The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

Biology Centre of the Czech Academy of Sciences CZ 37005 Ceske Budejovice Czech Republic

Department of Microbiology Faculty of Medicine Masaryk University and St Anne's Faculty Hospital CZ 65691 Brno Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences CZ 61264 Brno Czech Republic

Zobrazit více v PubMed

Donlan R.M., Costerton J.W. Biofilms: Survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 2002;15:167–193. doi: 10.1128/CMR.15.2.167-193.2002. PubMed DOI PMC

Costerton J.W., Stewart P.S., Greenberg E.P. Bacterial biofilms: A common cause of persistent infections. Science. 1999;284:1318–1322. doi: 10.1126/science.284.5418.1318. PubMed DOI

Alhede M., Qvortrup K., Liebrechts R., Hoiby N., Givskov M., Bjarnsholt T. Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition. FEMS Immunol. Med. Microbiol. 2012;65:335–342. doi: 10.1111/j.1574-695X.2012.00956.x. PubMed DOI

Ruzicka F., Horka M., Hola V. Extracellular Polysaccharides in Microbial Biofilm and Their Influence on the Electrophoretic Properties of Microbial Cells. In: Volpi N., editor. Capillary Electrophoresis of Carbohydrates. Humana Press; New York, NY, USA: 2011. pp. 105–126.

Donelli G. Microbial Biofilms: Methods and Protocols. Humana Press; New York, NY, USA: 2014.

Flemming H.C., Meier M., Schild T. Mini-review: Microbial problems in paper production. Biofouling. 2013;29:683–696. doi: 10.1080/08927014.2013.798865. PubMed DOI

Qin Z., Ou Y., Yang L., Zhu Y., Tolker-Nielsen T., Molin S., Qu D. Role of autolysin-mediated DNA release in biofilm formation of Staphylococcus epidermidis. Microbiology. 2007;153:2083–2092. doi: 10.1099/mic.0.2007/006031-0. PubMed DOI

Hola V., Ruzicka F., Tejkalova R., Votava M. Biofilm formation in nosocomial pathogens of respiratory tract. Int. J. Antimicrob. Agents. 2007;29:S142. doi: 10.1016/S0924-8579(07)70453-3. DOI

Wimpenny J. Microbial Metropolis. Adv. Microb. Physiol. 2009;56:29–84. PubMed

Marsh P.D. Plaque as a biofilm: Pharmacological principles of drug delivery and action in the sub- and supragingival environment. Oral Dis. 2003;9:16–22. doi: 10.1034/j.1601-0825.9.s1.4.x. PubMed DOI

Francolini I., Donelli G. Prevention and control of biofilm-based medical-device-related infections. FEMS Immunol. Med. Microbiol. 2010;59:227–238. doi: 10.1111/j.1574-695X.2010.00665.x. PubMed DOI

Barghi A., Sadati R., Larki R.A. Biological Wastewater Treatment through Biofilm. Iran. J. Public Health. 2016;45:101.

Voběrková S., Hermanová S., Hrubanová K., Krzyžánek V. Biofilm formation and extracellular polymeric substances (EPS) production by Bacillus subtilis depending on nutritional conditions in the presence of polyester film. Folia Microbiol. 2015 doi: 10.1007/s12223-015-0406-y. PubMed DOI

Cresson R., Dabert P., Bernet N. Microbiology and performance of a methanogenic biofilm reactor during the start-up period. J. Appl. Microbiol. 2009;106:863–876. doi: 10.1111/j.1365-2672.2008.04055.x. PubMed DOI

Bao J., Liu N., Zhu L., Xu Q., Huang H., Jiang L. Programming a Biofilm-Mediated Multienzyme-Assembly-Cascade System for the Biocatalytic Production of Glucosamine from Chitin. J. Agric. Food Chem. 2018;66:8061–8068. doi: 10.1021/acs.jafc.8b02142. PubMed DOI

De la Fuente-Nunez C., Cardoso M.H., de Souza Candido E., Franco O.L., Hancock R.E. Synthetic antibiofilm peptides. Biochim. Biophys. Acta. 2016;1858:1061–1069. doi: 10.1016/j.bbamem.2015.12.015. PubMed DOI PMC

Huq A., Whitehouse C.A., Grim C.J., Alam M., Colwell R.R. Biofilms in water, its role and impact in human disease transmission. Curr. Opin. Biotechnol. 2008;19:244–247. doi: 10.1016/j.copbio.2008.04.005. PubMed DOI

Sutherland I.W. Biofilm exopolysaccharides: A strong and sticky framework. Microbiology. 2001;147:3–9. doi: 10.1099/00221287-147-1-3. PubMed DOI

Branda S.S., Vik A., Friedman L., Kolter R. Biofilms: The matrix revisited. Trends Microbiol. 2005;13:20–26. doi: 10.1016/j.tim.2004.11.006. PubMed DOI

Sutherland I.W. The biofilm matrix—An immobilized but dynamic microbial environment. Trends Microbiol. 2001;9:222–227. doi: 10.1016/S0966-842X(01)02012-1. PubMed DOI

Adam B., Baillie G.S., Douglas L.J. Mixed species biofilms of Candida albicans and Staphylococcus epidermidis. J. Med. Microbiol. 2002;51:344–349. doi: 10.1099/0022-1317-51-4-344. PubMed DOI

Holá V., Růžička F., Votava M. The dynamics of Staphylococcus epidermis biofilm formation in relation to nutrition, temperature, and time. Scr. Med. Fac. Med.Univ. Brun. Masaryk. 2006;79:169–174.

