Dissertations / Theses on the topic 'Whole genome sequences (WGS)'

The rare disease pulmonary arterial hypertension (PAH) results in high blood pressure in the lung caused by narrowing of lung arteries. Genes causative in PAH were discovered through family studies and very often harbour rare variants. However, the genetic cause in heritable (31%) and idiopathic (79%) PAH cases is not yet known but are speculated to be caused by rare variants. Advances in high-throughput sequencing (HTS) technologies made it possible to detect variants in 98% of the human genome. A drop in sequencing costs made it feasible to sequence 10,000 individuals including 1,250 subjects diagnosed with PAH and relatives as part of the NIHR Bioresource - Rare (BR-RD) disease study. This large cohort allows the genome-wide identification of rare variants to discover novel causative genes associated with PAH in a case-control study to advance our understanding of the underlying aetiology. In the first part of my thesis, I establish a phenotype capture system that allows research nurses to record clinical measurements and other patient related information of PAH patients recruited to the NIHR BR-RD study. The implemented extensions provide a programmatic data transfer and an automated data release pipeline for analysis ready data. The second part is dedicated to the discovery of novel disease genes in PAH. I focus on one well characterised PAH disease gene to establish variant filter strategies to enrich for rare disease causing variants. I apply these filter strategies to all known PAH disease genes and describe the phenotypic differences based on clinically relevant values. Genome-wide results from different filter strategies are tested for association with PAH. I describe the findings of the rare variant association tests and provide a detailed interrogation of two novel disease genes. The last part describes the data characteristics of variant information, available non SQL (NoSQL) implementations and evaluates the suitability and scalability of distributed compute frameworks to store and analyse population scale variation data. Based on the evaluation, I implement a variant analysis platform that incrementally merges samples, annotates variants and enables the analysis of 10,000 individuals in minutes. An incremental design for variant merging and annotation has not been described before. Using the framework, I develop a quality score to reduce technical variation and other biases. The result from the rare variant association test is compared with traditional methods.

Staphylococcus aureus continues to present challenges to modern healthcare, due to its acquisition of antimicrobial resistance factors, its ability to cause invasive infections associated with significant morbidity and mortality, and its propensity for person-to-person transmission resulting in outbreaks. For outbreak investigation, current typing methods lack resolution, and the relatively slow turnaround times may hinder effective infection control intervention. Whole-genome sequencing (WGS) is rapidly becoming faster and more affordable, offering increased resolution in a comparable timeframe. Having the entire genome means the data can also be explored for resistance and virulence testing. In this thesis, I explore the use of WGS for investigating outbreaks and for predicting antimicrobial resistance phenotype. First, I establish the genetic diversity in the nasal S. aureus population at acquisition and after long-term carriage, to determine the effect of within-host diversity on outbreak WGS interpretation. I then use 15 well characterised S. aureus outbreaks to develop an WGS approach for outbreak investigation. I test this approach by applying WGS to a further 5 outbreaks where the infection control investigation was inconclusive. Combining the epidemiological and WGS data from all 20 outbreaks, I then evaluate whether the WGS data can be used to predict the possibility of a long-term carrier involved in maintaining an outbreak, and apply this in real-time using a rapid turnaround benchtop sequencer. Finally, I explore the use of WGS for predicting antimicrobial resistance phenotype. I conclude that, for outbreaks, WGS has enhanced resolution compared to standard techniques and can give additional information to aid the outbreak investigation. For antimicrobial resistance, WGS is as sensitive and specific as routine testing methods. WGS provides a promising alternative to traditional culture and typing methods for enhancing our understanding of S. aureus and ultimately other pathogens.

