancy was named few years later by Staley and Konopka as the “great plate count
101 anomaly” [15]. It was the difference between “what we can see” on direct microscopic 102 observation and “what’s growing in our plate”. This MANUSCRIPT was indeed confirmed 20 years later as 103 only 1% of bacteria can be easily grown in vitro [16].
104 Anaerobes were considered to be the major component of gut microflora [13], however
105 this seems biased as a great majority of studies concentrated their efforts on these specific
106 bacteria [5]. In 1969, Hungate revolutionized the anaerobic culture in developing the roll tube
107 technique [17], thus allowing isolation of extremely oxygen-sensitive (EOS) bacteria. Several
108 species (within genera Bacteroides , Clostridium , Veillonella , Ruminococcus , Eubacterium ,
109 Bifidobacterium , Lactobacillus , Fusobacterium , Peptococcus and Peptostreptococcus ) were 110 considered toACCEPTED dominate the gut microbiota. Finally, before molecular tools were incorporated, 111 it was estimated that 400-500 different species composed the gut microflora [13, 18], which
112 remained partially characterized due to the technical limitations.
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113 2.2. The molecular revolution: how improved technologies enhanced our
114 knowledge of prokaryotic diversity
115 Introduced fifteen years ago, 16S rDNA sequence analysis is still the basic tool for
116 studying bacterial taxonomy and phylogenic relationships between microorganisms. In the
117 2000s, the introduction of high-throughput sequencing techniques based on the amplification
118 of the 16S rRNA gene improved understanding of bacterial diversity from complex
119 microbiota, and demonstrated that 80% of bacteria detected with molecular tools were
120 uncultured [5]. However, it is important to understand the biases and limitations of 16S rRNA
121 gene profiling. Firstly, 16S rRNA gene lacks sensitivity within specific genera and cannot
122 delineate between two species with high interspecies similarity [19]. Secondly, gene sequence
123 heterogeneity can be encountered in species having more than one copy [20]. Regarding DNA
124 extraction kits [21, 22], the hypervariable region targeted in 16S rRNA gene and primer
125 choices [23], the depth bias [24], several studies reported the serious impact on the microbiota 126 abundance and diversity these factors could play. MMANUSCRIPTore recently, a study performed on 16 127 stool samples revealed that pyrosequencing performed on the V6 region on 16S rRNA gene
128 has neglected some of the gram-negative bacteria detected using transmission electron
129 microscopy [25].
130 Regarding prokaryotic diversity in humans, more than 120 different prokaryotic phyla have
131 been identified and only 31 phyla included cultured species [8]. Moreover, 12 bacterial phyla
132 with cultured representatives have been recorded in humans (Firmicutes , Bacteroidetes ,
133 Actinobacteria , Proteobacteria , Chlamydiae , Deinococcus-Thermus , Fusobacteria , 134 Tenericutes , LentisphaeraeACCEPTED, Spirochaetes , Synergistetes and Verrucomicrobia ) (Figure 1, 135 Table 1)[8], where each phylum represents species that have also been isolated in the human
136 gut. Moreover, the majority of species isolated in the gut belong to four phyla, Firmicutes ,
137 Proteobacteria , Actinobacteria and Bacteroidetes and dominant species from the families
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138 Bacillaceae , Enterobacteriaceae , Corynebacteriaceae and Bacteroidaceae respectively [8]. In
139 addition to cultured bacteria, several phyla have only been detected in the gut and remain as
140 yet uncultured [26]: species belonging to TM7 have been detected in both healthy persons and
141 patients suffering from inflammatory bowel disease [27]; Melainabacteria , a new candidate
142 phylum sibling to Cyanobacteria [28], and the Gemmatimonadetes phylum [26].
143 High-throughput sequencing studies performed the last ten years [29-33] showed a
144 majority of reads belonging to two dominant phyla (Firmicutes and Bacteroides ),
145 corresponding to species belonging to the Ruminococcaceae , Clostridiaceae ,
146 Lachnospiraceae , Bacteroidaceae families that contain a majority of anaerobic species as yet
147 uncultured. Moreover, several members of Lachnospiraceae family (Eubacterium spp .,
148 Anaerostipes spp ., Roseburia spp. , Coprococcus spp. for example) are butyrate-producing
149 bacteria that are difficult to cultivate from faecal samples [34, 35]. Butyrate-producing
150 bacteria have recently been associated with health status in several diseases [36-38] as anti- 151 inflammatory and anticarcinogenic properties have MANUSCRIPT been identified [39]. These discrepancies 152 between molecular and culture-dependant studies emphasize the need to improve the
153 anaerobic culture to understand the specific role these species are playing in the human gut
154 microbiota. Finally, species belonging to the highly diverse Proteobacteria phylum are
155 commonly detected in molecular studies [26]. Due to the specific primers the phylum
156 Actinobacteria needs to be amplified [40] and it often remains underrepresented by large
157 sequencing studies [26], whereas its species are easily cultivated.
158 2.3. Culturomics: a third shift in understanding gut microbiota? 159 Matrix-assistedACCEPTED laser desorption/ionization-time of flight mass spectrometry (MALDI- 160 TOF-MS) has emerged as a time- and cost-effective method for the identification of bacteria
161 [41]. This efficient identification method has been the main key to developing high-
162 throughput culture-dependent studies. Indeed, since 1995 with the development of culture-
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163 independent methods, notably 16S rRNA amplification and sequencing and more recently
164 metagenomics studies, bacterial culture was considered as outdated. In the last four years, a
165 new strategy named “culturomics” was developed to study the human gut microbiota [24].
166 This method is based on the diversification of culture conditions (by varying atmosphere,
167 temperature, incubation time, passive and active filtration or antibiotics in order to culture the
168 minority populations) with the objective of mimicking the natural conditions within the
169 digestive tract, using notably rumen fluid [24, 42].
170 In their pioneering study, Lagier et al. , using 212 culture conditions, testing 32,500
171 colonies by MALDI-TOF, identified 340 bacterial species, including 31 putative new species
172 [24]. In addition, they demonstrated the complementarity of metagenomics and culturomics
173 because only 15% of the bacterial species were concomitantly detected. A stringent analysis
174 of the culture conditions tested in the first study showed that all the bacteria identified were
175 cultured by testing 70 culture conditions [24]. These 70 culture conditions were applied for 176 the study of supplementary stool samples from other MANUSCRIPT patient populations such as those treated 177 with antibiotics, suffering from anorexia nervosa or obesity, and healthy subjects from various
178 geographic areas, extending the repertoire [24, 42-45]. For the ongoing studies we selected 18
179 different culture conditions in liquid media with subcultures on solid media every 3 days [42].
180 This resulted in culturing 717 bacterial species from 7 phyla ( Actinobacteria , Bacteroidetes ,
181 Deinococcus-Thermus , Firmicutes , Fusobacteria , Proteobacteria , Synergistetes ) including
182 several new bacterial species [42]. In order to describe these new isolates, we developed a
183 polyphasic strategy combining phenotypic and genotypic characteristics obtainable and 184 comparable byACCEPTED most laboratories, and named taxono-genomics [46]. The genome sequencing 185 and the MALDI-TOF spectra of the new bacterial species were systematically included. In
186 addition, we determined the Average Genomic Identity of Orthologous gene Sequences
187 (AGIOS) of studied strains by comparison with their closest phylogenetic species. To date,
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188 we have described 13 bacterial species using taxono-genomics (Aeromicrobium massiliense
189 sp. nov., Alistipes timonensis sp. nov., Anaerococcus senegalensis sp. nov., Brevibacillus
190 massiliensis sp. nov., Brevibacterium senegalense sp. nov., Enterobacter massiliensis sp.
191 nov., Herbaspirillum massiliense sp. nov. Kurthia massiliensis, Senegalimassilia anaerobia
192 gen. nov., sp. nov., Paenibacillus senegalensis , Cellulomonas massiliensis , Peptoniphilus
193 timonensis , and Clostridium senegalense). These have officially been recognized as new
194 genera and/or species in validation lists no. 153 [47], no. 155 [48] and no. 165 [49]. By
195 concomitantly using culturomics, 16S rRNA and MALDI-TOF-MS, the number of bacterial
196 species cultured in clinical microbiology has dramatically increased [42].
197 3. The archaeal diversity
198 The Archaea community constitutes the third domain of life, separate from bacteria and
199 eukaryote kingdoms [50], although archaea share many characteristics with both bacteria and
200 eukaryotes [51]. Extremophile species, which live in extreme environments under high salt 201 concentrations and extreme pH and temperatures, werMANUSCRIPTe the first to be discovered in the late 202 1970s [52]. However, we now know that archaeal mesophilic species also represents a large
203 part of the diversity and inhabit non-extreme environments [53]. Moreover, the human gut
204 microbiota include non-methanogenic and methanogenic species, with the latter representing
205 up to 10 10 cells per gram of faeces [54] and having a significant role in the gut microbiota
206 ecosystem through methane production during anaerobic fermentations [55].
