Identification of Variant Compositions in Related Strains Without Reference

Peer reviewe

Identification of variant compositions in related strains without reference

The genomes of all animals, plants and fungi are organized into chromosomes, which contain a sequence of the four nucleotides A, T, C and G. Chromosomes are further arranged into homologous groups, where two or more chromosomes are almost exact copies of each others. Species whose homologous groups contain pairs of chromosomes, such as humans, are called diploid. Species with more than two chromosomes in a homologous group are called polyploid. DNA sequencing technologies do not read an entire chromosome from end to end. Instead, the results of DNA sequencing are small sequences called reads or fragments. Due to the difficulty of assembling the full genome from reads, a reference genome is not always available for a species. For this reason, reference-free algorithms which do not use a reference genome are useful for poorly understood genomes. A common variation between the chromosomes in a homologous group is the single nucleotide polymorhpism (SNP), where the sequences differ by exactly one nucleotide at a location. Genomes are sometimes represented as a consensus sequence and a list of SNPs, without information about which variants of a SNP belong in which chromosome. This discards useful information about the genome. Identification of variant compositions aims to correct this. A variant composition is an assignment of the variants in a SNP to the chromosomes. Identification of variant compositions is closely related to haplotype assembly, which aims to solve the sequences of an organism's chromosomes, and variant detection, which aims to solve the sequences of a population of bacterial strains and their frequencies in the population. This thesis extends an existing exact algorithm for haplotype assembly of diploid species (Patterson et al, 2014) to the reference-free, polyploid case. Since haplotype assembly is NP-hard, the algorithm's time complexity is exponential to the maximum coverage of the input. Coverage means the number of reads which cover a position in the genome. Lowering the coverage of the input is necessary. Since the algorithm does not use a reference genome, the reads must be ordered in some other way. Ordering reads is an NP-hard problem and the technique of matrix banding (Junttila, PhD thesis, 2011) is used to approxiately order the reads to lower coverage. Some heuristics are also presented for merging reads. Experiments with simulated data show that the algorithm's accuracy is promising. The source code of the implementation and scripts for running the experiments are available online at https://github.com/maickrau/haplotyper

Population structures in the SARA and SARB reference collections of Salmonella enterica according to MLST, MLEE and microarray hybridization

In the 1980's and 1990's, population genetic analyses based on Multilocus Enzyme Electrophoresis (MLEE) provided an initial overview of the genetic diversity of multiple bacterial species, including Salmonella enterica. The genetic diversity within S. enterica subspecies enterica according to MLEE is represented by the SARA and SARB reference collections, each consisting of 72 isolates, which have been extensively used for comparative analyses. MLEE has subsequently been replaced by Multilocus Sequence Typing (MLST). Our initial MLST results indicated that some strains within the SARB collection differed from their published descriptions. We therefore performed MLST on four versions of the SARB collection from different sources and one collection of SARA, and found that multiple isolates in SARB and SARA differ in serovar from their original description, and other SARB isolates differed between different sources. Comparisons with a global MLST database allowed a plausible reconstruction of the serovars of the original collection. MLEE, MLST and microarrays were largely concordant at recognizing closely related strains. MLST was particularly effective at recognizing discrete population genetic groupings while the two other methods provided hints of higher order relationships. However, quantitative pair-wise phylogenetic distances differed considerably between all three methods. Our results provide a translation dictionary from MLEE to MLST for the extant SARA and SARB collections which can facilitate genomic comparisons based on archival insights from MLEE

Characterization and Comparison of 2 Distinct Epidemic Community-Associated Methicillin-Resistant Staphylococcus aureus Clones of ST59 Lineage.

Sequence type (ST) 59 is an epidemic lineage of community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) isolates. Taiwanese CA-MRSA isolates belong to ST59 and can be grouped into 2 distinct clones, a virulent Taiwan clone and a commensal Asian-Pacific clone. The Taiwan clone carries the Panton-Valentine leukocidin (PVL) genes and the staphylococcal chromosomal cassette mec (SCCmec) VT, and is frequently isolated from patients with severe disease. The Asian-Pacific clone is PVL-negative, carries SCCmec IV, and a frequent colonizer of healthy children. Isolates of both clones were characterized by their ability to adhere to respiratory A549 cells, cytotoxicity to human neutrophils, and nasal colonization of a murine and murine sepsis models. Genome variation was determined by polymerase chain reaction of selected virulence factors and by multi-strain whole genome microarray. Additionally, the expression of selected factors was compared between the 2 clones. The Taiwan clone showed a much higher cytotoxicity to the human neutrophils and caused more severe septic infections with a high mortality rate in the murine model. The clones were indistinguishable in their adhesion to A549 cells and persistence of murine nasal colonization. The microarray data revealed that the Taiwan clone had lost the ø3-prophage that integrates into the β-hemolysin gene and includes staphylokinase- and enterotoxin P-encoding genes, but had retained the genes for human immune evasion, scn and chps. Production of the virulence factors did not differ significantly in the 2 clonal groups, although more α-toxin was expressed in Taiwan clone isolates from pneumonia patients. In conclusion, the Taiwan CA-MRSA clone was distinguished by enhanced virulence in both humans and an animal infection model. The evolutionary acquisition of PVL, the higher expression of α-toxin, and possibly the loss of a large portion of the β-hemolysin-converting prophage likely contribute to its higher pathogenic potential than the Asian-Pacific clone