From an ecological perspective, knowledge of demographic history is highly valuable because population size fluctuations can be matched to known climatic events, thereby revealing great insight into a species’ reaction to past climate change. This in turn enables us to predict how they might respond to future climate scenarios. Prominently, with the advent of high-throughput sequencing it is now becoming possible to assemble genomes of non-model organisms thereby providing unprecedented resolution to the study of demographic history and speciation. This thesis utilises four species of grouse (Aves, subfamily Tetraoninae) in order to explore the demographic history and speciation within this lineage; the willow grouse, red grouse, rock ptarmigan and the black grouse. I, and my co-authors, begin by reviewing the plethora of methods used to estimate contemporary effective population size (Ne) and demographic history that are available to animal conservation practitioners. We find that their underlying assumptions and necessary input data can bias in their application, and thus we provide a summary of their applicability. I then use the whole genomes of the black grouse, willow grouse and rock ptarmigan to infer their population dynamics within the last million years. I find three dominant periods that shape their demographic history: early Pleistocene cooling (3-0.9 Mya), the mid-Brunhes event (430 kya) and the last glacial period (110-10 kya). I also find strong signals of local population history – recolonization and subdivision events – affecting their demography. In the subsequent study, I explore the grouse dynamics within the last glacial period in more detail by including more distant samples and using ecological modelling to track habitat distribution changes. I further uncover strong signals of local population history, with multiple fringe populations undergoing severe bottlenecks. I also determine that future climate change is expected to drastically constrict the distribution of the studied grouse. Lastly, I use whole genome sequencing to uncover 6 highly differentiated regions, containing 7 genes, hinting at their role in adaptation and speciation in three grouse taxa. I also locate a region of low differentiation, containing the Agouti pigmentation gene, indicating its role in the grouse plumage coloration.

Chicken is a major protein source and intensively selected for economically important traits by humans. As such, this generated a huge range of phenotypes that representing a diverse spectrum of genetic variation. Understanding the functional basis of the genetic variants that underlie these traits, however, remains a formidable endeavour particularly for complex traits. Nonetheless, molecular phenotyping of an organism from sequenced data is doable with the advances in bioinformatics analysis and unparalleled surveys of genome wide genetic variants. This provides the opportunity to gain insights into the genome architecture and assists in identifying chromosomal regions underlying selection through a “sequences to consequences” approach. Combining a whole genome re-sequencing (WGS) approach with the knowledge of selection history, this thesis aimed to study the chromosomal regions and genetic variants underlying traits of interest in various selected chicken populations. To achieve this, genetic (quantitative and population genetics), genomic and bioinformatics approaches were employed and integrated to investigate the genome wide selection signatures in a number of different lines of chicken selected for different complex traits. This includes analysing: (i) divergently selected broilers for fatness traits (Chapter 2), (ii) a closed population of layer chickens (Chapter 3), (iii) selection signatures unique to broiler or layer chickens (Chapter 4) and (iv) selection signatures in colony stimulating factor 1 (CSF1) associated with gene expression differences in broiler and layer populations (Chapter 5). Candidate genes and nucleotides underlying potential selection regions were identified, and attempts were made to further elucidate the potential interplay between genes and the biological pathways involved in regulating traits in these selected chicken lines. Incorporating integrative approaches, variants within selection signatures were annotated to provide further evidence of their functional consequences. Overall, non-coding regions were enriched in selection signatures implied that causative variants may have regulatory roles. Capitalising on the millions of genetic variants discovered from WGS, chromosomal regions subject to selection were detected using a number of population genetics statistics. In broiler chicken lines divergently selected for very low-density plasma lipoprotein (VLDL) (Chapter 2), incorporating signatures of selection helped to improve the resolution of previously mapped quantitative traits loci (QTL) intervals. This research demonstrated that the integration of the analysis of selection signatures with functional annotation of genetic variants enabled refinement and characterisation of the QTL for fatness traits. In a closed population of brown leghorn layers (Chapter 3), evidence of selection signatures was found through Tajima’s D analysis. The analysis unravelled selection signatures encoding genes involved in numerous pathways and genes having key roles such as in behaviour, including feather pecking. Combining population differentiation statistic (FST) and Tajima’s D, a number of regions subject to divergent selection between broilers and white egg layers were identified (Chapter 4). Selection signatures were found to harbour mutations involved in cellular and tissue development, including genes having important roles in growth, fatness, egg shell strength and muscle development. These regions and the overlapping genes thereby may be potentially contributing to the different phenotypic variations observed between broilers and layers. In Chapter 5, a revised gene model for colony stimulating factor 1 (CSF1) showing complex pattern of alternate transcripts was predicted from transcriptome analysis of RNA isolated from 21 different tissues. In parallel, selection signatures analysis with the FST statistic, identified selection signatures that differentiate broilers to white egg layers (3 regions) or brown egg layers to white egg layers (4 regions). All these selection signatures were located within non-coding regions, indicating potential divergent selection of CSF1 within regulatory regions. Overall, the results presented in this thesis using the “sequences to consequences” approach, link several genomic regions and genes to phenotypic variation in domesticated chicken lines. The work reported here serves as a foundation for further study to decipher the relationship between “genotype and phenotype” and its functional consequences due to selection.

