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 microorganism 1 . The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms 2 . 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 identification 2 . 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 studies 3-5 . 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.

Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44–1.51 Mb linear double-strand DNA coding for 1276–1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.

BackgroundDespite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated.Methodology/Principal FindingsIn this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks.Conclusion/SignificanceThese data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks?

BackgroundThe identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas.Methods/resultsBased on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs.ConclusionThis discovery expands our knowledge of Mimiviridae evolution and ecology.

Background SARS-CoV-2 predisposes patients to secondary infections; however, a better understanding of the impact of coinfections on the outcome of hospitalized COVID-19 patients is still necessary. Aim To analyse death risk due to coinfections in COVID-19 patients. Methods We evaluated the Odds of death of 212 severely ill COVID-19 patients, with detailed focus on the risks for each pathogen, site of infection, comorbidities and length of hospitalization. Findings The mortality rate was 50.47%. Fungal and/or bacterial isolation occurred in 89 patients, of which 83.14% died. Coinfected patients stayed hospitalized longer and had an increased Odds of dying (OR = 13.45, R 2 =0.31). The risk of death was increased by bacterial (OR=11.28) and fungal (OR=5.97) coinfections, with increased levels of creatinine, leukocytes, urea and C-reactive protein. Coinfections increased the risk of death if patients suffer from cardiovascular disease (OR= 11.53), diabetes (OR=6.00) or obesity (OR=5.60) in comparison with patients with these comorbidities but without pathogen isolation. The increased risk of death was detected for negative-coagulase Staphylococcus (OR=25.39), Candida non- albicans (OR=11.12), S. aureus (OR=10.72), Acinetobacter spp. (OR=6.88), Pseudomonas spp. (OR=4.77) and C. albicans (OR=3.97). The high-risk sites of infection were blood, tracheal aspirate and urine. Patients with coinfection undergoing invasive mechanical ventilation were 3.8 times more likely to die than those without positive cultures. Conclusions Severe COVID-19 patients with secondary coinfections required longer hospitalization and had higher risk of death. The early diagnosis of coinfections is essential to identify high-risk patients and to determine the right interventions to reduce mortality.

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This study confirms the high clinical frequency of human VACV infection, even among vaccinated individuals. The infection was related to detection of IgG- or IgM-specific antibodies that correlates in most of the cases with positive PRNT. The DNAemia suggests viremia during VACV natural infections. Our data indicate that patients vaccinated against smallpox may no longer be protected.

Anaerosalibacter massiliensis sp. nov. strain ND1T (= CSUR P762 = DSM 27308) is the type strain of A. massiliensis sp. nov., a new species within the genus Anaerosalibacter. This strain, the genome of which is described here, was isolated from the faecal flora of a 49-year-old healthy Brazilian man. Anaerosalibacter massiliensis is a Gram-positive, obligate anaerobic rod and member of the family Clostridiaceae. With the complete genome sequence and annotation, we describe here the features of this organism. The 3 197 911 bp long genome (one chromosome but no plasmid) contains 3271 protein-coding and 62 RNA genes, including six rRNA genes.