1. 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

2. 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)

3. Norway
Population: ~5 million

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

5. 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:
Karl Johan:
Løren:

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

7. 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

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

9. 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 – ng

10. 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

11. 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

12. Questions?

13. 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

14. 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]