Characterisation and relative abundance of bacteria present in the urine of a frog
Data files
Feb 28, 2024 version files 59.40 KB
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README.md
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Urinary_bacteria_ID_and_relative_abundance.xlsx
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Abstract
Reproductive technologies are increasingly being adopted to improve the Q6 conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however, both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.
README: Urinary bacteria ID and relative abundance
https://doi.org/10.5061/dryad.vt4b8gv0b
Bacterial species composition and relative abundance present in urine samples collected from individual male Common eastern froglets, Crinia signifera (n=14). All frogs were collected from a single, large population, located adjacent to the Ecological Research Centre (ERC) at the University of Wollongong (34.4048° S, 150.8717° E). Collections occurred on October 19 and 26, 2022. Each row represents a species of bacteria identified to the finest scale that was able to be identified according to the SILVA rRNA database by the Australian Genome Research Facility. Unassigned species are species that were not able to be identified to taxonomic order. Total absolute bacterial abundance is the sum of all absolute abundance raw reads counts for each species within each urine sample; data for each species is the relative abundance of each species within the sample, displayed as a percentage (total number of reads assigned to a species divided by the total number of reads in the sample * 100).
Data sets included
Relative abundance of bacteria in the urine of each male individual
- Phylum level relative abundance
- Species level relative abundance
Species identification data and ID confidence
Description of the data and file structure
To identify the composition and abundance of bacterial species within urine, urine samples were collected from 14 individual male frogs within 4-5 days of collection from the field. Urine was collected from 14 male frogs according to protocols previously developed (Silla & Roberts 2012), whereby a fire-polished microcapillary tube was inserted into the cloaca to stimulate urination. Each urine sample was absorbed onto a sterile dry rayon-tip swabs (CLASSIQ swabs 167KS01, Copan Diagnostics, Inc., USA), placed in a 1.5 mL Eppendorf tube, and stored in a -80 °C freezer until sample processing (microbiome extraction and profiling). Frozen samples were stored for 23 weeks before being transported on dry ice to the Australian Genome Research Facility in Urrbrae, South Australia for genome sequencing and species identification. DNA extraction was then performed using the DNeasy® 96 PowerSoil® Pro Kit (QIAGEN) as per the manufacturer’s instructions, for manual high-throughput isolation of microbial genomic DNA. PCR amplification was then performed, and PCR amplicons were generated using the primers and conditions; the first stage PCR was cleaned using magnetic beads, and samples were visualised on 2% Sybr Egel (Thermo-Fisher). A secondary PCR to index the amplicons was performed with Platinum SuperFi II mastermix (Life Technologies, Australia). The resulting amplicons were cleaned again using magnetic beads, quantified by fluorometry (Promega Quantifluor), and normalised. The eqimolar pool was cleaned a final time using magnetic beads to concentrate the pool and then measured using a High-Sensitivity D1000 Tape on an Agilent 2200 TapeStation. The pool was diluted to 5nM and molarity was confirmed again using a Qubit High Sensitivity dsDNA assay (ThermoFisher). This was followed by sequencing on an Illumina MiSeq (San Diego, CA, USA) with a V3, 600 cycle kit (2 x 300 base pairs paired-end).
Output generated included absolute abundance and relative abundance of bacterial species in each sample, identified using the SILVA rRNA database.
Data sharing/Access
This data set is to be used with permission from the corresponding author: Aimee Silla asilla@uow.edu.au
Funding
This study was funded by a grant from the Australian Research Council, Discovery Early Career Researcher Award (DE210100812) awarded to A.Silla