The dialogue between protozoa and bacteria in a microfluidic device

Autoři: Anna Gaines aff001;  Miranda Ludovice aff001;  Jie Xu aff001;  Marc Zanghi aff001;  Richard J. Meinersmann aff002;  Mark Berrang aff002;  Wayne Daley aff001;  Doug Britton aff001
Působiště autorů: Aerospace, Transportation and Advanced Systems Laboratory, Georgia Tech Research Institute, Georgia Institute of Technology, Atlanta, Georgia, United States of America aff001;  Richard B. Russell Research Center, Agricultural Research Service, United States Department of Agriculture, Athens, Georgia, United States of America aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria.

Klíčová slova:

Flow rate – Chemotaxis – Listeria – Microfluidics – Predation – Protozoans – Peptones – Nitrocellulose


1. Vaerewijck MJM, Baré J, Lambrecht E, Sabbe K, Houf K. Interactions of Foodborne Pathogens with Free-living Protozoa: Potential Consequences for Food Safety. Comprehensive Reviews in Food Science and Food Safety. 2014;13(5):924–44. doi: 10.1111/1541-4337.12100

2. Zubkov MV, Sleigh MA. Bacterivory by the ciliate Euplotes in different states of hunger. FEMS Microbiology Ecology. 1996;20(3):137–47.

3. Pachiadaki MG, Taylor C, Oikonomou A, Yakimov MM, Stoeck T, Edgcomb V. In situ grazing experiments apply new technology to gain insights into deep-sea microbial food webs. Deep Sea Research Part II: Topical Studies in Oceanography. 2016;129:223–31.

4. Sherr EB, Sherr BF. Bacterivory and herbivory: Key roles of phagotrophic protists in pelagic food webs. Microbial Ecology. 1994;28(2):223–35. doi: 10.1007/BF00166812 24186449

5. Pernthaler J. Predation on prokaryotes in the water column and its ecological implications. Nat Rev Micro. 2005;3(7):537–46.

6. Menon P, Billen G, Servais P. Mortality rates of autochthonous and fecal bacteria in natural aquatic ecosystems. Water Research. 2003;37(17):4151–8. doi: 10.1016/S0043-1354(03)00349-X 12946897

7. Strom SL, Loukos H. Selective feeding by protozoa: model and experimental behaviors and their consequences for population stability. Journal of Plankton Research. 1998;20(5):831–46. doi: 10.1093/plankt/20.5.831

8. Montagnes DJ, Barbosa AB, Boenigk J, Davidson K, Jurgens K, Macek M, et al. Selective feeding behaviour of key free-living protists: avenues for continued study. Aquatic Microbial Ecology. 2008;53(1):83–98.

9. Cohen MF, Mazzola M. Effects of Brassica napus seed meal amendment on soil populations of resident bacteria and Naegleria americana, and the unsuitability of arachidonic acid as a protozoan-specific marker J Protozool Res. 2006;16:16–25.

10. Jezbera J, Horňák K, Šimek K. Food selection by bacterivorous protists: insight from the analysis of the food vacuole content by means of fluorescence in situ hybridization. FEMS Microbiology Ecology. 2005;52(3):351–63. doi: 10.1016/j.femsec.2004.12.001 16329920

11. Gonsalves M-J, Fernandes SO, Priya ML, LokaBharathi PA. Grazing of particle-associated bacteria—an elimination of the non-viable fraction. Brazilian Journal of Microbiology. 2017;48(1):37–42. doi: 10.1016/j.bjm.2016.10.009 PMC5221368. 27939850

12. Saleem M, Fetzer I, Harms H, Chatzinotas A. Diversity of protists and bacteria determines predation performance and stability. The ISME Journal. 2013;7(10):1912–21. doi: 10.1038/ismej.2013.95 PMC3965320. 23765100

13. Boenigk J, Matz C, Jürgens K, Arndt H. Food concentration-dependent regulation of food selectivity of interception-feeding bacterivorous nanoflagellates. Aquatic Microbial Ecology. 2002;27(2):195–202.

