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Live-imaging of Bacillus subtilis spore germination and outgrowth
Pandey, R.
Publication date
2014
Document Version
Final published version
Link to publication
Citation for published version (APA):
Pandey, R. (2014). Live-imaging of Bacillus subtilis spore germination and outgrowth.
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Summary
Bacterial spores are metabolically dormant and are extremely resistant to envi-ronmental stresses for long periods of time. In favorable condition, germination and outgrowth initiates vegetative cell growth. Bacterial spores are ubiquitously present in fresh vegetables, milk as well as ingredients for processed foods such as herbs and spices or dairy products. Spores of Gram-positive bacteria such as Bacillus and Clostridium cause huge economic losses to the food industry. In food products spores survive under food preservation conditions and subsequent germination and outgrowth eventually causes food spoilage. Hence efforts are being made to eliminate or inactivate these bacterial spores in foods. In this re-gard food industry uses different preservation methods such as thermal-treatment, weak acids, antimicrobial compounds etc. Complete thermal inactivation of mi-croorganisms in food products has become less popular due to the adverse effects on food quality and flavour. To fulfill the consumer’s demand whilst maintaining safety of food products the “hurdle preservation” approach is favoured to control the outgrowth of these bacteria in foods. The spore germination and outgrowth is often very heterogeneous. Heterogeneity makes it difficult to pinpoint which phases of germination and outgrowth are specifically affected upon a given preser-vation treatment. Therefore, the work presented in the thesis was focused on to the development of an image analysis tools for the measurement of germination and outgrowth of spores as well as the internal pH of the cells at single spore/cell level, which is described in detail in chapter 2 and chapter 6. Chapter 6 also describes the effect of sorbic acid and acetic acid on the internal pH of p B. subtilis cells at single cell level. Chapter 3, chapter 4, chapter 5, chapter 6 focused on the establishment of a system to analyze the behaviour of single spores and bacillus cells in order to quantify the degree of heterogeneity in each phase of the spore’s life cycle, upon exposure to specific tea compounds, sorbic
and acetic acids, heat treatments as well as combinations of heat and sorbic acids. Chapter 3 also describes the design of an air-contaning chamber for actual ex-perimentation with the aerobic bacteria as well as an image analysis tools for the quantification of the various phases of germination and outgrowth of spores and the behavior of their internal pH during these phases. Hence, the presented studies of live-imaging of B. subtilis spore germination and outgrowth, contribute to new insight with respect to improve or design new preservation concepts of microbial food stability and food safety.
Samenvatting
Bacteriële sporen zijn metabool inactieve bacteriële structuren en zijn zeer re-sistent tegen kortdurende maar ook veelal langdurige omgevingsstress. Onder gunstige omgevingscondities qua nutriëntconcentratie en temperatuur vindt kiem-ing plaats en initieert uitgroei van de sporen de vegetatieve celgroei. Bacteriële sporen zijn alom aanwezig in verse groenten, melk en ingrediënten voor geproceste (behandelde)voedingsmiddelen zoals kruiden en specerijen of zuivelproducten. In voedingsproducten overlevende sporen kunnen na kieming en uitgroei uitein-delijk voedselbederf en ziekte veroorzaken. Daardoor kunnen Bacilli en Clostridia enorme economische verliezen voor de voedingsindustrie veroorzaken en hebben ze tevens een significante impact op de volksgezondheid. Vandaar dat de in-dustrie zich inspant om deze sporen te elimineren of te inactiveren door gebruik te maken van verschillende conserveringsmethoden zoals thermische behandeling, zwakke zuren, antimicrobiële verbindingen etc. Compleet thermische inactivatie van micro-organismen in voedingsmiddelen is minder populair geworden als gevolg van de negatieve effecten op de kwaliteit van het voedsel eten en de smaak van de producten. Bacteriële sporen kieming en uitgroei zijn vaak zeer heterogeen. Heterogeniteit bemoeilijkt het vaststelen van de precieze kiemings en uitgroeifase waarop conserverende strategieën invloed hebben. Daarom hebben we ons in het hier gepresenteerde werk gericht op het opzetten van een analyse methode om het gedrag van enkele sporen / cellen te analyseren teneinde het effect van ene gegeven behandeling en de mate van heterogeniteit in de respons te kwantifi-ceren voor iedere fase van de levenscyclus van Bacillus subtilis sporen. De exper-imenten werden uitgevoerd in aanwezigheid van theecomponenten, sorbinezuur, azijnzuur, en met een oog op de zogenaamde âĂŸhorde conserveringsstrategieâĂŹ aan sporen die werden blootgesteld aan een hitte behandeling in combinatie met het toevoegen van sorbinezuur aan het kweekmedium. Daarnaast is een systeem
opgezet om het gedrag van de intracellulaire pH(zuurgraad) op enkelcelniveau te kunnen bepalen.
