Large genomic deletions delineate Mycobacterium tuberculosis L4 sublineages in South American countries.

Mycobacterium tuberculosis (Mtb) is still one of the primary pathogens of humans causing tuberculosis (TB) disease. Mtb embraces nine well-defined phylogenetic lineages with biological and geographical disparities. The lineage L4 is the most globally widespread of all lineages and was introduced to America with European colonization. Taking advantage of many genome projects available in public repositories, we undertake an evolutionary and comparative genomic analysis of 522 L4 Latin American Mtb genomes. Initially, we performed careful quality control of public read datasets and applied several thresholds to filter out low-quality data. Using a genome de novo assembly strategy and phylogenomic methods, we spotted novel south American clades that have not been revealed yet. Additionally, we describe genomic deletion profiles of these strains from an evolutionary perspective and report Mycobacterium tuberculosis L4 sublineages signature-like gene deletions, some of the novel. One is a specific deletion of 6.5 kbp that is only present in sublineage 4.1.2.1. This deletion affects a complex group of 10 genes with putative products annotated, among others, as a lipoprotein, transmembrane protein, and toxin/antitoxin system proteins. The second novel deletion spans for 4.9 kbp and specific of a particular clade of the 4.8 sublineage and affects 7 genes. The last novel deletion affects 4 genes, extends for 4.8 kbp., and is specific to some strains within the 4.1.2.1 sublineage that are present in Colombia, Peru and Brasil.

Complete mitochondrial genome of Phoneutria depilata (Araneae, Ctenidae): New insights into the phylogeny and evolution of spiders.

Spiders (Araneae) are the most abundant terrestrial predators and megadiverse on earth. In recent years, the mitochondrial genome of a great diversity of species has been sequenced, mainly for ecological and commercial purposes. These studies have uncovered the existence of a variety of mitochondrial genome rearrangements. However, there is poor genetic information in several taxonomic families of spiders. We have sequenced the complete genome of Phoneutria depilata (Ctenidae) and, based on this, extract the mitogenomes of other ctenid species from published transcriptomes to perform a comparative study among spider species to determine the relationship between the level of mitochondrial rearrangements and its possible relationship with molecular variability in spiders. Complete mitochondrial genomes of eighteen spiders (including eight Ctenidae species) were obtained by two different methodologies (sequencing and transcriptome extraction). Fifty-eight spider mitochondrial genomes were downloaded from the NCBI database for gene order analysis. After verifying the annotation of each mitochondrial gene, a phylogenetic and a gene order analysis from 76 spider mitochondrial genomes were carried out. Our results show a high rate of annotation error in the published spider mitochondrial genomes, which could lead to errors in phylogenetic inference. Moreover, to provide new mitochondrial genomes in spiders by two different methodologies to obtain them, our analysis identifies six different mitochondrial architectures among all spiders. Translocation or tandem duplication random loss (TDRL) events in tRNA genes were identified to explain the evolution of the spider mitochondrial genome. In addition, our findings provide new insights into spider mitochondrial evolution.

Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host-pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host-pathogen interaction.

Metagenomic Analysis of Biocide-Treated Neotropical Oil Reservoir Water Unveils Microdiversity of Thermophile Tepidiphilus.

