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Cited by 20Bacterial biofilms and quorum sensingCell Image Analysis TechniquesListeria monocytogenes in Food Safety

Imaging Flow Cytometry to Study Biofilm-Associated Microbial Aggregates

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Konieczny, Michał, Rhein, Peter, Czaczyk, Katarzyna, Białas, Wojciech, Juzwa, Wojciech·2021·Molecules
DOI 10.3390/molecules26237096PMC PMC8659131
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Abstract

The aim of the research was to design an advanced analytical tool for the precise characterization of microbial aggregates from biofilms formed on food-processing surfaces. The approach combined imaging flow cytometry with a machine learning-based interpretation protocol. Biofilm samples were collected from three diagnostic points of the food-processing lines at two independent time points. The samples were investigated for the complexity of microbial aggregates and cellular metabolic activity. Thus, aggregates and singlets of biofilm-associated microbes were simultaneously examined for the percentages of active, mid-active, and nonactive (dead) cells to evaluate the physiology of the microbial cells forming the biofilm structures. The tested diagnostic points demonstrated significant differences in the complexity of microbial aggregates. The significant percentages of the bacterial aggregates were associated with the dominance of active microbial cells, e.g., 75.3% revealed for a mushroom crate. This confirmed the protective role of cellular aggregates for the survival of active microbial cells. Moreover, the approach enabled discriminating small and large aggregates of microbial cells. The developed tool provided more detailed characteristics of bacterial aggregates within a biofilm structure combined with high-throughput screening potential. The designed methodology showed the prospect of facilitating the detection of invasive biofilm forms in the food industry environment.

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  • Flow cytometry application to study antimicrobial activity against biofilms may necessitate additional considerations and adaptations.

    “while flow cytometry is well-suited for evaluating planktonic microbial cells, its application to study antimicrobial activity against biofilms may necessitate additional considerations and adaptations [88].”

    From Methods for screening and evaluation of antimicrobial activity: A review of protocols, advantages, and limitations (2024) · cites this paper as [88]

    The Discussion (page unavailable) says conventional flow cytometry has difficulty detecting and functionally analyzing biofilm bacteria because of their small size and aggregates, and describes imaging flow cytometry as a way to overcome those obstacles. This supports the need for adaptations when analyzing biofilms, but the paper does not evaluate flow cytometry specifically for measuring antimicrobial activity against them. (LLM, conf=0.85)