Hoofdstuk 2 beschrijft in detail de workflow van het imageanalysesysteem "SporeTracker" en de uitbreiding ervan, “multikanaal SporeTracker”, bedoeld voor analyse van de dynamiek van de intracellulaire pH van enkele sporen/cellen. Deze analyse-instrumenten werden gebruikt in hoofdstuk 3 en hoofdstuk 6 voor de analyse van de verschillende fasen van de kieming en uitgroei van de sporen en vegetatieve cellen. Hoofdstuk 3 beschrijft het ontwerp van een lucht bevattende observatiekamer, geschikt voor continue microscopische analyse van de (uit)groei van sporen en aërobe bacteriën. Een belangrijk kenmerk van deze kamer is dat er voldoende zuurstof en celkweekmedium is om optimale groeiomstandigheden van aërobe Bacilli te waarborgen. Dit hoofdstuk belicht ook de heterogeniteit in groeigedrag wanneer onbehandelde sporen werden ontkiemd en gekweekt in minimaal kweekmedium met een gedefinieerde hoeveelheid nutriÃńnten.
Thee (Camellia sinensis) is een van de meest geconsumeerde niet-alcoholische dranken in de wereld. Studies hebben aangetoond dat theepolyfenolen de groei van diverse Gram-positieve bacteriÃńn kan remmen. Het effect van deze verbindin-gen op ontkieming en uitgroei van bacteriesporen heeft tot nog toe weinig aan-dacht heeft gekregen. Hoofdstuk 4 beschrijft de kwantitatieve analyse van het effect van specifieke theeverbindingen op ontkieming en uitgroei van Bacillus sub-tilis sporen op enkel-cel resolutie. De studie gaf een beter begrip van het effect van de antimicrobiële in thee voorkomende stoffen gallocatechine gallaat, Teav-igo (> 90% epigallocatechine-3-gallaat), theaflavin 3,3’- digallate en galluszuur, op sporenontkieming en uitgroei. De geteste verbindingen hadden een significant effect op de meeste stadia van de kieming en uitgroei. Galluszuur had het sterk-ste effect op het vermogen tot uitgroei. De kiemingsefficiëntie (vermogen van de sporen om water op te nemen, correlerend met de fase-helder naar fase-donker overgang) werd niet beïnvloed. Bovendien beïnvloedden alle verbindingen, met name theaflavin 3,3 ’digallate duidelijk de groei van nieuwe vegetatieve cellen.
Zwakke zuren worden gewoonlijk gebruikt als, veelal natuurlijke, conserveer-middelen in de voedingsindustrie. Sorbinezuur is bekend om de groeiremmende effecten op verschillende micro-organismen waaronder bacteriën, gisten en schim-mels. Hoofdstuk 5 beschrijft de studie van het effect van de aanwezigheid van sorbinezuur (3 mM), de blootstelling aan warmte (85◦C/10 min) en de combinatie van beide op ontkieming en uitgroei van Bacillus subtilis sporen op enkel-cel res-olutie. De geteste concentratie van sorbinezuur (3 mM) heeft in de eerste plaats invloed op de uitgroei en generatietijd van de bacteriën die uit sporen ontstaan. De hittestress beïnvloedt vooral het kiemingsproces. De blootstelling verlengt de tijd tot het begin van de kieming en verlengt de kiemingstijd zelf. Het laatste wil zeggen dat de tijd die het duurt om van fase-helder tot fase-donker te komen
langer wordt. Er was echter geen effect op zowel uitgroei als generatietijd van de vegetatieve cellen die uit de sporen ontstonden. De combinatie van zowel een thermische behandeling en sorbinezuur gaf een synergistisch antimicrobieel effect. Hoofdstuk 6beschrijft de studie van het effect van zwakke zuren op de intra-cellulaire pH dynamica in vegetatieve cellen van Bacillus subtilis. In deze studie werd een verbeterde versie van de, genetisch gecodeerde, ratiometrische IpHluo-rin (afgeleid van GFP) gebruikt samen met live-imaging. Zwak organisch zuur zoals sorbinezuur en azijnzuur veroorzaakte concentratieafhankelijke intracellu-laire verlaging van de intracelluintracellu-laire pH die correleerde met een verlaging van de groeisnelheid. Interessant is dat er meer azijnzuur nodig was om de interne pH van de cel te verminderen dan sorbinezuur.
