MetaSUB and CSD in Oslo, Norway Marius Dybwad, Ph.D., Senior Scientist MetaSUB PI Oslo, Norway Protection and Societal Security Division Norwegian Defence Research Establishment FFI marius.dybwad@ffi.no

About me 33 years old B.Sc. Microbiology (3 yrs) «Strong» Bioaerosols Aerobiology Air sampling DNA isolation Biological background Microbiology «Not very strong» High-throughput sequencing Pipelined bioinformatics «Not strong at all» Advanced and custom bioinformatics 33 years old B.Sc. Microbiology (3 yrs) M.Sc. Molecular medicine (2 yrs) Ph.D. Molecular biology (4 yrs)

Norway Population: ~5 million

Oslo Population: 650 000 Number of lines: 5 Number of stations: 101 (17 underground) Length (km): 80 Riders per year: 100 million

Interesting features The subway network extends from the bustling city via residential neighbourhoods to the peaceful woods. Løren station is a new addition to the subway network and offers an opportunity to monitor a station over time from its commissioning (commissioned late-spring 2016). The city of Oslo is relatively small in size and has a low crowding factor compared to many other MetaSUB cities “Bustling city” “Peaceful woods” Løren station Frognerseteren: https://media-cdn.tripadvisor.com/media/photo-s/08/ef/15/05/frognerseteren-restaurant.jpg Karl Johan: http://www.kulturverk.com/wp-content/uploads/2011/05/folkemengde_Karl_Johan_.jpg Løren: https://commons.wikimedia.org/wiki/File:L%C3%B8ren_subway_station,_Oslo,_Norway_01.JPG

MetaSUB City Sampling Day (CSD) on June 21 44 stations 132 swab samples 15 air samples

Stations selected for CSD 44 stations covering all lines 13 underground stations and 31 outdoor stations Swab team consisting of 7 persons Air sampling team consisting of 2 persons

Sampling Volumetric air sampling (SASS 3100 electret filter sampler) at station platforms collection time 30 min @ 300 LPM Three wet swabs were collected on each station collection time 3 min Prioritized surfaces for swabs Ticket machines Railings (stair case/escalator) Benches

Preliminary results Station Object Surface Yield (ng) Ellingsrudåsen Kiosk Glass/plastic/metal 6.3 Railing Metal 2.7 Bench Wood 2.6 Haugerud 84.0 19.8 50.1 Tøyen 117.6 7.2 5.4 Carl Berners 12.9 11.4 6.6 Hasle 7.7 8.0 17.1 Økern 15.4 29.6 3.3 Vestli 18.4 177.6 5.1 Kalbakken 257.6 36.3 MetaSUB CSD 2016 in Oslo, Norway was executed successfully DNA is already extracted from 90/147 samples DNA yields from 1.4 – 257.6 ng

Potential comparisons/study groups Indoor (17) vs outdoor stations High (6) vs. low traffic stations Stations located close to major hospitals (9) vs. not Urban vs. rural areas Oslo East vs. Oslo West

Future perspectives During CSD duplicate swab sets were collected from each object and could be used for additional analyses Quantitative PCR (qPCR) using universal 16S rRNA gene primers RNA sequencing (stored at -80, but no preservative) Additional assay methods being considered Cultivation-based assays (TSA/R2A) MALDI-TOF MS for bacterial and fungal ID Antibiotic resistance profiling CFU pooling for cultivation-based metagenomics Future sampling campaigns Seasonal coverage – fall, winter, spring and summer High temporal resolution sampling – Diurnal/multi-day/week/month CSD 2017

Questions?

Method development and validation Dry vs. wet swab The same surfaces were swabbed with dry and wet swabs (dry swab first) Wet swabbing gave up to 15-fold higher DNA yields Dry [ng/µl] Wet [ng/µl] Ticket machine 0.24 3.78 Railing 0.30 0.46 Bench 0.13 1.63

Powersoil bead tubes vs. in-house bead tubes Method development and validation Powersoil bead tubes vs. in-house bead tubes Gram(+) vegetative cells (BG) Mobio PowerSoil/PathoMap protocol PowerSoil bead tubes Bead vortexing for 10 minutes In-house tubes and bead beating 2 ml bead beating tubes 600 mg 0.1 mm zirconium-silica beads and 550 µl PowerSoil Bead Solution Bead beating for 1 minute with Mini BeadBeater-8 Experiment was repeated twice with three replicate tubes for each organism Vortex Bead beating PowerSoil bead tubes 1.15 ± 0.07 6.47 ± 0.67 In-house bead tubes 7.39 ± 0.56 9.57 ± 0.86 [ng/µl] Gram(-) vegetative cells (EC) Vortex Bead beating PowerSoil bead tubes 1.86 ± 0.20 1.97 ± 0.36 In-house bead tubes 2.75 ± 0.13 2.96 ± 0.03 [ng/µl]