PPIntegrator: semantic integrative system for protein-protein interaction and application for host-pathogen datasets.

Summary: Semantic web standards have shown importance in the last 20 years in promoting data formalization and interlinking between the existing knowledge graphs. In this context, several ontologies and data integration initiatives have emerged in recent years for the biological area, such as the broadly used Gene Ontology that contains metadata to annotate gene function and subcellular location. Another important subject in the biological area is protein-protein interactions (PPIs) which have applications like protein function inference. Current PPI databases have heterogeneous exportation methods that challenge their integration and analysis. Presently, several initiatives of ontologies covering some concepts of the PPI domain are available to promote interoperability across datasets. However, the efforts to stimulate guidelines for automatic semantic data integration and analysis for PPIs in these datasets are limited. Here, we present PPIntegrator, a system that semantically describes data related to protein interactions. We also introduce an enrichment pipeline to generate, predict and validate new potential host-pathogen datasets by transitivity analysis. PPIntegrator contains a data preparation module to organize data from three reference databases and a triplification and data fusion module to describe the provenance information and results. This work provides an overview of the PPIntegrator system applied to integrate and compare host-pathogen PPI datasets from four bacterial species using our proposed transitivity analysis pipeline. We also demonstrated some critical queries to analyze this kind of data and highlight the importance and usage of the semantic data generated by our system. Availability and implementation: https://github.com/YasCoMa/ppintegrator, https://github.com/YasCoMa/ppi_validation_process and https://github.com/YasCoMa/predprin.

Analysis of a novel class A ß-lactamase OKP-B-6 of Klebsiella quasipneumoniae: structural characterisation and interaction with commercially available drugs.

BACKGROUND: Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum ß-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES: So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS: We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS: From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS: This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.

The gene regulatory network of Staphylococcus aureus ST239-SCCmecIII strain Bmb9393 and assessment of genes associated with the biofilm in diverse backgrounds.

Introduction: Staphylococcus aureus is one of the most prevalent and relevant pathogens responsible for a wide spectrum of hospital-associated or community-acquired infections. In addition, methicillin-resistant Staphylococcus aureus may display multidrug resistance profiles that complicate treatment and increase the mortality rate. The ability to produce biofilm, particularly in device-associated infections, promotes chronic and potentially more severe infections originating from the primary site. Understanding the complex mechanisms involved in planktonic and biofilm growth is critical to identifying regulatory connections and ways to overcome the global health problem of multidrug-resistant bacteria. Methods: In this work, we apply literature-based and comparative genomics approaches to reconstruct the gene regulatory network of the high biofilm-producing strain Bmb9393, belonging to one of the highly disseminating successful clones, the Brazilian epidemic clone. To the best of our knowledge, we describe for the first time the topological properties and network motifs for the Staphylococcus aureus pathogen. We performed this analysis using the ST239-SCCmecIII Bmb9393 strain. In addition, we analyzed transcriptomes available in the literature to construct a set of genes differentially expressed in the biofilm, covering different stages of the biofilms and genetic backgrounds of the strains. Results and discussion: The Bmb9393 gene regulatory network comprises 1,803 regulatory interactions between 64 transcription factors and the non-redundant set of 1,151 target genes with the inclusion of 19 new regulons compared to the N315 transcriptional regulatory network published in 2011. In the Bmb9393 network, we found 54 feed-forward loop motifs, where the most prevalent were coherent type 2 and incoherent type 2. The non-redundant set of differentially expressed genes in the biofilm consisted of 1,794 genes with functional categories relevant for adaptation to the variable microenvironments established throughout the biofilm formation process. Finally, we mapped the set of genes with altered expression in the biofilm in the Bmb9393 gene regulatory network to depict how different growth modes can alter the regulatory systems. The data revealed 45 transcription factors and 876 shared target genes. Thus, the gene regulatory network model provided represents the most up-to-date model for Staphylococcus aureus, and the set of genes altered in the biofilm provides a global view of their influence on biofilm formation from distinct experimental perspectives and different strain backgrounds.

