![Page 1: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/1.jpg)
Fireflies and superbugs: when science and nature collide
Dr Siouxsie Wiles
Dept. Molecular Medicine & Pathology
University of Auckland
![Page 2: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/2.jpg)
![Page 3: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/3.jpg)
Nuria Andreu
Brian Robertson
Paul Elkington
Taryn Fletcher
Nitya Krishnan
Andrea Zelmer
Ulrich Schaible
Greg Bancroft
John Fraser
Bill Wilson
Fiona Clow
Simon Swift
Debbie Williamson
Fiona Radcliff
Bill Denny
Brian Palmer
Phil Crosier
David Jenkins
Shaun Lott
Jimmy Dalton
Benedict Uy
Hannah Read
Sarah Johnson
Elin Rojdeby
Grant Mills
Christina Fehlmann
John Boikov
Greg Cook
Joanna Kirman
Vic Arcus
![Page 4: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/4.jpg)
The detection of light by specialised
charge coupled device (CCD) cameras
Biophotonic imaging (BPI)
NZMS
![Page 5: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/5.jpg)
Generating light: optical reporter genes
Bioluminescence
Enzyme –catalysed reaction
Fluorescence
Excitation of fluorophore
GFP ‘mFruits’
Shaner et al (2004) Nature Biotechnology 22:1567-1572
NZMS
![Page 6: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/6.jpg)
Bioluminescence Enzyme catalysed reaction
D-Luciferin Light
(560nm)
Luciferase
ATP AMP
CO2 O2
Oxyluciferin +
FFluc
Firefly (Photinus pyralis)
lux operon
C D A B E
R T S
R T S
-
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
? ?
Luciferase
FMNH FMN
NAD(P) NAD(P)H H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
lux operon
C D A B E
R T S
R T S
-
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
? ?
Luciferase
FMNH 2
FMN
NAD(P) H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
lux operon
C D A B E
R T S
R T S
R T S
Fatty acid reductase complex -
Fatty Acid
(RCOOH)
Aldehyde
(RHO)
? ?
Luciferase
FMNH FMN
NAD(P) H +
Flavin oxidoreductase
Light (490nm)
O 2 H
2 O
Photorhabdus luminescens
NZMS
![Page 7: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/7.jpg)
Why is bioluminescence so useful?
• No light unless luciferase genes present
• Dead cells don’t glow!
• The more bacteria there are, the more light there is
• Light travels through things – like flesh & skin!
• Potential for non-destructive real-time measurements
NZMS
![Page 8: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/8.jpg)
BPI Equipment
• Light-tight box
• Sensitive charge coupled device camera
• Number of machines commercially available
NZMS
![Page 9: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/9.jpg)
Procedure
• Animal anaesthetised for restraint purposes
• Add substrate if required
• Reference (grey-scale) photographic image taken under low illumination
• Image taken with/without illumination
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
100000
90000
80000
70000
60000
50000
40000
30000
ImageMin = -44712
Max = 5.9625e+05p/sec/cm 2̂/sr
Color BarMin = 30000
Max = 100000
bkg subflat-fieldedcosmic
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (2), FOV12, f8, 0.21307sFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
Click # SW2007041315155413 Apr 2007 15:16:06Bin:M (4), FOV12, f1, 1mFilter: OpenCamera: IVIS 82, DW34
Series: pre cage 2IACUC #: pre cage 2Experiment: pre cage 2Label: 1x4Comment: Analysis Comment:
100000
90000
80000
70000
60000
50000
40000
30000
ImageMin = -44712
Max = 5.9625e+05p/sec/cm 2̂/sr
Color BarMin = 30000
Max = 100000
bkg subflat-fieldedcosmic
NZMS
![Page 10: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/10.jpg)
Multi-parameter imaging: spectral unmixing
GT
RT
NZMS
![Page 11: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/11.jpg)
Biophotonic imaging
NZMS
![Page 12: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/12.jpg)
Monitoring biological processes
• Where are cells of interest?
• Are they dead or alive?
• What genes are being expressed?
