Biology subjects

Glenn, T.

Publications and source records attributed to Glenn, T..

6 recordsLinked to original sources

bioRxiv2025

Amplicon sequencing detects, identifies, and quantifies minority variants in mixed-species infections of Cryptosporidium parasites

Cryptosporidium is a globally endemic parasite genus with over 40 recognized species. While C. hominis and C. parvum are responsible for most human infections, human cases involving other species have also been reported. Furthermore, there is increasing evidence of simultaneous infections with multiple species. Therefore, we devised a new means to identify various species of Cryptosporidium in mixed infections by sequencing a 431 bp amplicon of the 18S rRNA gene encompassing two variable regions. Using the DADA2 pipeline, amplicons were first identified to a genus using the SILVA 132 reference database; then Cryptosporidium amplicons to a species using a custom database. This approach demonstrated sensitivity, successfully detecting and accurately identifying as little as 0.001 ng of C. parvum DNA in a complex stool background. Notably, we differentiated mixed infections and demonstrated the ability to identify potentially novel species of Cryptosporidium both in situ and in vitro. Using this method, we identified Cryptosporidium parvum in Egyptian rabbits with three samples showing minor mixed infections. By contrast, no mixed infections were detected in Egyptian children, who were primarily infected with C. hominis. Thus, this pipeline provides a sensitive tool for Cryptosporidium species-level identification, allowing for the detection and accurate identification of minor variants and mixed infections.

microbiology↗

bioRxiv2024

The repetitive genome of the Ixodes ricinus tick reveals transposable elements have driven genome evolution in ticks

Ticks are obligate blood-feeding parasites associated with a huge diversity of diseases globally. The hard tick Ixodes ricinus is the key vector of Lyme borreliosis and tick-borne encephalitis in Western Eurasia. Ixodes ticks have large and repetitive genomes that are not yet well characterized. Here we generate two high-quality I. ricinus genome assemblies, with haploid genome sizes of approximately 2.15 Gbp. We find transposable elements comprise at least 69% of the two I. ricinus genomes, amongst the highest proportions found in animals. The transposable elements in ticks are highly diverse and novel, so we constructed a repeat library for ticks using our I. ricinus genomes and the genome of I. scapularis, another major tick vector of Lyme borreliosis. To understand the impact of transposable elements on tick genomes we compared their accumulation in the two Ixodes sister species. We find transposable elements in these two species to be drivers of genome evolution in ticks. The I. ricinus genome assemblies and our tick repeat library will be valuable resources for biological insights into this important ectoparasite. Our findings highlight that further research into the impact of transposable elements on the genomes of blood-feeding parasites is required.

genomics↗

bioRxiv2024

A new chromosome-level genome assembly and annotation of Cryptosporidium meleagridis

Cryptosporidium spp. are medically and scientifically relevant protozoan parasites that cause severe diarrheal illness in infants and immunosuppressed populations as well as animals. Although most human Cryptosporidium infections are caused by C. parvum and C. hominis, there are several other human-infecting species including C. meleagridis, which is commonly observed in developing countries. Here, we polished and annotated a long-read genome sequence assembly for C. meleagridis TU1867, a species which infects birds and humans. The genome sequence was generated using a combination of whole genome amplification (WGA) and long-read Oxford Nanopore Technologies sequencing. The assembly was then polished with Illumina data. The chromosome-level genome assembly is 9.2 Mbp with a contig N50 of 1.1 Mb. Annotation revealed 3,923 protein-coding genes. A BUSCO analysis indicates a completeness of 96.6% (n=446), including 430 (96.4%) single-copy and 1 (0.224%) duplicated apicomplexan conserved gene(s). The new C. meleagridis genome assembly is nearly gap-free and provides a valuable new resource for the Cryptosporidium community and future studies on evolution and host-specificity.

genomics↗

bioRxiv2021

Escaping the Fate of Sisyphus: Assessing Resistome Hybridization Baits for Antimicrobial Resistance Gene Capture