In this dissertation I addressed the problem of SNP array bias when finding runs of ho- mozygosity. I demonstrated the pitfalls of using uninformed methods for finding runs of homozygosity and provide better alternatives, including a more reliable algorithm for identi- fying runs of homozygosity than the most commonly used program. I then provide a review of Ashkenazi population genetics. Next, I developed software to efficiently run millions of whole chromosome simulations, which is publicly available through GitHub, DockerHub, and on the CyVerse Discovery Environment. I applied my computational method to use Approximate Bayesian Computation to test models of Ashkenazi Jewish demographic his- tory. I found that the Ashkenazi Jews are comprised of genetically distinct subgroups from Eastern and Western Europe, as a result of massive population growth in the Eastern Ashkenazi Jews, but not in the Western Ashkenazi Jews. I further confirmed that the Ashkenazi Jews do not primarily originate from Khazaria. Finally, I created a correction for SNP array ascertainment bias in the median and total length of runs of homozygosity, and applied this correction to world-wide human populations. However, I found that ascertainment bias plays a minor role compared to SNP array bias in human populations.

Whole-Genome Sequencing (WGS) is ushering a new era in healthcare and research in identifying genetic variation in all populations. However, the African populations are still under-represented. Since African populations are being the most genetically diverse with high heterogeneity rate, we need to benchmark the Whole Genome Sequence (WGS) analysis pipeline to ensure reliable mutation detection. Therefore, it is essential to ensure that all steps of WGS downstream analysis are accurate, mainly the variant calling (VC). Current VC tools may produce falsepositive/negative results; such result may produce misleading conclusions in prioritisation of mutation, clinical relevancy and actionability of genes. With such many VC tools, two questions have arisen. Firstly, which tool has a high rate of sensitivity and precision in low either high coverage African sequences, given they have high genetic diversity and heterogeneity? Secondly, does the improvement of the VC result will advance the accuracy of detecting mutation and incidental finding (actionable genes) in African populations? In this project, a total of 100 DNA sequence samples was simulated (of which every 50 samples mimicked the genetics background of African and European, respectively) at different coverage (high and low). In particular, the sensitivity to discover polymorphisms was done by nine different VC tools. These tools were assessed in term of false positive/negative call rate given the simulated golden variants. Combining our result on sensitivity and positive predictive value (PPV). Lofreq performs best in African population data (sens=0.85, PPV=0.983, F-score=0.91) on high/low coverage data; as a result, we chose Lofreq to perform variant calling, and Gene-based annotation is performed to conduct in-sillico predication of mutation on publicly available data (the African Genome Variation and 1000 Genome Project). In doing so, we have leveraged WGS to examine and validate four of burden diseases in the African content, such as communicable diseases: HIV/AIDS, Malaria, Tuberculosis (TB), and Non-communicable diseases: such as Sickle cell disease, these diseases have uniquely shaped ethnic-specific and continental genomics variation and therefore provides unprecedented opportunities to map disease genes across the African continent. Moreover, the current actionable gene recommended by The American College of Medical Genetics and Genomics (ACMG) in the African population and update on additional African-specific actionable genes. Our result suggests African and African diaspora ethnic groups, particularly Bantu and Khoesan ethnics have gene diversity, high proportion of derived allele at low minor allele frequency (0.0 − 01) and the highest proportion of pathogenic variants within HIV, TB, Malaria, Sickle-Cell disease, while non-African ethnic groups including Latin America, Afro-Asiatic European related ethnic groups have high proportion of pathogenic variants within current actionable gene list. Overall, given the observed highest genetic diversity found in African ethnics and African diaspora related ethnics at these four Africa burden diseases and current actionable gene associated, our results support (1) the use of personalised medicine as beneficial to both African continent and worldwide; (2) a recommendation for African-specific actionable list of genes to further improve African and diaspora healthcare.

