Improving phylogeny reconstruction at the strain level using peptidome datasets

Typical bacterial strain differentiation methods are often challenged by high genetic similarity between strains. To address this problem, we introduce a novel in silico peptide fingerprinting method based on conventional wet-lab protocols that enables the identification of potential strain-specific peptides. These can be further investigated using in vitro approaches, laying a foundation for the development of biomarker detection and application-specific methods. This novel method aims at reducing large amounts of comparative peptide data to binary matrices while maintaining a high phylogenetic resolution. The underlying case study concerns the Bacillus cereus group, namely the differentiation of Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus strains. Results show that trees based on cytoplasmic and extracellular peptidomes are only marginally in conflict with those based on whole proteomes, as inferred by the established Genome-BLAST Distance Phylogeny (GBDP) method. Hence, these results indicate that the two approaches can most likely be used complementarily even in other organismal groups. The obtained results confirm previous reports about the misclassification of many strains within the B. cereus group. Moreover, our method was able to separate the B. anthracis strains with high resolution, similarly to the GBDP results as benchmarked via Bayesian inference and both Maximum Likelihood and Maximum Parsimony. In addition to the presented phylogenomic applications, whole-peptide fingerprinting might also become a valuable complementary technique to digital DNA-DNA hybridization, notably for bacterial classification at the species and subspecies level in the future.This research was funded by Grant AGL2013-44039-R from the Spanish “Plan Estatal de I+D+I”, and by Grant EM2014/046 from the “Plan Galego de investigación, innovación e crecemento 2011-2015”. BS was recipient of a Ramón y Cajal postdoctoral contractfrom the Spanish Ministry of Economyand Competitiveness. This work was also partially funded by the [14VI05] Contract-Programme from the University of Vigo and the Agrupamento INBIOMED from DXPCTSUG-FEDER unha maneira de facer Europa (2012/273).The research leading to these results has also received funding from the European Union’s Seventh Framework Programme FP7/REGPOT-2012-2013.1 under grant agreement n˚ 316265, BIOCAPS. This document reflects only the authors’ views and the European Union is not liable for any use that may be made of the information contained herein. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Modelling, prediction and classification of student academic performance using artificial neural networks

The conventional statistical evaluations are limited in providing good predictions of the university educational quality. This paper presents an approach with both conventional statistical analysis and neural network modelling/prediction of students’ performance. Conventional statistical evaluations are used to identify the factors that likely affect the students’ performance. The neural network is modelled with 11 input variables, two layers of hidden neurons, and one output layer. Levenberg–Marquardt algorithm is employed as the backpropagation training rule. The performance of neural network model is evaluated through the error performance, regression, error histogram, confusion matrix and area under the receiver operating characteristics curve. Overall, the neural network model has achieved a good prediction accuracy of 84.8%, along with limitation

Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections

<p>Abstract</p> <p>Background</p> <p>The number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission.</p> <p>Methods</p> <p>Baseline HIV-1 <it>pol </it>sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics.</p> <p>Results</p> <p>Multivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with <it>Chlamydia </it>(p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), <it>Chlamydia </it>infection (p = 0.013) and primary HIV (p = 0.017).</p> <p>Conclusions</p> <p>Combination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.</p

Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales)