207 In total, eight archaeal species have been isolated in the human gut microbiota [26], all of
208 which belong to the Euryarchaeota phylum (Figure 1). Methanobrevibacter ruminantium [56] 209 was the first speciesACCEPTED isolated from stool samples. This was probably a misidentification [26] 210 as Methanobrevibacter smithii dominates in the human gut [57]. The atypical methanogenic
211 Methanosphaera stadtmanae reduces the methanol [58] as does Methanomassiliicoccus
212 luminyensis , discovered more recently, but is phylogenetically distant from M stadtmanae
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213 [59]. Methanobrevibacter oralis was first isolated in the 1990s from human subgingival
214 plaque [60]. Its genome was only sequenced recently from a stool sample strain [61].
215 Methanomethylophilus alvus [62] and Methanomassiliicoccus intestinalis [63], two species
216 phylogenetically related to the Thermoplasmatales , were isolated and their genomes
217 sequenced, highlighting a new order named Methanomassiliicoccales shared with M
218 luminyensis [55]. Very recently, Methanobrevibacter arboriphilicus has been isolated from
219 the human gut [64]. Surprisingly it showed the lowest GC content in archaeal species [64].
220 Additionally, substantial diversity of archaeal species have been detected in the human gut
221 microbiota [26, 55] using16S rRNA, mcrA or amoA gene sequences [55]. Two halophilic
222 bacteria belonging to the Euryarchaeota phylum were detected in 2008 using the denaturing
223 gradient gel electrophoresis (DGGE) [65] method, and several archaeal representatives
224 associated with the human gut microbiota belonging to Sulfolobales , Nitrososphaerales ,
225 Methanosarcinales , Methanomicrobiales were identified [55]. 226 4. The existence of a human gut microbial virome MANUSCRIPT 227 By contrast with the bacterial microbiota, only a few studies have explored the “human
228 gut virome”, which represents a fairly new concept [66, 67]. This appears paradoxical, as the
229 existence of pathogenic viruses was discovered in human faeces a long time ago [68].
230 Studying viral diversity in the gut appears more difficult than for cellular organisms because
231 they are, for most of them, not visible under a light microscope, and they are devoid of any
232 conserved gene that is shared by all of them (that may be an equivalent to 16S-18S rRNA in
233 cells) [69]. Thus, viruses have been sought in the human gut using cell culture, electron 234 microscopy, PCRACCEPTED and finally metagenomics [67]. In current technology advances, high- 235 throughput sequencing highlighted groups of viruses (or Virus Like Particle (VLPs))
236 associated with the human gut microbiota [70], equivalent to 10 9 VLPs per gram of faeces
237 [71]. Both DNA and RNA viruses were investigated in human stools by metagenomics, from
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238 its very onset [66, 72]. Recent work on the viral community provided a better understanding
239 of the role viruses from the gut could play in its host [73, 74], how it could interact with the
240 complex gut ecosystem to influence food digestion [75], and also how it could actively
241 participate in human health through the prevention of invasion by pathogens [67, 76].
242 New sequencing strategies have shown that the gut virome was composed of both
243 eukaryotic viruses and prokaryotic viruses (i.e. viruses that infect bacteria, known as
244 bacteriophages), and that the latter dominate this virus community (Figure 1) [67, 75]. Tailed
245 bacteriophages with double-stranded DNA (dsDNA) that belong to the order Caudovirales
246 appear to be the most abundant in the gut with the single-strand DNA (ssDNA)
247 bacteriophages belonging to the family Microviridae (Figure 1) [70]. Many DNA or RNA
248 viruses, including rotaviruses, caliciviruses, astroviruses or adenoviruses, have been
249 associated with gastroenteritis [77]. In addition, several human viral pathogens such as
250 enteroviruses, as well as viruses that are transmitted via the faecal-oral route and are excreted 251 by the gut, including, for instance, hepatitis A an d MANUSCRIPTE viruses, can be found in stools. Many 252 viruses were unexpectedly identified in human stools. This was also the case for plant viruses,
253 among which Pepper mild mottle virus was found at the greatest titre, estimated to be up to
254 10 9 per gram of dried faeces, and whose presence seems to be directly correlated with
255 ingested food [72]. These findings suggest that viral diversity in the gut is probably still
256 largely untapped. Surprisingly, bacteriophages residing in the human gut have been estimated
257 at having a titre of the same order of magnitude as that of their bacterial hosts (ratio 1:1) [67,
258 74]. In contrast, in many habitats, oceans for example, bacteriophages are 10-fold more 259 abundant thanACCEPTED their bacterial hosts (ratio 10:1) [75, 78]. This could reflect the temperate 260 lifestyle bacteriophages may adopt in the human gut, contrasting with the “kill the winner
261 strategy” they use in marine habitats [75].
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262 In the same way as for the gut microbiota community, our knowledge on viruses from the
263 gut comes from evolving technologies, bearing in mind than each method provides benefits
264 and has limitations [67]: culture-based techniques (phage isolation) and microscopic
265 observations (TEM or fluorescence) were the first methods used, whereas PCR testing and
266 Sanger sequencing form the current majority, and an increasing number of investigations
267 conducted apply high-throughput sequencing to metagenomics [67]. Recently, the CRISPR
268 (Cluster Regularly Interspaced Short Palindromic Repeats)/Cas system was exploited to target
269 phage sequences in the gut microbiome in order to discover new phages associated with the
270 human body; this shed lights on a common phage reservoir associated with the human gut
271 microbiome. Thus, bacteriophage searches have opened a new field of interest and potential
272 applications in therapeutics, diagnostics and biotechnology [67, 79].
273 Giant viruses of amoebas have been discovered over the 12 last years, the first
274 representative being Mimivirus [10, 80]. To date, six new or putative viral families have been 275 uncovered, which have been linked by phylogenomics MANUSCRIPT to other double-stranded DNA viruses, 276 including poxiviruses, asfarviruses, irido-/ascoviruses, or phycodnaviruses [81, 82]. These
277 giant viruses were shown to be ubiquitous worldwide and common in the human
278 environment, primarily water and soil [83]. Unlike other viruses, giant viruses are visible
279 under a light microscope [84]. The size of giant virions is indeed similar to that of small
280 parasitic prokaryotes. In addition, their genome is as large as those of these intracellular
281 microorganisms and comprises a tremendous and unique gene repertoire among viruses.
282 Therefore, they can be considered as microbes [11]. Most of these viruses have been isolated 283 using co-cultureACCEPTED on free-living amoebas from the genera Acanthamoeba [83]. Their detection 284 as part of the virome has long been neglected due to their giant size, as samples were usually
285 filtered prior to analysis to separate viruses from cellular organisms; through this procedure,
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286 giant viruses, in contrast with the majority of viruses, are not filtered and remain within the
287 cellular fraction [85].
288 The first giant virus of amoeba isolated from the human gut, in 2012, was from the stools
289 of a young healthy Senegalese man [24, 85]. This was a serendipitous discovery as amoebal
290 co-culture of this virus, which was named Senegalvirus, was triggered by the detection in the
291 faeces of reads matching Marseillevirus, the prototype member of the second giant virus
292 family identified [86]. In 2013, a second giant virus that is a close relative of Mimivirus
293 named Shan virus, was isolated from the faeces of a Tunisian patient with pneumonia [87].
294 This strengthens the interest in expanding our knowledge of giant virus diversity and
295 prevalence in human stools. Moreover, metagenomic reads generated from human stools were
296 identified as matching sequences from virophages, which infect mimiviruses and were
297 discovered in 2008 as the first viruses of viruses [88, 89].
298 Eukaryotes 299 Research on this subdominant gut component MANUSCRIPT is fairly recent, as evidenced by the first 300 molecular study published in 2008 [90]. Eukaryotes, defined by the presence of a nucleus and
301 organelles, represent the third domain of life besides Bacteria and Archaea [91]. The
302 taxonomy of human gut eukaryotes is highly complex [91, 92] and highlights five major
303 groups (Amoebozoa, Opisthokonta, Excavata, Sar and Archaeplastida) based on recent
304 molecular phylogenetic classification [92]. Fungal species including yeasts and filamentous
305 fungi are the most abundant group of eukaryotes in the human gut. However parasites,
306 including unicellular organisms (protozoa) and multicellular species (helminths) (Figure 1), 307 could also beACCEPTED present in the human gut but in lower numbers. Their presence is mostly known 308 to have a pathogenic consequence to the host [26].