Analysis of a novel class A β-lactamase OKP-B-6 of Klebsiella quasipneumoniae: structural characterisation and interaction with commercially available drugs

BACKGROUND Gram-negative and Gram-positive bacteria produce beta-lactamase as factors to overcome beta-lactam antibiotics, causing their hydrolysis and impaired antimicrobial action. Class A beta-lactamase contains the chromosomal sulfhydryl reagent variable (SHV, point mutation variants of SHV-1), LEN (Klebsiella pneumoniae strain LEN-1), and other K. pneumoniae beta-lactamase (OKP) found mostly in Klebsiella's phylogroups. The SHV known as extended-spectrum β-lactamase can inactivate most beta-lactam antibiotics. Class A also includes the worrisome plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC-2), a carbapenemase that can inactivate most beta-lactam antibiotics, carbapenems, and some beta-lactamase inhibitors. OBJECTIVES So far, there is no 3D crystal structure for OKP-B, so our goal was to perform structural characterisation and molecular docking studies of this new enzyme. METHODS We applied a homology modelling method to build the OKP-B-6 structure, which was compared with SHV-1 and KPC-2 according to their electrostatic potentials at the active site. Using the DockThor-VS, we performed molecular docking of the SHV-1 inhibitors commercially available as sulbactam, tazobactam, and avibactam against the constructed model of OKP-B-6. FINDINGS From the point of view of enzyme inhibition, our results indicate that OKP-B-6 should be an extended-spectrum beta-lactamase (ESBL) susceptible to the same drugs as SHV-1. MAIN CONCLUSIONS This conclusion advantageously impacts the clinical control of the bacterial pathogens encoding OKP-B in their genome by using any effective, broad-spectrum, and multitarget inhibitor against SHV-containing bacteria.

A Systematic Strategy to Find Potential Therapeutic Targets for Pseudomonas aeruginosa Using Integrated Computational Models.

Pseudomonas aeruginosa is an opportunistic human pathogen that has been a constant global health problem due to its ability to cause infection at different body sites and its resistance to a broad spectrum of clinically available antibiotics. The World Health Organization classified multidrug-resistant Pseudomonas aeruginosa among the top-ranked organisms that require urgent research and development of effective therapeutic options. Several approaches have been taken to achieve these goals, but they all depend on discovering potential drug targets. The large amount of data obtained from sequencing technologies has been used to create computational models of organisms, which provide a powerful tool for better understanding their biological behavior. In the present work, we applied a method to integrate transcriptome data with genome-scale metabolic networks of Pseudomonas aeruginosa. We submitted both metabolic and integrated models to dynamic simulations and compared their performance with published in vitro growth curves. In addition, we used these models to identify potential therapeutic targets and compared the results to analyze the assumption that computational models enriched with biological measurements can provide more selective and (or) specific predictions. Our results demonstrate that dynamic simulations from integrated models result in more accurate growth curves and flux distribution more coherent with biological observations. Moreover, identifying drug targets from integrated models is more selective as the predicted genes were a subset of those found in the metabolic models. Our analysis resulted in the identification of 26 non-host homologous targets. Among them, we highlighted five top-ranked genes based on lesser conservation with the human microbiome. Overall, some of the genes identified in this work have already been proposed by different approaches and (or) are already investigated as targets to antimicrobial compounds, reinforcing the benefit of using integrated models as a starting point to selecting biologically relevant therapeutic targets.

Large-Scale Protein Interactions Prediction by Multiple Evidence Analysis Associated With an In-Silico Curation Strategy.

Predicting the physical or functional associations through protein-protein interactions (PPIs) represents an integral approach for inferring novel protein functions and discovering new drug targets during repositioning analysis. Recent advances in high-throughput data generation and multi-omics techniques have enabled large-scale PPI predictions, thus promoting several computational methods based on different levels of biological evidence. However, integrating multiple results and strategies to optimize, extract interaction features automatically and scale up the entire PPI prediction process is still challenging. Most procedures do not offer an in-silico validation process to evaluate the predicted PPIs. In this context, this paper presents the PredPrIn scientific workflow that enables PPI prediction based on multiple lines of evidence, including the structure, sequence, and functional annotation categories, by combining boosting and stacking machine learning techniques. We also present a pipeline (PPIVPro) for the validation process based on cellular co-localization filtering and a focused search of PPI evidence on scientific publications. Thus, our combined approach provides means to extensive scale training or prediction of new PPIs and a strategy to evaluate the prediction quality. PredPrIn and PPIVPro are publicly available at https://github.com/YasCoMa/predprin and https://github.com/YasCoMa/ppi_validation_process.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Structural analysis of a novel N-carbamoyl-d-amino acid amidohydrolase from a Brazilian Bradyrhizobium japonicum strain: In silico insights by molecular modelling, docking and molecular dynamics.