The Microsporidia are obligate intracellular parasites that have jettisoned oxidation phosphorylative capabilities during their early evolutionary history and so rely on ATP import from their host and glycolysis for their energy needs. Some species form tight associations with the host’s mitochondria and this is thought to facilitate ATP sequestration by the developing intracellular microsporidian. The human parasite, Enterocytozoon bieneusi has however lost glycolytic capabilities and may rely entirely on ATP import from its host for energy. E. bieneusi belongs to the Enterocytozoonidae microsporidian family and recent rDNA-based phylogenetic studies have suggested it has close evolutionary ties with Enterospora canceri, a crab-infecting intranuclear parasite. Such a close evolutionary relationship implied that glycolysis might also be absent in the intranuclear parasite raising questions as to how this parasite obtains energy from its unusual niche that is physically walled off from the host mitochondria, the main source of ATP in the host cell. In this study, draft genomes of four species of the Enterocytozoonidae namely, Ent. canceri, E. hepatopenaei, Hepatospora eriocheir and Hepatospora eriocheir canceri and one non-Enterocytozoonidae species, Thelohania sp. were assembled and annotated (The genome assembly of Hepatospora eriocheir was provided by Dr. Bryony Williams). Phylogenomics performed with this and publicly available genomic data confirmed the close evolutionary ties between Ent. canceri and E. bieneusi. Comparative genomic analyses also revealed that glycolysis is indeed lost in all members of the Enterocytozoonidae family sequenced in this study, hinting to the relaxation of evolutionary pressures to maintain this pathway at the base of this microsporidian family. Despite this absence, the hexokinase gene was retained in all aglycolytic genomes analysed, and that of Ent. canceri was fused to a PTPA gene. Functional assays and yeast complementation assays suggest that this chimera is able to recognise glucose as a substrate but the heterologously expressed homolog of H. eriocheir cannot. Finally, phylogenomics have been used here to demonstrate that despite the morphological differences between three Hepatospora-like organisms parasitizing different crab hosts, they are the same species. This finding adds more weight to current evidence suggesting that morphology is not an ideal marker for taxonomical classification in the Microsporidia.

La població romaní és el grup ètnic minoritari transnacional més gran d'Europa. Tenen un origen sud asiàtic i, durant la seva diàspora cap a Europa, van experimentar múltiples efectes fundadors i una barreja genètica extensa. En aquesta tesi, l’anàlisi de marcadors genètics autosòmics mostren que tots els romaní comparteixen un component genètic comú del sud d’Àsia, de l’Orient Mitjà i dels Balcans, mentre que els grups romaní de la Península Ibèrica i del Bàltic han tingut una barreja addicional amb les altres poblacions de l'entorn. Després de caracteritzar el seu panorama genètic, l’estudi d’exomes complets de romanís espanyols suggereix que el flux de gens no romanís ha contrarestat l’augment de mutacions deletèries causada pels efectes fundadors. A més, les variants clínicament rellevants es troben tant a haplotips europeus com sud-asiàtics consistent amb l’extensa barreja genètica. Aquest treball representa un pas endavant per entendre de manera exhaustiva la història demogràfica romaní i posa de manifest la necessitat d’estudiar poblacions històricament excloses per poder descriure la variació humana en la seva totalitat.
The Romani people are the largest transnational minority ethnic group in Europe. They have a South Asian origin and during their diaspora to Europe, they experienced multiple founder effects and gene flow events. In this thesis, the analysis of genome-wide array data shows that Romani groups share a common South Asian, Middle Eastern and Balkan ancestry, while Iberian and Baltic groups experienced additional admixture with the surrounding non-Roma European populations. After characterising their genetic landscape, the study of whole-exome sequences from Spanish Romani suggests that non-Roma gene flow has counteracted the increase in mutational load caused by the founder effects. In addition, clinically relevant variants are traced back to both European and South Asian ancestral haplotypes consistent with the extensive gene flow. Thus, the present work represents a step forward to comprehensively depict the Romani demographic history and emphasises the need to study other underrepresented and historically excluded populations to fully capture human variation.