309 4.1. Fungal diversity
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310 Studying the human gut mycobiome consists of studying how the entire fungal
311 community inhabits the human gastrointestinal tract. Very few studies have focused on the
312 fungal diversity, however recent technological advances can provide deep understanding on
313 this neglected component [93]. Both filamentous fungi and yeasts are cultivated on different
314 solid media, and the majority of them can be identified using MALDI-TOF mass
315 spectrometry [94, 95]. As for bacteria, MALDI-TOF mass spectrometry has revolutionized
316 the diagnosis of fungal infection in mycology laboratories, allowing both rapid and accurate
317 identification [96]. New culture-independent methods, which remain under-developed for
318 fungal population [97], are targeting mainly the Internal Transcribed Spacer ITS1 and ITS2
319 [91]. This region (i.e. ITS) appears to be more appropriate than 18S rDNA for analysing
320 fungal population at the level of clades and species thanks to its high sequence variability
321 [91]. However, many difficulties arise during fungal metagenomic analyses including the low
322 presence of fungal species in the complex biota of the human intestinal tract, requiring the 323 application of ultra-deep sequencing strategy, and MANUSCRIPTthe absence of a well established fungal 324 ITS database [97].
325 The human gut mycobiome displays a very small fungal community divided into three
326 major phyla (Ascomycota 63%, Basidiomycota 32%, Zygomycota 3%) [26], including 273
327 different species from more than 140 genera [91, 93] (Table 2). Recently, a fourth phylum
328 named Microsporidia has been reclassified as fungi [98]. It contains members associated with
329 intestinal diseases [98] including Encephalitozoon hellem, Encephalitozoon intestinalis,
330 Enterocytozoon bieuneusi and Enterocytozoon hellem . Regarding the actual fungal repertoire, 331 216 different ACCEPTEDspecies were detected by molecular tools, 86 species were isolated by culture 332 methods and 29 species were identified using both approaches [91, 93]. Among the yeast
333 community, Candida spp. is the most abundant species [26] and C. albicans and C. rugosa
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334 are in their natural environment. However, they can become pathogens and induce candidiasis
335 under specific circumstances [26].
336 Additionally, Saccharomyces cerevisiae and Galactomyces geotrichum [90] are two
337 yeasts detected frequently in human faecal samples. The second has been cultivated recently
338 from two different stool samples [99, 100]. Evidence has shown that many fungal species are
339 directly associated with food ingestion prior to sampling. This may explain the presence of G.
340 geotrichum and S. cerevisiae in human stools due to their use in food production [26].
341 However, determining the role fungi play in digestion remains to be illustrated. Finally,
342 several filamentous species belonging to Penicillium and Aspergillus genera have been
343 detected [90] and cultured more recently [99, 101]. It is likely that these fungi could represent
344 food contamination, or could just come from food ingestion [26]. Although they have been
345 found in many studies [18, 99, 102], it is not certain that they play any significant role in the
346 human gut microbiota. 347 5.2. Other Eukaryote diversity MANUSCRIPT 348 More than 50 helminthic genera (complex and multicellular organisms) belonging to
349 nematodes, trematoda and cestoda groups are known to parasitize the human gut [103] and
350 cause infections in millions of people worldwide. In addition, 15 different commensal or
351 parasitic protozoa genera [91] have also been described. Only a few of intestinal protozoa
352 (defined as unicellular eukaryotes) playing a role in disease in humans, while the majority of
353 protozoa exhibit a free-living lifestyle in water and soil environments [91]. Giardia
354 intestinalis (flagellates), Cryptosporidium parvum , Cyclospora cayetanensis and Isospora 355 belli (Coccidia),ACCEPTED Blastocystis sp. (stemenopile), Entamoeba histolytica (Amoeba) and 356 Balantidium coli (ciliates) are the most abundant protozoa parasitizing the human gut through
357 a cyst and trophozoite life cycle [91] (Table 2).
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358 Despite the drawbacks and the poor sensitivity associated with the microscopic
359 observation of stool samples, it remains the gold standard used by most parasitological
360 diagnostic laboratories [104]. Depending on the microscopist’s skill, diagnosis will target the
361 presence of protozoan cysts and trophozoites, as well as microscopic identification of
362 helminthic eggs [91]. Because there are sometimes too few eggs in samples, this tool must be
363 replaced by other methods such as serology-based ones including ELISA (Enzyme-Linked
364 Immunosorbent Assay) [105] and DFA (Direct Fluorescent Antibody) assays [106]. Parasitic
365 coproculture remains a challenging technique [91], although it demonstrates good efficiency
366 for some helminths [107] and protozoa [108, 109]. Molecular approaches based on
367 conventional PCR, Real-Time-PCR [110], PCR-restriction fragment length polymorphism
368 (RFLP) [111] and more recently the Loop-mediated isothermal amplification (LAMP) assay
369 [112], are alternative and promising methods for differentiating parasites [113]. Finally, next
370 generation sequencing using pyrosequencing methods has been used to detect and genotype 371 several protozoans [114, 115]. However, more metage MANUSCRIPTnomic and culturomic studies targeting 372 the eukaryome are required at this time to determine the occurrence of both fungi and
373 parasites in the human gut [91].
374 5. Perspectives
375 As described by Robert Koch, “a pure culture remains the foundation of all research in
376 microbiology” [42]. The rebirth of the culture through the example of culturomics due to the
377 efficient, cost-effective and rapid MALDI-TOF identification method opens broad
378 perspectives for the study of complex ecosystems [42]. This revolution now largely 379 recognized inACCEPTED bacterial culture is relevant for eukaryote identification [91]. 380 However, we have a major lack of tools that can list both prokaryotes and eukaryotes
381 isolated in humans. The recent repertoire proposed by Hugon et al. is an important first step,
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382 providing researchers and clinicians with the bacterial species associated with humans [8].
383 This may be implemented with eukaryotes.
384 Finally, this rebirth of culture highlighted a major discrepancy, that the taxonomic
385 description is not currently adapted to this dramatic increase of new prokaryotic species [8].
386 Taxono-genomics has proposed for three years modification of this taxonomic classification.
387 This new concept is based on current microbiological tools such as MALDI-TOF (spectra and
388 comparison with the closest bacterial species) and genome sequencing (including genome
389 comparison) to be in accordance with this reemerging field [42, 46, 116]. Moreover, the
390 taxonomic classification remains unclear for fungi and a few explored fields for the
391 eukaryotes. Finally, the emerging recognition of giant viruses of amoebas as inhabitants of
392 environments and humans still expands the range of micro-organisms to be characterized and
393 enumerated from human gut, and this would involve new high-throughput strategies that have
394 started being implemented [11]. Thinking beyond that, amoebal viruses are an incentive to 395 readdress microbe classification [11]. MANUSCRIPT