14. Matz C, Kjelleberg S. Off the hook–how bacteria survive protozoan grazing. Trends in Microbiology. 2005;13(7):302–7. doi: 10.1016/j.tim.2005.05.009 15935676

15. Pohnert G, Steinke M, Tollrian R. Chemical cues, defence metabolites and the shaping of pelagic interspecific interactions. Trends in Ecology & Evolution. 22(4):198–204. doi: 10.1016/j.tree.2007.01.005 17275948

16. Denoncourt AM, Paquet VE, Charette SJ. Potential role of bacteria packaging by protozoa in the persistence and transmission of pathogenic bacteria. Frontiers in Microbiology. 2014;5:240. doi: 10.3389/fmicb.2014.00240 PMC4033053. 24904553

17. Trigui H, Paquet VE, Charette SJ, Faucher SP. Packaging of Campylobacter jejuni into Multilamellar Bodies by the Ciliate Tetrahymena pyriformis. Applied and Environmental Microbiology. 2016;82(9):2783–90. doi: 10.1128/AEM.03921-15 26921427

18. Bronowski C, James CE, Winstanley C. Role of environmental survival in transmission of Campylobacter jejuni. FEMS Microbiology Letters. 2014;356(1):8–19. doi: 10.1111/1574-6968.12488 24888326

19. And VL, Hellung-Larsen P. Chemosensory behaviour of Tetrahymena. BioEssays. 1992;14(1):61–6. doi: 10.1002/bies.950140113 1546982

20. Hellung-Larsen P, Leick V, Tommerup N. Chemoattraction in Tetrahymena: On the Role of Chemokinesis. Biological Bulletin. 1986;170(3):357–67. doi: 10.2307/1541847

21. Jousset A, Rochat L, Scheu S, Bonkowski M, Keel C. Predator-Prey Chemical Warfare Determines the Expression of Biocontrol Genes by Rhizosphere-Associated Pseudomonas fluorescens. Applied and Environmental Microbiology. 2010;76(15):5263–8. doi: 10.1128/AEM.02941-09 20525866

22. and DH, Keel C. REGULATION OF ANTIBIOTIC PRODUCTION IN ROOT-COLONIZING PSEUDOMONAS SPP. AND RELEVANCE FOR BIOLOGICAL CONTROL OF PLANT DISEASE. Annual Review of Phytopathology. 2003;41(1):117–53. doi: 10.1146/annurev.phyto.41.052002.095656 12730389.

23. Dopheide A, Lear G, Stott R, Lewis G. Preferential Feeding by the Ciliates Chilodonella and Tetrahymena spp. and Effects of These Protozoa on Bacterial Biofilm Structure and Composition. Applied and Environmental Microbiology. 2011;77(13):4564–72. doi: 10.1128/AEM.02421-10 PMC3127703. 21602372

24. Schmidt CE, Shringi S, Besser TE. Protozoan Predation of Escherichia coli O157:H7 Is Unaffected by the Carriage of Shiga Toxin-Encoding Bacteriophages. PLoS ONE. 2016;11(1):1–11. doi: 10.1371/journal.pone.0147270 112634214.

25. Wanjugi P, Fox GA, Harwood VJ. The Interplay Between Predation, Competition, and Nutrient Levels Influences the Survival of Escherichia coli in Aquatic Environments. Microbial Ecology. 2016;72(3):526–37. doi: 10.1007/s00248-016-0825-6 27484343

26. Oguri S, Hanawa T, Matsuo J, Ishida K, Yamazaki T, Nakamura S, et al. Protozoal ciliate promotes bacterial autoinducer-2 accumulation in mixed culture with Escherichia coli. The Journal of General and Applied Microbiology. 2015;61(5):203–10. doi: 10.2323/jgam.61.203 26582290

27. Arnold JW, Spacht D, Koudelka GB. Determinants that govern the recognition and uptake of Escherichia coli O157: H7 by Acanthamoeba castellanii. Cellular Microbiology. 2016;18(10):1459–70. doi: 10.1111/cmi.12591 26990156

28. Fenchel T, Blackburn N. Motile Chemosensory Behaviour of Phagotrophic Protists: Mechanisms for and Efficiency in Congregating at Food Patches. Protist. 1999;150(3):325–36. 10575704

29. Mao H, Cremer PS, Manson MD. A sensitive, versatile microfluidic assay for bacterial chemotaxis. PNAS. 2003;100(9):5449–54. doi: 10.1073/pnas.0931258100 12704234

30. Mahdavifar A, Xu J, Hovaizi M, Hesketh P, Daley W, Britton D. A Nitrocellulose-Based Microfluidic Device for Generation of Concentration Gradients and Study of Bacterial Chemotaxis. Journal of The Electrochemical Society. 2014;161(2):B3064–B70. doi: 10.1149/2.009402jes

