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From nano-ZnOenabled antimicrobial surfaces to propidium iodide staining underestimating biofilm viability. Presentation by Merilin Rosenberg
- 1. From nano-ZnO enabled antimicrobial surfaces to propidium iodide staining underestimating biofilm viability Merilin Rosenberg COST AMICI ECI and STSM Workshop Riga 07.03.2019
- 2. • Background: – PhD project in National Institute of Chemical Physics and Biophysics (NICPB) • Laboratory of Environmental Toxicology • Dr Anne Kahru’s group – PhD student in Tallinn University of Technology (TalTech) • COST AMICI Short Term Scientific Mission (STSM) to – University of Porto • LEPABE - Laboratory for Process Engineering, Environment, Biotechnology and Energy • Dr. Nuno F. Azevedo
- 3. „Assessment of anti-biofilm activity of antimicrobial surfaces based on ZnO/Ag heterostructures“ • STSM duration: • 20/11/2017 to 19/12/2017, 1 month • Data analysis planned after STSM duration • STSM objective • Combining the expertise of NICPB and UoP to evaluate the anti-biofilm activity of surfaces covered with ZnO or ZnO/Ag heterostructure nanoparticles • Well defined experimental plan • Pre- ordered reagents • LIVE/DEAD™ BacLight™ Bacterial Viability Kit (Molecular Probes) • 3 well-described photocatalytic antimicrobial surface coatings and a control surface – Described in Visnapuu et al. 2018 • 2 bacterial species – Staphylococcus epidermidis DSM-20044 – Escherichia coli MG1655 • 4 time points – 2, 8, 24, 48 h – Dark incubation • 1 oligotrophic test medium – 1:500 diluted nutrient broth in deionized water • Main methods: – In situ viability staining of biofilm formation • Propidium iodide and SYTO 9 (LIVE/DEAD™ BacLight™ ) – Planktonic plate counts to assess dissolution toxicity – Planktonic samples for elemental analysis to assess dissolution – Possibly FISH or post-harvesting plate counts 1,8x1,8 cm glass surface 5 ml inoculum OD600=0,01
- 4. • S. epidermidis on control surface • S. epidermidis on toxic surface • E. coli on control surface • E. coli on toxic surface • Predictable total count results ↑ • Unexpected staining pattern! → • Harvested cells are metabolically active (FDA staining) and proved to be cultivable (plate counts)! • Osmotic stress? – Medium switched to PBS – Same phenomenon in PBS Red = dead Green = alive
- 5. DEAD – permanently damaged membrane PI - red Syto 9 - green VIABLE - reversibly permeable membrane VIABLE & CULTIVABLE – intact membrane VIABLE, but NOT CULTIVABLE (VBNC) PI staining eDNA Syto 9 staining DNA OR • dormant cells, • cultivable only in specific conditions, • decreased metabolic activity, • environmental stress, • starvation • reversible membrane damage • eDNA in biofilm ECM, • environmental stress, • specific growth phases, • gain of competence Propidium iodide (PI) Syto 9 chromosomal DNA, cell membrane
- 6. Rosenberg et al., submitted, preprint: https://doi.org/10.1101/475145 • 76% of Staphylococcus epidermidis and 96% of Escherichia coli cells in 24-hour initial biofilms on untreated glass are PI- positive ‘dead’ red cells. • 68% the cells of either species in these aggregates are metabolically active based on fluorescein diacetate (FDA) staining. • PI and SYTO 9 co-staining pattern is reversed by ultrasonication possibly by (partially) removing the extracellular matrix. • Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting by ultrasonication. • Most PI-stained ‘dead’ red cells are viable and cultivable!
- 7. Rosenberg et al., submitted, preprint: https://doi.org/10.1101/475145
- 8. Staphylococcus epidermidis and gram-negative Escherichia coli 24-hour initial biofilms on glass consist of 76 and 96% PI-positive red cells in situ, respectively, even though 68% the cells of either species in these aggregates are metabolically active. Furthermore, 82% of E. coli and 89% S. epidermidis are cultivable after harvesting. Rosenberg et al., submitted, preprint: https://doi.org/10.1101/475145 Confocal microscopy reveals mostly double-stained cells with red PI-stained eDNA coronas around green intracellular SYTO 9 signals. However, this explains only half of initial STSM conundrum of the undead microbes: the (extracellular) PI signal absence on ZnO-coated surfaces.
- 9. https://doi.org/10.1111/j.1471-4159.2009.06269.x • Similar staining pattern totally masking intracellular SYTO9 signal is achieved with SYTO9 and Congo red co-staining • Congo red is a well known amyloid stain staining also bacterial surface-bound amyloid fibrils (SAFs) e.g. E. coli curlis. • SAFs are abundantly present in biofilms, important in attachment to solid surfaces and able to bind DNA. • Zn(II) has been shown to inhibit amyloid fibril formation in a dose-dependent manner (Tõugu et al. 2009) in similar concentration range that is released from our ZnO-coated surfaces (≈15 µM). • Zn(II) released from ZnO may inhibit amyloid fibril formation and thereby restore green signals that are masked on surfaces that do not release Zn(II). That would also explain delayed biofilm formation on ZnO-coated surfaces. ThioflavinTfluorescence
- 10. Take-home message: • Select STSM host carefully – support at the lab is of utmost importance • Practical everyday lab questions • Background knowledge in the field • Laboratory infrastructure • Working language • Plan ahead as much as possible • Experimental plan, possible additional tests depending on results etc. • Transport of samples, reagents – are there restrictions? • Logistics, accommodation in case of late night lab sessions i.e. WIFI at the accommodation for literature searches is essential. • Pre-order or take with you everything needed • to avoid waiting for shipping dates during STSM • STSM budget is more limiting than longest possible time-frame without additional funding • Be prepared to work independently • while making use of all the help you can find • Ask questions! • Be prepared to manage major changes to experimental plan! • Find a way to make use of failed experiments as well as successful ones – questions make science! • Take the most out of professional networking and local expertise as well as sightseeing and cultural experiences!
- 11. https://doi.org/10.1111/j.1471-4159.2009.06269.x Be passionate! Ask questions! Do science! rosenbergmerilin@gmail.com