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396 Acknowledgments
397 We thank TradOnline (http://www.tradonline.fr/en/) and Karolina Griffiths for providing 398 English corrections.
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399 Figure legends
400 Figure 1: Diversity of Prokaryotes, Eukaryotes and viruses in the human gut microbiota
401
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402 Table 1: Prokaryotes cultivated in the human gut microbiota
Phylum Family Actinomycetaceae Bifidobacteriaceae Bogoriellaceae Brevibacteriaceae Cellulomonadaceae Coriobacteriaceae Corynebacteriaceae Dermabacteraceae Dermacoccaceae Dermatophilaceae Dietziaceae Actinobacteria Geodermatophilaceae Gordoniaceae Intrasporangiaceae Microbacteriaceae Micrococcaceae Micromonosporaceae Mycobacteriaceae Nocardiaceae Nocardioidaceae PromicromonosporaceaeMANUSCRIPT Propionibacteriaceae Streptomycetaceae Bacteroidaceae Flavobacteriaceae Porphyromonadaceae Bacteroidetes Prevotellaceae Rikenellaceae Sphingobacteriaceae Acidaminococcaceae Aerococcaceae Bacillaceae Carnobacteriaceae Catabacteriaceae Christensenellaceae Firmicutes Clostridiaceae ACCEPTEDClostridiales Enterococcaceae Erysipelotrichaceae Eubacteriaceae Lachnospiraceae Lactobacillaceae
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Leuconostocaceae Listeriaceae Paenibacillaceae Peptococcaceae Peptostreptococcaceae Planococcaceae Ruminococcaceae Staphylococcaceae Streptococcaceae Thermoactinomycetaceae Veillonellaceae Acetobacteraceae Aeromonadaceae Alcaligenaceae Brucellaceae Burkholderiaceae Campylobacteraceae Caulobacteraceae Chromobacteriaceae Comamonadaceae Desulfovibrionaceae Enterobacteriaceae Francisellaceae HalomonadaceaeMANUSCRIPT Helicobacteraceae Legionellaceae Proteobacteria Methylobacteriaceae Moraxellaceae Neisseriaceae Oxalobacteraceae Pasteurellaceae Pseudomonadaceae Rhizobiaceae Rhodobacteraceae Salinisphaeraceae Shewanellaceae Sphingomonadaceae Succinivibrionaceae ACCEPTEDSutterellaceae Vibrionaceae Xanthobacteraceae Xanthomonadaceae Chlamydiae Chlamydiaceae Deinococcus- Thermus Deinococcaceae
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Fusobacteria Fusobacteriaceae Lentisphaerae Victivallaceae Brachyspiraceae Spirochaetes Leptospiraceae Synergistetes Synergistaceae Tenericutes Mycoplasmataceae Verrucomicrobia Verrucomicrobiaceae Halobacteriaceae Euryarchaeota Methanobacteriaceae 403
404
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405 Table 2: Eukaryotes cultivated in the human gut microbiota
Taxa Species Acremonium falciforme Acremonium strictum Aspergilluis niger Aspergillus flavipes Aspergillus flavus Aspergillus fumigatus Aspergillus ruber Aspergillus spp. Aspergillus sydowii Aspergillus versicolor Beauveria bassiana Blastoschizomyces capitatus Candida albicans Candida famata Candida glabrata Candida guilliermondii Candida kefyr Candida krusei Candida lambica Candida lusitaniae Candida norvogensis Fungi Ascomycota CandidaMANUSCRIPT parapsilosis Candida pararugosa Candida rugosa Candida sp. Candida sphaerica Candida tropicalis Candida utilis Candida zeynaloides Cladosporidium bruhnei Cladosporium spp. Clavispsora lusitaniae Davidiella sp. Davidiella tassiana Debaryomyces hansenii Fusarium sp. Galactomyces candidum ACCEPTEDGalactomyces geotrichum Geothricum spp. Geotrichum candida Geotrichum candidum Hansenula anomala Hypocrea lixii
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Isaria farinosa Kluyveromyces marxianus Penicillium allii Penicillium brevicompactum Penicillium camemberti Penicillium citrinum Penicillium decumbens Penicillium dipodomyicola Penicillium marneffei Penicillium notatum Penicillium solitum Penicillium sp. Penicilliumn steckii Saccharomyces cerevisiae Saccharomyces sp. Torulaspora delbrueckii Yarrowia lipolytica Zygosaccharomyces bisporus Climacocystis sp. Cryptococcus albidus Cryptococcus luteolus Cryptococcus spp. Cystofilobasidium capitatum Exophiala dermatitidis MalasseziaMANUSCRIPT globosa Malassezia pachydermatis Fungi Basidiomycota Malassezia restricta Malassezia sp. Pityrosporum sp. Rhodotorula glutinis Rhodotorula rubra Rhodotorula sp. Trichosporon asahii Trichosporon beigelii Trichosporon spp. Basidiobolus ranarum Mucor racemosus Fungi Zygomycota Mucor spp. Rhizopus spp. ACCEPTEDAncylostoma duedonale Helminths Nematodes Necator americanus Strongyloides stercoralis Endolimax nana Entamoeba coli Protozoa Amoebozoa Entamoeba dispar Entamoeba histolytica
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Iodamoeba bütschlii Dientamoeba fragilis Protozoa Parabasalia Pentatrichomonas hominis Protozoa Stemenopile Blastocystis hominis 406
407
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408 References
409 [1] Marchesi JR, Adams DH, Fava F, Hermes GD, Hirschfield GM, Hold G, et al. The gut 410 microbiota and host health: a new clinical frontier. Gut. 2015. doi: 10.1136/gutjnl-2015- 411 309990. PubMed PMID: 26338727. 412 [2] Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes 413 associated with obesity. Nature. 2006;444(7122):1022-3. doi: 10.1038/4441022a. PubMed 414 PMID: 17183309. 415 [3] Li Q, Wang C, Tang C, Li N, Li J. Molecular-phylogenetic characterization of the 416 microbiota in ulcerated and non-ulcerated regions in the patients with Crohn's disease. PloS 417 one. 2012;7(4):e34939. doi: 10.1371/journal.pone.0034939. PubMed PMID: 22529960; 418 PubMed Central PMCID: PMCPMC3329531. 419 [4] Malinen E, Krogius-Kurikka L, Lyra A, Nikkila J, Jaaskelainen A, Rinttila T, et al. 420 Association of symptoms with gastrointestinal microbiota in irritable bowel syndrome. World 421 journal of gastroenterology : WJG. 2010;16(36):4532-40. PubMed PMID: 20857523; 422 PubMed Central PMCID: PMCPMC2945484. 423 [5] Lagier JC, Million M, Hugon P, Armougom F, Raoult D. Human gut microbiota: 424 repertoire and variations. Frontiers in cellular and infection microbiology. 2012;2:136. Epub 425 2012/11/07. doi: 10.3389/fcimb.2012.00136. PubMed PMID: 23130351; PubMed Central 426 PMCID: PMC3487222. 427 [6] Claesson MJ, Cusack S, O'Sullivan O, Greene-Diniz R, de Weerd H, Flannery E, et al. 428 Composition, variability, and temporal stability of the intestinal microbiota of the elderly. 429 Proceedings of the National Academy of Sciences of the United States of America. 2011;108 430 Suppl 1:4586-91. Epub 2010/06/24. doi: 10.1073/pnas.1000097107. PubMed PMID: 431 20571116; PubMed Central PMCID: PMC3063589. 432 [7] Sullivan A, Edlund C, Nord CE. Effect of antimicrobial agents on the ecological 433 balance of human microflora. The Lancet InfectiousMANUSCRIPT diseases. 2001;1(2):101-14. Epub 434 2002/03/02. doi: 10.1016/S1473-3099(01)00066-4. PubMed PMID: 11871461. 435 [8] Hugon P, Dufour JC, Colson P, Fournier PE, Sallah K, Raoult D. A comprehensive 436 repertoire of prokaryotic species identified in human beings. The Lancet Infectious diseases. 437 2015;15(10):1211-9. doi: 10.1016/S1473-3099(15)00293-5. PubMed PMID: 26311042. 438 [9] Boyer M, Madoui MA, Gimenez G, La Scola B, Raoult D. Phylogenetic and phyletic 439 studies of informational genes in genomes highlight existence of a 4 domain of life including 440 giant viruses. PloS one. 2010;5(12):e15530. doi: 10.1371/journal.pone.0015530. PubMed 441 PMID: 21151962; PubMed Central PMCID: PMCPMC2996410. 442 [10] Raoult D. TRUC or the need for a new microbial classification. Intervirology. 443 2013;56(6):349-53. doi: 10.1159/000354269. PubMed PMID: 23867259. 444 [11] Raoult D. How the virophage compels the need to readdress the classification of 445 microbes. Virology. 2015;477:119-24. doi: 10.1016/j.virol.2014.11.014. PubMed PMID: 446 25497204. 447 [12] Gossling J, Slack JM. Predominant gram-positive bacteria in human feces: numbers, 448 variety, and persistence. Infect Immun. 1974;9(4):719-29. PubMed PMID: 4595760; PubMed 449 Central PMCID: PMCPMC414872. 450 [13] Mata LJ,ACCEPTED Carrillo C, Villatoro E. Fecal microflora in health persons in a preindustrial 451 region. Appl Microbiol. 1969;17(4):596-602. PubMed PMID: 4890749; PubMed Central 452 PMCID: PMCPMC377749. 453 [14] Moore WE, Holdeman LV. Human fecal flora: the normal flora of 20 Japanese- 454 Hawaiians. Appl Microbiol. 1974;27(5):961-79. PubMed PMID: 4598229; PubMed Central 455 PMCID: PMCPMC380185.