31. Velve-Casquillas G, Le Berre M, Piel M, Tran PT. Microfluidic tools for cell biological research. Nano Today. 2010;5(1):28–47. doi: 10.1016/j.nantod.2009.12.001 21152269

32. Ahmed T, Shimizu TS, Stocker R. Microfluidics for bacterial chemotaxis. Integrative Biology. 2010;2(11–12):604–29. doi: 10.1039/c0ib00049c 20967322

33. Li J, Lin F. Microfluidic devices for studying chemotaxis and electrotaxis. Trends in Cell Biology. 21(8):489–97. doi: 10.1016/j.tcb.2011.05.002 21665472

34. Krajčovič J, Matej V, Schwartzbach SD. Euglenoid flagellates: A multifaceted biotechnology platform. Journal of Biotechnology. 2015;202:135–45. doi: 10.1016/j.jbiotec.2014.11.035 25527385

35. Gissibl A, Sun A, Care A, Nevalainen H, Sunna A. Bioproducts From Euglena gracilis: Synthesis and Applications. Frontiers in Bioengineering and Biotechnology. 2019;7(108). doi: 10.3389/fbioe.2019.00108 31157220

36. Radoshevich L, Cossart P. Listeria monocytogenes: towards a complete picture of its physiology and pathogenesis. Nature Reviews Microbiology. 2017;16:32. doi: 10.1038/nrmicro.2017.126 29176582

37. Lambrecht E, Baré J, Chavatte N, Bert W, Sabbe K, Houf K. Protozoan Cysts Act as a Survival Niche and Protective Shelter for Foodborne Pathogenic Bacteria. Applied and Environmental Microbiology. 2015;81(16):5604–12. doi: 10.1128/AEM.01031-15 26070667

38. Team S-l. scikit-learn: Machine Learning in Python 2017. Available from:

39. Diao J, Young L, Kim S, Fogarty EA, Heilman SM, Zhou P, et al. A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis. Lab on a Chip. 2006;6(3):381–8. doi: 10.1039/b511958h 16511621

40. Connell JL, Wessel AK, Parsek MR, Ellington AD, Whiteley M, Shear JB. Probing Prokaryotic Social Behaviors with Bacterial “Lobster Traps”. mBio. 2010;1(4):e00202–10. doi: 10.1128/mBio.00202-10 21060734

41. Ge Z, Girguis PR, Buie CR. Nanoporous microscale microbial incubators. Lab on a Chip. 2016;16(3):480–8. doi: 10.1039/c5lc00978b 26584739

42. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, et al. Use of Ichip for High-Throughput In Situ Cultivation of “Uncultivable” Microbial Species. Applied and Environmental Microbiology. 2010;76(8):2445–50. doi: 10.1128/AEM.01754-09 20173072

43. Wolfram CJ, Rubloff GW, Luo X. Perspectives in flow-based microfluidic gradient generators for characterizing bacterial chemotaxis. Biomicrofluidics. 2016;10(6):061301–. doi: 10.1063/1.4967777 27917249.

44. Barry MT, Rusconi R, Guasto JS, Stocker R. Shear-induced orientational dynamics and spatial heterogeneity in suspensions of motile phytoplankton. Journal of The Royal Society Interface. 2015;12(112). doi: 10.1098/rsif.2015.0791 26538558

45. Lee JW. The Effect of Temperature on Forward Swimming in Euglena and Chilomonas. Physiological Zoology. 1954;27(3):275–80.

46. Ogawa T, Shoji E, Suematsu NJ, Nishimori H, Izumi S, Awazu A, et al. The Flux of Euglena gracilis Cells Depends on the Gradient of Light Intensity. PLOS ONE. 2016;11(12):e0168114. doi: 10.1371/journal.pone.0168114 28033336

47. Giometto A, Altermatt F, Maritan A, Stocker R, Rinaldo A. Generalized receptor law governs phototaxis in the phytoplankton Euglena gracilis. Proceedings of the National Academy of Sciences. 2015;112(22):7045–50. doi: 10.1073/pnas.1422922112 25964338

48. Fellous S, Duncan A, Coulon A, Kaltz O. Quorum Sensing and Density-Dependent Dispersal in an Aquatic Model System. PLoS One. 2012;7(11). 1326737917.