Microorganisms are capable of colonizing extreme environments like deep biosphere and oil reservoirs. The prokaryotes diversity in exploited oil reservoirs is composed of indigenous microbial communities and artificially introduced microbes. In the present work, high throughput sequencing techniques were applied to analyze the microbial community from the injected and produced water in a neotropical hyper-thermophile oil reservoir located in the Orinoquia region of Colombia, South America. Tepidiphilus is the dominant bacteria found in both injection and produced waters. The produced water has a higher microbial richness and exhibits a Tepidiphilus microdiversity. The reservoir injected water is recycled and treated with the biocides glutaraldehyde and tetrakis-hydroxymethyl-phosphonium sulfate (THPS) to reduce microbial load. This process reduces microbial richness and selects a single Tepidiphilus genome (T. sp. UDEAICP_D1) as the dominant isolate. Thermus and Hydrogenobacter were subdominants in both water systems. Phylogenomic analysis of the injection water dominant Tepidiphilus positioned it as an independent branch outside T. succinatimandens and T. thermophilus lineage. Comparative analysis of the Tepidiphilus genomes revealed several genes that might be related to the biocide-resistant phenotype and the tolerance to the stress conditions imposed inside the oil well, like RND efflux pumps and type II toxin-antitoxin systems. Comparing the abundance of Tepidiphilus protein-coding genes in both water systems shows that the biocide selected Tepidiphilus sp. UDEAICP_D1 genome has enriched genes annotated as ABC-2 type transporter, ABC transporter, Methionine biosynthesis protein MetW, Glycosyltransferases, and two-component system NarL.

Cryptosporidium hominis Phylogenomic Analysis Reveals Separate Lineages With Continental Segregation.

Cryptosporidium is a leading cause of waterborne outbreaks globally, and Cryptosporidium hominis and C. parvum are the principal cause of human cryptosporidiosis on the planet. Thanks to the advances in Next-Generation Sequencing (NGS) sequencing and bioinformatic software development, more than 100 genomes have been generated in the last decade using a metagenomic-like strategy. This procedure involves the parasite oocyst enrichment from stool samples of infected individuals, NGS sequencing, metagenomic assembly, parasite genome computational filtering, and comparative genomic analysis. Following this approach, genomes of infected individuals of all continents have been generated, although with striking different quality results. In this study, we performed a thorough comparison, in terms of assembly quality and purity, of 100+ de novo assembled genomes of C. hominis. Remarkably, after quality genome filtering, a comprehensive phylogenomic analysis allowed us to discover that C. hominis encompasses two lineages with continental segregation. These lineages were named based on the observed continental distribution bias as C. hominis Euro-American (EA) and the C. hominis Afro-Asian (AA) lineages.

Comparative genomic analysis of the principal Cryptosporidium species that infect humans.

Cryptosporidium parasites are ubiquitous and can infect a broad range of vertebrates and are considered the most frequent protozoa associated with waterborne parasitic outbreaks. The intestine is the target of three of the species most frequently found in humans: C. hominis, C. parvum, and. C. meleagridis. Despite the recent advance in genome sequencing projects for this apicomplexan, a broad genomic comparison including the three species most prevalent in humans have not been published so far. In this work, we downloaded raw NGS data, assembled it under normalized conditions, and compared 23 publicly available genomes of C. hominis, C. parvum, and C. meleagridis. Although few genomes showed highly fragmented assemblies, most of them had less than 500 scaffolds and mean coverage that ranged between 35X and 511X. Synonymous single nucleotide variants were the most common in C. hominis and C. meleagridis, while in C. parvum, they accounted for around 50% of the SNV observed. Furthermore, deleterious nucleotide substitutions common to all three species were more common in genes associated with DNA repair, recombination, and chromosome-associated proteins. Indel events were observed in the 23 studied isolates that spanned up to 500 bases. The highest number of deletions was observed in C. meleagridis, followed by C. hominis, with more than 60 species-specific deletions found in some isolates of these two species. Although several genes with indel events have been partially annotated, most of them remain to encode uncharacterized proteins.

Contrasting microbiota profiles observed in children carrying either Blastocystis spp. or the commensal amoebas Entamoeba coli or Endolimax nana.