ACCEPTED MANUSCRIPT
456 [15] Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic 457 microorganisms in aquatic and terrestrial habitats. Annual review of microbiology. 458 1985;39:321-46. Epub 1985/01/01. doi: 10.1146/annurev.mi.39.100185.001541. PubMed 459 PMID: 3904603. 460 [16] Vartoukian SR, Palmer RM, Wade WG. Strategies for culture of 'unculturable' 461 bacteria. FEMS Microbiol Lett. 2010;309(1):1-7. doi: 10.1111/j.1574-6968.2010.02000.x. 462 PubMed PMID: 20487025. 463 [17] Nottingham PM, Hungate RE. Methanogenic fermentation of benzoate. Journal of 464 bacteriology. 1969;98(3):1170-2. Epub 1969/06/01. PubMed PMID: 5788702; PubMed 465 Central PMCID: PMC315310. 466 [18] Finegold SM, Sutter VL, Sugihara PT, Elder HA, Lehmann SM, Phillips RL. Fecal 467 microbial flora in Seventh Day Adventist populations and control subjects. The American 468 journal of clinical nutrition. 1977;30(11):1781-92. Epub 1977/11/01. PubMed PMID: 920638. 469 [19] Ash C, Farrow JA, Dorsch M, Stackebrandt E, Collins MD. Comparative analysis of 470 Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase 471 sequencing of 16S rRNA. International journal of systematic bacteriology. 1991;41(3):343-6. 472 Epub 1991/07/01. doi: 10.1099/00207713-41-3-343. PubMed PMID: 1715736. 473 [20] Chen J, Miao X, Xu M, He J, Xie Y, Wu X, et al. Intra-Genomic Heterogeneity in 16S 474 rRNA Genes in Strictly Anaerobic Clinical Isolates from Periodontal Abscesses. PloS one. 475 2015;10(6):e0130265. Epub 2015/06/24. doi: 10.1371/journal.pone.0130265. PubMed PMID: 476 26103050; PubMed Central PMCID: PMC4477887. 477 [21] Kennedy NA, Walker AW, Berry SH, Duncan SH, Farquarson FM, Louis P, et al. The 478 impact of different DNA extraction kits and laboratories upon the assessment of human gut 479 microbiota composition by 16S rRNA gene sequencing. PloS one. 2014;9(2):e88982. Epub 480 2014/03/04. doi: 10.1371/journal.pone.0088982. PubMed PMID: 24586470; PubMed Central 481 PMCID: PMC3933346. 482 [22] Walker AW, Martin JC, Scott P, Parkhill J,MANUSCRIPT Flint HJ, Scott KP. 16S rRNA gene-based 483 profiling of the human infant gut microbiota is str ongly influenced by sample processing and 484 PCR primer choice. Microbiome. 2015;3:26. Epub 2015/06/30. doi: 10.1186/s40168-015- 485 0087-4. PubMed PMID: 26120470; PubMed Central PMCID: PMC4482049. 486 [23] Claesson MJ, Wang Q, O'Sullivan O, Greene-Diniz R, Cole JR, Ross RP, et al. 487 Comparison of two next-generation sequencing technologies for resolving highly complex 488 microbiota composition using tandem variable 16S rRNA gene regions. Nucleic acids 489 research. 2010;38(22):e200. Epub 2010/10/01. doi: 10.1093/nar/gkq873. PubMed PMID: 490 20880993; PubMed Central PMCID: PMC3001100. 491 [24] Lagier JC, Armougom F, Million M, Hugon P, Pagnier I, Robert C, et al. Microbial 492 culturomics: paradigm shift in the human gut microbiome study. Clinical microbiology and 493 infection : the official publication of the European Society of Clinical Microbiology and 494 Infectious Diseases. 2012;18(12):1185-93. Epub 2012/10/05. doi: 10.1111/1469-0691.12023. 495 PubMed PMID: 23033984. 496 [25] Hugon P, Lagier JC, Robert C, Lepolard C, Papazian L, Musso D, et al. Molecular 497 studies neglect apparently gram-negative populations in the human gut microbiota. Journal of 498 clinical microbiology. 2013;51(10):3286-93. Epub 2013/07/26. doi: 10.1128/JCM.00473-13. 499 PubMed PMID:ACCEPTED 23885002; PubMed Central PMCID: PMC3811629. 500 [26] Rajilic-Stojanovic M, de Vos WM. The first 1000 cultured species of the human 501 gastrointestinal microbiota. FEMS microbiology reviews. 2014;38(5):996-1047. Epub 502 2014/05/28. doi: 10.1111/1574-6976.12075. PubMed PMID: 24861948; PubMed Central 503 PMCID: PMC4262072.
ACCEPTED MANUSCRIPT
504 [27] Kuehbacher T, Rehman A, Lepage P, Hellmig S, Folsch UR, Schreiber S, et al. 505 Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease. J Med Microbiol. 506 2008;57(Pt 12):1569-76. doi: 10.1099/jmm.0.47719-0. PubMed PMID: 19018031. 507 [28] Di Rienzi SC, Sharon I, Wrighton KC, Koren O, Hug LA, Thomas BC, et al. The 508 human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate 509 phylum sibling to Cyanobacteria. Elife. 2013;2. doi: ARTN e01102 510 10.7554/eLife.01102. PubMed PMID: WOS:000328635200002. 511 [29] Andersson AF, Lindberg M, Jakobsson H, Backhed F, Nyren P, Engstrand L. 512 Comparative analysis of human gut microbiota by barcoded pyrosequencing. PloS one. 513 2008;3(7):e2836. doi: 10.1371/journal.pone.0002836. PubMed PMID: 18665274; PubMed 514 Central PMCID: PMCPMC2475661. 515 [30] Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, et al. 516 Enterotypes of the human gut microbiome. Nature. 2011;473(7346):174-80. doi: 517 10.1038/nature09944. PubMed PMID: 21508958; PubMed Central PMCID: 518 PMCPMC3728647. 519 [31] Claesson MJ, O'Sullivan O, Wang Q, Nikkila J, Marchesi JR, Smidt H, et al. 520 Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring 521 microbial community structures in the human distal intestine. PloS one. 2009;4(8):e6669. doi: 522 10.1371/journal.pone.0006669. PubMed PMID: 19693277; PubMed Central PMCID: 523 PMCPMC2725325. 524 [32] Nam YD, Jung MJ, Roh SW, Kim MS, Bae JW. Comparative analysis of Korean 525 human gut microbiota by barcoded pyrosequencing. PloS one. 2011;6(7):e22109. doi: 526 10.1371/journal.pone.0022109. PubMed PMID: 21829445; PubMed Central PMCID: 527 PMCPMC3146482. 528 [33] Zupancic ML, Cantarel BL, Liu Z, Drabek EF, Ryan KA, Cirimotich S, et al. Analysis 529 of the gut microbiota in the old order Amish and its relation to the metabolic syndrome. PloS 530 one. 2012;7(8):e43052. doi: 10.1371/journal.pone.00MANUSCRIPT43052. PubMed PMID: 22905200; 531 PubMed Central PMCID: PMCPMC3419686. 532 [34] Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. 533 Diversity of the human intestinal microbial flora. Science. 2005;308(5728):1635-8. doi: 534 10.1126/science.1110591. PubMed PMID: 15831718; PubMed Central PMCID: 535 PMCPMC1395357. 536 [35] Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, et al. 537 Metagenomic analysis of the human distal gut microbiome. Science. 2006;312(5778):1355-9. 538 doi: 10.1126/science.1124234. PubMed PMID: 16741115; PubMed Central PMCID: 539 PMCPMC3027896. 540 [36] Murri M, Leiva I, Gomez-Zumaquero JM, Tinahones FJ, Cardona F, Soriguer F, et al. 541 Gut microbiota in children with type 1 diabetes differs from that in healthy children: a case- 542 control study. BMC Med. 2013;11:46. doi: 10.1186/1741-7015-11-46. PubMed PMID: 543 23433344; PubMed Central PMCID: PMCPMC3621820. 544 [37] Qin J, Li Y, Cai Z, Li S, Zhu J, Zhang F, et al. A metagenome-wide association study 545 of gut microbiota in type 2 diabetes. Nature. 2012;490(7418):55-60. doi: 546 10.1038/nature11450. PubMed PMID: 23023125. 547 [38] WangACCEPTED T, Cai G, Qiu Y, Fei N, Zhang M, Pang X, et al. Structural segregation of gut 548 microbiota between colorectal cancer patients and healthy volunteers. The ISME journal. 549 2012;6(2):320-9. doi: 10.1038/ismej.2011.109. PubMed PMID: 21850056; PubMed Central 550 PMCID: PMCPMC3260502. 551 [39] Hamer HM, Jonkers D, Venema K, Vanhoutvin S, Troost FJ, Brummer RJ. Review 552 article: the role of butyrate on colonic function. Aliment Pharmacol Ther. 2008;27(2):104-19. 553 doi: 10.1111/j.1365-2036.2007.03562.x. PubMed PMID: 17973645.