BACKGROUND: The epsilon proteobacteria, which include many important human pathogens, are presently recognized solely on the basis of their branching in rRNA trees. No unique molecular or biochemical characteristics specific for this group are known. RESULTS: Comparative analyses of proteins in the genomes of Wolinella succinogenes DSM 1740 and Campylobacter jejuni RM1221 against all available sequences have identified a large number of proteins that are unique to various epsilon proteobacteria (Campylobacterales), but whose homologs are not detected in other organisms. Of these proteins, 49 are uniquely found in nearly all sequenced epsilon-proteobacteria (viz. Helicobacter pylori (26695 and J99), H. hepaticus, C. jejuni (NCTC 11168, RM1221, HB93-13, 84-25, CF93-6, 260.94, 11168 and 81-176), C. lari, C. coli, C. upsaliensis, C. fetus, W. succinogenes DSM 1740 and Thiomicrospira denitrificans ATCC 33889), 11 are unique for the Wolinella and Helicobacter species (i.e. Helicobacteraceae family) and many others are specific for either some or all of the species within the Campylobacter genus. The primary sequences of many of these proteins are highly conserved and provide novel resources for diagnostics and therapeutics. We also report four conserved indels (i.e. inserts or deletions) in widely distributed proteins (viz. B subunit of exinuclease ABC, phenylalanyl-tRNA synthetase, RNA polymerase β '-subunit and FtsH protein) that are specific for either all epsilon proteobacteria or different subgroups. In addition, a rare genetic event that caused fusion of the genes for the largest subunits of RNA polymerase (rpoB and rpoC) in Wolinella and Helicobacter is also described. The inter-relationships amongst Campylobacterales as deduced from these molecular signatures are in accordance with the phylogenetic trees based on the 16S rRNA and concatenated sequences for nine conserved proteins. CONCLUSION: These molecular signatures provide novel tools for identifying and circumscribing species from the Campylobacterales order and its subgroups in molecular terms. Although sequence information for these signatures is presently limited to Campylobacterales species, it is likely that many of them will also be found in other epsilon proteobacteria. Functional studies on these proteins and conserved indels should reveal novel biochemical or physiological characteristics that are unique to these groups of epsilon proteobacteria

Survival or Revival: Long-Term Preservation Induces a Reversible Viable but Non-Culturable State in Methane-Oxidizing Bacteria

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium

Pseudomonas aeruginosa Population Structure Revisited

At present there are strong indications that Pseudomonas aeruginosa exhibits an epidemic population structure; clinical isolates are indistinguishable from environmental isolates, and they do not exhibit a specific (disease) habitat selection. However, some important issues, such as the worldwide emergence of highly transmissible P. aeruginosa clones among cystic fibrosis (CF) patients and the spread and persistence of multidrug resistant (MDR) strains in hospital wards with high antibiotic pressure, remain contentious. To further investigate the population structure of P. aeruginosa, eight parameters were analyzed and combined for 328 unrelated isolates, collected over the last 125 years from 69 localities in 30 countries on five continents, from diverse clinical (human and animal) and environmental habitats. The analysed parameters were: i) O serotype, ii) Fluorescent Amplified-Fragment Length Polymorphism (FALFP) pattern, nucleotide sequences of outer membrane protein genes, iii) oprI, iv) oprL, v) oprD, vi) pyoverdine receptor gene profile (fpvA type and fpvB prevalence), and prevalence of vii) exoenzyme genes exoS and exoU and viii) group I pilin glycosyltransferase gene tfpO. These traits were combined and analysed using biological data analysis software and visualized in the form of a minimum spanning tree (MST). We revealed a network of relationships between all analyzed parameters and non-congruence between experiments. At the same time we observed several conserved clones, characterized by an almost identical data set. These observations confirm the nonclonal epidemic population structure of P. aeruginosa, a superficially clonal structure with frequent recombinations, in which occasionally highly successful epidemic clones arise. One of these clones is the renown and widespread MDR serotype O12 clone. On the other hand, we found no evidence for a widespread CF transmissible clone. All but one of the 43 analysed CF strains belonged to a ubiquitous P. aeruginosa “core lineage” and typically exhibited the exoS+/exoU− genotype and group B oprL and oprD alleles. This is to our knowledge the first report of an MST analysis conducted on a polyphasic data set

Molecular Epidemiology of Endemic Human T-Lymphotropic Virus Type 1 in a Rural Community in Guinea-Bissau

Human T-Lymphotropic Virus type 1 (HTLV-1) affects millions of people worldwide. It is very similar to Simian T-Lymphotropic Virus, a virus that circulates in monkeys. HTLV-1 causes a lethal form of leukemia (Adult T-cell Leukemia) and a debilitating neurological syndrome (HTLV-associated myelopathy/tropical spastic paraparesis) in approximately 5% of infected people. Based on sequence variation, HTLV-1 can be divided into 7 subtypes (1a–1g) with the Cosmopolitan subtype 1a further subdivided into subgroups (A–E). We examined HTLV-1 diversity in a rural area in Guinea-Bissau, a country in West Africa with a high HTLV-1 prevalence (5%). We found that most viruses belong to the Cosmopolitan subtype 1a, subgroup D, but 2 viruses belonged to subtype 1g. This subtype had thus far only been found in monkey hunters in Cameroon, who were probably recently infected by monkeys. Our findings indicate that this subtype has spread beyond Central Africa. An important, unresolved question is whether persons with this subtype were infected by monkeys or through human-to-human transmission

Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4

<p>Abstract</p> <p>Background</p> <p>Human T-lymphotropic virus type 4 (HTLV-4) is a new deltaretrovirus recently identified in a primate hunter in Cameroon. Limited sequence analysis previously showed that HTLV-4 may be distinct from HTLV-1, HTLV-2, and HTLV-3, and their simian counterparts, STLV-1, STLV-2, and STLV-3, respectively. Analysis of full-length genomes can provide basic information on the evolutionary history and replication and pathogenic potential of new viruses.</p> <p>Results</p> <p>We report here the first complete HTLV-4 sequence obtained by PCR-based genome walking using uncultured peripheral blood lymphocyte DNA from an HTLV-4-infected person. The HTLV-4(1863LE) genome is 8791-bp long and is equidistant from HTLV-1, HTLV-2, and HTLV-3 sharing only 62–71% nucleotide identity. HTLV-4 has a prototypic genomic structure with all enzymatic, regulatory, and structural proteins preserved. Like STLV-2, STLV-3, and HTLV-3, HTLV-4 is missing a third 21-bp transcription element found in the long terminal repeats of HTLV-1 and HTLV-2 but instead contains unique c-Myb and pre B-cell leukemic transcription factor binding sites. Like HTLV-2, the PDZ motif important for cellular signal transduction and transformation in HTLV-1 and HTLV-3 is missing in the C-terminus of the HTLV-4 Tax protein. A basic leucine zipper (b-ZIP) region located in the antisense strand of HTLV-1 and believed to play a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-4. Detailed phylogenetic analysis shows that HTLV-4 is clearly a monophyletic viral group. Dating using a relaxed molecular clock inferred that the most recent common ancestor of HTLV-4 and HTLV-2/STLV-2 occurred 49,800 to 378,000 years ago making this the oldest known PTLV lineage. Interestingly, this period coincides with the emergence of <it>Homo sapiens sapiens </it>during the Middle Pleistocene suggesting that early humans may have been susceptible hosts for the ancestral HTLV-4.</p> <p>Conclusion</p> <p>The inferred ancient origin of HTLV-4 coinciding with the appearance of <it>Homo sapiens</it>, the propensity of STLVs to cross-species into humans, the fact that HTLV-1 and -2 spread globally following migrations of ancient populations, all suggest that HTLV-4 may be prevalent. Expanded surveillance and clinical studies are needed to better define the epidemiology and public health importance of HTLV-4 infection.</p

Impacts of Poultry House Environment on Poultry Litter Bacterial Community Composition

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly Gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens

Bacteria-inducing legume nodules involved in the improvement of plant growth, health and nutrition

Bacteria-inducing legume nodules are known as rhizobia and belong to the class Alphaproteobacteria and Betaproteobacteria. They promote the growth and nutrition of their respective legume hosts through atmospheric nitrogen fixation which takes place in the nodules induced in their roots or stems. In addition, rhizobia have other plant growth-promoting mechanisms, mainly solubilization of phosphate and production of indoleacetic acid, ACC deaminase and siderophores. Some of these mechanisms have been reported for strains of rhizobia which are also able to promote the growth of several nonlegumes, such as cereals, oilseeds and vegetables. Less studied are the mechanisms that have the rhizobia to promote the plant health; however, these bacteria are able to exert biocontrol of some phytopathogens and to induce the plant resistance. In this chapter, we revised the available data about the ability of the legume nodule-inducing bacteria for improving the plant growth, health and nutrition of both legumes and nonlegumes. These data showed that rhizobia meet all the requirements of sustainable agriculture to be used as bio-inoculants allowing the total or partial replacement of chemicals used for fertilization or protection of crops