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Abstract Bacteriophages are recognized as the most abundant members of microbiomes and have therefore a profound impact on microbial communities through the interactions with their bacterial hosts. The International Metagenomics and Metadesign of Subways and Urban Biomes Consortium (MetaSUB) has sampled mass-transit systems in 60 cities over 3 years using metagenomics, throwing light into these hitherto largely unexplored urban environments. MetaSUB focused primarily on the bacterial community. In this work, we explored MetaSUB metagenomic data in order to recover and analyze bacteriophage genomes. We recovered and analyzed 1714 phage genomes with size at least 40 kbp, from the class Caudoviricetes, the vast majority of which (80%) are novel. The recovered genomes were predicted to belong to temperate (69%) and lytic (31%) phages. Thirty-three of these genomes have more than 200 kbp, and one of them reaches 572 kbp, placing it among the largest phage genomes ever found. In general, the phages tended to be site-specific or nearly so, but 194 genomes could be identified in every city from which phage genomes were retrieved. We predicted hosts for 48% of the phages and observed general agreement between phage abundance and the respective bacterial host abundance, which include the most common nosocomial multidrug-resistant pathogens. A small fraction of the phage genomes are carriers of antibiotic resistance genes, and such genomes tended to be particularly abundant in the sites where they were found. We also detected CRISPR-Cas systems in five phage genomes. This study expands the previously reported MetaSUB results and is a contribution to the knowledge about phage diversity, global distribution, and phage genome content.

Summary: Background: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. Methods: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. Findings: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. Interpretation: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. Funding: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.

Abstract We investigated the nascent application and efficacy of sampling and sequencing environmental DNA (eDNA) in terrestrial environments using rainwater that filters through the forest canopy and understory vegetation (i.e., throughfall). We demonstrate the utility and potential of this method for measuring microbial communities and forest biodiversity. We collected pure rainwater (open sky) and throughfall, successfully extracted DNA, and generated over 5000 unique amplicon sequence variants. We found that several taxa including Mycoplasma sp., Spirosoma sp., Roseomonas sp., and Lactococcus sp. were present only in throughfall samples. Spiroplasma sp., Methylobacterium sp., Massilia sp., Pantoea sp., and Sphingomonas sp. were found in both types of samples, but more abundantly in throughfall than in rainwater. Throughfall samples contained Gammaproteobacteria that have been previously found to be plant-associated, and may contribute to important functional roles. We illustrate how this novel method can be used for measuring microbial biodiversity in forest ecosystems, foreshadowing the utility for quantifying both prokaryotic and eukaryotic lifeforms. Leveraging these methods will enhance our ability to detect extant species, describe new species, and improve our overall understanding of ecological community dynamics in forest ecosystems.

Citrus canker is one of the main bacterial diseases that affect citrus crops and is caused by Xanthomonas citri which affects all citrus species worldwide. New strategies to control citrus canker are necessary and the use of bacteriophages as biocontrol agent could be an alternative. Phages that infect Xanthomonas species have been studied, such as XacN1, a myovirus that infects X. citri. Here we report the isolation and characterization of a new jumbo phage, vb_XciM_LucasX, which infects X. citri and X. fuscans. Transmission electron microscopy allowed classification of LucasX in the Myoviridae family, which was corroborated by its genomic sequencing, annotation, and proteome clustering. LucasX has a 305,651 bp-long dsDNA genome. ORF prediction and annotation revealed 157 genes encoding putative structural proteins such as capsid and tail related proteins and phage assembly associated proteins, however, for most of the structural proteins it was not possible assign specific functions. Its genome encodes several proteins related to DNA replication and nucleotide metabolism, five putative RNA polymerases, at least one homing endonuclease mobile element, a terminase large subunit (TerL), an endolysin and many proteins classified as beneficial to the host. Proteome clustering and phylogeny analyses showed that LucasX is a new jumbo phage having as its closest neighbor the Xanthomonas jumbo phage Xoo-sp14. LucasX presented a burst size of 40 PFU/infected cell of X. citri 306, was completely inactivated at temperatures above 50°C, presented survival lower than 25% after 80 s of exposition to artificial UV light and had practically no tolerance to concentrations above 2.5 g/L NaCl or 40% ethanol. LucasX presented optimum pH at 7 and a broad range of Xanthomonas hosts, infecting twenty-one of the twenty-three strains tested. Finally, the LucasX yield was dependent on the host strain utilized, resulting one order of magnitude higher in X. fuscans C 752 than in X. citri 306, which points out to the possibility of phage yield improvement, an usual challenge for biocontrol purposes.

