Fadi Bittar

Microbial culturomics: paradigm shift in the human gut microbiome study

Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

Microbial diversity in the sputum of a cystic fibrosis patient studied with 16S rDNA pyrosequencing

Recent studies using 16S rRNA gene amplification followed by clonal Sanger sequencing in cystic fibrosis demonstrated that cultured microorganisms are only part of the infecting flora. The purpose of this paper was to compare pyrosequencing and clonal Sanger sequencing on sputum. The sputum of a patient with cystic fibrosis was analysed by culture, Sanger clone sequencing and pyrosequencing after 16S rRNA gene amplification. A total of 4,499 sequencing reads were obtained, which could be attributed to six consensus sequences, but the length of reads leads to fastidious data analysis. Compared to clonal Sanger sequencing and to cultivation results, pyrosequencing recovers greater species richness and gives a more reliable estimate of the relative abundance of bacterial species. The 16S pyrosequencing approach expands our knowledge of the microbial diversity of cystic fibrosis sputum. The current lack of phylogenetic resolution at the species level for the GS 20 sequencing reads will be overcome with the next generation of pyrosequencing apparatus.

Molecular Detection of Eukaryotes in a Single Human Stool Sample from Senegal

Background Microbial eukaryotes represent an important component of the human gut microbiome, with different beneficial or harmful roles; some species are commensal or mutualistic, whereas others are opportunistic or parasitic. The diversity of eukaryotes inhabiting humans remains relatively unexplored because of either the low abundance of these organisms in human gut or because they have received limited attention from a whole-community perspective. Methodology/Principal Finding In this study, a single fecal sample from a healthy African male was studied using both culture-dependent methods and extended molecular methods targeting the 18S rRNA and ITS sequences. Our results revealed that very few fungi, including Candida spp., Galactomyces spp., and Trichosporon asahii, could be isolated using culture-based methods. In contrast, a relatively a high number of eukaryotic species could be identified in this fecal sample when culture-independent methods based on various primer sets were used. A total of 27 species from one sample were found among the 977 analyzed clones. The clone libraries were dominated by fungi (716 clones/977, 73.3%), corresponding to 16 different species. In addition, 187 sequences out of 977 (19.2%) corresponded to 9 different species of plants; 59 sequences (6%) belonged to other micro-eukaryotes in the gut, including Entamoeba hartmanni and Blastocystis sp; and only 15 clones/977 (1.5%) were related to human 18S rRNA sequences. Conclusion Our results revealed a complex eukaryotic community in the volunteer’s gut, with fungi being the most abundant species in the stool sample. Larger investigations are needed to assess the generality of these results and to understand their roles in human health and disease.

Non-contiguous finished genome sequence and description of Bacillus massiliogorillae sp. nov.

Strain G2T sp. nov. is the type strain of B. massiliogorillae, a proposed new species within the genus Bacillus. This strain, whose genome is described here, was isolated in France from the fecal sample of a wild western lowland gorilla from Cameroon. B. massiliogorillae is a facultative anaerobic, Gram-variable, rod-shaped bacterium. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 5,431,633 bp long genome (1 chromosome but no plasmid) contains 5,179 protein-coding and 98 RNA genes, including 91 tRNA genes.

Repertory of eukaryotes (eukaryome) in the human gastrointestinal tract: taxonomy and detection methods

Eukaryotes are an important component of the human gut, and their relationship with the human host varies from parasitic to commensal. Understanding the diversity of human intestinal eukaryotes has important significance for human health. In the past few decades, most of the multitudes of techniques that are involved in the diagnosis of the eukaryotic population in the human intestinal tract were confined to pathological and parasitological aspects that mainly rely on traditionally based methods. However, development of culture‐independent molecular techniques comprised of direct DNA extraction from faeces followed by sequencing, offer new opportunities to estimate the occurrence of eukaryotes in the human gut by providing data on the entire eukaryotic community, particularly not‐yet‐cultured or fastidious organisms. Further broad surveys of the eukaryotic communities in the gut based on high throughput tools such as next generation sequencing might lead to uncovering the real diversity of these ubiquitous organisms in the human intestinal tract and discovering the unrecognized roles of these eukaryotes in modulating the host immune system and inducing changes in host gut physiology and ecosystem.

Wild Gorillas as a Potential Reservoir of Leishmania major

Vector-borne parasites of the genus Leishmania are responsible for severe human diseases. Cutaneous leishmaniasis, a common form of the disease, is most often caused by the transmission of Leishmania major to humans by female phlebotomine sand flies. Apes are increasingly being seen as a source of zoonotic diseases, including malaria and rickettsiosis. To examine whether gorillas harbor Leishmania species, we screened fecal samples from wild western lowland gorillas (Gorilla gorilla gorilla) in Cameroon for the presence of these pathogens. Of 91 wild gorilla fecal samples, 12 contained Leishmania parasites, and 4 contained phlebotomine sand fly vectors. The molecular identity was determined by running 3 different polymerase chain reaction tests for detection of L. major. Next, fluorescence in situ hybridization was performed to visualize L. major parasites in fecal samples from the gorillas. Both promastigote and amastigote forms of the parasite were found. This work strongly suggests that wild gorillas carry pathogenic Leishmania parasites.

Mansonella, including a Potential New Species, as Common Parasites in Children in Gabon.

Background Like other tropical African countries, Gabon is afflicted by many parasitic diseases, including filariases such as loiasis and mansonellosis. This study aimed to assess the prevalence of these two filarial diseases in febrile and afebrile children using quantitative real-time PCR and standard PCR assays coupled with sequencing. Methodology/Principal Findings DNA from blood specimens of 1,418 Gabonese children (1,258 febrile and 160 afebrile) were analyzed. Overall, filarial DNA was detected in 95 (6.7%) children, including 67 positive for M. perstans (4.7%), which was the most common. M. perstans was detected in 61/1,258 febrile children (4.8%) and 6/160 afebrile children (3.8%, P = 0.6). Its prevalence increased statistically with age: 3.5%, 7.7% and 10.6% in children aged ≤5, 6–10 and 11–15 years, respectively. M. perstans prevalence was significantly higher in Koulamoutou and Lastourville (12% and 10.5%, respectively) than in Franceville and Fougamou (2.6% and 2.4%, respectively). Loa loa was detected in seven febrile children including one co-infection with M. perstans. Finally, 21 filarial DNA positive were negative for M. perstans and Loa loa, but ITS sequencing could be performed for 12 and allowed the identification of a potential new species of Mansonella provisionally called “DEUX”. Mansonella sp. “DEUX” was detected only in febrile children. Conclusions/Significance Further study should be performed to characterize Mansonella sp. “DEUX” and evaluate the clinical significance of mansonellosis in humans.

Looking in apes as a source of human pathogens

Because of the close genetic relatedness between apes and humans, apes are susceptible to many human infectious agents and can serve as carriers of these pathogens. Consequently, they present a serious health hazard to humans. Moreover, many emerging infectious diseases originate in wildlife and continue to threaten human populations, especially vector-borne diseases described in great apes, such as malaria and rickettsiosis. These wild primates may be permanent reservoirs and important sources of human pathogens. In this special issue, we report that apes, including chimpanzees (Pan troglodytes), bonobos (Pan paniscus), gorillas (Gorilla gorilla and Gorilla beringei), orangutans (Pongo pygmaeus and Pongo abelii), gibbons (Hylobates spp., Hoolock spp. and Nomascus spp) and siamangs (Symphalangus syndactylus syndactylus and Symphalangus continentis), have many bacterial, viral, fungal and parasitic species that are capable of infecting humans. Serious measures should be adopted in tropical forests and sub-tropical areas where habitat overlaps are frequent to survey and prevent infectious diseases from spreading from apes to people.

Hugonella massiliensis gen. nov., sp. nov., genome sequence, and description of a new strictly anaerobic bacterium isolated from the human gut

The human gut is composed of a large diversity of microorganisms, which have been poorly described. Here, using culturomics, a new concept based on the variation in culture conditions and MALDI‐TOF MS identification, we proceed to explore the microbial diversity of the complex ecosystem of the human gut. Using this approach, we isolated strain AT8T (=CSUR P2118 = DSM 101782) from stool specimens collected from a 51‐year‐old obese French woman. Strain AT8T is a strictly anaerobic, nonmotile, nonspore‐forming gram‐positive coccus that do not exhibit catalase and oxidase activities. 16S rDNA‐based identification of strain AT8T demonstrated 92% gene sequence similarity with Eggerthella lenta DSM 2243, the phylogenetically closed validly named type species. Here, we present a set of features for the strain AT8T and the description of its complete genome sequence and annotation. The 2,091,845 bp long genome has a G+C content of 63.46% and encodes1,849 predicted genes; 1,781 were protein‐coding genes, and 68 were RNAs. On the basis of the characteristics reported here, we propose the creation of a new bacterial genus Hugonella gen. nov., belonging to the Eggerthellaceae family and including Hugonella massiliensis gen. nov., sp. nov., strain AT8T as the type strain.

Culturomics and Amplicon-based Metagenomic Approaches for the Study of Fungal Population in Human Gut Microbiota

Herein, the mycobiota was characterized in fecal samples from sick patients and healthy subjects, collected from different geographical locations and using both culturomics and amplicon-based metagenomics approaches. Using the culturomics approach, a total of 17,800 fungal colonies were isolated from 14 fecal samples, and resulted in the isolation of 41 fungal species, of which 10 species had not been previously reported in the human gut. Deep sequencing of fungal-directed ITS1 and ITS2 amplicons led to the detection of a total of 142 OTUs and 173 OTUs from the ITS1 and ITS2 regions, respectively. Ascomycota composed the largest fraction of the total OTUs analyzed (78.9% and 68.2% of the OTUs from the ITS1 and ITS2 regions, respectively), followed by Basidiomycota (16.9% and 30.1% of the OTUs from the ITS1 and ITS2 regions, respectively). Interestingly, the results demonstrate that the ITS1/ITS2 amplicon sequencing provides different information about gut fungal communities compared to culturomics, though both approaches complete each other in assessing fungal diversity in fecal samples. We also report higher fungal diversity and abundance in patients compared to healthy subjects. In conclusion, combining both culturomic and amplicon-based metagenomic approaches may be a novel strategy towards analyzing fungal compositions in the human gut.