ACCEPTED MANUSCRIPT
554 [40] Satokari RM, Vaughan EE, Akkermans AD, Saarela M, de Vos WM. Bifidobacterial 555 diversity in human feces detected by genus-specific PCR and denaturing gradient gel 556 electrophoresis. Appl Environ Microbiol. 2001;67(2):504-13. doi: 10.1128/AEM.67.2.504- 557 513.2001. PubMed PMID: 11157210; PubMed Central PMCID: PMCPMC92614. 558 [41] Seng P, Abat C, Rolain JM, Colson P, Lagier JC, Gouriet F, et al. Identification of rare 559 pathogenic bacteria in a clinical microbiology laboratory: impact of matrix-assisted laser 560 desorption ionization-time of flight mass spectrometry. Journal of clinical microbiology. 561 2013;51(7):2182-94. doi: 10.1128/JCM.00492-13. PubMed PMID: 23637301; PubMed 562 Central PMCID: PMCPMC3697718. 563 [42] Lagier JC, Hugon P, Khelaifia S, Fournier PE, La Scola B, Raoult D. The rebirth of 564 culture in microbiology through the example of culturomics to study human gut microbiota. 565 Clinical microbiology reviews. 2015;28(1):237-64. Epub 2015/01/09. doi: 566 10.1128/CMR.00014-14. PubMed PMID: 25567229; PubMed Central PMCID: 567 PMC4284300. 568 [43] Dubourg G, Lagier JC, Armougom F, Robert C, Hamad I, Brouqui P, et al. The gut 569 microbiota of a patient with resistant tuberculosis is more comprehensively studied by 570 culturomics than by metagenomics. European journal of clinical microbiology & infectious 571 diseases : official publication of the European Society of Clinical Microbiology. 572 2013;32(5):637-45. Epub 2013/01/08. doi: 10.1007/s10096-012-1787-3. PubMed PMID: 573 23291779. 574 [44] Dubourg G, Lagier JC, Robert C, Armougom F, Hugon P, Metidji S, et al. 575 Culturomics and pyrosequencing evidence of the reduction in gut microbiota diversity in 576 patients with broad-spectrum antibiotics. International journal of antimicrobial agents. 577 2014;44(2):117-24. Epub 2014/07/27. doi: 10.1016/j.ijantimicag.2014.04.020. PubMed 578 PMID: 25063078. 579 [45] Pfleiderer A, Lagier JC, Armougom F, Robert C, Vialettes B, Raoult D. Culturomics 580 identified 11 new bacterial species from a single anorexiaMANUSCRIPT nervosa stool sample. European 581 journal of clinical microbiology & infectious disea ses : official publication of the European 582 Society of Clinical Microbiology. 2013;32(11):1471-81. Epub 2013/06/04. doi: 583 10.1007/s10096-013-1900-2. PubMed PMID: 23728738. 584 [46] Ramasamy D, Mishra AK, Lagier JC, Padhmanabhan R, Rossi M, Sentausa E, et al. A 585 polyphasic strategy incorporating genomic data for the taxonomic description of novel 586 bacterial species. International journal of systematic and evolutionary microbiology. 587 2014;64(Pt 2):384-91. doi: 10.1099/ijs.0.057091-0. PubMed PMID: 24505076. 588 [47] Oren AG, G. M.;. Validation list N°153. International journal of systematic and 589 evolutionary microbiology. 2013;63:4. 590 [48] Oren AG, G. M.;. Validation list N°155. International journal of systematic and 591 evolutionary microbiology. 2014;64:5. 592 [49] Oren AG, G. M.;. Validation list N°165. International journal of systematic and 593 evolutionary microbiology. 2015;65:7. 594 [50] Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal 595 for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of 596 Sciences of the United States of America. 1990;87(12):4576-9. Epub 1990/06/01. PubMed 597 PMID: 2112744;ACCEPTED PubMed Central PMCID: PMC54159. 598 [51] Pereira SL, Reeve JN. Histones and nucleosomes in Archaea and Eukarya: a 599 comparative analysis. Extremophiles : life under extreme conditions. 1998;2(3):141-8. Epub 600 1998/10/23. PubMed PMID: 9783158. 601 [52] DeLong EF, Pace NR. Environmental diversity of bacteria and archaea. Systematic 602 biology. 2001;50(4):470-8. Epub 2002/07/16. PubMed PMID: 12116647.
ACCEPTED MANUSCRIPT
603 [53] DeLong EF. Everything in moderation: archaea as 'non-extremophiles'. Current 604 opinion in genetics & development. 1998;8(6):649-54. Epub 1999/01/23. PubMed PMID: 605 9914204. 606 [54] Miller TL, Wolin MJ. Methanogens in Human and Animal Intestinal Tracts. Syst Appl 607 Microbiol. 1986;7(2-3):223-9. PubMed PMID: WOS:A1986C607200010. 608 [55] Gaci N, Borrel G, Tottey W, O'Toole PW, Brugere JF. Archaea and the human gut: 609 new beginning of an old story. World journal of gastroenterology : WJG. 2014;20(43):16062- 610 78. Epub 2014/12/05. doi: 10.3748/wjg.v20.i43.16062. PubMed PMID: 25473158; PubMed 611 Central PMCID: PMC4239492. 612 [56] Nottingham PM, Hungate RE. Isolation of methanogenic bacteria from feces of man. 613 Journal of bacteriology. 1968;96(6):2178-9. Epub 1968/12/01. PubMed PMID: 4881707; 614 PubMed Central PMCID: PMC252579. 615 [57] Dridi B, Henry M, El Khechine A, Raoult D, Drancourt M. High prevalence of 616 Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using 617 an improved DNA detection protocol. PloS one. 2009;4(9):e7063. Epub 2009/09/18. doi: 618 10.1371/journal.pone.0007063. PubMed PMID: 19759898; PubMed Central PMCID: 619 PMC2738942. 620 [58] Miller TL, Wolin MJ. Methanosphaera-Stadtmaniae Gen-Nov, Sp-Nov - a Species 621 That Forms Methane by Reducing Methanol with Hydrogen. Arch Microbiol. 622 1985;141(2):116-22. doi: Doi 10.1007/Bf00423270. PubMed PMID: 623 WOS:A1985AHM2800003. 624 [59] Dridi B, Fardeau ML, Ollivier B, Raoult D, Drancourt M. Methanomassiliicoccus 625 luminyensis gen. nov., sp. nov., a methanogenic archaeon isolated from human faeces. 626 International journal of systematic and evolutionary microbiology. 2012;62(Pt 8):1902-7. 627 Epub 2012/08/04. doi: 10.1099/ijs.0.033712-0. PubMed PMID: 22859731. 628 [60] Ferrari A, Brusa T, Rutili A, Canzi E, Biavati B. Isolation and Characterization of 629 Methanobrevibacter-Oralis Sp-Nov. Curr Microbiol.MANUSCRIPT 1994;29(1):7-12. doi: Doi 630 10.1007/Bf01570184. PubMed PMID: WOS:A1994NM2160000 2. 631 [61] Khelaifia S, Garibal M, Robert C, Raoult D, Drancourt M. Draft Genome Sequencing 632 of Methanobrevibacter oralis Strain JMR01, Isolated from the Human Intestinal Microbiota. 633 Genome announcements. 2014;2(1). Epub 2014/02/22. doi: 10.1128/genomeA.00073-14. 634 PubMed PMID: 24558239; PubMed Central PMCID: PMC3931360. 635 [62] Borrel G, Harris HM, Tottey W, Mihajlovski A, Parisot N, Peyretaillade E, et al. 636 Genome sequence of "Candidatus Methanomethylophilus alvus" Mx1201, a methanogenic 637 archaeon from the human gut belonging to a seventh order of methanogens. Journal of 638 bacteriology. 2012;194(24):6944-5. Epub 2012/12/05. doi: 10.1128/JB.01867-12. PubMed 639 PMID: 23209209; PubMed Central PMCID: PMC3510639. 640 [63] Borrel G, Harris HM, Parisot N, Gaci N, Tottey W, Mihajlovski A, et al. Genome 641 Sequence of "Candidatus Methanomassiliicoccus intestinalis" Issoire-Mx1, a Third 642 Thermoplasmatales-Related Methanogenic Archaeon from Human Feces. Genome 643 announcements. 2013;1(4). Epub 2013/07/13. doi: 10.1128/genomeA.00453-13. PubMed 644 PMID: 23846268; PubMed Central PMCID: PMC3709145. 645 [64] Khelaifia S, Garibal M, Robert C, Raoult D, Drancourt M. Draft Genome Sequence of 646 a Human-AssociatedACCEPTED Isolate of Methanobrevibacter arboriphilicus, the Lowest-G+C-Content 647 Archaeon. Genome announcements. 2014;2(1). Epub 2014/01/25. doi: 648 10.1128/genomeA.01181-13. PubMed PMID: 24459264; PubMed Central PMCID: 649 PMC3900896. 650 [65] Nam YD, Chang HW, Kim KH, Roh SW, Kim MS, Jung MJ, et al. Bacterial, archaeal, 651 and eukaryal diversity in the intestines of Korean people. Journal of microbiology.
