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Relucenti, M. Microscopy Methods for Biofilm Imaging. Encyclopedia. Available online: https://encyclopedia.pub/entry/6952 (accessed on 21 December 2024).
Relucenti M. Microscopy Methods for Biofilm Imaging. Encyclopedia. Available at: https://encyclopedia.pub/entry/6952. Accessed December 21, 2024.
Relucenti, Michela. "Microscopy Methods for Biofilm Imaging" Encyclopedia, https://encyclopedia.pub/entry/6952 (accessed December 21, 2024).
Relucenti, M. (2021, February 01). Microscopy Methods for Biofilm Imaging. In Encyclopedia. https://encyclopedia.pub/entry/6952
Relucenti, Michela. "Microscopy Methods for Biofilm Imaging." Encyclopedia. Web. 01 February, 2021.
Microscopy Methods for Biofilm Imaging
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This entry is adapted from the peer-reviewed paper 10.3390/biology10010051

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure.

scanning electron microscopy variable pressure scanning electron microscopy

1. Introduction

Surface-attached microbial agglomerations were for the first time named as a “biofilm” by William J. Costerton in 1978 [1]. In the following years, he perfected this definition by also considering the host role and the three-dimensional (3-D) architecture. The definition of biofilm was thus implemented, expanding the concept toward a complex community of microorganisms living attached to a surface or interface, being enclosed in an exopolysaccharide matrix (Eps) of microbial and host origin arranged in a three-dimensional (3-D) architecture [2]. Bacterial species in biofilms exhibit cooperation [3], behaving as complex multi-cellular organisms [4]. Eps composition is complex and it may contain polysaccharides, proteins, nucleic acid, lipids, and metals. [5]. The complex array of chemically and functionally diverse biomolecules in the Eps has been termed the matrixome [6], which contributes to the peculiar characteristics of biofilm behavior. According to the National Institutes of Health (NIH), bacterial biofilms are responsible for up to 75% of infectious diseases in humans, as implant-related infections and/or tissue-associated infections [7]. In the European Union and European Economic Area, 8.9 million healthcare-associated infection episodes per year are due to biofilms [8]. These infections are often recurrent and resistant to antibiotic treatments [9][10] due to the particular characteristics of Eps that protect the resident microorganisms from the effects of host immunity or administered antimicrobial drugs [11]. It is of crucial importance nowadays to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm-related infections. In this kind of investigation, the use of different microscopy techniques is required to better understand the intimate details of the biofilms’ ultrastructure, their 3-D organization, cell population behavior, and reactions after drug treatments [12]. The development of novel morphological investigation approaches is therefore crucial.

2. Microscopy Techniques Applied to Biofilm Imaging

2.1. Light Microscopy (LM)

Light microscopy (LM) is a basic imaging technique that is useful for providing the visual identification of biofilm presence and also has significant prognostic value [13]. It can be used for quantitative assessment of biofilm biomass, being easy and low cost to perform [14][15]. However, light microscopy has limited magnification and resolution, so it cannot be applied to describe the finest details of biofilm cell morphology or Eps architecture, but it can be coupled with Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) in correlative studies as in [16]. In this study on teeth microflora, light microscopy observation of semi-thin sections from demineralized teeth provided the best overall perspective of the root canal, enabling larger areas to be observed at low magnification. Samples observed with SEM did not show bacteria in dentine tubules, in contrast, when the same samples were demineralized and included in resin, their semi-thin section LM images revealed the presence of bacteria, then TEM images confirmed the LM findings [16].