Genregulation beeinflusst Phänotypen im Kontext von Gesundheit und Krankheit. In Krebszellen regulieren genetische und epigenetische Faktoren die Genexpression in cis. Das Neuroblastom ist eine Krebserkrankung, die häufig im Kindesalter auftritt. Es ist gekennzeichnet durch eine geringe Anzahl exonischer Mutationen und durch häufige Veränderungen der somatischen Kopienzahl, einschließlich Genamplifikationen auf extrachromosomaler zirkulärer DNA. Bisher ist wenig darüber bekannt, wie lokale genetische und epigenetische Faktoren Gene im Neuroblastom regulieren. In dieser Arbeit kombiniere ich die allelspezifische Analyse ganzer Genome (WGS), Transkriptome und zirkulärer DNA von Neuroblastom-Patienten, um genetische und cis-regulatorische Effekte zu charakterisieren. Ich zeige, dass somatische Dosis-Effekte der Kopienzahl andere lokale genetische Effekte dominieren und wichtige Signalwege regulieren. Genamplifikationen zeigen starke Dosis-Effekte und befinden sich häufig auf großen extrachromosomalen zirkulären DNAs. Die vorgestellte Analyse zeigt, dass der Verlust von 11q zu einer Hochregulation von Histonvarianten H3.3 und H2A in Tumoren mit alternativer Verlängerung der Telomere (ALT) führt, und dass erhöhte somatische Kopienzahl die Expression der TERT Gens verstärken können. Weitere Erkenntnisse sind, dass 17p-Ungleichgewichte und die damit verbundene Herunterregulierung neuronaler Gene sowie die Hochregulierung des genomisch geprägten Gens RTL1 durch Kopienzahl-unabhängige allelische Dosis-Effekte mit einer ungünstigen Prognose verbunden sind. Die cis-QTL-Analyse bestätigt eine zuvor beschriebene Regulation des LMO1 Gens durch einen Enhancer-Polymorphismus und charakterisiert das regulatorische Potenzial weiterer GWAS-Risiko-Loci. Die Arbeit unterstreicht die Bedeutung von Dosis-Effekten im Neuroblastom und liefert eine detaillierte Übersicht regulatorischer Varianten, die in dieser Krankheit aktiv sind.
Gene regulation controls phenotypes in health and disease. In cancer, the interplay between germline variation, genetic aberrations and epigenetic factors modulate gene expression in cis. The childhood cancer neuroblastoma originates from progenitor cells of the sympathetic nervous system. It is characterized by a sparsity of recurrent exonic mutations but frequent somatic copy-number alterations, including gene amplifications on extrachromosomal circular DNA. So far, little is known on how local genetic and epigenetic factors regulate genes in neuroblastoma to establish disease phenotypes. I here combine allele-specific analysis of whole genomes, transcriptomes and circular DNA from neuroblastoma patients to characterize genetic and cis-regulatory effects, and prioritize germline regulatory variants by cis-QTLs mapping and chromatin profiles. The results show that somatic copy-number dosage dominates local genetic effects and regulates pathways involved in telomere maintenance, genomic stability and neuronal processes. Gene amplifications show strong dosage effects and are frequently located on large but not small extrachromosomal circular DNAs. My analysis implicates 11q loss in the upregulation of histone variants H3.3 and H2A in tumors with alternative lengthening of telomeres and cooperative effects of somatic rearrangements and somatic copy-number gains in the upregulation of TERT. Both 17p copy-number imbalances and associated downregulation of neuronal genes as well as upregulation of the imprinted gene RTL1 by copy-number-independent allelic dosage effects is associated with an unfavorable prognosis. cis-QTL analysis confirms the previously reported regulation of the LMO1 gene by a super-enhancer risk polymorphism and characterizes the regulatory potential of additional GWAS risk loci. My work highlights the importance of dosage effects in neuroblastoma and provides a detailed map of regulatory variation active in this disease.

Escherichia coli is one of the most important bacterial pathogens associated with extraintestinal infections in both humans and animals. In companion animals, it is associated with urinary, respiratory, gastrointestinal tract, joint, wound, skin and soft tissue infections. These infections vary significantly between cats and dogs: for example, extraintestinal infection in cats is infrequent, and they rarely have simple, uncomplicated bacterial UTIs compared to dogs. E. coli strains causing UTIs in both cats and dogs share pathotypic similarities and are phylogenetically related to ExPEC isolates from humans. It is, therefore, possible for humans to share pathogenic ExPEC strains with their companion animals, which suggests the possibility of both zoonotic (animal-to-human) and anthropozoonotic (human-to-animal) transmission. Among phylogenetic group B2 FQ-susceptible cat clinical isolates from an Australia-wide collection (n = 323), it was found that ST73 was the dominant B2-associated ST, based on both WGS (23/53, 43%) and ST-specific PCR (45/221, 20%). Less dominant STs identified by WGS included ST127, ST12, and ST372 (4/53, 8% each). By contrast, among group B2 FQ-susceptible dog clinical isolates from an Australia-wide collection (n = 449), ST372, an infrequent human pathogen, was found to be the predominant B2-associated ST according to both WGS (24/77, 31%) and ST-specific PCR (53/240, 22%). The other primary STs identified by WGS included ST73 (13/77, 17%), ST12 and ST80 (5/77, 7% each). To further compare the relatedness of ST73 isolates from dogs, cats, and humans, WGS-based phylogenetic comparison was performed. This comparison showed that there was considerable overall phylogenetic diversity among the isolates. Although most clusters were species-specific, some contained closely related human and animal (dog>cat) isolates. These results confirmed that while there is clonal commonality among ExPEC isolated from cats, dogs and humans, there are dominant host-specific clonal groups – ST372 and ST73 in dogs and ST73 in cats – and potential for bi-directional (i.e., companion animal-human and human-companion animal) transmission of ST73 