ACCEPTED MANUSCRIPT
652 2008;46(5):491-501. Epub 2008/11/01. doi: 10.1007/s12275-008-0199-7. PubMed PMID: 653 18974948. 654 [66] Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton J, Salamon P, et al. Metagenomic 655 analyses of an uncultured viral community from human feces. Journal of bacteriology. 656 2003;185(20):6220-3. PubMed PMID: 14526037; PubMed Central PMCID: 657 PMCPMC225035. 658 [67] Ogilvie LA, Jones BV. The human gut virome: a multifaceted majority. Frontiers in 659 microbiology. 2015;6:918. Epub 2015/10/07. doi: 10.3389/fmicb.2015.00918. PubMed 660 PMID: 26441861; PubMed Central PMCID: PMC4566309. 661 [68] Scarpellini E, Ianiro G, Attili F, Bassanelli C, De Santis A, Gasbarrini A. The human 662 gut microbiota and virome: Potential therapeutic implications. Digestive and liver disease : 663 official journal of the Italian Society of Gastroenterology and the Italian Association for the 664 Study of the Liver. 2015. Epub 2015/08/11. doi: 10.1016/j.dld.2015.07.008. PubMed PMID: 665 26257129. 666 [69] Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nature reviews 667 Microbiology. 2008;6(4):315-9. doi: 10.1038/nrmicro1858. PubMed PMID: 18311164. 668 [70] Foca A, Liberto MC, Quirino A, Marascio N, Zicca E, Pavia G. Gut inflammation and 669 immunity: what is the role of the human gut virome? Mediators of inflammation. 670 2015;2015:326032. Epub 2015/05/07. doi: 10.1155/2015/326032. PubMed PMID: 25944980; 671 PubMed Central PMCID: PMC4405218. 672 [71] Minot S, Bryson A, Chehoud C, Wu GD, Lewis JD, Bushman FD. Rapid evolution of 673 the human gut virome. Proceedings of the National Academy of Sciences of the United States 674 of America. 2013;110(30):12450-5. Epub 2013/07/10. doi: 10.1073/pnas.1300833110. 675 PubMed PMID: 23836644; PubMed Central PMCID: PMC3725073. 676 [72] Zhang T, Breitbart M, Lee WH, Run JQ, Wei CL, Soh SW, et al. RNA viral 677 community in human feces: prevalence of plant pathogenic viruses. PLoS biology. 678 2006;4(1):e3. Epub 2005/12/13. doi: 10.1371/journalMANUSCRIPT.pbio.0040003. PubMed PMID: 679 16336043; PubMed Central PMCID: PMC1310650. 680 [73] Ogilvie LA, Bowler LD, Caplin J, Dedi C, Diston D, Cheek E, et al. Genome 681 signature-based dissection of human gut metagenomes to extract subliminal viral sequences. 682 Nature communications. 2013;4:2420. Epub 2013/09/17. doi: 10.1038/ncomms3420. PubMed 683 PMID: 24036533; PubMed Central PMCID: PMC3778543. 684 [74] Reyes A, Haynes M, Hanson N, Angly FE, Heath AC, Rohwer F, et al. Viruses in the 685 faecal microbiota of monozygotic twins and their mothers. Nature. 2010;466(7304):334-8. 686 Epub 2010/07/16. doi: 10.1038/nature09199. PubMed PMID: 20631792; PubMed Central 687 PMCID: PMC2919852. 688 [75] Reyes A, Semenkovich NP, Whiteson K, Rohwer F, Gordon JI. Going viral: next- 689 generation sequencing applied to phage populations in the human gut. Nature reviews 690 Microbiology. 2012;10(9):607-17. Epub 2012/08/07. doi: 10.1038/nrmicro2853. PubMed 691 PMID: 22864264; PubMed Central PMCID: PMC3596094. 692 [76] Gorski A, Weber-Dabrowska B. The potential role of endogenous bacteriophages in 693 controlling invading pathogens. Cellular and molecular life sciences : CMLS. 694 2005;62(5):511-9. Epub 2005/03/05. doi: 10.1007/s00018-004-4403-6. PubMed PMID: 695 15747058. ACCEPTED 696 [77] Klein EJ, Boster DR, Stapp JR, Wells JG, Qin X, Clausen CR, et al. Diarrhea etiology 697 in a Children's Hospital Emergency Department: a prospective cohort study. Clin Infect Dis. 698 2006;43(7):807-13. doi: 10.1086/507335. PubMed PMID: 16941358. 699 [78] Suttle CA. Viruses in the sea. Nature. 2005;437(7057):356-61. Epub 2005/09/16. doi: 700 10.1038/nature04160. PubMed PMID: 16163346.
ACCEPTED MANUSCRIPT
701 [79] Stern A, Mick E, Tirosh I, Sagy O, Sorek R. CRISPR targeting reveals a reservoir of 702 common phages associated with the human gut microbiome. Genome research. 703 2012;22(10):1985-94. Epub 2012/06/27. doi: 10.1101/gr.138297.112. PubMed PMID: 704 22732228; PubMed Central PMCID: PMC3460193. 705 [80] La Scola B, Audic S, Robert C, Jungang L, de Lamballerie X, Drancourt M, et al. A 706 giant virus in amoebae. Science. 2003;299(5615):2033. doi: 10.1126/science.1081867. 707 PubMed PMID: 12663918. 708 [81] Legendre M, Lartigue A, Bertaux L, Jeudy S, Bartoli J, Lescot M, et al. In-depth study 709 of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba. Proceedings 710 of the National Academy of Sciences of the United States of America. 2015;112(38):E5327- 711 35. doi: 10.1073/pnas.1510795112. PubMed PMID: 26351664; PubMed Central PMCID: 712 PMCPMC4586845. 713 [82] Reteno DG, Benamar S, Khalil JB, Andreani J, Armstrong N, Klose T, et al. 714 Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae. J Virol. 715 2015;89(13):6585-94. doi: 10.1128/JVI.00115-15. PubMed PMID: 25878099; PubMed 716 Central PMCID: PMCPMC4468488. 717 [83] Pagnier I, Reteno DG, Saadi H, Boughalmi M, Gaia M, Slimani M, et al. A decade of 718 improvements in Mimiviridae and Marseilleviridae isolation from amoeba. Intervirology. 719 2013;56(6):354-63. doi: 10.1159/000354556. PubMed PMID: 24157882. 720 [84] Raoult D, La Scola B, Birtles R. The discovery and characterization of Mimivirus, the 721 largest known virus and putative pneumonia agent. Clin Infect Dis. 2007;45(1):95-102. doi: 722 10.1086/518608. PubMed PMID: 17554709. 723 [85] Colson P, Fancello L, Gimenez G, Armougom F, Desnues C, Fournous G, et al. 724 Evidence of the megavirome in humans. J Clin Virol. 2013;57(3):191-200. doi: 725 10.1016/j.jcv.2013.03.018. PubMed PMID: 23664726. 726 [86] Boyer M, Yutin N, Pagnier I, Barrassi L, Fournous G, Espinosa L, et al. Giant 727 Marseillevirus highlights the role of amoebae as a MANUSCRIPTmelting pot in emergence of chimeric 728 microorganisms. Proceedings of the National Academy of Sciences of the United States of 729 America. 2009;106(51):21848-53. doi: 10.1073/pnas.0911354106. PubMed PMID: 730 20007369; PubMed Central PMCID: PMCPMC2799887. 731 [87] Saadi H, Reteno DG, Colson P, Aherfi S, Minodier P, Pagnier I, et al. Shan virus: a 732 new mimivirus isolated from the stool of a Tunisian patient with pneumonia. Intervirology. 733 2013;56(6):424-9. doi: 10.1159/000354564. PubMed PMID: 24157888. 734 [88] La Scola B, Desnues C, Pagnier I, Robert C, Barrassi L, Fournous G, et al. The 735 virophage as a unique parasite of the giant mimivirus. Nature. 2008;455(7209):100-4. doi: 736 10.1038/nature07218. PubMed PMID: 18690211. 737 [89] Zhou J, Zhang W, Yan S, Xiao J, Zhang Y, Li B, et al. Diversity of virophages in 738 metagenomic data sets. J Virol. 2013;87(8):4225-36. doi: 10.1128/JVI.03398-12. PubMed 739 PMID: 23408616; PubMed Central PMCID: PMCPMC3624350. 740 [90] Scanlan PD, Marchesi JR. Micro-eukaryotic diversity of the human distal gut 741 microbiota: qualitative assessment using culture-dependent and -independent analysis of 742 faeces. The ISME journal. 2008;2(12):1183-93. Epub 2008/08/02. doi: 743 10.1038/ismej.2008.76. PubMed PMID: 18670396. 744 [91] HamadACCEPTED I, Raoult D, Bittar F. Repertory of Eukaryotes (Eukaryome) in the Human 745 Gastrointestinal Tract: Taxonomy and Detection Methods. Parasite immunology. 2015. Epub 746 2015/10/06. doi: 10.1111/pim.12284. PubMed PMID: 26434599. 747 [92] Adl SM, Simpson AG, Lane CE, Lukes J, Bass D, Bowser SS, et al. The revised 748 classification of eukaryotes. The Journal of eukaryotic microbiology. 2012;59(5):429-93. 749 Epub 2012/10/02. doi: 10.1111/j.1550-7408.2012.00644.x. PubMed PMID: 23020233; 750 PubMed Central PMCID: PMC3483872.