De gepresenteerde studies van beeldvorming van Bacillus subtilis sporen kiem-ing en uitgroei geven een unieke toolbox die nu zal kunnen dragen tot het verkri-jgen van nieuwe inzichten met betrekking tot het verbeteren of ontwerpen van nieuwe conserveringsconcepten teneinde beter kwaliteit voeding mogelijk te maken bij het behoud van de gewenste microbiële stabiliteit van ons voedsel.
List of Publications
1. Rachna Pandey, Alex Ter Beek, Norbert O. E. Vischer, Jan P.P.M. Smelt, Rober Kemperman, Erik M.M. Manders and Stanley Brul (2014). Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution. Food mi-crobiology. doi: 10.1016/j.fm.2014.03.006
2. Wishwas Abhyankar, Rachna Pandey, Alexander Ter Beek b, Stanley Brul, Leo J. de Koning, Chris G. de Koster (2014). Monitoring the progress in cross-linking of spore coat proteins during maturation of Bacillus subtilis spores. Food microbiology. doi: 10.1016/j.fm.2014.03.007
3. Rachna Pandey, Norbert O. E. Vischer, Jan P.P.M. Smelt, Alex Ter Beek, Stanley Brul, Erik M.M. Manders.(2013). Live cell imaging of germi-nation and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker. PLoS One. 8(3), e58972. doi: 10.1371/journal.pone.0058972
4. Alex Ter Beek, Luc M Hornstra, Rachna Pandey, Wouter W Kalle-meijn, Jan P P M Smelt, Erik M M Manders, Stanley Brul (2011). Models of the behaviour of (thermally stressed) microbial spores in foods: tools to study mechanisms of damage and repair. Food Microbiology 28, 678-684. doi: 10.1016/j.fm.2010.07.003
Acknowledgements
You’ll meet more angels on a winding path than on a straight one. Terri Guillemets. The successful completion of this thesis was made possible through the invalu-able contribution of a number of people. I am here to give my sincere thanks to all those who contributed in many ways to the success of this study and made it an unforgettable experience for me.
First and foremost, I would like to express my deepest and sincere gratitude to my supervisor (promoter) Professor Dr. Stanley Brul for having faith and patience with me. Your continuous optimism, enthusiasm, encouragement and support concerning this work helped me in successful completion of this thesis.
I would like to extend my sincere gratitude to my co-promoter Dr. Erik Menders, thank you for your understanding, encouragement and personal atten-tion, which have provided me a good and smooth basis for my PhD. If becoming an independent scientist is one of the main goals of doing a PhD, then I think you have done a great job. I like to give my thanks to Dr. Alex Ter Beek for suggestions on the experiments and comments and criticism whenever needed. I also thank you for your company in conferences. I had great time with you and will always cherries those times. Thank you!
I am deeply grateful to all members of the doctoral committee Prof. Dr. Mike Peck, Prof. Dr. C. G. De Koster, Prof. Dr. Tjakko Abee, Dr. Robèr Kemperman, Dr. Winnok de Vos and Dr. Joost Teixeira de Mattos for finding time to review the thesis and provide there valuable and fare comments. I want to thank my collaborator from Unilever and STW for showing interested in my work. Also, many thanks to all the co-authors from all the chapters and publications. Specially Norbert Vischer, Thank you for all the help and guidence for making SporeTracker and also for all the comments and criticism whenever needed. Thank
you from bottom of my heart.
I take this opportunity to sincerely acknowledge the Erasmas Mundas of Eu-ropean Union for the financial assistance in the form of scholarship in initial three year and University of Amsterdam for supporting me in the last year of my PhD, which buttressed me to perform my work comfortably.
I want to thank my students Marta and Gerard, whom I supervised during my PhD. Your work made it into the thesis. Thank you and good luck with the rest of your careers!
I would like to show my greatest appreciation to Niels Molennar, Jasper Faber, Ines Crespo and Casper Huijser for helping me whenever I asked for whether it is residence permit, housing or other official matter. Thank you! With this I thank Christel, Laura, Claire, Ans, Bondien, Dennis, Muss and Joost. Thanks for all the necessary “non-research-stuff”. Without you there would not be a running lab! Cristal.. I am very big fan of your well-organized attitude. Trying to adopt it form you.
