Viral Metagenomics Reveals Widely Diverse Viral Community of Freshwater Amazonian Lake.

Despite the importance of understanding the ecology of freshwater viruses, there are not many studies on the subject compared to marine viruses. The microbiological interactions in these environments are still poorly known, especially between bacteriophages and their host bacteria and between cyanophages and cyanobacteria. Lake Bologna, Belém, capital of the Brazilian State of Pará, is a water source that supplies the city and its metropolitan region. However, it remains unexplored regarding the contents of its virome and viral diversity composition. Therefore, this work aims to explore the taxonomic diversity of DNA viruses in this lake, especially bacteriophages and cyanophages, since they can act as transducers of resistance genes and reporters of water quality for human consumption. We used metagenomic sequencing data generated by previous studies. We analyzed it at the taxonomic level using the tools Kraken2, Bracken, and Pavian; later, the data was assembled using Genome Detective, which performs the assembly of viruses. The results observed here suggest the existence of a widely diverse viral community and established microbial phage-regulated dynamics in Lake Bolonha. This work is the first ever to describe the virome of Lake Bolonha using a metagenomic approach based on high-throughput sequencing, as it contributes to the understanding of water-related public health concerns regarding the spreading of antibiotic resistance genes and population control of native bacteria and cyanobacteria.

Proteins from the core genome of Corynebacterium ulcerans respond for pathogenicity and reveal promising vaccine targets for diphtheria.

Corynebacterium ulcerans is an emerging pathogen able to transmit the acute infection diphtheria to humans. Although there is a well-established vaccine based on the toxin produced by Corynebacterium diphtheriae, another species of this genus known to cause the disease, there is still no vaccine formulations described for C. ulcerans; this fact contributes to the increase in cases of infection that has been observed. In this study, we want to provide information at the genomic level of this bacterium in order to suggest proteins as possible vaccine targets. We carried out an in silico prospection of vaccine candidates through reverse vaccinology for targets that exhibit antigenic potential against diphtheria. We found important virulence factors, such as adhesion-related ones, that are responsible for pathogen-host interaction after infection, but we did not find the diphtheria toxin, which is the main component of the currently available vaccine. This study provides detailed information about the exoproteome and hypothetical proteins from the core genome of C. ulcerans, suggesting vaccine targets to be further tested in vitro for the development of a new vaccine against diphtheria.

Essential Oil Composition and DNA Barcode and Identification of Aniba species (Lauraceae) Growing in the Amazon Region.

Lauraceae species are widely represented in the Amazon, presenting a significant essential oil yield, large chemical variability, various biological applications, and high economic potential. Its taxonomic classification is difficult due to the accentuated morphological uniformity, even among taxa from a different genus. For this reason, the present work aimed to find chemical and molecular markers to discriminate Aniba species collected in the Pará State (Brazil). The chemical composition of the essential oils from Aniba canelilla, A. parviflora, A. rosaeodora, and A. terminalis were grouped by multivariate statistical analysis. The major compounds were rich in benzenoids and terpenoids such as 1-nitro-2-phenylethane (88.34-70.85%), linalool (15.2-75.3%), α-phellandrene (36.0-51.8%), and ß-phellandrene (11.6-25.6%). DNA barcodes were developed using the internal transcribed spacer (ITS) nuclear region, and the matK, psbA-trnH, rbcL, and ycf1 plastid regions. The markers psbA-trnH and ITS showed the best discrimination for the species, and the phylogenic analysis in the three- (rbcL + matK + trnH - psbA and rbcL + matK + ITS) and four-locus (rbcL + matK + trnH - psbA + ITS) combination formed clades with groups strongly supported by the Bayesian inference (BI) (PP:1.00) and maximum likelihood (ML) (BS ≥ 97%). Therefore, based on statistical multivariate and phylogenetic analysis, the results showed a significant correlation between volatile chemical classes and genetic characteristics of Aniba species.

In silico functional prediction of hypothetical proteins from the core genome of Corynebacterium pseudotuberculosis biovar ovis.