strains between host species. A collection of FQR E. coli isolates from the same Australia-wide collection of isolates from extraintestinal infections in Australian cats and dogs (n = 59) was also investigated to determine the prevalence of the globally disseminated MDR pandemic clonal groups ST131 and ST1193. The main STs identified in this study were ST224 (10/59, 17%), ST744 (8/59, 14%), and ST38 (8/59, 14%). The overall prevalence of ST131 was (6/59, 10%), however, no ST1193 isolates were identified in contrast to a 5-year-earlier report which identified two dog-source ST1193 isolates closely related to human source isolates. Furthermore, the findings showed that the prevalence of ST131 as a cause of FQR infections in Australian companion animals was relatively constant between this study and the 5-year-earlier study of Platell et al. (2010) (9/125 isolates, 7.2%). To investigate the clonal commonality of human and companion animal ST131, 20 Australian cat- and dog-source ST131 genomes were compared to 173 reference human-source ST131 genomes. This analysis revealed that the animal-sourced isolates were widely distributed throughout the ST131 phylogeny. There was also some minor sub-clustering of dog- and cat-source isolates, though most were closely related to human-source ST131 isolates. The absence of a separate clade containing only companion animal ST131 isolates combined with the higher prevalence of ST131 and ST1193 in humans reported in the literature suggests that companion animals act as spillover hosts rather than primary reservoirs for these lineages. The high degree of clonal commonality among these FQR clinical isolates from humans and companion animals further suggests the possibility of bi-directional between-species transmission. In addition to being both a commensal and pathogen in companion animals, E. coli is also the main facultative anaerobic bacteria colonising the pig gastrointestinal tract. While being a beneficial gut commensal, some E. coli pathotypes cause both intestinal and extraintestinal infections in pigs with AMR of increasing concern in both commensals and pathogens. On this basis the frequency of antimicrobial non-susceptibility among E. coli (n = 201) and Salmonella spp. (n = 69) isolated from rectal contents of healthy Australian finisher pigs at slaughter was determined. Only low levels of non-susceptibility to CIAs (ESCs and FQs) were identified, with non-susceptibility to both cefoxitin and ciprofloxacin observed in only 1% (2/201) of E. coli isolates. In addition, all E. coli isolates were fully susceptible to ceftiofur. None of the Salmonella spp. isolates showed any non-susceptibility to these CIAs. As the use of fluoroquinolones in food-producing animals in Australia is banned, the identification of fluoroquinolone resistance in E. coli from Australian pigs is significant as it demonstrates that even in the absence of local antimicrobial selection pressure resistance can occur. FQR E. coli can enter livestock production facilities despite strict biosecurity possibly via human carriers or wild birds. Other potential sources include feed, water, rodents, and insects. The results from these studies highlight the need for further surveillance of AMR and identification of prominent resistant clonal lineages in both companion and food-producing animals in Australia.
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 2021

Listeria monocytogenes is a Gram-positive bacterium that is found ubiquitously throughout nature and is the etiologic agent of listeriosis. The majority of human listeriosis is foodborne, resulting from the consumption of unpasteurized and ready-to-eat foods that are contaminated during food processing. During the 2008 nationwide outbreak, the Gilmour laboratory performed the first real-time application of high-throughput whole-genome sequencing (WGS) of outbreak strains. Within this genomic data, the 77 kb plasmid, pLM5578, was newly identified in a clinical isolate, and additional Listeria plasmids (the 80 kb pLM5026 and the 60 kb pLM0813) were subsequently identified after WGS was completed on an expanded panel of outbreak isolates. Little was known regarding how plasmids contribute to persistence and virulence of L. monocytogenes, and to investigate these potential relationships, a panel of 147 L. monocytogenes food, environmental, and clinical isolates from Canadian public health events from the last three decades was selected for further study of the plasmids they might contain. Strain carriage of plasmids was determined using conventional PCR targeting known plasmid gene targets. Bioinformatic analyses were then used to predict the functions of individual genes encoded by each sequenced plasmid. These analyses were then used to direct experiments investigating the functions and associated phenotypes conferred by plasmid carriage. Phenotypic analyses included antimicrobial susceptibility testing, heavy metal resistance, and biofilm formation assays. Finally, WGS analyses was performed on isolates with plasmid screening patterns that indicated carriage of potential novel plasmids. Screening revealed that 75 of 147 isolates were positive for the presence of a plasmid, for which WGS analysis identified 24 unique newly identified L. monocytogenes plasmids. Phenotypically, 15 of these plasmids were found to contribute to a decreased susceptibility to the heavy metal cadmium, whereas 4 conferred resistance to the sanitizer benzalkonium chloride. Plasmid carriage was also found to affect biofilm formation. Nine plasmids correlated with stronger biofilm formation phenotypes; whereas 5 plasmids were correlated with weaker biofilm formation phenotypes. No known virulence factors or antibiotic resistance determinants were present in the DNA sequences of these 24 newly identified plasmids. Numerous coding sequences predicted to assist with survival under environmental stress were identified, and it is hypothesized that these plasmids likely contributed to persistence of L. monocytogenes within food processing environments.