2.2. Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) allows for the quantitative evaluation of structural parameters as biovolume (cells overall volume in the observation field), thickness, and roughness. Sample 3-D architecture representation and its time-dependent variation (real-time 4-D) can also be achieved [17]. CLSM was used in combination with a fluorescent stain and was successfully applied on different biofilms species [18][19][20][21]. The CLSM resolution level is singe cell dimension and using pathogen-specific probes labeled with different fluorescent dyes (FISH followed by CLSM) as described in [22], identification of a single species in multispecies samples is allowed. With the same approach, interspecies competition assessment as well as interference in-between species were analyzed [23]. In studies assessing drug antimicrobial effects, CLSM was used, together with specific fluorophores, to discriminate between live or dead bacterial cells, localizing also their spatial distribution [24][25][26][27][28]. CLSM is a method of choice for biofilm visualization and quantification. Unfortunately, CLSM biofilm analysis has limitations due to the use of fluorophores, the existence of a limited number of reporter molecules, and the signal of interest might be hidden by the interference of intrinsic biofilm fluorescence with that of the probe.

2.3. Atomic Force Microscopy (AFM)

Bacteria respond to different mechanical signals [29] like adhesion forces originating during adhesion processes. During these events, bacterial surfaces deform [30], modifying the intra-bilayer pressure profile [31], which, in turn, changes bacterial gene expressions, transforming a planktonically growing cell into a biofilm growing one. Atomic force microscopy (AFM) allows for the quantification of adhesion forces existing among living cells, and between cells and surfaces [32][33]. The knowledge of how adhesion and viscoelasticity can modulate biofilm development may be important in the design of biofilm control strategies. Viscoelastic properties of biofilms influence antimicrobial penetration and removal of biofilm from surfaces and therefore performs a role in their protection against mechanical and chemical challenges [34]. This approach was recently used to demonstrate how amyloid protein production dramatically increases the stiffness of Pseudomonas biofilms [35]. AFM has been applied to obtain insights into biofilm 3-D developmental patterns [36][37][38][39][40][41]. Atomic force microscopy (AFM) allows for the quantification of biofilm biomass in terms of thickness and Eps amount based on height and roughness analyses from AFM images [42][43][44][45][46][47][48][49]. Vantages, disadvantages and application fields of non-electron microscopic techniques are summarized in Table 1.

Table 1. Most widely used non-electron microscopic techniques for biofilm study.

 

Light Microscopy

CLSM

AFM

Pros

Simple protocols

Cheap and easy to perform

Large investigation area

Allows single cell visualization and 3-D imaging

Nondestructive technique that works under physiological-like conditions, allowing living biofilms qualitative and quantitative assessment with few treatments, sample 3-D structure reconstruction at nanometer scale.

Cons

Low resolution and magnification power, need for sample staining, gross morphological differentiation, finest details not visible

Use of fluorophores, limited number of reporter molecules, intrinsic biofilm fluorescence can interfere with probes fluorescence

Small scan area (max 150 × 150 µm), no image of bacterial cells sidewalls, possible surface damage during imaging due to tip interactions.

Applications

Visualization of biofilm formation and quantitative assessment of its biomass

Assessment of biofilm structural parameters, Biofilm 3D structure, identification and localization of living and death cells

Quantitative biofilm analysis, determination of adhesion forces, biofilm topography, in situ imaging.