Survival of thermophilic spore-forming bacteria in a 90+ year old milk powder from Ernest Shackelton's Cape Royds Hut in Antarctica

Milk powder taken to Antarctica on Shackelton's British Antarctic Expedition in 1907 was produced in New Zealand by a roller drying process in the first factory in the world dedicated to this process. Thermophilic bacilli are the dominant contaminants of modern spray-dried milk powders and the 1907 milk powder allows a comparison to be made of contaminating strains in roller-dried and spray-dried powders. Samples of milk powder obtained from Shackelton's Hut at Cape Royds had low levels of thermophilic contamination (<500 cfu ml−1) but the two dominant strains (Bacillus licheniformis strain F and Bacillus subtilis) were typical of those found in spray-dried powders. Soil samples from the floor of the hut also contained these strains, whereas soils distant from the hut did not. Differences in the RAPD profiles of isolates from the milk powder and the soils suggest that contamination of the milk from the soil was unlikely. It is significant that the most commonly encountered contaminant strain in modern spray-dried milk (Anoxybacillus flavithermus strain C) was not detected in the 1907 sample

Strain-resolved analysis of the human intestinal microbiota

The gut microbiota is ascribed a crucial role in human health, particularly in regulating immune and inflammatory responses, which is why it is being associated with a wide range of diseases, including obesity, diabetes, and cancer. Nonetheless, fundamental ecological questions of microbiome establishment, stability and resilience, as well as its transmission across hosts and generations remain incompletely understood, partly due to the lack of methods for high-resolution microbiome profiling. New insights in this field can therefore directly contribute to the development of bacterial and microbiota-based therapies. This work introduces SameStr, a novel bioinformatic program for strain-resolved metagenomics that allows for the specific tracking of microbes across samples, enabling the detection and quantification of microbial transmission and persistence, as well as the observation of direct strain competition. Deployed across cohorts to process over 4200 metagenomes, SameStr enabled analysis of the microbiome with unprecedented phylogenetic resolution. The data included both publicly available metagenomes and sequence data generated in collaboration with our research partners, and was examined using multivariate statistics and machine learning frameworks. First, the establishment and development of the neonatal microbiota was studied, revealing a birth mode-dependent vertical transmission of the maternal microbiota. The microbiota of neonates born by cesarean section was characterized by increased relative abundance of oxygen-tolerant and atypical organisms and showed signs of a delayed establishment of a strictly anaerobic gut environment in these children. Such birth mode-dependent differences diminished over time, yet were measurable within the first two years of life. Furthermore, strain analysis verified the transmission and colonization of parental microbes, which indicated a possible lifelong colonization by microbes from selected species. The temporal persistence of microbes was also characterized in healthy adults, revealing similar taxonomy-dependent patterns of stability. For some species, persistence has been demonstrated both in children and in adults over a period of at least two years. These species are known for their capability to metabolize host-derived glycans found both in breastmilk and intestinal mucus, pointing to a potential strategy for effective cross-generational microbiota transmission, and warranting additional research to assess the implications of their disturbed transfer for