49. Audrain B, Farag MA, Ryu C-M, Ghigo J-M. Role of bacterial volatile compounds in bacterial biology. FEMS Microbiology Reviews. 2015;39(2):222–33. doi: 10.1093/femsre/fuu013 25725014

50. Weise T, Kai M, Gummesson A, Troeger A, von Reuß S, Piepenborn S, et al. Volatile organic compounds produced by the phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria 85–10. Beilstein Journal of Organic Chemistry. 2012;8:579–96. doi: 10.3762/bjoc.8.65 22563356

51. Schulz-Bohm K, Geisen S, Wubs ERJ, Song C, de Boer W, Garbeva P. The prey’s scent–Volatile organic compound mediated interactions between soil bacteria and their protist predators. The Isme Journal. 2016;11:817. doi: 10.1038/ismej.2016.144 27911440

52. Schulz-Bohm K, Zweers H, de Boer W, Garbeva P. A fragrant neighborhood: volatile mediated bacterial interactions in soil. Frontiers in Microbiology. 2015;6(1212). doi: 10.3389/fmicb.2015.01212 26579111

53. Sharifi R, Ryu C-M. Are Bacterial Volatile Compounds Poisonous Odors to a Fungal Pathogen Botrytis cinerea, Alarm Signals to Arabidopsis Seedlings for Eliciting Induced Resistance, or Both? Frontiers in Microbiology. 2016;7(196). doi: 10.3389/fmicb.2016.00196 26941721

54. Vigneau E, Courcoux P, Symoneaux R, Guérin L, Villière A. Random forests: A machine learning methodology to highlight the volatile organic compounds involved in olfactory perception. Food Quality and Preference. 2018;68:135–45.

55. Tait E, Perry JD, Stanforth SP, Dean JR. Identification of Volatile Organic Compounds Produced by Bacteria Using HS-SPME-GC–MS. Journal of Chromatographic Science. 2014;52(4):363–73. doi: 10.1093/chromsci/bmt042 23661670

56. Bos LDJ, Sterk PJ, Schultz MJ. Volatile Metabolites of Pathogens: A Systematic Review. PLOS Pathogens. 2013;9(5):e1003311. doi: 10.1371/journal.ppat.1003311 23675295

57. Gao J, Zou Y, Wang Y, Wang F, Lang L, Wang P, et al. Breath analysis for noninvasively differentiating Acinetobacter baumannii ventilator-associated pneumonia from its respiratory tract colonization of ventilated patients. Journal of Breath Research. 2016;10(2):027102. doi: 10.1088/1752-7155/10/2/027102 27272697

58. Lazazzara V, Perazzolli M, Pertot I, Biasioli F, Puopolo G, Cappellin L. Growth media affect the volatilome and antimicrobial activity against Phytophthora infestans in four Lysobacter type strains. Microbiological Research. 2017;201:52–62. doi: 10.1016/j.micres.2017.04.015 28602402

59. Alfonzo A, Urso V, Corona O, Francesca N, Amato G, Settanni L, et al. Development of a method for the direct fermentation of semolina by selected sourdough lactic acid bacteria. International Journal of Food Microbiology. 2016;239:65–78. doi: 10.1016/j.ijfoodmicro.2016.06.027 27374130

60. Citron CA, Rabe P, Dickschat JS. The Scent of Bacteria: Headspace Analysis for the Discovery of Natural Products. Journal of Natural Products. 2012;75(10):1765–76. doi: 10.1021/np300468h 22994159

61. Gu Y-Q, Mo M-H, Zhou J-P, Zou C-S, Zhang K-Q. Evaluation and identification of potential organic nematicidal volatiles from soil bacteria. Soil Biology and Biochemistry. 2007;39(10):2567–75.

62. Bertram JH, Mulliner KM, Shi K, Plunkett MH, Nixon P, Serratore NA, et al. Five Fatty Aldehyde Dehydrogenase Enzymes from Marinobacter and Acinetobacter spp. and Structural Insights into the Aldehyde Binding Pocket. Applied and Environmental Microbiology. 2017;83(12). doi: 10.1128/aem.00018-17 28389542

63. Antipa GA, Martin K, Rintz MT. A Note on the Possible Ecological Significance of Chemotaxis in Certain Ciliated Protozoa1. The Journal of Protozoology. 1983;30(1):55–62. doi: 10.1111/j.1550-7408.1983.tb01033.x

64. DeMarco SF, Saada EA, Lopez MA, Hill KL. Identification of positive chemotaxis in the protozoan pathogen Trypanosoma brucei. bioRxiv. 2019:667378. doi: 10.1101/667378

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