I would like to thank all my colleagues from department of Molecular Biology & Microbial Food Safety as well as from department of Van Leeuwenhoek Centre for Advanced Microscopy Section of Moleular Cytology. Particularly. Jan Smelt, thank you for all the help and social talks that allowed me to know more about the Netherlands and it’s culture. I really appreciate your enthusiasm also kind and helping nature. I will always miss you wherever I will be. Johan van Beilen, thank you for being nice colleague, I appreciate your punctuality. Best of luck! Yanfang Feng, thank you for nice friendship and nice talk over coffee also for the jogging time was awesome. Hope our friendship remains forever. All the Best Dear. Daniel, thank you being good friend and nice coffee/tea time. Best wishes for future. Wishwas, I am deeply grateful to you for all the help and support you provided and also for all the trips and conference we attented during all these year as well as tea/coffee and dinner times. I wish you happy and successful life ahead. Ronald, thank you very much for your help with microscope and it’s booking. My deepest appreciation goes to Linli, Richard, Nadine, Reuben, Macro, Soraya, Laura, Gertein, Hans, Yelena, Andrea, Jeroen, Que, Sacha. I also want to thank Anna Ada, Linda, Giulia, Kobus. Thanks and best wishes to you all. Jolanda, I am deeply grateful to you for all the help you did when I was working in your lab.
I would like to thank all the Erasmus Mundus grantees whom I met in Ams-terdam. Thank you all for all the parties and enjoyable moments. I would also like to extend huge, warm thanks to all my roommates in all the three years. I also had the opportunity to meet many other persons in various conferences and I would like to thank all of them for giving me inspiring ideas or raising challenging questions.
Sjuual and Harm, I cannot forget to thank you for your unconditional support and valuable help. I can say I always had someone to look to. Thank you and I appreciate your support.
I am very grateful to Hansuk Buncherd, Weina Du, Abeer Hussain, Mouri Faruque who was wonderful accompany and very helpful all the time. I enjoyed all the dinners, trips and coffee time along with jokes and informative talks with you. Hansuk I appreciate your way of looking at life. You changed my perspective toward life. Thank you from deep of my heart. Abeer, thank you for being so nice to me. I can say you are really a good human being. Best wishes for future! Mouri, you joined lately in the lab but still the time we spent was awesome. I cannot forget those days. You are really a nice person. I wish you success and happiness in life. Aatish Kumar, thank you for all your help and suggestions you gave whenever needed. I am grateful to you from bottom of my heart. Thank you and I wish you best of luck for your defense. Pranav Puri, I met lately but still the help and support you gave is unforgettable. I am very grateful to you. Thank you very much! I would like to thank Edwin Gnanakumar, Mihir Jain and Kandan Ramakrishnan. Thank you guys!
Thanks to all my Indian friends other then the science for their help, support and motivation. Thank you for nice celebration be it Diwali or Navratri or some other occasions. I always enjoyed those dance parties and delicious food. Thank you for keeping me as part of the group and I will always remember those days as I captured it in my camera.
I met some wonderful friends with whom I shared all me happiness and frus-trations. Nutan-Ashish and Hemant-Mani thanks you for dinners, trips, ‘friendly’ games and listening to all my complaints about work. Great to meet outside work as well as thanks for being good friends! Hemant-Mani thanks you for giving me very young friend Yehvi. She is bundle of happiness to me. God bless her. I miss you all and will be missed wherever I will be. Chandan, what can I say about you! You are like angel to me. Thank you very much for all your great support and help. I appreciate your photography that helped me in capturing all my memories and times in the camera. I am fan of your Biriyani too. Even though you like Chicken Biryani but you always cooked Veg-Biriyan for me. Thank you from bottom of my heart and hope to get same kind of support in future too.
This acknowledgements list will not be completed without all my teachers who showed me correct path. Particularly Dr. J.B. Prajapati, thank you for encouragement and support. Without you it would not have been possible for me to come here to Netherlands. With this I would like to thank all my friends back in India specially, Dipal Soni, thank you for being these great everlasting friend everybody needs! I like to thank my Uncle and Aunty for love and support.
Thank you both of you.
Last but not least, I give deepest and sincere gratitude to my Parents Dr. R. N. Pandey and Gayatri Pandey. You brought me to life, raised me and supported me throughout my education. Thank you for your support, unconditional love and having faith and confidence in me. You are best parents in the world. Brother “Rohit” and sister “Richa” thank you for being nice siblings. You are like my friend and I thank you for being there for me all the time. I wish both of you a great, successful and happy life ahead. God bless you!
How can I forget to thank my Chinu-Minusss (Bacteria-Bacillus subtilis). You were part of my life for all these years. I spent most of my time with you then anyone else. Thank you! Finally, Thank you God for good care, support and strength gave to me throughout these years. Thank you very much.
Rachna
Amsterdam, 2014