long-term health. Since their specificity allows assignment to individual hosts, fingerprints of individual microbial strains offer the potential to be used in forensics and data quality control applications. Finally, to gain new insights into the microbiota dynamics during Fecal Microbiota Transplantation (FMT), microbial strain transmission was analyzed in the context of a diverse set of patient, microbiome, and clinical conditions. In the analyzed studies, FMT was used for the experimental treatment of a variety of diseases, including colonization with drug-resistant and pathogenic microbes, metabolic and inflammatory bowel diseases, and as an adjunct to the immunotherapeutic treatment of cancer. Analyses uncovered what appear to be the universal drivers of post-FMT microbiota assembly, including clinical and ecological factors that are important for successful transplantation of donor strains. In particular, the relevance of the microbiota dysbiosis of the recipient was emphasized, which was inducible by pre-treating the patient with antibiotics or laxatives. Presumably, this can open up ecological niches in the patients intestines, which favors colonization with donor strains. Colonization rates did not play a role for the treatment success of recurrent C. difficile infections and inflammatory bowel disease, but indicated a trend associated with an improved immune response in cancer patients. Concerningly, the transfer of an atypical and potentially pro-inflammatory microbial community from one donor was also observed, calling for further investigations into the immediate and long-term clinical consequences of FMT. These analyses demonstrate the advantages of a strain-based microbiome analysis. Due to the achieved methodological accuracy, strain-resolved microbial dynamics could be precisely disentangled when comparing longitudinal samples from healthy adults as well as parent-child and patient-donor pairs. This revealed taxonomic, clinical, and ecological factors that are critical to microbiome assembly, including microbial transmission, persistence, and competition. Together, these findings lay the groundwork for future developments of precision personalized microbiota modulation therapies.Der Darmmikrobiota wird eine entscheidende Rolle für die menschliche Gesundheit zugeschrieben, was insbesondere die Regulation von Immun- und Entzündungsreaktionen betrifft, weshalb sie mit einer Vielzahl von Krankheiten wie etwa Fettleibigkeit, Diabetes oder Krebs in Verbindung gebracht wird. Nichtsdestotrotz sind grundlegende ökologische Fragen der Etablierung, Stabilität und Resilienz von Mikrobiomen sowie ihrer Übertragung über Wirte und Generationen hinweg noch immer unvollständig untersucht, was teilweise auf das Fehlen von Methoden zur hochauflösenden Mikrobiom-Profilierung zurückzuführen ist. Neue Erkenntnisse auf diesem Gebiet können daher unmittelbar zur Entwicklung von Bakterien- und Mikrobiota-basierten Therapien beitragen. Diese Arbeit stellt SameStr vor, ein neues bioinformatisches Programm für stammaufgelöste Metagenomik, das die spezifische probenübergreifende Untersuchung von Mikroorganismen ermöglicht. Hiermit können der Nachweis und die Quantifizierung der Übertragung und Persistenz, sowie die Beobachtung der direkten Konkurrenz mikrobieller Stämme erfolgen. SameStr wurde kohortenübergreifend für die Analyse von über 4200 Metagenomen eingesetzt und ermöglichte die Profilierung des Mikrobioms mit einer beispiellosen phylogenetischen Auflösung. Die Metagenome, welche sowohl öffentlich verfügbare als auch in Zusammenarbeit mit unseren Forschungspartnern generierte Daten beinhalteten, konnten mittels multivariater Statistik und maschinellen Lernens beleuchtet werden. Zunächst wurde die Etablierung und Entwicklung der neonatalen Mikrobiota analysiert, was eine vom Geburtsmodus abhängige