Optimization of Low-Biomass Sample Collection and Quantitative PCR-Based Titration Impact 16S rRNA Microbiome Resolution

Clokie, Benjamin Gregory James; Elsheshtawy, Ahmed; Albalat, Amaya; Nylund, Are; Beveridge, Allan; Payne, Chris J.; Mackenzie, Simon

doi:10.1128/spectrum.02255-22

Cited by 10 publications

(7 citation statements)

References 43 publications

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“…To date, very few comparative studies focused on mucosalassociated microbiota when using different sampling methods have been performed in fish mucosal tissues, such as gills (57)(58)(59), skin (60), and gut (61), which have obtained some controversial results. In the gastrointestinal tract, there has been shown evidence of a divergent composition between the bacteria weakly adhering to the intestinal mucosa collected by washing and those obtained when scraping the mucosa in European whitefish (Coregonus lavaretus) (61).…”

Section: Resultsmentioning

confidence: 99%

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies

Ruiz,

Torrecillas,

Kashinskaya

et al. 2024

Front. Vet. Sci.

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In the present study, we propose the use of swabs in non-lethal sampling procedures to collect the mucosa-adhered gut microbiota from the posterior intestine of fish, and therefore, we compare the bacterial communities collected by conventional scraping and by swabbing methods. For this purpose, samples of the posterior intestine of rainbow trout (Oncorhynchus mykiss) were collected first using the swabbing approach, and after fish euthanasia, by mucosa scraping. Finally, bacterial communities were compared by 16S rRNA gene Illumina sequencing. Results from the current study revealed that similar values of bacterial richness and diversity were found for both sampling procedures. Similarly, there were no differences between procedures when using qualitative metrics (Jaccard and unweighted UniFrac) for estimating inter-individual diversity, but the quantitative metrics (Bray-Curtis and weighted UniFrac) showed a higher dispersion when samples were obtained by swabbing compared to scraping. In terms of bacterial composition, there were differences in abundance for the phyla Firmicutes and Proteobacteria. The cause of these differential abundances may be the inability of the swab to access to certain areas, such as the basal region of the intestinal villi. Moreover, swabbing allowed a higher representation of low abundant taxa, which may also have an important role in host microbiome regardless of their low abundance. Overall, our results demonstrate that the sampling method is a factor to be considered in experimental design when studying gut bacterial communities to avoid potential biases in the interpretation or comparison of results from different studies. In addition, the advantages and disadvantages of each procedure (swabbing vs scraping) are discussed in detail, concluding that swabbing can be implemented as a reliable and non-lethal procedure for posterior gut microbiota studies, which is of particular interest for animal welfare and the 3Rs principle, and may offer a wide range of novel applications.

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Section: Resultsmentioning

confidence: 99%

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies

Ruiz,

Torrecillas,

Kashinskaya

et al. 2024

Front. Vet. Sci.

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“…Further refinement of non‐lethal methods and validation studies of these methods would also be beneficial. There has been some movement in the past few years toward fish‐specific microbiome method evaluations (see Clinton et al, 2021; Clokie et al, 2022; Hildonen et al, 2019; Nyholm et al, 2022). Ideally, the research community will converge on a set of best practices that provide repeatable and reproducible results for different fish body compartments for microbiome analysis.…”

Section: Discussionmentioning

confidence: 99%

“…Further, fish gut samples have very different chemical and enzymatic profiles, which may result in differing degrees of PCR inhibition (Hildonen et al, 2019). Fish gill samples also prove to be rich in PCR inhibitors; however, gill biopsies are likely more problematic than gill swabs due to being a blood‐rich tissue (Clokie et al, 2022). Inhibitors can also be introduced during sample preservation and storage.…”

Section: Challenges With Non‐lethal Microbiome Samplingmentioning

confidence: 99%

Opportunities and challenges with transitioning to non‐lethal sampling of wild fish for microbiome research

