The gut microbiota of a patient with resistant tuberculosis is more comprehensively studied by culturomics than by metagenomics

Abstract

Gut microbiota consists of 10(10) bacteria per gram of stool. Many antibiotic regimens induce a reduction in both the diversity and the abundance of the gut flora. We analyzed one stool sample collected from a patient treated for drug-resistant Mycobacterium tuberculosis and who ultimately died from pneumonia due to a Streptococcus pneumoniae 10 months later. We performed microscopic observation, used 70 culture conditions (microbial culturomics) with identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and 16S rRNA amplification and sequencing, pyrosequencing, and 18S rRNA amplification and clone sequencing. Electron and optical microscopic observations revealed the presence of yeast, but no bacterial species were observed. By culture, only 39 bacterial species were identified, including one new species, as well as three species that have not been previously observed in the human gut. The pyrosequencing showed only 18 phylotypes, detecting a lower number of bacterial species than the culture techniques. Only two phylotypes overlapped with culturomics. In contrast, an amount of chloroplasts was found. Additionally, specific molecular eukaryote detection found three fungal species. We recovered, for the first time, more cultivable than non-cultivable bacterial species in a patient with a low bacterial load in the gut, demonstrating the depth bias of pyrosequencing. We propose that the desertification of gut microbiota in this patient is a reflection of the total body microbiota and may have contributed to the invasive infection of S. pneumoniae. This finding suggests that caution should be applied when treating patients with broad-spectrum antibiotics, and preventive measures should be taken in order to avoid invasive infection.

The proof of concept that culturomics can be superior to metagenomics to study atypical stool samples

“…Dear Editor, We read with interest the comments regarding our article [1]. We are grateful to the authors for their remarks, and we agree with the biases linked to metagenomics explaining the absence of reproducibility between the different studies about the gut microbiota repertoire and have reported this elsewhere [2][3][4].…”

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

“…In addition, all the bacteria identified in the first study were cultured at least once using one of the 70 culture conditions (4) and were used for the following studies with various stool samples. Thus far, between 3,000 and Ͼ34,000 different colonies were analyzed in each stool sample (5)(6)(7)121). With 14 stool samples already reported and completely analyzed by culturomics (4,5,7,121), with a large study using a few conditions applied to 347 stool samples (220), and with the analysis of Ͼ170,000 colonies by MALDI-TOF MS, 559 bacterial species were cultured, including 281 species from the Firmicutes phylum, 135 from the Actinobacteria phylum, 82 from the Proteobacteria phylum, 52 from the Bacteroidetes phylum, 6 from the Fusobacterium phylum, 2 from the Synergistetes phylum, and 1 from the Deinococcus-Thermus phylum (4-6, 121).…”

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

“…Seventy-one of 116 (61%) bacterial species were previously absent in our MALDI-TOF database. Among 45 (39%) species present in our MALDI-TOF database, 24 (20%) have only 1 reference spectrum, and only one serovar of 18 serovars of Acinetobacter pittii has more than 10 spectra in the database (59)(60)(61). We used an incremental database with each spectra identified by 16S rRNA gene sequencing from the first three stool samples that allowed us to use the culturomics study of Dubourg et al (61) for the fourth stool sample; in the study of Dubourg et al, only 4 of 4,000 bacterial colonies needed molecular identification (61).…”