Corynebacterium pseudotuberculosis is a pathogen of veterinary relevance diseases, being divided into two biovars: equi and ovis; causing ulcerative lymphangitis and caseous lymphadenitis, respectively. The isolation and sequencing of C. pseudotuberculosis biovar ovis strains in the Northern and Northeastern regions of Brazil exhibited the emergence of this pathogen, which causes economic losses to small ruminant producers, and condemnation of carcasses and skins of animals. Through the pan-genomic approach, it is possible to determine and analyze genes that are shared by all strains of a species-the core genome. However, many of these genes do not have any predicted function, being characterized as hypothetical proteins (HP). In this study, we considered 32 C. pseudotuberculosis biovar ovis genomes for the pan-genomic analysis, where were identified 172 HP present in a core genome composed by 1255 genes. We are able to functionally annotate 80 sequences previously characterized as HP through the identification of structural features as conserved domains and families. Furthermore, we analyzed the physicochemical properties, subcellular localization and molecular function. Additionally, through RNA-seq data, we investigated the differential gene expression of the annotated HP. Genes inserted in pathogenicity islands had their virulence potential evaluated. Also, we have analyzed the existence of functional associations for their products based on protein-protein interaction networks, and perform the structural prediction of three targets. Due to the integration of different strategies, this study can underlie deeper in vitro researches in the characterization of these HP and the search for new solutions for combat this pathogen.

Co-Expression Networks for Causal Gene Identification Based on RNA-Seq Data of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that causes caseous lymphadenitis, a disease that predominantly affects sheep, goat, cattle, buffalo, and horses, but has also been recognized in other animals. This bacterium generates a severe economic impact on countries producing meat. Gene expression studies using RNA-Seq are one of the most commonly used techniques to perform transcriptional experiments. Computational analysis of such data through reverse-engineering algorithms leads to a better understanding of the genome-wide complexity of gene interactomes, enabling the identification of genes having the most significant functions inferred by the activated stress response pathways. In this study, we identified the influential or causal genes from four RNA-Seq datasets from different stress conditions (high iron, low iron, acid, osmosis, and PH) in C. pseudotuberculosis, using a consensus-based network inference algorithm called miRsigand next identified the causal genes in the network using the miRinfluence tool, which is based on the influence diffusion model. We found that over 50% of the genes identified as influential had some essential cellular functions in the genomes. In the strains analyzed, most of the causal genes had crucial roles or participated in processes associated with the response to extracellular stresses, pathogenicity, membrane components, and essential genes. This research brings new insight into the understanding of virulence and infection by C. pseudotuberculosis.

Resistome in Lake Bolonha, Brazilian Amazon: Identification of Genes Related to Resistance to Broad-Spectrum Antibiotics.

Resistance to antibiotics is one of the most relevant public health concerns in the world. Aquatic environments play an important role because they are reservoirs for antibiotic resistance genes and antibiotic-resistant strains, contributing to the spread of resistance. The present study investigated the resistome in Lake Bolonha (three sampling sites) in the Amazon region using a metagenomics approach and culture-dependent methods. Whole-metagenome-based results showed that the most abundant phyla were Protobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Cyanobacteria. The composition of the resistome demonstrated that the genes that confer resistance to ß-lactams were prevalent at all sampling sites, followed by genes conferring resistance to aminoglycosides and tetracycline. Acquired genes encoding extended-spectrum ß-lactamases (e.g., bla CTX-M) and resistance to carbapenems (e.g., bla IMP and bla VIM) were detected through metagenome analysis. Bacteria were isolated from culture medium supplemented with cefotaxime or imipenem, and isolates were identified and analyzed for their antibiotic susceptibility profiles and resistance genes. In total, 98 bacterial isolates belonging to the genera Pseudomonas (37), Acinetobacter (32), Klebsiella (13), Enterobacter (9), Pantoe (3), Stenotrophomonas (3), and Methylobacterium (1) were obtained. Among isolates, the most abundant genes were bla CTX-M (28.3%), bla SHV (22.6%) and bla TEM (18.8%) in isolates from cefotaxime-supplemented medium and bla VIM (28.8%) and bla IMP (22.2%) in isolates recovered from imipenem-supplemented medium. The genes intl1 and intl2 were detected in 19.3% and 7.1% of isolates. Antibiograms showed that 94.9% (from cefotaxime-supplemented medium) and 85.7% (from imipenem-supplemented medium) of the isolates were multidrug resistant. Besides cefotaxime and imipenem, isolates were mostly resistant to aztreonam (91.8%), amoxicillin (98.8%), ampicillin (82.6%), and nalidixic acid (77.5%). Hence, the present study demonstrates that Lake Bolonha is a reservoir of bacteria resistant to antibiotics and resistance genes, some of which are of critical importance to human health.

Prediction of new vaccine targets in the core genome of Corynebacterium pseudotuberculosis through omics approaches and reverse vaccinology.

Corynebacterium pseudotuberculosis is the etiologic agent of veterinary relevance diseases, such as caseous lymphadenitis, affecting different animal species causing damage to the global agribusiness. So far, there are no completely effective treatment methods to overcome the impacts caused by this pathogen. Several genomes of the species are deposited on public databases, allowing the execution of studies related to the pan-genomic approach. In this study, we used an integrated in silico workflow to prospect novel putative targets using the core genome, a set of shared genes among 65 C. pseudotuberculosis strains. Subsequently, through RNA-Seq data of the same abiotic stresses in two strains, we selected only induced genes to compose the reverse vaccinology workflow based in two different strategies. Our results predicted six probable antigens in both analysis, which indicates that they have a strong potential to be used in further studies as vaccine targets against this bacterium.

Extended Spectrum Beta-Lactamase-Producing Gram-Negative Bacteria Recovered From an Amazonian Lake Near the City of Belém, Brazil.

Aquatic systems have been described as antibiotic resistance reservoirs, where water may act as a vehicle for the spread of resistant bacteria and resistance genes. We evaluated the occurrence and diversity of third generation cephalosporin-resistant gram-negative bacteria in a lake in the Amazonia region. This water is used for human activities, including consumption after appropriate treatment. Eighteen samples were obtained from six sites in October 2014. Water quality parameters were generally within the legislation limits. Thirty-three bacterial isolates were identified as Escherichia (n = 7 isolates), Acinetobacter, Enterobacter, and Klebsiella (n = 5 each), Pseudomonas (n = 4), Shigella (n = 3), and Chromobacterium, Citrobacter, Leclercia, Phytobacter (1 isolate each). Twenty nine out of 33 isolates (88%) were resistant to most beta-lactams, except carbapenems, and 88% (n = 29) were resistant to antibiotics included in at least three different classes. Among the beta-lactamase genes inspected, the bla CTX-M was the most prevalent (n = 12 positive isolates), followed by bla TEM (n = 5) and bla SHV (n = 4). bla CTX-M-15 (n = 5), bla CTX-M-14 (n = 1) and bla CTX-M-2 (n = 1) variants were detected in conserved genomic contexts: bla CTX-M-15 flanked by ISEcp1 and Orf477; bla CTX-M-14 flanked by ISEcp1 and IS903; and bla CTX-M-2 associated to an ISCR element. For 4 strains the transfer of bla CTX-M was confirmed by conjugation assays. Compared with the recipient, the transconjugants showed more than 500-fold increases in the MICs of cefotaxime and 16 to 32-fold increases in the MICs of ceftazidime. Two isolates (Escherichia coli APC43A and Acinetobacter baumannii APC25) were selected for whole genome analysis. APC43A was predicted as a E. coli pathogen of the high-risk clone ST471 and serotype O154:H18. bla CTX-M-15 as well as determinants related to efflux of antibiotics, were noted in APC43A genome. A. baumannii APC25 was susceptible to carbapenems and antibiotic resistance genes detected in its genome were intrinsic determinants (e.g., bla OXA-208 and bla ADC-like). The strain was not predicted as a human pathogen and belongs to a new sequence type. Operons related to metal resistance were predicted in both genomes as well as pathogenicity and resistance islands. Results suggest a high dissemination of ESBL-producing bacteria in Lake Água Preta which, although not presenting characteristics of a strongly impacted environment, contains multi-drug resistant pathogenic strains.

Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress.

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

Functional annotation of hypothetical proteins from the Exiguobacterium antarcticum strain B7 reveals proteins involved in adaptation to extreme environments, including high arsenic resistance.

Exiguobacterium antarcticum strain B7 is a psychrophilic Gram-positive bacterium that possesses enzymes that can be used for several biotechnological applications. However, many proteins from its genome are considered hypothetical proteins (HPs). These functionally unknown proteins may indicate important functions regarding the biological role of this bacterium, and the use of bioinformatics tools can assist in the biological understanding of this organism through functional annotation analysis. Thus, our study aimed to assign functions to proteins previously described as HPs, present in the genome of E. antarcticum B7. We used an extensive in silico workflow combining several bioinformatics tools for function annotation, sub-cellular localization and physicochemical characterization, three-dimensional structure determination, and protein-protein interactions. This genome contains 2772 genes, of which 765 CDS were annotated as HPs. The amino acid sequences of all HPs were submitted to our workflow and we successfully attributed function to 132 HPs. We identified 11 proteins that play important roles in the mechanisms of adaptation to adverse environments, such as flagellar biosynthesis, biofilm formation, carotenoids biosynthesis, and others. In addition, three predicted HPs are possibly related to arsenic tolerance. Through an in vitro assay, we verified that E. antarcticum B7 can grow at high concentrations of this metal. The approach used was important to precisely assign function to proteins from diverse classes and to infer relationships with proteins with functions already described in the literature. This approach aims to produce a better understanding of the mechanism by which this bacterium adapts to extreme environments and to the finding of targets with biotechnological interest.

Genomic Architecture of the Two Cold-Adapted Genera Exiguobacterium and Psychrobacter: Evidence of Functional Reduction in the Exiguobacterium antarcticum B7 Genome.

Exiguobacterium and Psychrobacter are bacterial genera with several cold-adapted species. These extremophiles are commonly isolated from the same habitats in Earth's cryosphere and have great ecological and biotechnological relevance. Thus, through comparative genomic analyses, it was possible to understand the functional diversity of these psychrotrophic and psychrophilic species and present new insights into the microbial adaptation to cold. The nucleotide identity between Exiguobacterium genomes was >90%. Three genomic islands were identified in the E. antarcticum B7 genome. These islands contained genes involved in flagella biosynthesis and chemotaxis, as well as enzymes for carotenoid biosynthesis. Clustering of cold shock proteins by Ka/Ks ratio suggests the occurrence of a positive selection over these genes. Neighbor-joining clustering of complete genomes showed that the E. sibiricum was the most closely related to E. antarcticum. A total of 92 genes were shared between Exiguobacterium and Psychrobacter. A reduction in the genomic content of E. antarcticum B7 was observed. It presented the smallest genome size of its genus and a lower number of genes because of the loss of many gene families compared with the other genomes. In our study, eight genomes of Exiguobacterium and Psychrobacter were compared and analysed. Psychrobacter showed higher genomic plasticity and E. antarcticum B7 presented a large decrease in genomic content without changing its ability to grow in cold environments.

Heat shock stress: Profile of differential expression in Corynebacterium pseudotuberculosis biovar Equi.

