The use of next generation sequencing to study the environmental mycobiome and its potential health effects

Emma Marczylo Bioaerosols – June 2017 Overview

• Brief Background: • Why are CRCE interested in fungal bioaerosols? • Mycobiome analysis: • Why use next generation sequencing? • Ongoing work • What is the current focus of CRCE’s bioaerosol research?

2 Bioaerosols – June 2017 Why bioaerosols? • Respiratory health/toxicology a big focus within our department & bioaerosols represent a current respiratory health concern

3 Bioaerosols – June 2017 Why bioaerosols? • Common sources relevant to public health

4 Bioaerosols – June 2017 Why bioaerosols? • Public concern over health effects of living near composting and intensive farming sites

• Systematic reviews on exposures and health outcomes related to bioaerosol emissions from composting facilities (published 2015*) or intensive farming (ongoing) in collaboration with SAHSU

• Evidence on both exposure assessment and health effects are limited

• A big unknown is the microbial composition of such bioaerosols

*Pearson et al, 2015, J Toxicol Environ Health B Crit Rev,18:43-69

5 Bioaerosols – June 2017 Why fungi? Normally die rapidly due to water evaporation, although increased humidity and clumping can prolong survival

• Can remain viable for much longer periods, even at low humidity & high/low temperatures • Much less known about the fungal composition of bioaerosols (and other samples) • Fungi linked with development and exacerbation of asthma symptoms

6 Bioaerosols – June 2017 Mycobiome analysis

• Mycobiome = fungal composition (eg air, soil, oral swab) • Traditional methods for identifying fungi rely on culture or morphological identification by microscopy

• Culture-dependant methods are biased towards fungi that can be cultured (around 17% of known fungi) • Microscopy can be difficult as many fungi cannot be distinguished from each other based upon morphology alone

7 Bioaerosols – June 2017 Mycobiome: NGS method

Fungi/spores DNA: ribosomal RNA genes

5.8S Extract DNA PowerSoil® & PureLink™ DNA isolation kits Phusion™ P Ion high-fidelity C R Xpress™ DNA Sample 1 Sample 2 barcodes polymerase BC1 BC4 Aspergillus Sample 1 fumigatus Ion Torrent™ BC2 BC4 BC3 Ion S5 ™ BC2 BC1 BC1 BC5 BC1 Epicoccum BC1 BC2 BC2 BC2 BC4 nigrum BC3 BC3 BC2 BC5 Penicillium BC1 BC6 Sample 2 chrysogenum BC6 NGS + Bioinformatics BC5 BC4 BC3 BC6 BC4 USEARCH BC4 BC3 BC5 Alternaria BC5 BC5 BC6 BC3 BC6 alternata BC6 Database of sample mycobiomes Barcoded PCR products

8 Bioaerosols – June 2017 Mycobiome: Environmental & human samples Soil samples: Indoor: Human: • Woodland • Compost bin (in • Oral/dental (me) kitchen)

• Bathroom • Scrubland near (tiles/bath) river

9 Bioaerosols – June 2017 Mycobiome: Environmental & human samples

10 Bioaerosols – June 2017 Current bioaerosol research focus

11 Bioaerosols – June 2017 Acknowledgements

PHE SAHSU • Tim Gant • Anna Hansell • Sarah Robertson • Pippa Douglas • Gillian Smith • Alex Elliot University of Adelaide • Giovanni Leonardi • Martin Breed

Keele University • Daniel Tonge

THANKS!

12 Bioaerosols – June 2017