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  1. [1]Bengtsson-Palme J. Microbial model communities: To understand complexity, harness the power of simplicity Comput. Struct. Biotechnol. J. 2020 18 3987 4001 10.1016/j.csbj.2020.11.043 33363696 PMC7744646 · DOI
  2. [2]Comas-Riu J. Rius N. Flow cytometry applications in the food industry J. Ind. Microbiol. Biotechnol. 2009 36 999 1011 10.1007/s10295-009-0608-x 19557445 · DOI
  3. [3]Galié S. García-Gutiérrez C. Miguélez E.M. Villar C.J. Lombó F. Biofilms in the food industry: Health aspects and control methods Front. Microbiol. 2018 9 989 10.3389/fmicb.2018.00898 29867809 PMC5949339 · DOI
  4. [4]Carrascosa C. Raheem D. Ramos F. Saraiva A. Raposo A. Microbial biofilms in the food industry—a comprehensive review Int. J. Environ. Res. Public Health 2021 18 2014 10.3390/ijerph18042014 33669645 PMC7922197 · DOI
  5. [5]Iñiguez-Moreno M. Gutiérrez-Lomelí M. Avila-Novoa M.G. Kinetics of biofilm formation by pathogenic and spoilage microorganisms under conditions that mimic the poultry, meat, and egg processing industries Int. J. Food Microbiol. 2019 303 32 41 10.1016/j.ijfoodmicro.2019.04.012 31129476 · DOI
  6. [6]Lindsay D. Von Holy A. What food safety professionals should know about bacterial biofilms Br. Food, J. 2006 108 27 37 10.1108/00070700610637616 · DOI
  7. [7]Burmølle M. Ren D. Bjarnsholt T. Sørensen S.J. Interactions in multispecies biofilms: Do they actually matter? Trends Microbiol. 2014 22 84 91 10.1016/j.tim.2013.12.004 24440178 · DOI
  8. [8]Jiang Y. Geng M. Bai L. Targeting biofilms therapy: Current research strategies and development hurdles Microorganisms 2020 8 1222 10.3390/microorganisms8081222 32796745 PMC7465149 · DOI
  9. [9]Karygianni L. Ren Z. Koo H. Thurnheer T. Biofilm Matrixome: Extracellular Components in Structured Microbial Communities Trends Microbiol. 2020 28 668 681 10.1016/j.tim.2020.03.016 32663461 · DOI
  10. [10]Yang L. Liu Y. Wu H. Høiby N. Molin S. Song Z.J. Current understanding of multispecies biofilms Int. J. Oral Sci. 2011 3 74 81 10.4248/IJOS11027 21485311 PMC3469880 · DOI
  11. [11]Burmølle M. Webb J.S. Rao D. Hansen L.H. Sørensen S.J. Kjelleberg S. Enhanced biofilm formation and increased resistance to antimicrobial agents and bacterial invasion are caused by synergistic interactions in multispecies biofilms Appl Env. Microbiol 2006 72 3916 3923 10.1128/AEM.03022-05 16751497 PMC1489630 · DOI
  12. [12]Giaouris E. Chorianopoulos N. Doulgeraki A. Nychas G.J. Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride PLoS ONE 2013 8 e77276 10.1371/journal.pone.0077276 24130873 PMC3795059 · DOI
  13. [13]Luppens S.B.I. Kara D. Bandounas L. Jonker M.J. Wittink F.R.A. Bruning O. Breit T.M. Ten Cate J.M. Crielaard W. Effect of Veillonella parvula on the antimicrobial resistance and gene expression of Streptococcus mutans grown in a dual-species biofilm Oral Microbiol. Immunol. 2008 23 183 189 10.1111/j.1399-302X.2007.00409.x 18402603 · DOI
  14. [14]Parijs I. Steenackers H.P. Competitive inter-species interactions underlie the increased antimicrobial tolerance in multispecies brewery biofilms ISME J. 2018 12 2061 2075 10.1038/s41396-018-0146-5 29858577 PMC6052168 · DOI
  15. [15]Schwering M. Song J. Louie M. Turner R.J. Ceri H. Multi-species biofilms defined from drinking water microorganisms provide increased protection against chlorine disinfection Biofouling 2013 29 917 928 10.1080/08927014.2013.816298 23879183 · DOI
  16. [16]Sharma D. Misba L. Khan A.U. Antibiotics versus biofilm: An emerging battleground in microbial communities Antimicrob. Resist. Infect. Control 2019 8 1 10 10.1186/s13756-019-0533-3 31131107 PMC6524306 · DOI
  17. [17]Simões M. Simões L.C. Vieira M.J. Species association increases biofilm resistance to chemical and mechanical treatments Water Res. 2009 43 229 237 10.1016/j.watres.2008.10.010 18977505 · DOI
  18. [18]Van der Veen S. Abee T. Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid Int. J. Food Microbiol. 2011 144 421 431 10.1016/j.ijfoodmicro.2010.10.029 21084128 · DOI
  19. [19]Kaplan J.B. Biofilm Dispersal: Mechanisms, Clinical Implications, and Potential Therapeutic Uses J. Dent. Res. 2010 89 205 218 10.1177/0022034509359403 20139339 PMC3318030 · DOI · Paper page
