DOI: 10.5061/dryad.vt4b8gv0b
title: Characterisation and relative abundance of bacteria present in the urine of a frog

description: 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.
description: # Urinary bacteria ID and relative abundance
 [https://doi.org/10.5061/dryad.vt4b8gv0b](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 1. Relative abundance of bacteria in the urine of each male individual 1. Phylum level relative abundance 2. Species level relative abundance 2. 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](mailto:asilla@uow.e) ## Funding This study was funded by a grant from the Australian Research Council, Discovery Early Career Researcher Award (DE210100812) awarded to A.Silla