Finding, characterizing, and monitoring reservoirs for antimicrobial resistance (AMR) is vital to protecting public health. Hybridization capture baits are an accurate, sensitive, and cost-effective technique used to enrich and characterize DNA sequences of interest, including antimicrobial resistance genes (ARGs), in complex environmental samples. We demonstrate the continued utility of a set of 19,933 hybridization capture baits designed from the Comprehensive Antibiotic Resistance Database (CARD)v1.1.2 and Pathogenicity Island Database (PAIDB)v2.0, targeting 3,565 unique nucleotide sequences that confer resistance. We demonstrate the efficiency of our bait set on a custom-made resistance mock community and complex environmental samples to increase the proportion of on-target reads as much as >200-fold. However, keeping pace with newly discovered ARGs poses a challenge when studying AMR, because novel ARGs are continually being identified and would not be included in bait sets designed prior to discovery. We provide imperative information on how our bait set performs against CARDv3.3.1, as well as a generalizable approach for deciding when and how to update hybridization capture bait sets. This research encapsulates the full life cycle of baits for hybridization capture of the resistome from design and validation (both in silico and in vitro) to utilization and forecasting updates and retirement. Originality-Significance StatementThis work is applicable to a wide range of research. It helps to define conditions under which hybridization capture is useful regarding not only antimicrobial resistance specifically, but also more generally how to assess the ongoing utility of existing bait sets - giving objective criteria for when and by what strategies baits should be updated. We also provide a method for quantifying and comparing antimicrobial resistance genes (ARGs) similar to what is used for RNAseq experiments. This approach improves comparison of ARGs across environments. Thus, the work provides an improved foundation for ARG future studies, while cutting across traditional areas of microbiology and extending beyond.

microbiology↗

bioRxiv2020

Whole-Genome Sequence Typing shows extensive diversity of Listeria monocytogenes in the outdoor environment and poultry processing plants.

A reliable and standardized classification of Listeria monocytogenes (Lm) is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS) based approaches for strain characterization either lack standardization, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool Haplo-ST to improve standardization and provide maximum discriminatory potential to WGS data tied to an MLST (multi locus sequence typing) framework. Haplo-ST performs whole-genome MLST (wgMLST) for Lm while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely-used BIGSdb-Lm database. Tests on Haplo-STs performance with simulated reads from Lm reference strains yielded a high sensitivity of 97.5%, and coverage depths of [≥] 20x was found to be sufficient for wgMLST profiling. We used Haplo-ST to characterize and differentiate between two groups of Lm isolates, derived from the natural environment and poultry processing plants. Phylogenetic reconstruction showed sharp delineation of lineages within each group and no lineage-specificity was observed with isolate phenotypes (transient vs. persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of Lm within poultry processing plants. IMPORTANCEWe have developed an open-source tool that provides allele-based subtyping of Lm isolates at the whole genome level. Along with allelic profiles, this tool also generates allele sequences, and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of Lm isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlights the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates for routine surveillance, outbreak investigations and source tracking.

bioinformatics↗

bioRxiv2019

Dynamics between horizontal gene transfer and acquired antibiotic resistance in S. Heidelberg following in vitro incubation in broiler ceca

The chicken gastrointestinal tract harbors taxa of microorganisms that play a role in the health and disease status of the host. The cecum is the part of the gut that carries the highest microbial densities, has the longest residence time of digesta and is a vital site for urea recycling and water regulation. Therefore, the cecum provides a rich environment for bacteria to horizontally transfer genes between one another via mobile genetic elements such as plasmids and bacteriophages. In this study, we used broiler chicken cecum as a model to investigate antibiotic resistance genes that can be transferred in vitro from ceca flora to Salmonella enterica serovar Heidelberg (S. Heidelberg). We used whole genome sequencing and resistome enrichment to decipher the interactions between S. Heidelberg, gut microbiome and acquired antibiotic resistance. After 48 h incubation of ceca under microaerophilic conditions, one S. Heidelberg isolate was recovered with an acquired Inck2 plasmid (88 kb) encoding extended {beta}-lactamase producing gene (blaCMY-2). In vitro, this plasmid was transferrable between E. coli and S. Heidelberg strains, but transfer was unsuccessful between S. Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with P1-like phages. This study contributes to our understanding of the dynamics between an important food-borne pathogen and the chicken gut microbiome.\n\nImportanceS. Heidelberg is a clinically important serovar, linked to food-borne illness and among the top 5 serovars isolated from poultry in USA and Canada. Acquisition of new genetic material from microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding on the dynamic interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. In this study, we show that the native flora in the broiler ceca were capable of transferring mobile genetic elements carrying AmpC {beta}-lactamase (blaCMY-2) gene to an important food-borne pathogen S. Heidelberg. The potential role for P1-like bacteriophage transduction was also discussed.

microbiology↗