Recent studies have shown how intestinal parasites can modulate gut microbiota. This observation is not surprising since the human intestinal lumen, like any other niche, is a battlefield of microbial competition, and Eukaryotes can affect bacterial populations. Intestinal pathogenic protist has been associated with reshaping the microbial community structure; however, the interactions between the colonic bacterial communities and parasites like Blastocystis spp., Entamoeba coli, and Endolimax nana have been poorly studied. In this work, we studied the distal intestinal bacterial microbiota of 49 children attending 7 public daycare centers in Medellin, Colombia, and compared the bacterial microbiota structure in the presence or absence of the protists Blastocystis spp., E. coli, and E. nana. Parasite colonization was associated with an increase in bacterial richness. Moreover, Blastocystis spp. presented a positive relationship with Prevotella, since this bacterium was selectively enriched in children carrying it. Remarkably, the E. coli colonized children showed a microbial profile that was closer to uninfected controls, although some bacterial taxa displayed to be enriched. This is the case for Akkermansia, which showed to be favored in E. coli colonized individuals, while notably reduced in the Blastocystis spp. parasitized group.

DATMA: Distributed AuTomatic Metagenomic Assembly and annotation framework.

BACKGROUND: A prime objective in metagenomics is to classify DNA sequence fragments into taxonomic units. It usually requires several stages: read's quality control, de novo assembly, contig annotation, gene prediction, etc. These stages need very efficient programs because of the number of reads from the projects. Furthermore, the complexity of metagenomes requires efficient and automatic tools that orchestrate the different stages. METHOD: DATMA is a pipeline for fast metagenomic analysis that orchestrates the following: sequencing quality control, 16S rRNA-identification, reads binning, de novo assembly and evaluation, gene prediction, and taxonomic annotation. Its distributed computing model can use multiple computing resources to reduce the analysis time. RESULTS: We used a controlled experiment to show DATMA functionality. Two pre-annotated metagenomes to compare its accuracy and speed against other metagenomic frameworks. Then, with DATMA we recovered a draft genome of a novel Anaerolineaceae from a biosolid metagenome. CONCLUSIONS: DATMA is a bioinformatics tool that automatically analyzes complex metagenomes. It is faster than similar tools and, in some cases, it can extract genomes that the other tools do not. DATMA is freely available at https://github.com/andvides/DATMA.

Annual microbial community dynamics in a full-scale anaerobic sludge digester from a wastewater treatment plant in Colombia.

Anaerobic digestion is a microbe-driven process widely applied to treat activated sludge from municipal wastewater treatment plants. It is one of the most efficient solutions for sludge reduction along with biogas production. However, the knowledge of the microbial consortium involved in this process is still unknown in full-scale anaerobic digesters from Latin America. This study aimed to elucidate the dynamics of the microbial community of a full-scale anaerobic digester for a year using 16S rDNA amplicon sequencing with the Illumina Miseq platform. The results showed fluctuations in the frequencies of dominant phyla with a decrease of Proteobacteria and Bacteroidetes after a temporary suspension of anaerobic digester. The core community was affiliated with bacterial phyla Firmicutes, Actinobacteria, Proteobacteria, and Chloroflexi. The core community was represented by 154 OTUs that accounted for 74% of all the processed reads. The Anaerolineaceae family, within Chloroflexi phylum, was the most frequently observed taxonomic group in all samples analyzed. Despite the microbial fluctuations, the biogas production was stable over the studied year (average 66% methane production), which might indicate a functional redundancy in the microbial consortium.

Differential determinants of virulence in two Mycobacterium tuberculosis Colombian clinical isolates of the LAM09 family.

The heterogeneity of the clinical outcome of Mycobacterium tuberculosis (Mtb) infection may be due in part to different strategies used by circulating strains to cause disease. This heterogeneity is one of the main limitations to eradicate tuberculosis disease. In this study, we have compared the transcriptional response of two closely related Colombian clinical isolates (UT127 and UT205) of the LAM family under two axenic media conditions. These clinical isolates are phenotypically different at the level of cell death, cytokine production, growth kinetics upon in vitro infection of human tissue macrophages, and membrane vesicle secretion upon culture in synthetic medium. Using RNA-seq, we have identified different pathways that account for two different strategies to cope with the stressful condition of a carbon-poor media such as Sauton's. We showed that the clinical isolate UT205 focus mainly in the activation of virulence systems such as the ESX-1, synthesis of diacyl-trehalose, polyacyl-trehalose, and sulfolipids, while UT127 concentrates its efforts mainly in the survival mode by the activation of the DNA replication, cell division, and lipid biosynthesis. This is an example of two Mtb isolates that belong to the same family and lineage, and even though they have a very similar genome, its transcriptional regulation showed important differences. This results in summary highlight the necessity to reach a better understanding of the heterogeneity in the behavior of these circulating Mtb strains which may help us to design better treatments and vaccines and to identify new targets for drugs.