Transcriptome studies on Corynebacterium pseudotuberculosis have recently contributed to the understanding about this microorganism's survival mechanisms in various hostile conditions. The gene expression profile of the C. pseudotuberculosis strain 1002 (Ovis biovar), has revealed genes that are possible candidates responsible for its maintenance in adverse environments, such as those found in the host. In another strain of this bacterium, 258 (Equi biovar), a high temperature condition was simulated, in order to verify which genes are responsible for promoting the persistence of the bacterium in these conditions, since it tolerates temperatures higher than 40°C, despite being a mesophilic bacterium. It was possible to generate a list of genes using RNAseq technology that possibly contribute to the survival of the bacteria in this hostile environment. A total of 562 genes were considered as differentially expressed, then, after the fold-change cutoff, 113 were considered induced and 114 repressed, resulting in a total of 227 genes. Therefore, hypothetical proteins presented a fold change above 6, and genes characteristically in control for this type of stress, such as hspR, grpE, and dnaK, presented a fold change above 3. The clpB gene, a chaperone, drew attention due to presenting a fold change above 3 and located in a pathogenicity island. These genes may contribute towards efficient solutions to the effects caused by ulcerative lymphangitis in equines, thus attenuating the damage it causes to agribusiness.

Whole-Genome Sequence of Corynebacterium pseudotuberculosis PA04, Isolated from the Lymph Node of a Sheep in the Amazon, Brazil.

This study reports the complete genome sequence of Corynebacterium pseudotuberculosis strain PA04, isolated from a sheep in the Amazon, Brazil. This bacterium is the etiological agent of caseous lymphadenitis. This genome contains 2,338,093 bp, 52.2% G+C content, and a total of 2,104 coding sequences (CDSs), 41 pseudogenes, 12 rRNAs, and 49 tRNAs.

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA05 Isolated from an Ovine Host in Pará State, Brazil.

We report here the draft genome sequence of Corynebacterium pseudotuberculosis PA05, isolated from an ovine host in Pará State, Brazil. C. pseudotuberculosis is an etiological agent of diseases with veterinary and medical importance. The genome contains 2,435,137 bp, a G+C content of 52.2%, 2,295 coding sequences, five pseudogenes, 53 tRNAs, and six rRNAs.

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA06 Isolated from a Subauricular Abscess in an Ovine Host.

We report here the draft genome sequence of Corynebacterium pseudotuberculosis PA06, isolated from a subauricular abscess in an ovine host. C. pseudotuberculosis is a worldwide pathogen of small and large ruminants. The genome comprises 2,320,074 bp, with a G+C content of 52.2%, 2,195 coding sequences, 48 tRNAs, and three rRNAs.

A Proteomic Perspective on the Bacterial Adaptation to Cold: Integrating OMICs Data of the Psychrotrophic Bacterium Exiguobacterium antarcticum B7.

Since the publication of one of the first studies using 2D gel electrophoresis by Patrick H. O'Farrell in 1975, several other studies have used that method to evaluate cellular responses to different physicochemical variations. In environmental microbiology, bacterial adaptation to cold environments is a "hot topic" because of its application in biotechnological processes. As in other fields, gel-based and gel-free proteomic methods have been used to determine the molecular mechanisms of adaptation to cold of several psychrotrophic and psychrophilic bacterial species. In this review, we aim to describe and discuss these main molecular mechanisms of cold adaptation, referencing proteomic studies that have made significant contributions to our current knowledge in the area. Furthermore, we use Exiguobacterium antarcticum B7 as a model organism to present the importance of integrating genomic, transcriptomic, and proteomic data. This species has been isolated in Antarctica and previously studied at all three omic levels. The integration of these data permitted more robust conclusions about the mechanisms of bacterial adaptation to cold.

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA07 Biovar ovis, Isolated from a Sheep Udder in Amazonia.

In this work, we present the draft genome sequence of Corynebacterium pseudotuberculosis strain PA07 biovar ovis, isolated from a caseous secretion from a sheep udder in Pará, Brazil. The genome contains 2,320,235 bp, 52.2% G+C content, 2,191 coding sequences (CDSs), five pseudogenes, 48 tRNAs, and three rRNAs.

Genome Sequence of Corynebacterium pseudotuberculosis Strain PA02 Isolated from an Ovine Host in the Amazon.

In this work, we report the complete genome sequence of Corynebacterium pseudotuberculosis strain PA02 isolated from an ovine host. The genome contains 2,328,435 bp, a 52.2% G+C content, 2,035 coding sequences, 12 rRNA operons, 45 tRNAs, and 14 predicted pseudogenes.