In this work we performed several in silico analyses to describe the relevant structural aspects of an enzyme N-Carbamoyl-d-amino acid amidohydrolase (d-NCAase) encoded on the genome of the Brazilian strain CPAC 15 (=SEMIA 5079) of Bradyrhizobium japonicum, a nonpathogenic species belonging to the order Rhizobiales. d-NCAase has wide applications particularly in the pharmaceutical industry, since it catalyzes the production of d-amino acids such as D-p-hydroxyphenylglycine (D-HPG), an intermediate in the synthesis of ß-lactam antibiotics. We applied a homology modelling approach and 50 ns of molecular dynamics simulations to predict the structure and the intersubunit interactions of this novel d-NCAase. Also, in order to evaluate the substrate binding site, the model was subjected to 50 ns of molecular dynamics simulations in the presence of N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) (a d-NCAase canonical substrate) and water-protein/water-substrate interactions analyses were performed. Overall, the structural analysis and the molecular dynamics simulations suggest that d-NCAase of B. japonicum CPAC-15 has a homodimeric structure in solution. Here, we also examined the substrate specificity of the catalytic site of our model and the interactions with water molecules into the active binding site were comprehensively discussed. Also, these simulations showed that the amino acids Lys123, His125, Pro127, Cys172, Asp174 and Arg176 are responsible for recognition of ligand in the active binding site through several chemical associations, such as hydrogen bonds and hydrophobic interactions. Our results show a favourable environment for a reaction of hydrolysis that transforms N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) into the active compound D-p-hydroxyphenylglycine (D-HPG). This work envisage the use of d-NCAase from the Brazilian Bradyrhizobium japonicum strain CPAC-15 (=SEMIA 5079) for the industrial production of D-HPG, an important intermediate for semi-synthesis of ß-lactam antibiotics such as penicillins, cephalosporins and amoxicillin.

Characterization of Laguncularia racemosa transcriptome and molecular response to oil pollution.

Mangroves are ecosystems of economic and ecological importance. Laguncularia racemosa (Combretaceae), popularly known as white mangrove, is a species that greatly contributes to the community structure of neotropical and West African mangrove forests. Despite the significance of these ecosystems, they have been destroyed by oil spills that can cause yellowing of leaves, increased sensitivity to other stresses and death of trees. However, the molecular response of plants to oil stress is poorly known. In this work, Illumina reads were de novo assembled into 46,944 transcripts of L. racemosa roots and leaves, including putative isoform variants. In addition to improving the genomic information available for mangroves, the L. racemosa assembled transcriptome allowed us to identify reference genes to normalize quantitative real-time PCR (qPCR) expression data from oil-stressed mangrove plants, which were used in RNASeq validation. The analysis of expression changes induced by the oil exposure revealed 310 and 286 responsive transcripts of leaves and roots, respectively, mainly up-regulated. Enriched GO categories related to chloroplasts and photosynthesis were found among both leaf and root oil-responsive transcripts, while "response to heat" and "response to hypoxia" were exclusively enriched in leaves and roots, respectively. The comparison of L. racemosa 12-h-oil-stressed leaf expression profile to previous Arabidopsis heat-stress studies and co-expression evidence also pointed to similarities between the heat and oil responses, in which the HSP-coding genes seem to play a key role. A subset of the L. racemosa oil-responsive root genes exhibited similar up-regulation profiles to their Arabidopsis homologs involved in hypoxia responses, including the HRA1 and LBD41 TF-coding genes. Genes linked to the ethylene pathway such as those coding for ERF TFs were also modulated during the L. racemosa root response to oil stress. Taken together, these results show that oil contamination affects photosynthesis, protein metabolism, hypoxia response and the ethylene pathway in L. racemosa 12-h-oil-exposed leaves and roots.

Comparative metagenome of a stream impacted by the urbanization phenomenon

Abstract Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

The polymyxin B-induced transcriptomic response of a clinical, multidrug-resistant Klebsiella pneumoniae involves multiple regulatory elements and intracellular targets.

