B. La Scola

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.

Aerobic culture of anaerobic bacteria using antioxidants: a preliminary report

Antioxidants have been shown to help the growth of anaerobic bacteria. We were able to grow six anaerobe species (including Fusobacterium necrophorum and Ruminococcus gravus) and seven aerobic species all aerobically in Schaedler agar tubes and agar plates with high doses of ascorbic acid and/or glutathione. This may deeply change strategies for culturing bacteria.

A quasi-universal medium to break the aerobic/anaerobic bacterial culture dichotomy in clinical microbiology

In the mid-19th century, the dichotomy between aerobic and anaerobic bacteria was introduced. Nevertheless, the aerobic growth of strictly anaerobic bacterial species such as Ruminococcus gnavus and Fusobacterium necrophorum, in a culture medium containing antioxidants, was recently demonstrated. We tested aerobically the culture of 623 bacterial strains from 276 bacterial species including 82 strictly anaerobic, 154 facultative anaerobic, 31 aerobic and nine microaerophilic bacterial species as well as ten fungi. The basic culture medium was based on Schaedler agar supplemented with 1 g/L ascorbic acid and 0.1 g/L glutathione (R-medium). We successively optimized this media, adding 0.4 g/L uric acid, using separate autoclaving of the component, or adding haemin 0.1 g/L or α-ketoglutarate 2 g/L. In the basic medium, 237 bacterial species and ten fungal species grew but with no growth of 36 bacterial species, including 22 strict anaerobes. Adding uric acid allowed the growth of 14 further species including eight strict anaerobes, while separate autoclaving allowed the growth of all tested bacterial strains. To extend its potential use for fastidious bacteria, we added haemin for Haemophilus influenzae, Haemophilus parainfluenzae and Eikenella corrodens and α-ketoglutarate for Legionella pneumophila. This medium allowed the growth of all tested strains with the exception of Mycobacterium tuberculosis and Mycobacterium bovis. Testing primoculture and more fastidious species will constitute the main work to be done, but R-medium coupled with a rapid identification method (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) will facilitate the anaerobic culture in clinical microbiology laboratories.

Amoebae as Battlefields for Bacteria, Giant Viruses, and Virophages

ABSTRACT When amoebae are simultaneously infected with Acanthamoeba polyphaga Mimivirus (APM) and the strictly intracellular BABL1 bacterium, the latter is always lost after serial subculturing. We showed that the virophage Sputnik 1, by reducing APM fitness, preserved BABL1 growth in acute and chronic models. This capability of a virophage to modulate the virulence of mimiviruses highlights the competition that occurs between them during natural host infection.

Clostridium butyricum: from beneficial to a new emerging pathogen.

Clostridium butyricum, a strictly anaerobic spore-forming bacillus, is a common human and animal gut commensal bacterium, and is also frequently found in the environment. Whereas non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. In terms of the latter, within the same species, different strains have antagonist effects on the intestinal mucosa. In particular, short-chain fatty acids, which are products of carbohydrate fermentation, have a dose-dependent paradoxical effect. Moreover, toxin genes have been identified by genome sequencing in pathological strains. Asymptomatic carriage of these strains has also been reported. Herein, we provide an overview of the implications of C. butyricum for human health, from the beneficial to the pathogenic. We focus on pathogenic strains associated with the occurrence of necrotizing enterocolitis. We also discuss the need to use complementary microbiological methods, including culture, in order to better assess gut bacterial diversity and identify new emergent enteropathogens at the strain level.

Survey of laboratory-acquired infections around the world in biosafety level 3 and 4 laboratories

Laboratory-acquired infections due to a variety of bacteria, viruses, parasites, and fungi have been described over the last century, and laboratory workers are at risk of exposure to these infectious agents. However, reporting laboratory-associated infections has been largely voluntary, and there is no way to determine the real number of people involved or to know the precise risks for workers. In this study, an international survey based on volunteering was conducted in biosafety level 3 and 4 laboratories to determine the number of laboratory-acquired infections and the possible underlying causes of these contaminations. The analysis of the survey reveals that laboratory-acquired infections have been infrequent and even rare in recent years, and human errors represent a very high percentage of the cases. Today, most risks from biological hazards can be reduced through the use of appropriate procedures and techniques, containment devices and facilities, and the training of personnel.

Genomic analysis of three African strains of Bacillus anthracis demonstrates that they are part of the clonal expansion of an exclusively pathogenic bacterium.

Bacillus anthracis is the causative agent of anthrax and is classified as a ‘Category A’ biological weapon. Six complete genomes of B. anthracis (A0248, Ames, Ames Ancestor, CDC684, H0491, and Sterne) are currently available. In this report, we add three African strain genomes: Sen2Col2, Sen3 and Gmb1. To study the pan-genome of B. anthracis, we used bioinformatics tools, such as Cluster of Orthologous Groups, and performed phylogenetic analysis. We found that the three African strains contained the pX01 and pX02 plasmids, the nonsense mutation in the plcR gene and the four known prophages. These strains are most similar to the CDC684 strain and belong to the A cluster. We estimated that the B. anthracis pan-genome has 2893 core genes (99% of the genome size) and 85 accessory genes. We validated the hypothesis that B. anthracis has a closed pan-genome and found that the three African strains carry the two plasmids associated with bacterial virulence. The pan-genome nature of B. anthracis confirms its lack of exchange (similar to Clostridium tetani) and supports its exclusively pathogenic role, despite its survival in the environment. Moreover, thanks to the study of the core content single nucleotide polymorphisms, we can see that our three African strains diverged very recently from the other B. anthracis strains.

“Lachnoclostridium touaregense,” a new bacterial species isolated from the human gut microbiota

We report the main characteristics of “Lachnoclostridium touaregense” strain Marseille-P2415T (= CSUR P2415 = DSM 102219), a new bacterial species isolated from the gut microbiota of a healthy young girl from Niger.

‘Lachnoclostridium massiliosenegalense’, a new bacterial species isolated from the human gut microbiota

We report the main characteristics of ‘Lachnoclostridium massiliosenegalense’ strain mt23T (=CSUR P299 =DSM 102084), a new bacterial species isolated from the gut microbiota of a healthy young girl from Senegal.

‘Selenomonas massiliensis,’ a new anaerobic bacterial species isolated from human oral microbiota

We report the main characteristics of ‘Selenomonas massiliensis’ sp. nov., strain Marseille-P4036T (= CSUR P4036). The culturomic combined with taxonogenomic methods were used to identify and characterize this new anaerobic bacterial species, which was isolated from an oral sample of a 25-year-old healthy woman.