Metagenomic assessment of the microbial community and methanogenic pathways in biosolids from a municipal wastewater treatment plant in Medellín, Colombia.

Abundance and diversity of microbial communities in biosolids are variable and poorly studied in the tropics, and it is known that rainfall is one of the events that could affect the phylogenetic and functional microbial structure. In the present study, using NGS technics, we studied the microbial diversity as well as the methanogenesis pathway in one of the largest WWTP in Colombia. Besides, we sampled and analyzed biosolids from rainy season and dry season. Phylogenetic classification showed a predominance of bacteria in both samples and difference in the dominant groups depending on the rainfall season. Whereas Pseudomonas was the dominant bacteria in the dry season, Coprothermobacter was in the rainy season. Archaea abundance was higher in the rainy season (11.5%) doubling dry season proportion. The bioreactor biogas production and total solids content showed similar results between rainy and dry season at the sampling dates. The most abundant Archaea related with methanogenesis was Methanosaeta, which is a methanogenic microorganism that exclusively uses acetate to produce methane. Moreover, annotation of the methanogenic pathway in the metagenome showed abundance in genes encoding Acetyl-CoA synthetases (ACSS), an enzyme that catalyzes acetate activation. Our results suggest that the microbial diversity was stable among the two time points tested, rainy season and dry season; and, although there were changes in the microbial abundance of dominant bacterial species, anaerobic digester performance is not affected.

CLAME: a new alignment-based binning algorithm allows the genomic description of a novel Xanthomonadaceae from the Colombian Andes.

BACKGROUND: Hot spring bacteria have unique biological adaptations to survive the extreme conditions of these environments; these bacteria produce thermostable enzymes that can be used in biotechnological and industrial applications. However, sequencing these bacteria is complex, since it is not possible to culture them. As an alternative, genome shotgun sequencing of whole microbial communities can be used. The problem is that the classification of sequences within a metagenomic dataset is very challenging particularly when they include unknown microorganisms since they lack genomic reference. We failed to recover a bacterium genome from a hot spring metagenome using the available software tools, so we develop a new tool that allowed us to recover most of this genome. RESULTS: We present a proteobacteria draft genome reconstructed from a Colombian's Andes hot spring metagenome. The genome seems to be from a new lineage within the family Rhodanobacteraceae of the class Gammaproteobacteria, closely related to the genus Dokdonella. We were able to generate this genome thanks to CLAME. CLAME, from Spanish "CLAsificador MEtagenomico", is a tool to group reads in bins. We show that most reads from each bin belong to a single chromosome. CLAME is very effective recovering most of the reads belonging to the predominant species within a metagenome. CONCLUSIONS: We developed a tool that can be used to extract genomes (or parts of them) from a complex metagenome.

Early-Onset Invasive Infection Due to Corynespora cassiicola Associated with Compound Heterozygous CARD9 Mutations in a Colombian Patient.

