Biology subjects

Lipp, E. K.

Publications and source records attributed to Lipp, E. K..

4 recordsLinked to original sources

bioRxiv2024

Comparison of three tiled amplicon sequencing approaches for SARS-CoV-2 variant detection from wastewater

During the COVID-19 pandemic, the detection and sequencing of SARS-CoV-2 from wastewater proved to be a valuable tool in assessing trends at the community level. Several whole genome enrichment methods have been proposed for sequencing SARS-CoV-2 from the mixed wastewater community, but there is little consensus on the most appropriate sequencing methods for variant detection or abundance estimations. Few studies have elucidated the errors associated with these methods or have established minimum sequencing requirements for correct interpretation of the results. To address these needs, we systematically assessed the efficacy of three tiled amplicon enrichment methods (Freed/Midnight, ARTIC V4, NEB VarSkip) for whole genome sequencing of SARS-CoV-2 variants using mock wastewater communities with variants at known proportions. We found the ARTIC V4 approach yielded the most accurate results for variant identification and variant abundance estimation, followed by the NEB VarSkip approach. Conversely, the NEB VarSkip method obtained the highest genomic coverage, with the ARTIC V4 method achieving the second highest coverage. Finally, we determined that the Freed/Midnight library preparation methods are not well-suited for use with short read sequencing. Based on the present results, the ARTIC V4 workflow appears to be the most robust and cost-effective approach for monitoring circulating SARS-CoV-2 variants with wastewater surveillance. IMPORTANCEThis work is informative for practitioners of wastewater-based epidemiology. Here, we detail a systematic comparison of three tiled amplicon sequencing approaches for enrichment of SARS-CoV-2 variants from wastewater. Using mock communities of known variant composition, we validate the analysis methods previously published by Baaijens et al. in Genome Biology (2022) for estimating variant abundance from wastewater using an RNAseq pipeline, kallisto. We provide recommendations for minimum sequencing requirements for accurate abundance estimates of SARS-CoV-2 variants in wastewater. The sequences generated from the mock communities have been uploaded to NCBIs Sequence Read Archive and will be useful to other practitioners seeking to validate their sequencing methods or bioinformatic pipelines.

genomics↗

bioRxiv2023

Coral disease and ingestion: investigating the role of heterotrophy in the transmission of pathogenic Vibrio spp. using a sea anemone (Exaiptasia pallida) model system

Understanding disease transmission in corals can be complicated given the intracity of the holobiont and difficulties associated with ex situ coral cultivation. As a result, most of the established transmission pathways for coral disease are associated with perturbance (i.e., damage) rather than evasion of immune defenses. Here we investigate ingestion as a potential pathway for the transmission of coral pathogens that evades the mucus membrane. Using sea anemones (Exaiptasia pallida) and brine shrimp (Artemia sp.) to model coral feeding, we tracked the acquisition of the putative pathogens, Vibrio alginolyticus, V. harveyi, and V. mediterranei using GFP-tagged strains. Vibrio sp. were provided to anemones using three experimental exposures 1) direct water exposure alone, 2) water exposure in the presence of a food source (clean Artemia), and 3) through a "spiked" food source (Vibrio-colonized Artemia) created by exposing Artemia cultures to GFP-Vibrio via the ambient water overnight. Following a 3 h feeding/exposure duration, the level of acquired GFP-Vibrio was quantified from anemone tissue homogenate. Ingestion of spiked Artemia resulted in a significantly greater burden of GFP-Vibrio equating to an 829.7-fold, 3,108.2-fold, and 435.0-fold increase in CFU mL-1 when compared to water exposed trials and a 206.8-fold, 62.2-fold, and 27.3-fold increase in CFU mL-1 compared to water exposed with food trials for V. alginolyticus, V. harveyi, and V. mediterranei, respectively. These data suggest that ingestion can facilitate delivery of an elevated dose of pathogenic bacteria in cnidarians and may describe an important portal of entry for pathogens in the absence of perturbing conditions. ImportanceThe front line of pathogen defense in corals is the mucus membrane. This membrane coats the surface body wall creating a semi-impermeable layer that inhibits pathogen entry from the ambient water both physically and biologically through mutualistic antagonism from resident mucus microbes. To date, much of the coral disease transmission research has been focused on mechanisms associated with perturbance of this membrane such as direct contact, vector lesions (predation/biting), and waterborne exposure through preexisting lesions. The present research describes a transmission pathway that evades the defenses provided by this membrane allowing unencumbered entry of bacteria as in association with food. This pathway may explain an important portal of entry for emergence of idiopathic infections in otherwise healthy corals and can be used to improve management practices for coral conservation.

microbiology↗

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↗

bioRxiv2019

Gut microbiome shifts with urbanization and potentially facilitates a zoonotic pathogen in a wading bird

Microbial communities in the gastrointestinal tract influence many aspects of host health, including metabolism and susceptibility to pathogen colonization. These relationships and the environmental and individual factors that drive them are relatively unexplored for free-living wildlife. We quantified the relationships between urban habitat use, diet, and age with microbiome composition and diversity for 82 American white ibises (Eudocimus albus) captured along an urban gradient in south Florida and tested whether gut microbial diversity was associated with Salmonella enterica prevalence. Shifts in community composition were significantly associated with urban land cover and, to a lesser extent, diets higher in provisioned food. The diversity of genera was negatively associated with community composition associated with urban land cover, positively associated with age class, and negatively associated with Salmonella shedding. Our results suggest that shifts in both habitat use and diet for urban birds significantly alter gut microbial composition and diversity in ways that may influence health and pathogen susceptibility as species adapt to urban habitats.

ecology↗