Yes
Background: Mycobacterium tuberculosis rapid diagnostic tests (RDTs) are widely employed in routine laboratories and national surveys for detection of rifampicinresistant (RR)-TB. However, as next-generation sequencing technologies have become more commonplace in research and surveillance programs, RDTs are being increasingly complemented by whole genome sequencing (WGS). While comparison between RDTs is difficult, all RDT results can be derived from WGS data. This can facilitate continuous analysis of RR-TB burden regardless of the data generation technology employed. By converting WGS to RDT results, we enable comparison of data with different formats and sources particularly for low- and middle-income high TB-burden countries that employ different diagnostic algorithms for drug resistance surveys. This allows national TB control programs (NTPs) and epidemiologists to utilize all available data in the setting for improved RR-TB surveillance. Methods: We developed the Python-based MycTB Genome to Test (MTBGT) tool that transforms WGS-derived data into laboratory-validated results of the primary RDTs—Xpert MTB/RIF, XpertMTB/RIF Ultra, GenoType MDRTBplus v2.0, and GenoscholarNTM+MDRTB II. The tool was validated through RDT results of RR-TB strains with diverse resistance patterns and geographic origins and applied on routine-derived WGS data. Results: The MTBGT tool correctly transformed the single nucleotide polymorphism (SNP) data into the RDT results and generated tabulated frequencies of the RDT probes as well as rifampicin-susceptible cases. The tool supplemented the RDT probe reactions output with the RR-conferring mutation based on identified SNPs. The MTBGT tool facilitated continuous analysis of RR-TB and Xpert probe reactions from different platforms and collection periods in Rwanda. Conclusion: Overall, the MTBGT tool allows low- and middle-income countries to make sense of the increasingly generated WGS in light of the readily available RDT.
Erasmus Mundus Joint Doctorate Fellowship grant 2016- 1346.

Poultry meat is thought to harbour the most “human-like” E. coli strains, capable of causing both intestinal and extra-intestinal infections in humans. Furthermore, poultry meat is also known to harbour multi-drug resistant E. coli strains. In this study, extensive sampling of retail poultry meat products sold in major supermarkets and independent butchers in Canberra was undertaken. Phenotyping and genotyping of the E. coli isolated from the meat samples was then performed to better understand the evolution and diversity of these strains and their significance to human health. Using a whole genome sequence based approach, the strains were then compared to clinical and commensal human E. coli isolates to determine if there is transmission between the two hosts. A highly diverse population of E. coli in poultry meat was identified in this study. Although the exact contamination route is hard to determine, our findings suggest that poultry meat is frequently contaminated, not only with E. coli isolates from poultry birds but also significantly from other sources, possibly during processing chains and transport. In addition, the genetic structure comparison of human and poultry meat E. coli strains indicate that there is cross-transmission between the two hosts, but at low levels. Therefore, poultry meat serves as a low risk transmission route for zoonotic E. coli strains that can cause extra-intestinal infections. Our findings also suggest that the risk of exposure to intestinal or diarrheal pathogenic E. coli strains through poultry meat consumption and handling is even lower than exposure to extra-intestinal pathogenic E. coli (ExPEC) strains. Furthermore, poultry meat in Canberra region does not serve as a highly significant carrier of antimicrobial resistant E. coli unlike several other countries. Resistance was commonly observed to antibiotic classes approved for use in poultry industries (like ampicillin and tetracycline), but was low for critically important antibiotics to humans which are banned in the industry (like fluoroquinolone and 3rd generation cephalosporins). In addition, no carbapenem and colistin resistance was observed, which has recently been identified in poultry meat products in many countries including China, United Kingdom, Denmark. Poultry meat in Canberra is therefore more frequently contaminated with E. coli strains from the birds rather than from other contamination sources.