PURPOSE: CARD9 deficiency is an inborn error of immunity that predisposes otherwise healthy humans to mucocutaneous and invasive fungal infections, mostly caused by Candida, but also by dermatophytes, Aspergillus, and other fungi. Phaeohyphomycosis are an emerging group of fungal infections caused by dematiaceous fungi (phaeohyphomycetes) and are being increasingly identified in patients with CARD9 deficiency. The Corynespora genus belongs to phaeohyphomycetes and only one adult patient with CARD9 deficiency has been reported to suffer from invasive disease caused by C. cassiicola. We identified a Colombian child with an early-onset, deep, and destructive mucocutaneous infection due to C. cassiicola and we searched for mutations in CARD9. METHODS: We reviewed the medical records and immunological findings in the patient. Microbiologic tests and biopsies were performed. Whole-exome sequencing (WES) was made and Sanger sequencing was used to confirm the CARD9 mutations in the patient and her family. Finally, CARD9 protein expression was evaluated in peripheral blood mononuclear cells (PBMC) by western blotting. RESULTS: The patient was affected by a large, indurated, foul-smelling, and verrucous ulcerated lesion on the left side of the face with extensive necrosis and crusting, due to a C. cassiicola infectious disease. WES led to the identification of compound heterozygous mutations in the patient consisting of the previously reported p.Q289* nonsense (c.865C > T, exon 6) mutation, and a novel deletion (c.23_29del; p.Asp8Alafs10*) leading to a frameshift and a premature stop codon in exon 2. CARD9 protein expression was absent in peripheral blood mononuclear cells from the patient. CONCLUSION: We describe here compound heterozygous loss-of-expression mutations in CARD9 leading to severe deep and destructive mucocutaneous phaeohyphomycosis due to C. cassiicola in a Colombian child.

Inherited p40phox deficiency differs from classic chronic granulomatous disease.

Biallelic loss-of-function (LOF) mutations of the NCF4 gene, encoding the p40phox subunit of the phagocyte NADPH oxidase, have been described in only 1 patient. We report on 24 p40phox-deficient patients from 12 additional families in 8 countries. These patients display 8 different in-frame or out-of-frame mutations of NCF4 that are homozygous in 11 of the families and compound heterozygous in another. When overexpressed in NB4 neutrophil-like cells and EBV-transformed B cells in vitro, the mutant alleles were found to be LOF, with the exception of the p.R58C and c.120_134del alleles, which were hypomorphic. Particle-induced NADPH oxidase activity was severely impaired in the patients' neutrophils, whereas PMA-induced dihydrorhodamine-1,2,3 (DHR) oxidation, which is widely used as a diagnostic test for chronic granulomatous disease (CGD), was normal or mildly impaired in the patients. Moreover, the NADPH oxidase activity of EBV-transformed B cells was also severely impaired, whereas that of mononuclear phagocytes was normal. Finally, the killing of Candida albicans and Aspergillus fumigatus hyphae by neutrophils was conserved in these patients, unlike in patients with CGD. The patients suffer from hyperinflammation and peripheral infections, but they do not have any of the invasive bacterial or fungal infections seen in CGD. Inherited p40phox deficiency underlies a distinctive condition, resembling a mild, atypical form of CGD.

Transcriptome profiling of the cysticercus stage of the laboratory model Taenia crassiceps, strain ORF.

Neurocysticercosis (NC) is a serious public health problem mainly in developing countries. NC caused by the cysticercus stage from cestode Taenia solium is considered by the WHO and ITFDE as a potentially eradicable disease. Definitive diagnosis of NC is challenging because of the unspecific clinical manifestations such as the non-definitive evidence presented by neuroimaging (in most cases) and the lack of definitive serological test. Taenia crassiceps (ORF strain) is a cestode closely related to T. solium and it has frequently been used as a source of antigens for immunodiagnostics. A murine model to study host immune response to infection has also been established by using T. crassiceps. Despite the extensive use of T. crassiceps for research, molecular information for this cestode is scarce in public databases. With the aim of providing more extensive information on T. crassiceps biology, an RNA-seq experiment and subsequent bioinformatic transcriptome processing of this cestode parasite mRNA in its cysticercus stage were carried out. A total of 227,082 read/ESTs were sequenced using the 454-GS FLX Titanium technology and assembled into 10,787 contigs. This transcriptome dataset represents new and valuable molecular information of the cestode T. crassiceps (ORF). This information will substantially improve public information and will help to achieve a better understanding of the biology of T. crassiceps and to identify target proteins for serodiagnosis and vaccination.