References

  1. Costerton, J.W.; Geesey, G.G.; Cheng, K.-J. How Bacteria Stick. Sci. Am. 1978, 238, 86–95, doi:10.1038/scientificamerican0178-86.
  2. Costerton, J.W.; Lewandowski, Z.; Caldwell, D.E.; Korber, D.R.; Lappin-Scott, H.M. Microbial Biofilms. Annu. Rev. Microbiol. 1995, 49, 711–745, doi:10.1146/annurev.mi.49.100195.003431.
  3. Kolenbrander, P.E. Oral Microbial Communities: Biofilms, Interactions, and Genetic Systems. Annu. Rev. Microbiol. 2000, 54, 413–437, doi:10.1146/annurev.micro.54.1.413.
  4. Shapiro, J.A. Thinking about bacterial populations as multicellular organisms. Annu. Rev. Microbiol. 1998, 52, 81–104, doi:10.1146/annurev.micro.52.1.81.
  5. Di Martino, P. Extracellular polymeric substances, a key element in understanding biofilm phenotype. AIMS Microbiol. 2018, 4, 274–288, doi:10.3934/microbiol.2018.2.274.
  6. Karygianni, L.; Ren, Z.; Koo, H.; Thurnheer, T. Biofilm Matrixome: Extracellular Components in Structured Microbial Com-munities. Trends Microbiol. 2020, 28, 668–681, doi:10.1016/j.tim.2020.03.016.
  7. Yadav, M.K.; Song, J.-J.; Singh, B.P.; Vidal, J.E. Chapter 1—Microbial biofilms and human disease: A concise review. In New and Future Developments in Microbial-l Biotechnology and Bioengineering; Current Research and Future Trends in Microbial Biofilms; Yadav, M.K., Singh, B.P., Eds; Elsevier: Amsterdam, Netherlands; 2020; pp. 1–13.
  8. Suetens, C.; Latour, K.; Kärki, T.; Ricchizzi, E.; Kinross, P.; Moro, M.L.; Jans, B.; Hopkins, S.; Hansen, S.; Lyytikäinen, O.; et al. Prevalence of healthcare-associated infections, estimated incidence and composite antimicrobial resistance index in acute care hospitals and long-term care facilities: Results from two European point prevalence surveys, 2016 to 2017. Eurosurveillance 2018, 23, 1800516, doi:10.2807/1560-7917.es.2018.23.46.1800516.
  9. Breidenstein, E.B.; De La Fuente-Nunez, C.; Hancock, R.E. Pseudomonas aeruginosa: All roads lead to resistance. Trends Microbiol. 2011, 19, 419–426, doi:10.1016/j.tim.2011.04.005.
  10. Vestby, L.K.; Grønseth, T.; Simm, R.; Nesse, L.L. Bacterial Biofilm and its Role in the Pathogenesis of Disease. Antibiotics 2020, 9, 59, doi:10.3390/antibiotics9020059.
  11. Li, Y.; Xiao, P.; Wang, Y.; Hao, Y. Mechanisms and Control Measures of Mature Biofilm Resistance to Antimicrobial Agents in the Clinical Context. ACS Omega 2020, 5, 22684–22690, doi:10.1021/acsomega.0c02294.
  12. Papa, R.; Garzoli, S.; Vrenna, G.; Sabatino, M.; Sapienza, F.; Relucenti, M.; Donfrancesco, O.; Fiscarelli, E.V.; Artini, M.; Selan, L.; et al. Essential Oils Biofilm Modulation Activity, Chemical and Machine Learning Analysis. Application on Staphy-lococcus aureus Isolates from Cystic Fibrosis Patients. Int. J. Mol. Sci. 2020, 21, 9258, doi:10.3390/ijms21239258.
  13. Hong, S.D.; Dhong, H.-J.; Chung, S.-K.; Kim, H.Y.; Park, J.; Ha, S.Y. Hematoxylin and Eosin Staining for Detecting Biofilms: Practical and Cost-Effective Methods for Predicting Worse Outcomes After Endoscopic Sinus Surgery. Clin. Exp. Otorhinolaryngol. 2014, 7, 193–197, doi:10.3342/ceo.2014.7.3.193.