La digoxine est un cardiotonique largement employé pour contrôler les symptômes de l'insuffisance cardiaque et de la fibrillation auriculaire. Il est connu depuis les années 1980 que le métabolite principal de la digoxine, la dihydrodigoxine, est produit exclusivement par le microbiome intestinal (métabolisme de premier passage) et plus précisément la bactérie Eggerthella lenta. Aux États-Unis, c'est 14% des participants à une étude qui excrétaient 40% et plus de la dose sous la forme de ce métabolite rapidement éliminable et ayant perdu son affinité pour sa cible. De plus, chaque année, la digoxine est le médicament qui engendre le plus d'hospitalisations pour effets secondaires toxiques. Les effets secondaires très problématiques de la digoxine sont souvent déclenchés par l'ajout d'antibiotiques (surtout les macrolides) à la prescription de digoxine. La théorie explorée ici explique les évènements de toxicité chez les patients métabolisateurs. Ces derniers ont une dose quotidienne de maintien de digoxine plus élevée pour compenser l'action de la bactérie et, lorsque ces patients reçoivent un antibiotique pour une infection non reliée à leur condition cardiaque, l'arrêt du métabolisme par le microbiome engendre une augmentation de la biodisponibilité de la digoxine. Si la concentration plasmatique du médicament augmente trop, les effets secondaires peuvent aller jusqu'à causer la mort. Dans le présent projet, nous avons vérifié la sensibilité de E. lenta à deux antibiotiques de la famille des céphalosporines de seconde génération, in vivo et in vitro. Pour les 18 volontaires qui ont été exposés à 2x500mg de céfprozil durant une semaine, il y a une tendance à la baisse de l'abondance de la bactérie d'intérêt (par 58,3% par rapport au niveau initial), mais pas de significativité au niveau des tests statistiques. Pour les échantillons complets de microbiome fécal, mis en culture avec et sans antibiotiques, il y a une différence statistiquement significative avec une valeur-p de 0,0457, alors que la croissance de E. lenta a été impactée négativement par l'ajout de céfprozil au milieu de culture. Les résultats valident une prémisse importante pour la démonstration du rôle du microbiome dans la pharmacocinétique de la digoxine et la gestion clinique du médicament cardiotonique.
Digoxin is a widely used cardiotonic drug in the management of heart failure and atrial fibrillation. It has been known since the early 1980's that the main metabolite of digoxin, dihydrodigoxin, is synthesized by the gut microbiome during first pass metabolism and is exclusively produced by the bacteria Eggerthella lenta. In a clinical study done in the U.S.A., there were 14% of high metabolizers, for whom over 40% of the oral digoxin dose is transformed to the inactive metabolite and rapidly eliminated. Digoxin toxicity is the leading cause of hospitalization from medication's secondary effects. The toxicity events are often associated with the addition of an antibiotic (mostly from the macrolides class) to the patient's drugs regiments. The theory explored in this project could help explain the toxicity events in metabolizers. These patients have a higher daily digoxin maintenance dose to counteract the effects of the microbiome and are then prescribed antibiotics for an infection unrelated to their heart condition. The antibiotic alters E. lenta negatively, which cannot metabolize digoxin anymore and therefore augments the bioavailability of the cardiotonic. If the plasmatic concentration reaches dangerous levels (over 2ng/ml of plasma), the patients face adverse effects that include death. In the present project, we evaluated the susceptibility of E. lenta to two second generation cephalosporins, in vivo and in vitro. With the 18 healthy volunteers that were exposed to 2x500mg of cefprozil daily for 7 days, we observed a diminution of the abundance of the bacteria of interest by 58,3% from the initial levels. This change did not however produce statistically significant tests results. For the complete fecal microbiome that were cultivated in vitro, with or without cefprozil, the difference between the two conditions resulted in a statistically significant p-value of 0.0457, confirming the sensitivity of E. lenta to this cephalosporin. These results validate an important premise for the demonstration of the importance of the gut microbiome in the pharmacokinetics of digoxin and the clinical management of the drug to avoid toxicity events in clinical practice.