ACCEPTED MANUSCRIPT
751 [93] Gouba N, Drancourt M. Digestive tract mycobiota: a source of infection. Medecine et 752 maladies infectieuses. 2015;45(1-2):9-16. Epub 2015/02/17. doi: 753 10.1016/j.medmal.2015.01.007. PubMed PMID: 25684583. 754 [94] Marklein G, Josten M, Klanke U, Muller E, Horre R, Maier T, et al. Matrix-assisted 755 laser desorption ionization-time of flight mass spectrometry for fast and reliable identification 756 of clinical yeast isolates. Journal of clinical microbiology. 2009;47(9):2912-7. Epub 757 2009/07/03. doi: 10.1128/JCM.00389-09. PubMed PMID: 19571014; PubMed Central 758 PMCID: PMC2738125. 759 [95] Santos C, Paterson RR, Venancio A, Lima N. Filamentous fungal characterizations by 760 matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Journal of 761 applied microbiology. 2010;108(2):375-85. Epub 2009/08/08. doi: 10.1111/j.1365- 762 2672.2009.04448.x. PubMed PMID: 19659699. 763 [96] Patel R. MALDI-TOF MS for the diagnosis of infectious diseases. Clinical chemistry. 764 2015;61(1):100-11. Epub 2014/10/04. doi: 10.1373/clinchem.2014.221770. PubMed PMID: 765 25278500. 766 [97] Tang J, Iliev ID, Brown J, Underhill DM, Funari VA. Mycobiome: Approaches to 767 analysis of intestinal fungi. Journal of immunological methods. 2015;421:112-21. Epub 768 2015/04/22. doi: 10.1016/j.jim.2015.04.004. PubMed PMID: 25891793; PubMed Central 769 PMCID: PMC4451377. 770 [98] Berman JJ. Taxonomic guide to infectious diseases : understanding the biologic 771 classes of pathogenic organisms. 1st ed. London ; Waltham, MA: Elsevier/Academic Press; 772 2012. xvii, 355 p. p. 773 [99] Gouba N, Raoult D, Drancourt M. Plant and fungal diversity in gut microbiota as 774 revealed by molecular and culture investigations. PloS one. 2013;8(3):e59474. Epub 775 2013/04/05. doi: 10.1371/journal.pone.0059474. PubMed PMID: 23555039; PubMed Central 776 PMCID: PMC3598745. 777 [100] Hamad I, Sokhna C, Raoult D, Bittar F. MolecuMANUSCRIPTlar detection of eukaryotes in a single 778 human stool sample from Senegal. PloS one. 2012;7(7 ):e40888. Epub 2012/07/19. doi: 779 10.1371/journal.pone.0040888. PubMed PMID: 22808282; PubMed Central PMCID: 780 PMC3396631. 781 [101] Gouba N, Raoult D, Drancourt M. Eukaryote culturomics of the gut reveals new 782 species. PloS one. 2014;9(9):e106994. Epub 2014/09/12. doi: 10.1371/journal.pone.0106994. 783 PubMed PMID: 25210972; PubMed Central PMCID: PMC4161381. 784 [102] Finegold SM, Attebery HR, Sutter VL. Effect of diet on human fecal flora: 785 comparison of Japanese and American diets. The American journal of clinical nutrition. 786 1974;27(12):1456-69. Epub 1974/12/01. PubMed PMID: 4432829. 787 [103] Awasthi S, Bundy DA, Savioli L. Helminthic infections. Bmj. 2003;327(7412):431-3. 788 Epub 2003/08/23. doi: 10.1136/bmj.327.7412.431. PubMed PMID: 12933732; PubMed 789 Central PMCID: PMC188497. 790 [104] McHardy IH, Wu M, Shimizu-Cohen R, Couturier MR, Humphries RM. Detection of 791 intestinal protozoa in the clinical laboratory. Journal of clinical microbiology. 792 2014;52(3):712-20. Epub 2013/11/08. doi: 10.1128/JCM.02877-13. PubMed PMID: 793 24197877; PubMed Central PMCID: PMC3957779. 794 [105] UngarACCEPTED BL. Enzyme-linked immunoassay for detection of Cryptosporidium antigens in 795 fecal specimens. Journal of clinical microbiology. 1990;28(11):2491-5. Epub 1990/11/01. 796 PubMed PMID: 2254426; PubMed Central PMCID: PMC268212. 797 [106] Garcia LS, Shimizu RY. Evaluation of nine immunoassay kits (enzyme immunoassay 798 and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in 799 human fecal specimens. Journal of clinical microbiology. 1997;35(6):1526-9. Epub 800 1997/06/01. PubMed PMID: 9163474; PubMed Central PMCID: PMC229779.
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801 [107] Rayan ZS, H; Galal, M;. Detection of Strongyloides stercoralis in Fecal Samples 802 Using Conventional Parasitological Techniques and Real-Time PCR: A Comparative Study. 803 Parasitologists United Journal. 2012;(5):8. 804 [108] Sawangjaroen N, Luke R, Prociv P. Diagnosis by faecal culture of Dientamoeba 805 fragilis infections in Australian patients with diarrhoea. Transactions of the Royal Society of 806 Tropical Medicine and Hygiene. 1993;87(2):163-5. Epub 1993/03/01. PubMed PMID: 807 8337717. 808 [109] Yakoob J, Jafri W, Jafri N, Khan R, Islam M, Beg MA, et al. Irritable bowel 809 syndrome: in search of an etiology: role of Blastocystis hominis. The American journal of 810 tropical medicine and hygiene. 2004;70(4):383-5. Epub 2004/04/22. PubMed PMID: 811 15100450. 812 [110] Bell AS, Ranford-Cartwright LC. Real-time quantitative PCR in parasitology. Trends 813 in parasitology. 2002;18(8):337-42. Epub 2002/10/17. PubMed PMID: 12380021. 814 [111] Zavvar M, Sadraei J, Emadi H, Pirestani M. The use of a nested PCR-RFLP technique, 815 based on the parasite's 18S ribosomal RNA, to characterise Cryptosporidium isolates from 816 HIV/AIDS patients. Annals of tropical medicine and parasitology. 2008;102(7):597-601. 817 Epub 2008/09/27. doi: 10.1179/136485908X311876. PubMed PMID: 18817600. 818 [112] Tomita N, Mori Y, Kanda H, Notomi T. Loop-mediated isothermal amplification 819 (LAMP) of gene sequences and simple visual detection of products. Nature protocols. 820 2008;3(5):877-82. Epub 2008/05/03. doi: 10.1038/nprot.2008.57. PubMed PMID: 18451795. 821 [113] Gasser RB, Cantacessi C, Loukas A. DNA technological progress toward advanced 822 diagnostic tools to support human hookworm control. Biotechnology advances. 823 2008;26(1):35-45. Epub 2007/11/21. doi: 10.1016/j.biotechadv.2007.09.003. PubMed PMID: 824 18024057. 825 [114] Sreekumar C, Hill DE, Miska KB, Vianna MC, Yan L, Myers RL, et al. Genotyping 826 and detection of multiple infections of Toxoplasma gondii using Pyrosequencing. 827 International journal for parasitology. 2005;35(9):991-9.MANUSCRIPT Epub 2005/07/02. doi: 828 10.1016/j.ijpara.2005.03.017. PubMed PMID: 15990100 . 829 [115] Stensvold CR, Lebbad M, Verweij JJ, Jespersgaard C, von Samson-Himmelstjerna G, 830 Nielsen SS, et al. Identification and delineation of members of the Entamoeba complex by 831 pyrosequencing. Molecular and cellular probes. 2010;24(6):403-6. Epub 2010/08/10. doi: 832 10.1016/j.mcp.2010.07.008. PubMed PMID: 20691255. 833 [116] Fournier PEL, J.C.; Dubourg, G.; Raoult, D. From culturomics to taxonogenomics: a 834 need to change the taxonomy of prokaryotes in clinical microbiology. Anaerobe. 2015. Epub 835 In press. 836
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