Kelly,

Yost,

Cooke

2024

Journal of Fish Biology

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The microbial communities of fish are considered an integral part of maintaining the overall health and fitness of their host. Research has shown that resident microbes reside on various mucosal surfaces, such as the gills, skin, and gastrointestinal tract, and play a key role in various host functions, including digestion, immunity, and disease resistance. A second, more transient group of microbes reside in the digesta, or feces, and are primarily influenced by environmental factors such as the host diet. The vast majority of fish microbiome research currently uses lethal sampling to analyse any one of these mucosal and/or digesta microbial communities. The present paper discusses the various opportunities that non‐lethal microbiome sampling offers, as well as some inherent challenges, with the ultimate goal of creating a sound argument for future researchers to transition to non‐lethal sampling of wild fish in microbiome research. Doing so will reduce animal welfare and population impacts on fish while creating novel opportunities to link host microbial communities to an individual's behavior and survival across space and time (e.g., life‐stages, seasons). Current lethal sampling efforts constrain our ability to understand the mechanistic ecological consequences of variation in microbiome communities in the wild. Transitioning to non‐lethal sampling will open new frontiers in ecological and microbial research.

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“…In total, we sequenced 385 metagenomic and 365 metatranscriptomic swabs comprising ten body sites representing the oral, nasal, and skin microbiomes ( To account for variation due to database and algorithmic bias, we used a diverse set of shortread alignment and de novo assembly approaches to estimate the microbial community taxonomic and functional composition of our dataset (Supplementary Figure 1, Supplementary Tables 2-6, Methods). We observed that many of the swabs collected, especially those from the skin sites, comprised low biomass microbial communities; there are many documented challenges in analyzing these data 30,31 . To filter environmental contamination and the kitome 32 influencing our findings, we collected and sequenced negative controls of both (1) the water that sterile swabs were dipped in prior to use as well as (2) the ambient air around the sites of sample collection and processing for sequencing.…”

Section: Quantifying the Metagenomic Architecture Of Short-term Space...mentioning

confidence: 99%

Viral activation and ecological restructuring characterize a microbiome axis of spaceflight-associated immune activation

Mason,

Tierney,

Kim

et al. 2023

Preprint

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Maintenance of astronaut health during spaceflight will require monitoring and potentially modulating their microbiomes, which play a role in some space-derived health disorders. However, documenting the response of microbiota to spaceflight has been difficult thus far due to mission constraints that lead to limited sampling. Here, we executed a six-month longitudinal study centered on a three-day flight to quantify the high-resolution microbiome response to spaceflight. Via paired metagenomics and metatranscriptomics alongside single immune profiling, we resolved a microbiome “architecture” of spaceflight characterized by time-dependent and taxonomically divergent microbiome alterations across 750 samples and ten body sites. We observed pan-phyletic viral activation and signs of persistent changes that, in the oral microbiome, yielded plaque-associated pathobionts with strong associations to immune cell gene expression. Further, we found enrichments of microbial genes associated with antibiotic production, toxin-antitoxin systems, and stress response enriched universally across the body sites. We also used strain-level tracking to measure the potential propagation of microbial species from the crew members to each other and the environment, identifying microbes that were prone to seed the capsule surface and move between the crew. Finally, we identified associations between microbiome and host immune cell shifts, proposing both a microbiome axis of immune changes during flight as well as the sources of some of those changes. In summary, these datasets and methods reveal connections between crew immunology, the microbiome, and their likely drivers and lay the groundwork for future microbiome studies of spaceflight.

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Optimization of Low-Biomass Sample Collection and Quantitative PCR-Based Titration Impact 16S rRNA Microbiome Resolution

Cited by 10 publications

References 43 publications

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies

Opportunities and challenges with transitioning to non‐lethal sampling of wild fish for microbiome research

Viral activation and ecological restructuring characterize a microbiome axis of spaceflight-associated immune activation

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