vertikale Übertragung der mütterlichen Mikrobiota aufzeigte. Die Mikrobiota von Neugeborenen die durch einen Kaiserschnitt zur Welt gekommen waren, war vermehrt von Sauerstoff-toleranten und Darm-untypischen Organismen besiedelt und deutete darauf hin, dass sich ein strikt anaerobes Darmmilieu bei diesen Kindern mit einer gewissen Verzögerung einstellte. Derartige geburtsabhängige Veränderungen schwächten sich mit der Zeit ab, waren jedoch bis zum zweiten Lebensjahr messbar. Weiterhin konnte die Übertragung und Kolonisierung elterlicher Organismen mittels Stamm-Analyse nachgewiesen werden, was außerdem auf eine mögliche lebenslange Besiedlung durch Mikroben ausgewählter Spezies hindeutete. Die zeitliche Persistenz von Mikroorganismen wurde darüber hinaus auch bei gesunden Erwachsenen charakterisiert, was ebenfalls Taxonomie-abhängige Stabilitätsmuster zum Vorschein brachte. Bei einigen Spezies, die bekannt dafür sind vom menschlichen Wirt stammende Glykane zu metabolisieren, wurde die Persistenz sowohl bei Kindern als auch bei Erwachsenen über einen Zeitraum von mindestens zwei Jahren nachgewiesen. Diese Glykane kommen sowohl in der Muttermilch als auch im Darmschleim vor, was auf eine potenzielle Strategie für eine effektive generationsübergreifende Übertragung der Mikrobiota hinweist. Um die langfristigen Auswirkungen einer gestörten Mikrobiota-Übertragung auf die Gesundheit bewerten zu können, wird jedoch weitere Forschung benötigt. Da ihre Spezifität die Zuordnung zu individuellen Wirten ermöglicht, bieten mikrobielle Stämme zudem das Potenzial in der Forensik und bei Datenqualitätstests Anwendung zu finden. Um schließlich neue Erkenntnisse zur Mikrobiota-Dynamik während der fäkalen Mikrobiota-Transplantation (FMT) zu gewinnen, wurde die Stammübertragung im Kontext einer Vielzahl von Patienten-, Mikrobiom- und klinischen Parametern analysiert. FMT wurde in den vorliegenden Studien zur experimentellen Behandlung verschiedenster Erkrankungen eingesetzt, darunter Kolonisierung mit resistenten und pathogenen Keimen, metabolische Erkrankungen, entzündliche Erkrankungen des Darms, sowie begleitend zur immuntherapeutischen Behandlung von Krebs. Die Analysen zeigten scheinbar universelle klinische und ökologische Faktoren auf, welche für eine erfolgreiche Integration von Spenderstämmen von Bedeutung sind. Insbesondere wurde die Relevanz der Mikrobiota-Dysbiose des Empfängers hervorgehoben, welche zudem durch Vorbehandlung der Patienten mittels Gabe von Antibiotika oder Laxativa induziert werden kann. Vermutlich können hierdurch ökologische Nischen im Darm der Patienten eröffnet werden, was eine Kolonisierung mit Spenderstämmen begünstigt. Kolonisierungsraten spielten für den Behandlungserfolg wiederkehrender Clostridien-Infektionen und entzündlicher Darmerkrankungen keine Rolle, deuteten jedoch auf einen Trend hin, der mit einer verbesserten Immunantwort bei Krebspatienten einhergeht. Beunruhigenderweise wurde auch die Übertragung einer atypischen und potenziell entzündungsfördernden Mikrobiota eines Donoren beobachtet, was weitere Untersuchungen zu unmittelbaren und langfristigen klinischen Folgen der FMT erforderlich macht. Die Ergebnisse dieser Arbeit zeigen die Vorteile einer Stamm-basierten Mikrobiom-Analyse auf. Durch die erreichte methodische Genauigkeit konnten bei Vergleichen von Zeitverlaufsproben gesunder Erwachsener sowie Eltern-Kind- und Patienten-Spender-Paaren, die Dynamiken mikrobieller Stämme präzise entschlüsselt werden. Dabei kamen taxonomische, klinische und ökologische Faktoren zum Vorschein, welche für die Zusammensetzung der Mikrobiota, einschließlich der mikrobiellen Übertragung, Persistenz und Kompetition, maßgebend sind. Diese neuen Erkenntnisse bilden die Grundlage für künftige Entwicklungen von Therapien zur präzisen, personalisierten Modulation der Mikrobiota