BACKGROUND: The emergence of multidrug-resistant Klebsiella pneumoniae is a major public health concern. Many K. pneumoniae infections can only be treated when resorting to last-line drugs such as polymyxin B (PB). However, resistance to this antibiotic is also observed, although insufficient information is described on its mode of action as well as the mechanisms used by resistant bacteria to evade its effects. We aimed to study PB resistance and the influence of abiotic stresses in a clinical K. pneumoniae strain using whole transcriptome profiling. RESULTS: We sequenced 12 cDNA libraries of K. pneumoniae Kp13 bacteria, from two biological replicates of the original strain Kp13 (Kp13) and five derivative strains: induced high-level PB resistance in acidic pH (Kp13pH), magnesium deprivation (Kp13Mg), high concentrations of calcium (Kp13Ca) and iron (Kp13Fe), and a control condition with PB (Kp13PolB). Our results show the involvement of multiple regulatory loci that differentially respond to each condition as well as a shared gene expression response elicited by PB treatment, and indicate the participation of two-regulatory components such as ArcA-ArcB, which could be involved in re-routing the K. pneumoniae metabolism following PB treatment. Modules of co-expressed genes could be determined, which correlated to growth in acid stress and PB exposure. We hypothesize that polymyxin B induces metabolic shifts in K. pneumoniae that could relate to surviving against the action of this antibiotic. CONCLUSIONS: We obtained whole transcriptome data for K. pneumoniae under different environmental conditions and PB treatment. Our results supports the notion that the K. pneumoniae response to PB exposure goes beyond damaged membrane reconstruction and involves recruitment of multiple gene modules and intracellular targets.

Comparative metagenome of a stream impacted by the urbanization phenomenon.

Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

A unique SaeS allele overrides cell-density dependent expression of saeR and lukSF-PV in the ST30-SCCmecIV lineage of CA-MRSA.

ST30 (CC30)-SCCmec IV (USA1100) is one of the most common community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) lineages. ST30 isolates typically carry lukSF-PV genes encoding the Panton-Valentine leukocidin (PVL) and are responsible for outbreaks of invasive infections worldwide. In this study, twenty CC30 isolates were analyzed. All were very susceptible to non-ß-lactam antimicrobials, 18/20 harbored the lukSF-PV genes, only 1/20 exhibited agr-rnaIII dysfunction, and the majority was not able to form biofilm on inert surfaces. Analysis of lukSF-PV temporal regulation revealed that opposite to other CA-MRSA isolates, these genes were more highly expressed in early log phase than in stationary phase. This inverted lukSF-PV temporal expression was associated with a similar pattern of saeRS expression in the ST30 isolates, namely high level expression in log phase and reduced expression in stationary phase. Reduced saeRS expression in stationary phase was associated with low expression levels of the sae regulators, agr and agr-upregulator sarA, which activate the stationary phase sae-P1 promoter and overexpression of agr-RNAIII restored the levels of saeR and lukSF-PV trancripts in stationary phase. Altered SaeRS activity in the ST30 isolates was attributed to amino acid substitutions (N227S, E268K and S351T) in the HTPase_c domain of SaeS (termed SaeS(SKT)). Complementation of a USA300 saeS mutant with the saeS(SKT) and saeS alleles under the direction of the log phase sae-P3 promoter revealed that saeR and lukSF-PV transcription levels were more significantly activated by saeS(SKT) than saeS. In summary our data identify a unique saeS allele (saeS(SKT)) which appears to override cell-density dependent SaeR and PVL expression in ST30 CA-MRSA isolates. Further studies to determine the contribution of saeS(SKT) allele to the pathogenesis of infections caused by ST30 isolates are merited.

A whole genome bioinformatic approach to determine potential latent phase specific targets in Mycobacterium tuberculosis.

Current Tuberculosis treatment is long and expensive, faces the increasing burden of MDR/XDR strains and lack of effective treatment against latent form, resulting in an urgent need of new anti-TB drugs. Key to TB biology is its capacity to fight the host's RNOS mediated attack. RNOS are known to display a concentration dependent mycobactericidal activity, which leads to the following hypothesis "if we know which proteins are targeted by RNOS and kill TB, we we might be able to inhibit them with drugs resulting in a synergistic bactericidal effect". Based on this idea, we performed an Mtb metabolic network whole proteome analysis of potential RNOS sensitive and relevant targets which includes target druggability and essentiality criteria. Our results, available at http://tuberq.proteinq.com.ar yield new potential TB targets, like I3PS, while also providing and updated view of previous proposals becoming an important tool for researchers looking for new ways of killing TB.