  20. [20]Juzwa W. Duber A. Myszka K. Białas W. Czaczyk K. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting Biofouling 2016 32 841 851 10.1080/08927014.2016.1201657 27406324 · DOI
  21. [21]Tamburini S. Foladori P. Ferrentino G. Spilimbergo S. Jousson O. Accurate flow cytometric monitoring of Escherichia coli subpopulations on solid food treated with high pressure carbon dioxide J. Appl. Microbiol. 2014 117 440 450 10.1111/jam.12528 24766564 · DOI
  22. [22]Fux C.A. Wilson S. Stoodley P. Detachment Characteristics and Oxacillin Resistance of Staphyloccocus aureus Biofilm Emboli in an In Vitro Catheter Infection Model J. Bacteriol. 2004 186 4486 4491 10.1128/JB.186.14.4486-4491.2004 15231780 PMC438612 · DOI
  23. [23]Guilhen C. Forestier C. Balestrino D. Biofilm dispersal: Multiple elaborate strategies for dissemination of bacteria with unique properties Mol. Microbiol. 2017 105 188 210 10.1111/mmi.13698 28422332 · DOI
  24. [24]Baltenneck J. Reverchon S. Hommais F. Quorum sensing regulation in phytopathogenic bacteria Microorganisms 2021 9 239 10.3390/microorganisms9020239 33498890 PMC7912708 · DOI
  25. [25]Coughlan L.M. Cotter P.D. Hill C. Alvarez-Ordóñez A. New weapons to fight old enemies: Novel strategies for the (bio)control of bacterial biofilms in the food industry Front. Microbiol. 2016 7 1641 10.3389/fmicb.2016.01641 27803696 PMC5067414 · DOI
  26. [26]Nikolaev Y.A. Plakunov V.K. Biofilm-“city of microbes” or an analogue of multicellular organisms? Microbiology 2007 76 125 138 10.1134/S0026261707020014 17583210 · DOI
  27. [27]Srey S. Jahid I.K. Ha S.D. Biofilm formation in food industries: A food safety concern Food Control 2013 31 572 585 10.1016/j.foodcont.2012.12.001 · DOI
  28. [28]Cai Y.M. Non-surface Attached Bacterial Aggregates: A Ubiquitous Third Lifestyle Front. Microbiol. 2020 11 557035 10.3389/fmicb.2020.557035 33343514 PMC7746683 · DOI
  29. [29]Karatan E. Watnick P. Signals, Regulatory Networks, and Materials That Build and Break Bacterial Biofilms Microbiol. Mol. Biol. Rev. 2009 73 310 347 10.1128/MMBR.00041-08 19487730 PMC2698413 · DOI
  30. [30]Abdallah M. Benoliel C. Drider D. Dhulster P. Chihib N.E. Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments Archives of Microbiology Springer Berlin, Germany 2014 453 472 10.1007/s00203-014-0983-1 24744186 · DOI
  31. [31]Barteneva N.S. Fasler-Kan E. Vorobjev I.A. Imaging flow cytometry: Coping with heterogeneity in biological systems J. Histochem. Cytochem. Off. J. Histochem. Soc. 2012 60 723 733 10.1369/0022155412453052 PMC3524563 22740345 · DOI
  32. [32]Wilson C. Lukowicz R. Merchant S. Valquier-Flynn H. Caballero J. Sandoval J. Okuom M. Huber C. Durham Brooks T. Wilson E. Quantitative and Qualitative Assessment Methods for Biofilm Growth: A Mini-review HHS Public Access Res. Rev. J. Eng. Technol. 2017 6 PMC6133255 30214915
  33. [33]Heydorn A. Nielsen A.T. Hentzer M. Sternberg C. Givskov M. Ersboll B.K. Molin S. Quantification of biofilm structures by the novel computer program COMSTAT Microbiology 2000 146 2395 2407 10.1099/00221287-146-10-2395 11021916 · DOI
  34. [34]Hartmann R. Jeckel H. Jelli E. Singh P.K. Vaidya S. Bayer M. Rode D.K.H. Vidakovic L. Díaz-Pascual F. Fong J.C.N. Quantitative image analysis of microbial communities with BiofilmQ Nat. Microbiol. 2021 6 151 156 10.1038/s41564-020-00817-4 33398098 PMC7840502 · DOI
  35. [35]Mcevoy B. Lynch M. Rowan N.J. Opportunities for the application of real-time bacterial cell analysis using flow cytometry for the advancement of sterilization microbiology J. Appl. Microbiol. 2020 130 1794 1812 10.1111/jam.14876 33155740 · DOI
  36. [36]Macey M.G. Flow Cytometry: Principles and Applications 2007th ed. Humana Press New York, NY, USA 2007 10.1007/978-1-59745-451-3 · DOI
  37. [37]Kudernatsch R.F. Letsch A. Stachelscheid H. Volk H.D. Scheibenbogen C. Doublets pretending to be CD34+ T cells despite doublet exclusion Cytom. Part A 2013 83 173 176 10.1002/cyto.a.22247 23281028 · DOI
  38. [38]Pugsley H.R. Quantifying autophagy: Measuring LC3 puncta and autolysosome formation in cells using multispectral imaging flow cytometry Methods 2017 112 147 156 10.1016/j.ymeth.2016.05.022 27263026 · DOI
