Homoplasy in Bacterial Evolution

This study addressed the following research goals: (1) a reevaluation of Lifelong Learning (LLL) scale’s reliability and validity measuring LLL skills, (2) reevaluation of Engineering Attitude Survey’s (EAS) reliability and validity of measuring students’ attitudes toward STEM fields, (3) effects of an evidence-based pedological (EBP) treatment on students’ LLL and engineering attitudes (EA) during a Computer-Aided Design course. The appearance of homoplasy occurs when mutations are not derived from a common ancestor but arise independently in multiple branches of a phylogenetic tree. For bacteria, it suggests that genetic recombination events occur or positive selection exists during evolution, affecting the accuracy of phylogeny estimation. Without considering recombination, the reconstruction of phylogenetic trees based on an alignment of bacterial strains could be misleading. Hence, to better understand their true evolutionary histories among a bacterial population, it is essential to identify recombination breakpoints before estimating their phylogeny. We developed an average compatibility ratio method with a permutation test, ptACR, to detect recombination breakpoints in a multiple sequence alignment without requiring a tree. We use a sliding window to evaluate the local compatibility of adjacent polymorphic sites to locate potential breakpoints and then assess the statistical significance of candidate breakpoints by applying a permutation test. We evaluate the performance of ptACR on both simulated and empirical datasets. The simulation results show that it has similar sensitivity but higher specificity and better F1 score compared to existing methods. Also, ptACR detects recombination events in a collection of clinical isolates of Mycobacterium avium and Staphylococcus aureus, and identifies boundaries of regions with statistical significance, where the adjacent regions exhibit distinct phylogenies. For clonal species, since recombination is less likely to occur, the occurrence of homoplasy is a strong indicator of positive selection, such as antibiotic resistance. To identify mutations conferring resistance, genome-wide association studies are commonly applied to identify statistically significant associations between genotypes (polymorphisms) and phenotypes of interests (antibiotic resistance) across the entire genome. However, homoplasy is not well accounted for by most bacterial genome-wide association analyses, producing false positives or false negatives. Also, existing association methods usually use an individual site or group polymorphisms within a gene as genotypes without considering the frequency of evolutionary convergence and the mutation rate in different regions. To better exploit homoplasy, we developed a two-phase evolutionary cluster-based convergence test (ECC) to identify regions harboring mutations under selection pressure associated with antibiotic resistance. In the first-phase step, we apply a Poisson distribution to detect regions exhibiting more changes (distinct mutational events) than expected by optimizing the grouping of SNPs within windows. Next, we test associations between the clustered regions and drug resistance using a hypergeometric distribution based on the concept of convergence test in the second phase. We model the distribution of changes occurring in the resistant or sensitive branches for each clustered region and compare it to the background. We evaluate the ECC method on empirical datasets of clinical isolates of Mycobacterium tuberculosis with seven phenotypes from drug susceptibility tests. Our two-phase evolutionary cluster-based convergence method is able to identify known resistant-associated sites within genes or intergenic regions corresponding to seven anti-tuberculous drugs. It also identifies two novel clustered regions in Rv2571 and Rv1830, potentially linked to isoniazid resistance. It improves the potential over existing methods for association tests to find more novel resistant-associated mutations, which will ultimately help in developing new antibiotic treatments. In sum, we present two models for identifying genomic regions affected by recombination (ptACR) and clustered regions associated with antibiotic resistance driven by selection pressure (ECC) in bacterial genomes

Virus evolution in Wolbachia-infected Drosophila

Wolbachia, a common vertically transmitted symbiont, can protect insects against viral infection and prevent mosquitoes from transmitting viral pathogens. For this reason, Wolbachia-infected mosquitoes are being released to prevent the transmission of dengue and other arboviruses. An important question for the long-term success of these programmes is whether viruses can evolve to escape the antiviral effects of Wolbachia. We have found that Wolbachia altered the outcome of competition between strains of the DCV virus in Drosophila. However, Wolbachia still effectively blocked the virus genotypes that were favoured in the presence of the symbiont. We conclude that Wolbachia did cause an evolutionary response in viruses, but this has little or no impact on the effectiveness of virus blocking