  14. Bulut, F.; Meric, F.; Yorgancilar, E.; Nergiz, Y.; Akkus, M.; Nergiz, S.; Nasir, Y. Effects of N-acetyl-cysteine and acetylsalicylic acid on the tonsil bacterial biofilm tissues by light and electron microscopy. Eur. Rev. Med. Pharmacol. Sci. 2014, 18, 3720–3725.
  15. De Carvalho, C.C.C.R.; Da Fonseca, M.M.R.; Da Fonseca, M.M.R. Assessment of three-dimensional biofilm structure using an optical microscope. BioTechniques 2007, 42, 616–620, doi:10.2144/000112403.
  16. Richardson, N.; Mordan, N.J.; Figueiredo, J.A.P.; Ng, Y.; Gulabivala, K. Microflora in teeth associated with apical periodonti-tis: A methodological observational study comparing two protocols and three microscopy techniques. Int. Endod. J. 2009, 42, 908–921, doi:10.1111/j.1365-2591.2009.01594.x.
  17. Klausen, M.; Heydorn, A.; Ragas, P.; Lambertsen, L.; Aaes-Jørgensen, A.; Molin, S.; Tolker-Nielsen, T. Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol. Microbiol. 2003, 48, 1511–1524, doi:10.1046/j.1365-2958.2003.03525.x.
  18. Bridier, A.; Dubois-Brissonnet, F.; Boubetra, A.; Thomas, V.; Briandet, R. The biofilm architecture of sixty opportunistic path-ogens deciphered using a high throughput CLSM method. J. Microbiol. Methods 2010, 82, 64–70, doi:10.1016/j.mimet.2010.04.006.
  19. Guilbaud, M.; Piveteau, P.; Desvaux, M.; Brisse, S.; Briandet, R. Exploring the Diversity of Listeria monocytogenes Biofilm Architecture by High-Throughput Confocal Laser Scanning Microscopy and the Predominance of the Honeycomb-Like Morphotype. Appl. Environ. Microbiol. 2014, 81, 1813–1819, doi:10.1128/aem.03173-14.
  20. Sun, L.; Liao, K.; Wang, D. Effects of Magnolol and Honokiol on Adhesion, Yeast-Hyphal Transition, and Formation of Bio-film by Candida albicans. PLoS ONE 2015, 10, e0117695, doi:10.1371/journal.pone.0117695.
  21. Villacorte, L.O.; Ekowati, Y.; Neu, T.R.; Kleijn, J.M.; Winters, H.; Amy, G.; Schippers, J.; Kennedy, M. Characterisation of al-gal organic matter produced by bloom-forming marine and freshwater algae. Water Res. 2015, 73, 216–230, doi:10.1016/j.watres.2015.01.028.
  22. Thornton, R.B.; Rigby, P.J.; Wiertsema, S.P.; Filion, P.; Langlands, J.; Coates, H.; Vijayasekaran, S.; Keil, A.D.; Richmond, P.C. Multi-species bacterial biofilm and intracellular infection in otitis media. BMC Pediatr. 2011, 11, 94, doi:10.1186/1471-2431-11-94.
  23. Bridier, A.; Briandet, R.; Bouchez, T.; Jabot, F. A model-based approach to detect interspecific interactions during biofilm de-velopment. Biofouling 2014, 30, 761–771, doi:10.1080/08927014.2014.923409.
  24. Bridier, A.; Sanchez-Vizuete, M.D.P.; Le Coq, D.; Aymerich, S.; Meylheuc, T.; Maillard, J.-Y.; Thomas, V.; Dubois-Brissonnet, F.; Briandet, R. Biofilms of a Bacillus subtilis Hospital Isolate Protect Staphylococcus aureus from Biocide Action. PLoS ONE 2012, 7, e44506, doi:10.1371/journal.pone.0044506.
