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Fireflies & superbugs: when science & nature collide
 

Fireflies & superbugs: when science & nature collide

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Slides from my talk at the NZ Micro Society Annual conference in Dunedin, November 2012.

Slides from my talk at the NZ Micro Society Annual conference in Dunedin, November 2012.

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    Fireflies & superbugs: when science & nature collide Fireflies & superbugs: when science & nature collide Presentation Transcript

    • Fireflies and superbugs:when science and nature collideDr Siouxsie WilesDept. Molecular Medicine & PathologyUniversity of Auckland
    • Nuria Andreu John Fraser Jimmy DaltonBrian Robertson Bill Wilson Benedict UyPaul Elkington Fiona Clow Hannah ReadTaryn Fletcher Simon Swift Sarah JohnsonNitya Krishnan Debbie Williamson Elin Rojdeby Fiona Radcliff Grant Mills Bill Denny Christina FehlmannAndrea Zelmer Brian Palmer John BoikovUlrich SchaibleGreg Bancroft Phil Crosier David Jenkins Shaun Lott Greg Cook Vic Arcus Joanna Kirman
    • NZMS Biophotonic imaging (BPI) The detection of light by specialised charge coupled device (CCD) cameras
    • NZMS Generating light: optical reporter genes Bioluminescence Fluorescence Enzyme –catalysed reaction Excitation of fluorophore GFP ‘mFruits’ Shaner et al (2004) Nature Biotechnology 22:1567-1572
    • NZMS Bioluminescence Enzyme catalysed reaction Flavin O2 CO2 oxidoreductase FMNH FMN 2 H+ O2 NAD(P) NAD(P)H H2O Light LightD-Luciferin Oxyluciferin + (560nm)  ?  ? (490nm) Luciferase Luciferase ATP AMP  ?  ? FFluc Aldehyde Fatty Acid (RHO) C D A B E (RCOOH) lux operon R T S T S R - Fatty acid reductase complex Firefly (Photinus pyralis) Photorhabdus luminescens
    • NZMS 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 BPI Equipment • Light-tight box • Sensitive charge coupled device camera • Number of machines commercially available
    • NZMS Procedure • Animal anaesthetised for restraint purposes • Add substrate if required • Reference (grey-scale) photographic image taken under low illumination • Image taken with/without illumination Image Image Min = -44712 Min = -44712 Max = 5.9625e+05 Max = 5.9625e+05 p/sec/cm^2/sr p/sec/cm^2/sr 100000 100000 90000 90000 80000 80000 70000 70000 60000 60000 50000 50000 40000 40000 30000 30000 Color Bar Color Bar Min = 30000 Min = 30000 Max = 100000 Max = 100000 bkg sub bkg sub flat-fielded flat-fielded cosmic cosmic Click # SW20070413151554 Series: pre cage 2 Click # SW20070413151554 Click # SW20070413151554 Series: pre cage 22 Series: pre cage 13 Apr 2007 15:16:06 IACUC #: pre cage 2 Click # SW20070413151554 Series: pre cage 2 1313 Apr 2007 15:16:06 Apr 2007 15:16:06 IACUC #: pre cage 22 IACUC #: pre cage Bin:M (2), FOV12, f8, 0.21307s Experiment: pre cage 2 13 Apr 2007 15:16:06 IACUC #: pre cage 2 Bin:M (4), FOV12, f1, 1m Bin:M (2), FOV12, f8, 0.21307s Experiment: pre cage 2 2 Experiment: pre cage Bin:M (4), FOV12, f1, 1m Experiment: pre cage 2 Filter: Open Label: 1x4 Filter: Open Label: 1x4 Filter: Open Label: 1x4 Filter: Open Label: 1x4 Camera: IVIS 82, DW34 Comment: Camera: IVIS 82, DW34 Comment: Camera: IVIS 82, DW34 Comment: Camera: IVIS 82, DW34 Comment: Analysis Comment: Analysis Comment: Analysis Comment: Analysis Comment:
    • NZMS Multi-parameter imaging: spectral unmixing RT GT
    • NZMS Biophotonic imaging
    • MWC Symposium Monitoring biological processes • Where are cells of interest? • Are they dead or alive? • What genes are being expressed?
    • NZMS Real-time infection dynamics 103 106 103 Photons sec-1 cm Day 1 Day 3 Day 6 Day 8 Day 10 Day 14(10 min exp) (5 min exp) (1 min exp) (1 min exp) (1 min exp) (1 min exp) sr-1-2 Wiles et al (2006). Infection and Immunity 74:5391-5396.
    • NZMS Real-time infection dynamics Image Image Min = -8.7851e+05 = -8.7851e+05 Min Max = 4.2345e+07 = 4.2345e+07 Max p/sec/cm^2/sr p/sec/cm^2/sr d1 20 20 (Photons sec-1 cm2 -1 sr-1) 15 15 Luminescence Image Image Min = -3083.3 Mi Min == 3.4125e+05 Max Max -3271.7 Max p/sec/cm^2/sr = 1.212e+05 p/s x10 p/sec/cm^2/sr x10 50000 50000 6 10 6 10 40000 d2 40000 30000 30000 5 5 20000 20000 10000 10000 Color Bar Color Bar Color Bar Col Color = 5000 Min Bar ~108 cfu mouse-1 24h-1 Min Min = 6.375e+05 = 6.375e+05 Min Min = 5000 Max = 50000 Max Max = 2.125e+07 = 2.125e+07 Max Max = 50000 bkg sub bkg s bkg sub flat-fielded flat-fie flat-fielded cosmic cosm cosmic Infection by natural transmission Click # SW20051123130535 Click # SW20051123130535 Series: 1 Series: 1 Click # SW20051123132046 Series: 1 bkg sub bkg sub 23 Nov 2005 13:05:49 23 Nov 2005 13:21:00 23 Nov 2005 13:05:49 IACUC #: IACUC #: IACUC #: Bin:M (4), FOV12,Bin:M (4), FOV12, f1, 10m f1, 10m Experiment: ICC180 NT B Experiment: ICC180 NT B flat-fielded flat-fielded Bin:M (4), FOV12, f1, 10m Filter: Open Filter: Open Experiment: ICC180 NT B Label: d2 10min x4bin d2 10min x4bin Label: cosmic Filter: Open Bishop et al (2007). Microb. Infect. 9:1316-1324. Label: d2 10min x4bin cosmic Camera: IVIS 82, Camera: IVIS 82, DW34 DW34 Comment: G/Bl/U Comment: G/Bl/U GI GI Camera: IVIS 82, DW34 Comment: BG litle BR GI
    • NZMS 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 Light reflects cell numbers luc-expressing lux-expressing Light levelsLight levels Bacterial numbers Bacterial numbers Answer in minutes rather than weeks!
    • NZMS Non-invasive drug screening Treated Untreated 2 days treatment 8 days treatment Andreu et al . Submitted
    • NZMS Auckland - FMHS
    • NZMS
    • NZMS Massive reduction in animal use 1010(cfu per gram tissue) 108 Number of bacteria 106 104 102 0 5 10 15 20 25 Time (days) Wiles et al (2004). Cellular Microbiology 6:963-972.
    • NZMS Massive reduction in animal use Dennis et al (2008). Infection and Immunity. 76:4978-4988
    • NZMS 3Rs - Refinement Intraperitoneal administration Oral gavage Incorrectly dosed animals can be removed from study Andreu et al (2011). FEMS Microbiology Reviews. 35(2):360-394
    • NZMS Not just for mice….
    • Get in touch! s.wiles@auckland.ac.nz www.youtube.com/user/Skeptimoo @SiouxsieW