The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color

Background Theobroma cacao L. cultivar Matina 1-6 belongs to the most cultivated cacao type. The availability of its genome sequence and methods for identifying genes responsible for important cacao traits will aid cacao researchers and breeders. Results We describe the sequencing and assembly of the genome of Theobroma cacao L. cultivar Matina 1-6. The genome of the Matina 1-6 cultivar is 445 Mbp, which is significantly larger than a sequenced Criollo cultivar, and more typical of other cultivars. The chromosome-scale assembly, version 1.1, contains 711 scaffolds covering 346.0 Mbp, with a contig N50 of 84.4 kbp, a scaffold N50 of 34.4 Mbp, and an evidence-based gene set of 29,408 loci. Version 1.1 has 10x the scaffold N50 and 4x the contig N50 as Criollo, and includes 111 Mb more anchored sequence. The version 1.1 assembly has 4.4% gap sequence, while Criollo has 10.9%. Through a combination of haplotype, association mapping and gene expression analyses, we leverage this robust reference genome to identify a promising candidate gene responsible for pod color variation. We demonstrate that green/red pod color in cacao is likely regulated by the R2R3 MYB transcription factor TcMYB113, homologs of which determine pigmentation in Rosaceae, Solanaceae, and Brassicaceae. One SNP within the target site for a highly conserved trans-acting siRNA in dicots, found within TcMYB113, seems to affect transcript levels of this gene and therefore pod color variation. Conclusions We report a high-quality sequence and annotation of Theobroma cacao L. and demonstrate its utility in identifying candidate genes regulating traits

DECODING BACTERIAL GENOME WITH HIGH-THROUGHPUT SEQUENCING: GENES AND GENETIC MARKERS

Ph.DDOCTOR OF PHILOSOPH

Integrating whole-genome sequencing within the National Antimicrobial Resistance Surveillance Program in the Philippines

Funding: This work was funded by the Newton Fund, Medical Research Council (UK) grant MR/N019296/1, Philippine Council for Health Research and Development project number FP160007. J.S. was partially supported by research grants RR025040 and U01CA207167 from the National Institutes of Health (NIH). S.A. and D.M.A. were additionally supported by the National Institute for Health Research (UK) Global Health Research Unit on genomic Surveillance of AMR(16_136_111) and by the Centre for Genomic Pathogen Surveillance (http://pathogensurveillance.net).National networks of laboratory-based surveillance of antimicrobial resistance (AMR) monitor resistance trends and disseminate these data to AMR stakeholders. Whole-genome sequencing (WGS) can support surveillance by pinpointing resistance mechanisms and uncovering transmission patterns. However, genomic surveillance is rare in low- and middle-income countries. Here, we implement WGS within the established Antimicrobial Resistance Surveillance Program of the Philippines via a binational collaboration. In parallel, we characterize bacterial populations of key bug-drug combinations via a retrospective sequencing survey. By linking the resistance phenotypes to genomic data, we reveal the interplay of genetic lineages (strains), AMR mechanisms, and AMR vehicles underlying the expansion of specific resistance phenotypes that coincide with the growing carbapenem resistance rates observed since 2010. Our results enhance our understanding of the drivers of carbapenem resistance in the Philippines, while also serving as the genetic background to contextualize ongoing local prospective surveillance.Publisher PDFPeer reviewe

Global dissemination of a multidrug resistant Escherichia coli clone.

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum β-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen

Diversity, Virulence, and Antimicrobial Resistance in Isolates From the Newly Emerging Klebsiella pneumoniae ST101 Lineage

The global dissemination of Klebsiella pneumoniae and Klebsiella pneumoniae carbapenemase (KPC) has been largely attributed to a few high-risk sequence types (STs) (ST258, ST11, ST512) associated with human disease. ST101 is an emerging clone that has been identified in different parts of the world with the potential to become a global, persistent public health threat. Recent research suggests the ST101 lineage is associated with an 11% increase in mortality rate in comparison to non-ST101 infections. In this study, we generated a high-quality, near-finished genome assembly of a multidrug-resistant (MDR) isolate from Italy (isolate 4743) that is a single locus variant of ST101 (ST1685). We demonstrate that the 4743 genome contains virulence features such as an integrative conjugative element carrying the yersiniabactin siderophore (ICEKp3), the mannose-resistant Klebsiella-like (type III) fimbriae cluster (mrkABCDFHIJ), the ferric uptake system (kfuABC), the yersiniabactin receptor gene fyuA, a capsular K type K17, and an O antigen type of O1. K. pneumoniae 4743 carries the blaKPC-2 carbapenemase gene along with genes conferring resistance to aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, macrolides, lincosamides, and streptogramin B. A comparative genomics analysis of 44 ST101 genomes as well as newly sequenced isolate 4743 identified variable antimicrobial resistance (AMR) resistance profiles and incompatibility plasmid types, but similar virulence factor profiles. Using Bayesian methodologies, we estimate the common ancestor for the ST101 lineage emerged in 1990 (95% HPD: 1965 to 2007) and isolates within the lineage acquired blaKPC after the divergence from its parental clonal group and dissemination. The identification of virulence factors and antibiotic resistance genes acquired by this newly emerging clone provides insight into the reported increased mortality rates and highlights its potential success as a persistent nosocomial pathogen. With a combination of both colistin resistance, carbapenem resistance, and several known virulence factors, the ST101 genetic repertoire may be a “perfect storm” allowing for a newly emerging, high-risk, extensively antibiotic resistant clone. This high-risk clone appears adept at acquiring resistance and may perpetuate the dissemination of extensive antimicrobial resistance. Greater focus on the acquisition of virulence factors and antibiotic resistance genes is crucial for understanding the spread of antibiotic resistance