  25. Doroshenko, N.; Tseng, B.S.; Howlin, R.P.; Deacon, J.; Wharton, J.A.; Thurner, P.J.; Gilmore, B.F.; Parsek, M.R.; Stoodley, P. Extracellular DNA Impedes the Transport of Vancomycin in Staphylococcus epidermidis Biofilms Preexposed to Subinhibito-ry Concentrations of Vancomycin. Antimicrob. Agents Chemother. 2014, 58, 7273–7282, doi:10.1128/aac.03132-14.
  26. Marchal, M.; Briandet, R.; Halter, D.; Koechler, S.; Dubow, M.S.; Lett, M.-C.; Bertin, P.N. Subinhibitory Arsenite Concentra-tions Lead to Population Dispersal in Thiomonas sp. PLoS ONE 2011, 6, e23181, doi:10.1371/journal.pone.0023181.
  27. Verma, V.; Harjai, K.; Chhibber, S. Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm ofKlebsiella pneumoniae. Biofouling 2010, 26, 729–737, doi:10.1080/08927014.2010.511196.
  28. Hope, C.K.; Clements, D.; Wilson, M. Determining the spatial distribution of viable and nonviable bacteria in hydrated mi-crocosm dental plaques by viability profiling. J. Appl. Microbiol. 2002, 93, 448–455, doi:10.1046/j.1365-2672.2002.01703.x.
  29. Dufrêne, Y.F.; Persat, A. Mechanomicrobiology: How bacteria sense and respond to forces. Nat. Rev. Microbiol. 2020, 18, 227–240, doi:10.1038/s41579-019-0314-2.
  30. Li, J.; Busscher, H.J.; Swartjes, J.J.T.M.; Chen, Y.; Harapanahalli, A.K.; Norde, W.; Van Der Mei, H.C.; Sjollema, J. Resi-dence-time dependent cell wall deformation of different Staphylococcus aureus strains on gold measured using sur-face-enhanced-fluorescence. Soft Matter 2014, 10, 7638–7646, doi:10.1039/c4sm00584h.
  31. Perozo, E.; Kloda, A.; Cortes, D.M.; Martinac, B. Physical principles underlying the transduction of bilayer defor-mation forces during mechanosensitive channel gating. Nat. Struct. Biol. 2002, 9, 696–703.
  32. Baro, A.M.; Reifenberger, R.G. Atomic Force Microscopy in Liquid: Biological Applications; Wiley-VCH: Weinheim, Germany, 2012.
  33. Beaussart, A.; El-Kirat-Chatel, S.; Sullan, R.M.A.; Alsteens, D.; Herman, P.; Derclaye, S.; Dufrêne, Y.F. Quantifying the forces guiding microbial cell adhesion using single-cell force spectroscopy. Nat. Protoc. 2014, 9, 1049–1055, doi:10.1038/nprot.2014.066.
  34. Peterson, B.W.; He, Y.; Ren, Y.; Zerdoum, A.; Libera, M.R.; Sharma, P.K.; Van Winkelhoff, A.-J.; Neut, D.; Stoodley, P.; Van Der Mei, H.C.; et al. Viscoelasticity of biofilms and their recalcitrance to mechanical and chemical challenges. FEMS Microbiol. Rev. 2015, 39, 234–245, doi:10.1093/femsre/fuu008.
  35. Zeng, G.; Müller, T.; Meyer, R.L. Single-Cell Force Spectroscopy of Bacteria Enabled by Naturally Derived Proteins. Langmuir 2014, 30, 4019–4025, doi:10.1021/la404673q.
  36. Tarafdar, A.; Lee, J.-U.; Jeong, J.-E.; Lee, H.; Jung, Y.; Oh, H.-B.; Woo, H.Y.; Kwon, J.-H. Biofilm development of Bacillus sia-mensis ATKU1 on pristine short chain low-density polyethylene: A case study on microbe-microplastics interaction. J. Hazard. Mater. 2020, 124516, doi:10.1016/j.jhazmat.2020.124516.
  37. Boyd, C.D.; Smith, T.J.; El-Kirat-Chatel, S.; Newell, P.D.; Dufrêne, Y.F.; O’Toole, G.A. Structural Features of the Pseudomonas fluorescens Biofilm Adhesin LapA Required for LapG-Dependent Cleavage, Biofilm Formation, and Cell Surface Localization. J. Bacteriol. 2014, 196, 2775–2788, doi:10.1128/jb.01629-14.