Here we present and analyze the complete genome of Alcaligenes faecalis strain Mc250 (Mc250), a bacterium isolated from the roots of Mimosa calodendron, an endemic plant growing in ferruginous rupestrian grasslands in Minas Gerais State, Brazil. The genome has 4,159,911 bp and 3,719 predicted protein-coding genes, in a single chromosome. Comparison of the Mc250 genome with 36 other Alcaligenes faecalis genomes revealed that there is considerable gene content variation among these strains, with the core genome representing only 39% of the protein-coding gene repertoire of Mc250. Mc250 encodes a complete denitrification pathway, a network of pathways associated with phenolic compounds degradation, and genes associated with HCN and siderophores synthesis; we also found a repertoire of genes associated with metal internalization and metabolism, sulfate/sulfonate and cysteine metabolism, oxidative stress and DNA repair. These findings reveal the genomic basis for the adaptation of this bacterium to the harsh environmental conditions from where it was isolated. Gene clusters associated with ectoine, terpene, resorcinol, and emulsan biosynthesis that can confer some competitive advantage were also found. Experimental results showed that Mc250 was able to reduce (~60%) the virulence phenotype of the plant pathogen Xanthomonas citri subsp. citri when co-inoculated in Citrus sinensis, and was able to eradicate 98% of juveniles and stabilize the hatching rate of eggs to 4% in two species of agricultural nematodes. These results reveal biotechnological potential for the Mc250 strain and warrant its further investigation as a biocontrol and plant growth-promoting bacterium.

BackgroundThe performance of the microbiota is observed in several digestive tract diseases. Therefore, reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, tests and therapies in hepatobiliopancreatic diseases.MethodsBile samples will be collected in endoscopic retrograde cholangiopancreatography patients (case group) and living liver transplantation donors (control group). We will characterize the microbiome based on two types of sequence data: the V3/V4 regions of the 16S ribosomal RNA (rRNA) gene and total shotgun DNA. For 16S sequencing data a standard 16S processing pipeline based on the Amplicon Sequence Variant concept and the qiime2 software package will be employed; for shotgun data, for each sample we will assemble the reads and obtain and analyze metagenome-assembled genomes.ResultsThe primary expected results of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. In addition, it seeks to demonstrate the existence of changes in the case of illness and also possible disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases.Trial registrationNCT04391426. Registered 18 May 2020, https://clinicaltrials.gov/ct2/show/NCT04391426.

The genetic determinants of bacterial pathogenicity are highly variable between species and strains. However, a factor that is commonly associated with virulent Gram-negative bacteria, including many Aeromonas spp., is the type 3 secretion system (T3SS), which is used to inject effector proteins into target eukaryotic cells. In this study, we developed a bioinformatics pipeline to identify T3SS effector proteins, applied this approach to the genomes of 105 Aeromonas strains isolated from environmental, mutualistic, or pathogenic contexts and evaluated the cytotoxicity of the identified effectors through their heterologous expression in yeast. The developed pipeline uses a two-step approach, where candidate Aeromonas gene families are initially selected using Hidden Markov Model (HMM) profile searches against the Virulence Factors DataBase (VFDB), followed by strict comparisons against positive and negative control datasets, greatly reducing the number of false positives. This approach identified 21 Aeromonas T3SS likely effector families, of which 8 represent known or characterized effectors, while the remaining 13 have not previously been described in Aeromonas. We experimentally validated our in silico findings by assessing the cytotoxicity of representative effectors in Saccharomyces cerevisiae BY4741, with 15 out of 21 assayed proteins eliciting a cytotoxic effect in yeast. The results of this study demonstrate the utility of our approach, combining a novel in silico search method with in vivo experimental validation, and will be useful in future research aimed at identifying and authenticating bacterial effector proteins from other genera.

Hydrocarbons are important environmental pollutants, and the isolation and characterization of new microorganisms with the ability to degrade these compounds are important for effective biodegradation. In this work we isolated and characterized several bacterial isolates from compost, a substrate rich in microbial diversity. The isolates were obtained from selective culture medium containing n-hexadecane, aiming to recover alkane-degraders. Six isolates identified as Gordonia by MALDI-TOF and 16S rRNA sequencing had the ability to degrade n-hexadecane in three days. Two isolates were selected for genomic and functional characterization, Gordonia paraffinivorans (MTZ052) and Gordonia sihwensis (MTZ096). The CG-MS results showed distinct n-hexadecane degradation rates for MTZ052 and MTZ096 (86% and 100% respectively). The genome sequence showed that MTZ052 encodes only one alkane degrading gene cluster, the CYP153 system, while MTZ096 harbors both the Alkane Hydroxylase (AH) and the CYP153 systems. qPCR showed that both gene clusters are induced by the presence of n-hexadecane in the growth medium, suggesting that G. paraffinivorans and G. sihwensis use these systems for degradation. Altogether, our results indicate that these Gordonia isolates have a good potential for biotransformation of hydrocarbons.

Composting operations are a rich source for prospection of biomass degradation enzymes. We have analyzed the microbiomes of two composting samples collected in a facility inside the São Paulo Zoo Park, in Brazil. All organic waste produced in the park is processed in this facility, at a rate of four tons/day. Total DNA was extracted and sequenced with Roche/454 technology, generating about 3 million reads per sample. To our knowledge this work is the first report of a composting whole-microbial community using high-throughput sequencing and analysis. The phylogenetic profiles of the two microbiomes analyzed are quite different, with a clear dominance of members of the Lactobacillus genus in one of them. We found a general agreement of the distribution of functional categories in the Zoo compost metagenomes compared with seven selected public metagenomes of biomass deconstruction environments, indicating the potential for different bacterial communities to provide alternative mechanisms for the same functional purposes. Our results indicate that biomass degradation in this composting process, including deconstruction of recalcitrant lignocellulose, is fully performed by bacterial enzymes, most likely by members of the Clostridiales and Actinomycetales orders.