Most bacteria in the human gut are difficult to culture, and culturomics has been designed to overcome this issue. Culturomics makes it possible to obtain living bacteria for further experiments, unlike metagenomics. However, culturomics is work-intensive, which prevents its wide application. In this study, we performed a 30-day continuous enrichment in blood culture bottles and cultured bacterial isolates from pre-cultures removed at different time points. We compared the bacteria isolated from the enriched culture with or without adding fresh medium after each pre-culture was removed. We also compared "experienced" colony picking (i.e., picking two to three colonies for each recognized colony type) and picking all the colonies from each plate. In total, from five fecal samples, 106 species were isolated, including three novel species and six that have not previously been isolated from the human body. Adding fresh medium to the culture increased the rate of bacterial species isolation by 22% compared with the nonsupplemented culture. Picking all colonies increased the rate of bacterial isolation by only 8.5% compared with experienced colony picking. After optimization through statistical analysis and simulation, sampling aerobic and anaerobic enrichment cultures at six and seven time-points, respectively, is likely to isolate >90% of bacterial species, reducing the workload by 40%. In conclusion, an extended enrichment step ensures isolation of different bacterial species at different time-points, while adding the same quantity of fresh medium after sampling, the experienced picking and the optimized time-points favor the chance of isolating more bacterial species with less work.

Aims Hydroxychloroquine (HCQ), a widely used antimalarial drug, is proposed to treat coronavirus disease 2019 (COVID-19). However, no report is currently available regarding the direct effects of HCQ on gut microbiota, which is associated with the outcomes of elderly patients with COVID-19. Here, we first investigated the effects of HCQ on intestinal microecology in mice. Main methods Fifteen female C57BL/6J mice were randomly divided into two groups: HCQ group (n = 10) and control group (n = 5). Mice in the HCQ group were administered with HCQ at dose of 100 mg/kg by gavage daily for 14 days. The feces of mice were collected before and on the 7th and 14th days after HCQ challenge, and then analyzed by 16S rRNA amplicon sequencing. At the end of the experiment, the hematology, serum biochemistry and cytokines were determined, respectively. The mRNA expression of tight junction proteins in colonic tissues were also studied by RT-PCR. Key findings HCQ challenge had no effects on the counts of white blood cells, the levels of serum cytokines, and the gene expression of tight junction proteins in colon. HCQ also did not increase the content of serum d -lactate in mice. Notably, HCQ significantly decreased the diversity of gut microbiota, increased the relative abundance of phylum Bacteroidetes whereas decreased that of Firmicutes . Significance Short-term high dose HCQ challenge changes gut microbiota but not the intestinal integrity and immunological responses in mice. Special attention should be paid to the effects of HCQ on intestinal microecology in future clinical use.

Background Colorectal cancer (CRC) is the third most common cancer in the world, and a strong relationship exists between CRC and gut microbiota, which affects the occurrence, development, and metastasis of cancer. Bioinformatics-based analyses revealed that the abundance of Parvimonas micra (P. micra) in the feces of patients with cancer is significantly higher than that in healthy people. Therefore, an important relationship may exist between P. micra and CRC. Methods We first confirmed that P. micra can promote the proliferation of cell lines through cell experiments and mouse models. Then we selected the signaling pathways and content of exosomes to promote the development of CRC by transcriptomics and microRNA sequencing. Finally, we confirmed that P. micra promoted CRC development through miR-218-5p/Ras/ERK/c-Fos pathway through the in vivo and in vitro experiments. Results First, it was confirmed by in vitro and in vivo experiments that P. micra can promote the development of CRC. Transcriptome analysis after the coincubation of bacteria and cells revealed that P. micra promoted cell proliferation by activating the Ras/ERK/c-Fos pathway. Furthermore, microRNA sequencing analysis of the cells and exosomes showed that miR-218-5p and protein tyrosine phosphatase receptor R (PTPRR) were the key factors involved in activating the Ras/ERK/c-Fos pathway, and the miR-218-5p inhibitor was used to confirm the role of microRNA in xenograft mice. Conclusion This experiment confirmed that P. micra promoted the development of CRC by upregulating miR-218-5p expression in cells and exosomes, inhibiting PTPRR expression, and ultimately activating the Ras/ERK/c-Fos signaling pathway.

BackgroundCulturomics can ascertain traces of microorganisms to be cultivated using different strategies and identified by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry or 16S rDNA sequencing. However, to cater to all requirements of microorganisms and isolate as many species as possible, multiple culture conditions must be used, imposing a heavy workload. In addition, the fast-growing bacteria (e.g., Escherichia) surpass the slow-growing bacteria in culture by occupying space and using up nutrients. Besides, some bacteria (e.g., Pseudomonas) suppress others by secreting antibacterial metabolites, making it difficult to isolate bacteria with lower competence. Applying inhibitors to restrain fast-growing bacteria is one method to cultivate more bacterial species from human feces.ResultsWe applied CHIR-090, an LpxC enzyme inhibitor that has antibacterial activity against most Gram-negative bacteria, to culturomics of human fresh feces. The antibacterial activity of CHIR-090 was first assessed on five Gram-negative species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus vulgaris, and Bacteroides vulgatus), all of which are commonly isolated from the human gut. Then, we assessed suitable concentrations of the inhibitor. Finally, CHIR-090 was applied in blood culture bottles for bacterial cultivation. In total, 102 species from five samples were identified. Of these, we found one new species, two species not reported previously in the human gut, and 11 species not previously isolated from humans.ConclusionsCHIR-090 can suppress E. coli, P. aeruginosa, K. pneumoniae, Pro. vulgaris, but not B. vulgatus. Compared with the non-inhibitor group, CHIR-090 increased bacteria isolation by 23.50%, including four species not reported in humans and one new species. Application of LpxC enzyme inhibitor in culturomics increased the number of species isolated from the human gut.

External environmental factors can cause an imbalance in intestinal flora. For people living in the extremes of a plateau climate, lack of oxygen is a primary health challenge that leads to a series of reactions. We wondered how intestinal microorganisms might change in a simulated plateau environment, and what changes might occur in the host organism and intestinal microorganisms in the absence of hypoxia-related factors. In this study, mice carrying a knockout of hypoxia-inducible factor 1β (Hif-1β) in myeloid cells and wild-type mice were raised in a composite hypoxic chamber to simulate a plateau environment at 5,000 meters of elevation for 14 days. The mice carrying the myeloid Hif-1β deletion displayed aggravated hypoxic phenotypes, significantly greater weight loss, and significantly higher cardiac index values compared with the wild-type group. The levels of some cytokines increased under the hypoxic environment. Analysis of 16S rRNA sequencing results showed that hypoxia had a significant effect on the gut microbiota in both wild-type and Hif-1β-deficient mice, especially on the first day. The Bacteroidaceae family increased continuously from Day 1 to Day 14 in Hif-1β deletion mice, and they represented an obviously different group of bacteria at Day 14 compared with the wild-type mice. Butyrate-producing bacteria, such as Butyricicoccus, were only found in wild-type mice after 14 days in the hypoxic environment. In conclusion, hypoxia caused heart enlargement, greater weight loss and obvious microbial imbalance in myeloid Hif-1β deficient mice. This study reveals genetic and microecological pathways for research on mechanisms of hypoxia.