The structure and diversity of strain-level variation in vaginal bacteria

The vaginal microbiome plays an important role in human health and species of vaginal bacteria have been associated with reproductive disease. Strain-level variation is also thought to be important, but the diversity, structure and evolutionary history of vaginal strains is not as well characterized. We developed and validated an approach to measure strain variation from metagenomic data based on SNPs within the core genomes for six species of vaginal bacteria

Ex Vivo and In Vivo Methods and Related Compositions for Regenerating Hematopoietic Stem Cell Populations

Various embodiments provide methods and related compositions for increasing the population size ofhematopoietic stem cells (HSCs) in patients that may benefit from reconstitution of stem cells and/or differentiated cells of the blood lineage. The present methods enable the production ofHSCs ex vivo and in vivo by reducing latexin expression and/or latexin activity within HSC exposed to various antagonists. Inhibition of latexin expression and/or latexin activity by various antagonists can promote HSC proliferation and/or inhibit HSC apoptosis. Antagonists that can reduce latexin expression and/or latexin activity can be utilized to regenerate endogenous HSCs within patients affected with disorders, diseases, cancers, or therapies for such conditions, that result in the depletion or reduction in HSCs

Patent Landscape of Influenza A Virus Prophylactic Vaccines and Related Technologies

Executive Summary: This report focuses on patent landscape analysis of technologies related to prophylactic vaccines targeting pandemic strains of influenza. These technologies include methods of formulating vaccine, methods of producing of viruses or viral subunits, the composition of complete vaccines, and other technologies that have the potential to aid in a global response to this pathogen. The purpose of this patent landscape study was to search, identify, and categorize patent documents that are relevant to the development of vaccines that can efficiently promote the development of protective immunity against pandemic influenza virus strains. The search strategy used keywords which the team felt would be general enough to capture (or “recall”) the majority of patent documents which were directed toward vaccines against influenza A virus. After extensive searching of patent literature databases, approximately 33,500 publications were identified and collapsed to about 3,800 INPADOC families. Relevant documents, almost half of the total, were then identified and sorted into the major categories of vaccine compositions (about 570 families), technologies which support the development of vaccines (about 750 families), and general platform technologies that could be useful but are not specific to the problems presented by pandemic influenza strains (about 560 families). The first two categories, vaccines and supporting technologies, were further divided into particular subcategories to allow an interested reader to rapidly select documents relevant to the particular technology in which he or she is focused. This sorting process increased the precision of the result set. The two major categories (vaccines and supporting technologies) were subjected to a range of analytics in order to extract as much information as possible from the dataset. First, patent landscape maps were generated to assess the accuracy of the sorting procedure and to reveal the relationships between the various technologies that are involved in creating an effective vaccine. Then, filings trends are analyzed for the datasets. The country of origin for the technologies was determined, and the range of distribution to other jurisdictions was assessed. Filings were also analyzed by year, by assignee, and by inventor. Finally, the various patent classification systems were mapped to find which particular classes tend to hold influenza vaccine-related technologies. Besides the keywords developed during the searches and the landscape map generation, the classifications represent an alternate way for further researchers to identify emerging influenza technologies. The analysis included creation of a map of keywords, as shown above, describing the relationship of the various technologies involved in the development of prophylactic influenza A vaccines. The map has regions corresponding to live attenuated virus vaccines, subunit vaccines composed of split viruses or isolated viral polypeptides, and plasmids used in DNA vaccines. Important technologies listed on the map include the use of reverse genetics to create reassortant viruses, the growth of viruses in modified cell lines as opposed to the traditional methods using eggs, the production of recombinant viral antigens in various host cells, and the use of genetically-modified plants to produce virus-like particles. Another major finding was that the number of patent documents related to influenza being published has been steadily increasing in the last decade, as shown in the figure below. Until the mid-1990s, there were only a few influenza patent documents being published each year. The number of publications increased noticeably when TRIPS took effect, resulting in publication of patent applications. However, since 2006 the number of vaccine publications has exploded. In each of 2011 and 2012, about 100 references disclosing influenza vaccine technologies were published. Thus, interest in developing new and more efficacious influenza vaccines has been growing in recent years. This interest is probably being driven by recent influenza outbreaks, such as the H5N1 (bird flu) epidemic that began in the late 1990s and the 2009 H1N1 (swine flu) pandemic. The origins of the vaccine-related inventions were also analyzed. The team determined the country in which the priority application was filed, which was taken as an indication of the country where the invention was made or where the inventors intended to practice the invention. By far, most of the relevant families originated with patent applications filed in the United States. Other prominent priority countries were the China and United Kingdom, followed by Japan, Russia, and South Korea. France was a significant priority country only for supporting technologies, not for vaccines. Top assignees for these families were mostly large pharmaceutical companies, with the majority of patent families coming from Novartis, followed by GlaxoSmithKline, Pfizer, U.S. Merck (Merck, Sharpe, & Dohme), Sanofi, and AstraZeneca. Governmental and nonprofit institutes in China, Japan, Russia, South Korea and the United States also are contributing heavily to influenza vaccine research. Lastly, the jurisdictions were inventors have sought protection for their vaccine technologies were determined, and the number of patent families filing in a given country is plotted on the world map shown on page seven. The United States, Canada, Australia, Japan, South Korea and China have the highest level of filings, followed by Germany, Brazil, India, Mexico and New Zealand. However, although there are a significant number of filings in Brazil, the remainder of Central and South America has only sparse filings. Of concern, with the exception of South Africa, few other African nations have a significant number of filings. In summary, the goal of this report is to provide a knowledge resource for making informed policy decisions and for creating strategic plans concerning the assembly of efficacious vaccines against a rapidly-spreading, highly virulent influenza strain. The team has defined the current state of the art of technologies involved in the manufacture of influenza vaccines, and the important assignees, inventors, and countries have been identified. This document should reveal both the strengths and weaknesses of the current level of preparedness for responding to an emerging pandemic influenza strain. The effects of H5N1 and H1N1 epidemics have been felt across the globe in the last decade, and future epidemics are very probable in the near future, so preparations are necessary to meet this global health threat

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation