Jean-Christophe Lagier

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.

Identification of Rare Pathogenic Bacteria in a Clinical Microbiology Laboratory: Impact of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT During the past 5 years, matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has become a powerful tool for routine identification in many clinical laboratories. We analyzed our 11-year experience in routine identification of clinical isolates (40 months using MALDI-TOF MS and 91 months using conventional phenotypic identification [CPI]). Among the 286,842 clonal isolates, 284,899 isolates of 459 species were identified. The remaining 1,951 isolates were misidentified and required confirmation using a second phenotypic identification for 670 isolates and using a molecular technique for 1,273 isolates of 339 species. MALDI-TOF MS annually identified 112 species, i.e., 36 species/10,000 isolates, compared to 44 species, i.e., 19 species/10,000 isolates, for CPI. Only 50 isolates required second phenotypic identifications during the MALDI-TOF MS period (i.e., 4.5 reidentifications/10,000 isolates) compared with 620 isolates during the CPI period (i.e., 35.2/10,000 isolates). We identified 128 bacterial species rarely reported as human pathogens, including 48 using phenotypic techniques (22 using CPI and 37 using MALDI-TOF MS). Another 75 rare species were identified using molecular methods. MALDI-TOF MS reduced the time required for identification by 55-fold and 169-fold and the cost by 5-fold and 96-fold compared with CPI and gene sequencing, respectively. MALDI-TOF MS was a powerful tool not only for routine bacterial identification but also for identification of rare bacterial species implicated in human infectious diseases. The ability to rapidly identify bacterial species rarely described as pathogens in specific clinical specimens will help us to study the clinical burden resulting from the emergence of these species as human pathogens, and MALDI-TOF MS may be considered an alternative to molecular methods in clinical laboratories.

The Rebirth of Culture in Microbiology through the Example of Culturomics To Study Human Gut Microbiota

SUMMARY Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a “dark matter” of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the “great plate count anomaly,” which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire.

A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species

Currently, bacterial taxonomy relies on a polyphasic approach based on the combination of phenotypic and genotypic characteristics. However, the current situation is paradoxical in that the genetic criteria that are used, including DNA-DNA hybridization, 16S rRNA gene sequence nucleotide similarity and phylogeny, and DNA G+C content, have significant limitations, but genome sequences that contain the whole genetic information of bacterial strains are not used for taxonomic purposes, despite the decreasing costs of sequencing and the increasing number of available genomes. Recently, we diversified bacterial culture conditions with the aim of isolating uncultivated bacteria. To classify the putative novel species that we cultivated, we used a polyphasic strategy that included phenotypic as well as genomic criteria (genome characteristics as well as genomic sequence similarity). Herein, we review the pros and cons of genome sequencing for taxonomy and propose that the incorporation of genome sequences in taxonomic studies has the advantage of using reliable and reproducible data. This strategy, which we name taxono-genomics, may contribute to the taxonomic classification of bacteria.

Culture of previously uncultured members of the human gut microbiota by culturomics

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization–time of flight and 16S rRNA for identification2. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies3–5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.

Human Gut Microbiota: Repertoire and Variations

The composition of human gut microbiota and their relationship with the host and, consequently, with human health and disease, presents several challenges to microbiologists. Originally dominated by culture-dependent methods for exploring this ecosystem, the advent of molecular tools has revolutionized our ability to investigate these relationships. However, many biases that have led to contradictory results have been identified. Microbial culturomics, a recent concept based on a use of several culture conditions with identification by MALDI-TOF followed by the genome sequencing of the new species cultured had allowed a complementarity with metagenomics. Culturomics allowed to isolate 31 new bacterial species, the largest human virus, the largest bacteria, and the largest Archaea from human. Moreover, some members of this ecosystem, such as Eukaryotes, giant viruses, Archaea, and Planctomycetes, have been neglected by the majority of studies. In addition, numerous factors, such as age, geographic provenance, dietary habits, antibiotics, or probiotics, can influence the composition of the microbiota. Finally, in addition to the countless biases associated with the study techniques, a considerable limitation to the interpretation of studies of human gut microbiota is associated with funding sources and transparency disclosures. In the future, studies independent of food industry funding and using complementary methods from a broad range of both culture-based and molecular tools will increase our knowledge of the repertoire of this complex ecosystem and host-microbiota mutualism.

Systemic Tropheryma whipplei: clinical presentation of 142 patients with infections diagnosed or confirmed in a reference center.

Culture of Tropheryma whipplei, the agent of Whipple disease (WD), was achieved in our laboratory in 2000, allowing new perspectives for the diagnosis of this disease and for the description of other potential clinical manifestations caused by this microorganism. Since 2000, we have developed new tools in our center in Marseille, France, to optimize the diagnosis of T whipplei infections. Classic WD was characterized by positive periodic acid-Schiff performed on duodenal biopsy. In the absence of duodenal histologic involvement, localized infections were defined by specific positive T whipplei polymerase chain reaction (PCR) results obtained using samples of other tissues and body fluids. The physicians in charge of patients were asked to complete a questionnaire. A total of 215 diagnoses were performed or confirmed and, among these, 142 patients with sufficient clinical data were included. Herein, we report epidemiologic data, clinical manifestations, and diagnostic tools of T whipplei infections. In the 113 patients with classic WD, the main symptom was arthralgia (88/113, 78%), which explains the many cases misdiagnosed as inflammatory rheumatoid disease (56/113, 50%). Frequently immunosuppressive treatments, more recently including tumor necrosis factor inhibitor, had been previously prescribed (50%) and were often responsible for more rapid clinical progression (43%). Sometimes a short course of antibiotics improved the clinical status. Endocarditis was the second most frequent manifestation of T whipplei, with 16 cases. The clinical picture of this entity corresponds to cardiovascular involvement with acute heart failure (50%) occurring without fever (75%) or previous valvular disease (69%). Neurologic symptoms were the third major manifestation. Other localized infections such as adenopathy, uveitis, pulmonary involvement, or joint involvement were sporadic. Infection with T whipplei resulted in multifaceted conditions. Some localized infections due to this agent have recently been reported and may correspond to emerging entities. Patients with inflammatory rheumatoid disease must be systematically interviewed to determine the efficacy of previous immunosuppressive and antibiotic therapies. Abbreviations: CSF = cerebrospinal fluid, PAS = periodic acid-Schiff, PCR = polymerase chain reaction, WD = Whipple disease.

Gut bacterial microbiota and obesity

Although probiotics and antibiotics have been used for decades as growth promoters in animals, attention has only recently been drawn to the association between the gut microbiota composition, its manipulation, and obesity. Studies in mice have associated the phylum Firmicutes with obesity and the phylum Bacteroidetes with weight loss. Proposed mechanisms linking the microbiota to fat content and weight include differential effects of bacteria on the efficiency of energy extraction from the diet, and changes in host metabolism of absorbed calories. The independent effect of the microbiota on fat accumulation has been demonstrated in mice, where transplantation of microbiota from obese mice or mice fed western diets to lean or germ-free mice produced fat accumulation among recipients. The microbiota can be manipulated by prebiotics, probiotics, and antibiotics. Probiotics affect the microbiota directly by modulating its bacterial content, and indirectly through bacteriocins produced by the probiotic bacteria. Interestingly, certain probiotics are associated with weight gain both in animals and in humans. The effects are dependent on the probiotic strain, the host, and specific host characteristics, such as age and baseline nutritional status. Attention has recently been drawn to the association between antibiotic use and weight gain in children and adults. We herein review the studies describing the associations between the microbiota composition, its manipulation, and obesity.

Non contiguous-finished genome sequence and description of Alistipes obesi sp. nov.

Alistipes obesi sp. nov. strain ph8T is the type strain of A. obesi, a new species within the genus Alistipes. This strain, whose genome is described here, was isolated from the fecal flora of a 26-year-old woman suffering from morbid obesity. A. obesi is an obligately anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,162,233 bp long genome (1 chromosome but no plasmid) contains 2,623 protein-coding and 49 RNA genes, including three rRNA genes.

Non-contiguous finished genome sequence and description of Anaerococcus senegalensis sp. nov.

Anaerococcus senegalensis strain JC48T sp. nov. is the type strain of A. senegalensis sp. nov. a new species within the genus Anaerococcus. This strain whose genome is described here was isolated from the fecal flora of a healthy patient. A. senegalensis is an obligate anaerobic coccus. Here we describe the features of this organism together with the complete genome sequence and annotation. The 1,790,835 bp long genome (1 chromosome but no plasmid) contains 1,721 protein-coding and 53 RNA genes including 5 rRNA genes