  38. Cabral, V.; Znaidi, S.; Walker, L.A.; Martin-Yken, H.; Dague, E.; Legrand, M.; Lee, K.; Chauvel, M.; Firon, A.; Rossignol, T.; et al. Targeted Changes of the Cell Wall Proteome Influence Candida albicans Ability to Form Single- and Multi-strain Bio-films. PLOS Pathog. 2014, 10, e1004542, doi:10.1371/journal.ppat.1004542.
  39. Lim, J.; Cui, Y.; Oh, Y.J.; Park, J.R.; Jo, W.; Cho, Y.-H.; Park, S. Studying the effect of alginate overproduction on Pseudomo-nas aeruginosa biofilm by atomic force microscopy. J. Nanosci. Nanotechnol. 2011, 11, 5676–5681, doi:10.1166/jnn.2011.4491.
  40. Ovchinnikova, E.S.; Krom, B.P.; Harapanahalli, A.K.; Busscher, H.J.; Van Der Mei, H.C. Surface Thermodynamic and Adhe-sion Force Evaluation of the Role of Chitin-Binding Protein in the Physical Interaction between Pseudomonas aeruginosaand Candida albicans. Langmuir 2013, 29, 4823–4829, doi:10.1021/la400554g.
  41. Potthoff, E.; Ossola, D.; Zambelli, T.; Vorholt, J.A. Bacterial adhesion force quantification by fluidic force microscopy. Nanoscale 2015, 7, 4070–4079, doi:10.1039/c4nr06495j.
  42. Ansari, M.J.; Al-Ghamdi, A.; Usmani, S.; Al-Waili, N.; Sharma, D.; Nuru, A.; Al-Attal, Y. Effect of Jujube Honey on Candida albicans Growth and Biofilm Formation. Arch. Med. Res. 2013, 44, 352–360, doi:10.1016/j.arcmed.2013.06.003.
  43. Chatterjee, S.; Biswas, N.; Datta, A.; Dey, R.; Maiti, P. Atomic force microscopy in biofilm study. Microscopy (Oxf. Engl.) 2014, 63, 269–278, doi:10.1093/jmicro/dfu013.
  44. Li, L.; Mendis, N.; Trigui, H.; Oliver, J.D.; Faucher, S.P. The importance of the viable but non-culturable state in human bac-terial pathogens. Front. Microbiol. 2014, 5, 258, doi:10.3389/fmicb.2014.00258.
  45. Danin, P.- Éric; Girou, E.; Legrand, P.; Louis, B.; Fodil, R.; Christov, C.; Devaquet, J.; Isabey, D.; Brochard, L. Description and Microbiology of Endotracheal Tube Biofilm in Mechanically Ventilated Subjects. Respir. Care 2014, 60, 21–29, doi:10.4187/respcare.02722.
  46. Mangalappalli-Illathu, A.K.; Vidović, S.; Korber, D. Differential Adaptive Response and Survival of Salmonella enterica Serovar Enteritidis Planktonic and Biofilm Cells Exposed to Benzalkonium Chloride. Antimicrob. Agents Chemother. 2008, 52, 3669–3680, doi:10.1128/aac.00073-08.
  47. Nandakumar, K.; Obika, H.; Utsumi, A.; Ooie, T.; Yano, T. In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength. Biotechnol. Bioeng. 2004, 86, 729–736, doi:10.1002/bit.10829.
  48. Qin, Z.; Zhang, J.; Hu, Y.; Chi, Q.; Mortensen, N.P.; Qu, D.; Molin, S.; Ulstrup, J. Organic compounds inhibiting S. epider-midis adhesion and biofilm formation. Ultramicroscopy 2009, 109, 881–888, doi:10.1016/j.ultramic.2009.03.040.
  49. Sharma, S.; Cross, S.E.; Hsueh, C.; Wali, R.P.; Stieg, A.Z.; Gimzewski, J.K. Nanocharacterization in Dentistry. Int. J. Mol. Sci. 2010, 11, 2523–2545, doi:10.3390/ijms11062523.
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