De novo transcriptome analysis of Hevea brasiliensis tissues by RNA-seq and screening for molecular markers.

BACKGROUND: The rubber tree, Hevea brasiliensis, is a species native to the Brazilian Amazon region and it supplies almost all the world's natural rubber, a strategic raw material for a variety of products. One of the major challenges for developing rubber tree plantations is adapting the plant to biotic and abiotic stress. Transcriptome analysis is one of the main approaches for identifying the complete set of active genes in a cell or tissue for a specific developmental stage or physiological condition. RESULTS: Here, we report on the sequencing, assembling, annotation and screening for molecular markers from a pool of H. brasiliensis tissues. A total of 17,166 contigs were successfully annotated. Then, 2,191 Single Nucleotide Variation (SNV) and 1.397 Simple Sequence Repeat (SSR) loci were discriminated from the sequences. From 306 putative, mainly non-synonymous SNVs located in CDS sequences, 191 were checked for their ability to characterize 23 Hevea genotypes by an allele-specific amplification technology. For 172 (90%), the nucleotide variation at the predicted genomic location was confirmed, thus validating the different steps from sequencing to the in silico detection of the SNVs. CONCLUSIONS: This is the first study of the H. brasiliensis transcriptome, covering a wide range of tissues and organs, leading to the production of the first developed SNP markers. This process could be amplified to a larger set of in silico detected SNVs in expressed genes in order to increase the marker density in available and future genetic maps. The results obtained in this study will contribute to the H. brasiliensis genetic breeding program focused on improving of disease resistance and latex yield.

Comparative analysis of the complete genome of KPC-2-producing Klebsiella pneumoniae Kp13 reveals remarkable genome plasticity and a wide repertoire of virulence and resistance mechanisms.

BACKGROUND: Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial and community-acquired infections. A wide repertoire of virulence and antimicrobial resistance genes is present in K. pneumoniae genomes, which can constitute extra challenges in the treatment of infections caused by some strains. K. pneumoniae Kp13 is a multidrug-resistant strain responsible for causing a large nosocomial outbreak in a teaching hospital located in Southern Brazil. Kp13 produces K. pneumoniae carbapenemase (KPC-2) but is unrelated to isolates belonging to ST 258 and ST 11, the main clusters associated with the worldwide dissemination of KPC-producing K. pneumoniae. In this report, we perform a genomic comparison between Kp13 and each of the following three K. pneumoniae genomes: MGH 78578, NTUH-K2044 and 342. RESULTS: We have completely determined the genome of K. pneumoniae Kp13, which comprises one chromosome (5.3 Mbp) and six plasmids (0.43 Mbp). Several virulence and resistance determinants were identified in strain Kp13. Specifically, we detected genes coding for six beta-lactamases (SHV-12, OXA-9, TEM-1, CTX-M-2, SHV-110 and KPC-2), eight adhesin-related gene clusters, including regions coding for types 1 (fim) and 3 (mrk) fimbrial adhesins. The rmtG plasmidial 16S rRNA methyltransferase gene was also detected, as well as efflux pumps belonging to five different families. Mutations upstream the OmpK35 porin-encoding gene were evidenced, possibly affecting its expression. SNPs analysis relative to the compared strains revealed 141 mutations falling within CDSs related to drug resistance which could also influence the Kp13 lifestyle. Finally, the genetic apparatus for synthesis of the yersiniabactin siderophore was identified within a plasticity region. Chromosomal architectural analysis allowed for the detection of 13 regions of difference in Kp13 relative to the compared strains. CONCLUSIONS: Our results indicate that the plasticity occurring at many hierarchical levels (from whole genomic segments to individual nucleotide bases) may play a role on the lifestyle of K. pneumoniae Kp13 and underlie the importance of whole-genome sequencing to study bacterial pathogens. The general chromosomal structure was somewhat conserved among the compared bacteria, and recombination events with consequent gain/loss of genomic segments appears to be driving the evolution of these strains.

Complete Genome Sequence of a Variant of the Methicillin-Resistant Staphylococcus aureus ST239 Lineage, Strain BMB9393, Displaying Superior Ability To Accumulate ica-Independent Biofilm.

Biofilm is considered an important virulence factor in nosocomial infections. Herein, we report the complete genome sequence of a variant of methicillin-resistant Staphylococcus aureus, strain BMB9393, which is highly disseminated in Brazil. This strain belongs to the lineage ST239 and displays increased ability to accumulate ica-independent biofilm and to invade human epithelial cells.