Prevalence and relationship of endosymbiotic Wolbachia in the butterfly genus Erebia

Wolbachia is an endosymbiont common to most invertebrates, which can have significant evolutionary implications for its host species by acting as a barrier to gene flow. Despite the importance of Wolbachia, still little is known about its prevalence and diversification pattern among closely related host species. Wolbachia strains may phylogenetically coevolve with their hosts, unless horizontal host-switches are particularly common. We address these issues in the genus Erebia, one of the most diverse Palearctic butterfly genera.; We sequenced the Wolbachia genome from a strain infecting Erebia cassioides and showed that it belongs to the Wolbachia supergroup B, capable of infecting arthropods from different taxonomic orders. The prevalence of Wolbachia across 13 closely related Erebia host species based on extensive population-level genetic data revealed that multiple Wolbachia strains jointly infect all investigated taxa, but with varying prevalence. Finally, the phylogenetic relationships of Wolbachia strains are in some cases significantly associated to that of their hosts, especially among the most closely related Erebia species, demonstrating mixed evidence for phylogenetic coevolution.; Closely related host species can be infected by closely related Wolbachia strains, evidencing some phylogenetic coevolution, but the actual pattern of infection more often reflects historical or contemporary geographic proximity among host species. Multiple processes, including survival in distinct glacial refugia, recent host shifts in sympatry, and a loss of Wolbachia during postglacial range expansion seem to have jointly shaped the complex interactions between Wolbachia evolution and the diversification of its host among our studied Erebia species

X chromosome as a polarising signal in asymmetric cell division

In the nematode genus of Auanema, sex is determined by X chromosome dosage, females and self-fertilising hermaphrodites are XX and males are XO. Surprisingly, cross between XX female and XO male results in mostly XX progeny. This occurs because males only produce one functional sperm bearing an X chromosome, whereas nullo-X sperm is discarded. The exclusive formation of X-bearing sperm by males is attributed to the asymmetric segregation of essential sperm components, necessary for sperm function and motility, with the X chromosome. Whereas non-essential cytoplasmic materials (e.g., Golgi complex, ribosomes) disposed of in the nullo-X cell that takes the role of a residual body. This unique system permits easy-to-score phenotype on the organismal level to study asymmetric cell division; because the sex ratio of a cross-progeny can be used to monitor the type of divisions occurring during male spermatogenesis. Here I report that during Auanema spermatogenesis, the X chromosome acts as a polarising signal for sperm components to segregate with the cell inheriting the X chromosome. Sperm components also segregate with the X chromosomes in other A. rhodensis spermatogenesis models. During the spermatogenesis of A. rhodensis XX hermaphrodites, sperm components segregate to the cell inheriting the X chromosome. Similarly, I identified that during the spermatogenesis of A. rhodensis masculiniser mutant Arh-mas-1 (Genetically XX but with a male morphology), sperm components co-segregate with X chromosomes in asymmetric division or equally with X chromosomes to both daughter cells in symmetric division. I demonstrated that shuffling X chromosome haplotype blocks in A. freiburgensis RIAILs from two strains with different genetic background resulted in a new transgressive phenotype. Where, males from RIAILs produced a high number of males, due to their ability to form a functional nullo-X sperm. Genetic mapping using BSA analysis identified four candidate regions on the X chromosome involved in the spatial localisation of cytoplasmic components. Those findings led to hypothesised that interaction between a group of polygenes on the X chromosome in Auanema polarises sperm components to segregate with the X chromosome. Mechanisms describing how intrinsic factors regulate cell polarity are poorly understood, mostly because it is difficult to establish in the absence of extrinsic factors influence. Here, I present a novel mechanism for an intrinsic polarisation of asymmetrically dividing cells initiated by the X chromosome