  39. [40]Azeredo J. Azevedo N.F. Briandet R. Cerca N. Coenye T. Costa A.R. Desvaux M. Di Bonaventura G. Hébraud M. Jaglic Z. Critical review on biofilm methods Crit. Rev. Microbiol. 2017 43 313 351 10.1080/1040841X.2016.1208146 27868469 · DOI
  40. [41]Li L. Mendis N. Trigui H. Oliver J.D. Faucher S.P. The importance of the viable but non-culturable state in human bacterial pathogens Front. Microbiol. 2014 5 258 10.3389/fmicb.2014.00258 24917854 PMC4040921 · DOI
  41. [42]Park Y. Abihssira-García I.S. Thalmann S. Wiegertjes G.F. Barreda D.R. Olsvik P.A. Kiron V. Imaging Flow Cytometry Protocols for Examining Phagocytosis of Microplastics and Bioparticles by Immune Cells of Aquatic Animals Front. Immunol. 2020 11 203 10.3389/fimmu.2020.00203 32133001 PMC7039858 · DOI
  42. [43]Power A.L. Barber D.G. Groenhof S.R.M. Wagley S. Liu P. Parker D.A. Love J. The Application of Imaging Flow Cytometry for Characterisation and Quantification of Bacterial Phenotypes Front. Cell. Infect. Microbiol. 2021 11 716592 10.3389/fcimb.2021.716592 34368019 PMC8335544 · DOI
  43. [44]Vorobjev I.A. Natasha S. Barteneva N.S. Quantitative Functional Morphology by Imaging Flow Cytometry Imaging Flow Cytometry Methods and Protocols Methods in Molecular Biology Barteneva N.S. Vorobjev I.A. Humana Press New York, NY, USA 2016 3 11 10.1007/978-1-4939-3302-0 27460234 · DOI
  44. [45]Ferone M. Gowen A. Fanning S. Scannell A.G.M. Microbial detection and identification methods: Bench top assays to omics approaches Compr. Rev. Food Sci. Food Saf. 2020 19 3106 3129 10.1111/1541-4337.12618 33337061 · DOI
  45. [46]Müller S. Nebe-Von-Caron G. Functional single-cell analyses: Flow cytometry and cell sorting of microbial populations and communities FEMS Microbiol. Rev. 2010 34 554 587 10.1111/j.1574-6976.2010.00214.x 20337722 · DOI
  46. [47]Wille J. Coenye T. Biofilm dispersion: The key to biofilm eradication or opening Pandora’s box? Biofilm 2020 2 100027 10.1016/j.bioflm.2020.100027 33447812 PMC7798462 · DOI
  47. [48]Chua S.L. Yang-Yi Tan S. Rybtke M.T. Chen Y. Rice S.A. Kjelleberg S. Tolker-Nielsen T. Yang L. Givskov M. Bis-(3′-5′)-cyclic dimeric GMP regulates antimicrobial peptide resistance in Pseudomonas aeruginosa Antimicrob. Agents Chemother. 2013 57 2066 2075 10.1128/AAC.02499-12 23403434 PMC3632963 · DOI
  48. [49]Chua S.L. Liu Y. Yam J.K.H. Chen Y. Vejborg R.M. Tan B.G.C. Kjelleberg S. Tolker-Nielsen T. Givskov M. Yang L. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles Nat. Commun. 2014 5 4462 10.1038/ncomms5462 25042103 · DOI
  49. [50]Gray D. Yue R.S. Chueng C.Y. Godfrey W. Bacterial vitality detected by a novel fluorogenic redox dye using flow cytometry Proceedings of the Abstract of 105th General Meeting of the American Society for Microbiology Atlanta, GA, USA 5–9 June 2005 Volume 105 331
  50. [51]Díaz M. Herrero M. García L.A. Quirós C. Application of flow cytometry to industrial microbial bioprocesses Biochem. Eng. J. 2010 48 385 407 10.1016/j.bej.2009.07.013 · DOI
  51. [52]Juzwa W. Myszka K. Białas W. Investigation of the population dynamics within a Pseudomonas aeruginosa biofilm using a flow based biofilm model system and flow cytometric evaluation of cellular physiology Biofouling 2018 34 835 850 10.1080/08927014.2018.1508569 30332894 · DOI

Cited by

Sorted by citations

Authors
Konieczny, M., Rhein, P., Czaczyk, K., Białas, W., & Juzwa, W.
Affiliations
  • Poznań University of Life Sciences (PL)
  • Luminex B.V., A DiaSorin Company, 5215 MV ‘s-Hertogenbosch, The Netherlands
Corresponding author
Juzwa, Wojciech
ORCID
Venue
Molecules
Publisher
MDPI AG
Published
2021-11-24
Volume
26
Issue
23
Pages
7096
ISSN
1420-3049 (ISSN-L)
DOI
10.3390/molecules26237096
PMC
PMC8659131
OpenAlex
W3215148857
Cited by
20
References
1
Citations by year
2026: 2 · 2025: 5 · 2024: 3 · 2023: 6 · 2022: 4
FWCI
1.02
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecules (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bacterial biofilms and quorum sensing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Listeria monocytogenes in Food Safety
Keywords
imaging flow cytometry, food processing surfaces, bacterial metabolic activity, cell viability, food safety
MeSH terms
Bacteria, Flow Cytometry, Food Handling, Food Microbiology, Biofilms, High-Throughput Screening Assays

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