Genomic basis of broad host range and environmental adaptability of Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 which are used in inoculants for common bean (Phaseolus vulgaris L.).

BACKGROUND: Rhizobium tropici CIAT 899 and Rhizobium sp. PRF 81 are α-Proteobacteria that establish nitrogen-fixing symbioses with a range of legume hosts. These strains are broadly used in commercial inoculants for application to common bean (Phaseolus vulgaris) in South America and Africa. Both strains display intrinsic resistance to several abiotic stressful conditions such as low soil pH and high temperatures, which are common in tropical environments, and to several antimicrobials, including pesticides. The genetic determinants of these interesting characteristics remain largely unknown. RESULTS: Genome sequencing revealed that CIAT 899 and PRF 81 share a highly-conserved symbiotic plasmid (pSym) that is present also in Rhizobium leucaenae CFN 299, a rhizobium displaying a similar host range. This pSym seems to have arisen by a co-integration event between two replicons. Remarkably, three distinct nodA genes were found in the pSym, a characteristic that may contribute to the broad host range of these rhizobia. Genes for biosynthesis and modulation of plant-hormone levels were also identified in the pSym. Analysis of genes involved in stress response showed that CIAT 899 and PRF 81 are well equipped to cope with low pH, high temperatures and also with oxidative and osmotic stresses. Interestingly, the genomes of CIAT 899 and PRF 81 had large numbers of genes encoding drug-efflux systems, which may explain their high resistance to antimicrobials. Genome analysis also revealed a wide array of traits that may allow these strains to be successful rhizosphere colonizers, including surface polysaccharides, uptake transporters and catabolic enzymes for nutrients, diverse iron-acquisition systems, cell wall-degrading enzymes, type I and IV pili, and novel T1SS and T5SS secreted adhesins. CONCLUSIONS: Availability of the complete genome sequences of CIAT 899 and PRF 81 may be exploited in further efforts to understand the interaction of tropical rhizobia with common bean and other legume hosts.

Pyrosequencing-based analysis reveals a novel capsular gene cluster in a KPC-producing Klebsiella pneumoniae clinical isolate identified in Brazil.

BACKGROUND: An important virulence factor of Klebsiella pneumoniae is the production of capsular polysaccharide (CPS), a thick mucus layer that allows for evasion of the host's defense and creates a barrier against antibacterial peptides. CPS production is driven mostly by the expression of genes located in a locus called cps, and the resulting structure is used to distinguish between different serotypes (K types). In this study, we report the unique genetic organization of the cps cluster from K. pneumoniae Kp13, a clinical isolate recovered during a large outbreak of nosocomial infections that occurred in a Brazilian teaching hospital. RESULTS: A pyrosequencing-based approach showed that the cps region of Kp13 (cpsKp13) is 26.4 kbp in length and contains genes common, although not universal, to other strains, such as the rmlBADC operon that codes for L-rhamnose synthesis. cpsKp13 also presents some unique features, like the inversion of the wzy gene and a unique repertoire of glycosyltransferases. In silico comparison of cpsKp13 RFLP pattern with 102 previously published cps PCR-RFLP patterns showed that cpsKp13 is distinct from the C patterns of all other K serotypes. Furthermore, in vitro serotyping showed only a weak reaction with capsular types K9 and K34. We confirm that K9 cps shares common genes with cpsKp13 such as the rmlBADC operon, but lacks features like uge and Kp13-specific glycosyltransferases, while K34 capsules contain three of the five sugars that potentially form the Kp13 CPS. CONCLUSIONS: We report the first description of a cps cluster from a Brazilian clinical isolate of a KPC-producing K. pneumoniae. The gathered data including K-serotyping support that Kp13's K-antigen belongs to a novel capsular serotype. The CPS of Kp13 probably includes L-rhamnose and D-galacturonate in its structure, among other residues. Because genes involved in L-rhamnose biosynthesis are absent in humans, this pathway may represent potential targets for the development of antimicrobial agents. Studying the capsular serotypes of clinical isolates is of great importance for further development of vaccines and/or novel therapeutic agents. The distribution of K-types among multidrug-resistant isolates is unknown, but our findings may encourage scientists to perform K-antigen typing of KPC-producing strains worldwide.