MWC Symposium
![Page 13: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/13.jpg)
Real-time infection dynamics
Day 1 (10 min exp)
Day 3 (5 min exp)
Day 6 (1 min exp)
Day 8 (1 min exp)
Day 10 (1 min exp)
Day 14 (1 min exp)
10
3
10
6
10
3
Photo
ns s
ec
-1 cm
-2 s
r-1
Wiles et al (2006). Infection and Immunity 74:5391-5396.
NZMS
![Page 14: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/14.jpg)
Real-time infection dynamics
Click # SC20041208101555
08 Dec 2004 10:16:24
Bin:1, FOV12, f1, 1m
Filter: Open
Camera: IVIS 82, DW34
Series:
IACUC #:
Experiment: cage
Label: 11
Comment:
Analysis Comment: 1x1
20
15
10
5
x10
6
Image
Min = -8.7851e+05
Max = 4.2345e+07
p/sec/cm^2/sr
Color Bar
Min = 6.375e+05
Max = 2.125e+07
bkg sub
flat-fielded
cosmic
Click # SC20041208101555
08 Dec 2004 10:16:24
Bin:1, FOV12, f1, 1m
Filter: Open
Camera: IVIS 82, DW34
Series:
IACUC #:
Experiment: cage
Label: 11
Comment:
Analysis Comment: 1x1
20
15
10
5
x10
6
Image
Min = -8.7851e+05
Max = 4.2345e+07
p/sec/cm^2/sr
Color Bar
Min = 6.375e+05
Max = 2.125e+07
bkg sub
flat-fielded
cosmic
~108 cfu mouse-1 24h-1
Lum
inescence
(P
hoto
ns s
ec
-1 cm
2 -1 s
r-1)
d1
Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3083.3
Max = 3.4125e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
Click # SW2005112313053523 Nov 2005 13:05:49Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: G/Bl/U GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3083.3
Max = 3.4125e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
Click # SW2005112313204623 Nov 2005 13:21:00Bin:M (4), FOV12, f1, 10mFilter: OpenCamera: IVIS 82, DW34
Series: 1IACUC #: Experiment: ICC180 NT BLabel: d2 10min x4binComment: BG litle BR GIAnalysis Comment:
50000
40000
30000
20000
10000
ImageMin = -3271.7
Max = 1.212e+05p/sec/cm^2/sr
Color BarMin = 5000
Max = 50000
bkg subflat-fieldedcosmic
d2
Infection by natural transmission
Bishop et al (2007). Microb. Infect. 9:1316-1324.
NZMS
![Page 15: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/15.jpg)
Mycobacterium tuberculosis: the captain of death
• 1/3 people latently infected
• 4,500 people die every day
• Resistant to common antibiotics
• Grows very slowly…
Desperate need for new antibiotics and faster ways to test them!
NZMS
![Page 16: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/16.jpg)
Light reflects cell numbers
Lig
ht
levels
Answer in minutes rather than weeks!
Bacterial numbers Bacterial numbers
Lig
ht
levels
lux-expressing luc-expressing
NZMS
![Page 17: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/17.jpg)
Untreated Treated
2 days treatment
8 days treatment
Non-invasive drug screening
Andreu et al . Submitted
NZMS
![Page 18: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/18.jpg)
Auckland - FMHS
NZMS
![Page 19: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/19.jpg)
NZMS
![Page 20: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/20.jpg)
Massive reduction in animal use
1010
108
106
102
104
0 5 10 15 20 25
Time (days)
Num
ber
of
bacte
ria
(cfu
per
gra
m t
issue)
Wiles et al (2004). Cellular Microbiology 6:963-972.
NZMS
![Page 21: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/21.jpg)
Massive reduction in animal use
Dennis et al (2008). Infection and Immunity. 76:4978-4988
NZMS
![Page 22: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/22.jpg)
3Rs - Refinement
Incorrectly dosed animals can be removed from study
Intraperitoneal administration Oral gavage
Andreu et al (2011). FEMS Microbiology Reviews. 35(2):360-394
NZMS
![Page 23: Fireflies & superbugs: when science & nature collide](https://reader034.vdocument.in/reader034/viewer/2022050808/54902715b4795963488b4d35/html5/thumbnails/23.jpg)
Not just for mice….
NZMS