Liu H.Y., Zhao Y.F., Zhao D., Gong T., Wu Y.C., Han H.Y., Xu T., Peschel A., Han S.Q., Qu D. Antibacterial and anti-biofilm activities of thiazolidione derivatives against clinical staphylococcus strains. Emerg. Microbes Infect. 2015;4:e1. doi: 10.1038/emi.2015.1. PubMed DOI PMC

Ruzicka F., Horka M., Hola V., Kubesova A., Pavlik T., Votava M. The differences in the isoelectric points of biofilm-positive and biofilm-negative Candida parapsilosis strains. J. Microbiol. Methods. 2010;80:299–301. doi: 10.1016/j.mimet.2010.01.007. PubMed DOI

Deleo F., Otto M.W. Bacterial Pathogenesis: Methods and Protocols. Humana Press; Totowa, NJ, USA: 2008.

Paiva L.C.F., Vidigal P.G., Donatti L., Svidzinski T.I.E., Consolaro M.E.L. Assessment of in vitro biofilm formation by Candida species isolates from vulvovaginal candidiasis and ultrastructural characteristics. Micron. 2012;43:497–502. doi: 10.1016/j.micron.2011.09.013. PubMed DOI

Bandara H.M.H.N., Lam O.L.T., Watt R.M., Jin L.J., Samaranayake L.P. Bacterial lipopolysaccharides variably modulate in vitro biofilm formation of Candida species. J. Med. Microbiol. 2010;59:1225–1234. doi: 10.1099/jmm.0.021832-0. PubMed DOI

Lattif A.A., Mukherjee P.K., Chandra J., Swindell K., Lockhart S.R., Diekema D.J., Pfaller M.A., Ghannoum M.A. Characterization of biofilms formed by Candida parapsilosis, C. metapsilosis, and C. orthopsilosis. Int. J. Med. Microbiol. 2010;300:265–270. doi: 10.1016/j.ijmm.2009.09.001. PubMed DOI

Dohnalkova A.C., Marshall M.J., Arey B.W., Williams K.H., Buck E.C., Fredrickson J.K. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy. Appl. Environ. Microb. 2011;77:1254–1262. doi: 10.1128/AEM.02001-10. PubMed DOI PMC

Schaudinn C., Stoodley P., Hall-Stoodley L., Gorur A., Remis J., Wu S., Auer M., Hertwig S., Guerrero-Given D., Hu F.Z., et al. Death and Transfiguration in Static Staphylococcus epidermidis Cultures. PLoS ONE. 2014;9:e100002. doi: 10.1371/journal.pone.0100002. PubMed DOI PMC

Lawrence J.R., Swerhone G.D.W., Leppard G.G., Araki T., Zhang X., West M.M., Hitchcock A.P. Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms. Appl. Environ. Microb. 2003;69:5543–5554. doi: 10.1128/AEM.69.9.5543-5554.2003. PubMed DOI PMC

Karcz J., Bernas T., Nowak A., Talik E., Woznica A. Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy. Scanning. 2012;34:26–36. doi: 10.1002/sca.20275. PubMed DOI

Krzyzanek V., Sporenberg N., Keller U., Guddorf J., Reichelt R., Schonhoff M. Polyelectrolyte multilayer capsules: Nanostructure and visualisation of nanopores in the wall. Soft Matter. 2011;7:7034–7041. doi: 10.1039/c1sm05406f. DOI

Hrubanova K., Nebesarova J., Ruzicka F., Krzyzanek V. The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm. Micron. 2018;110:28–35. doi: 10.1016/j.micron.2018.04.006. PubMed DOI

Biel S.S., Wilke K., Dunckelmann K., Wittern K.P., Wepf R. Light and electron microscopy: Histochemistry on the identical biopsy after high-pressure freezing. J. Histochem. Cytochem. 2004;52:S62.

Hawser S.P., Douglas L.J. Biofilm Formation by Candida Species on the Surface of Catheter Materials in-Vitro. Infect. Immunity. 1994;62:915–921. PubMed PMC

Kuo J. Electron Microscopy: Methods and Protocols. Volume 369 Springer Science & Business Media; Berlin, Germany: 2007.

Montesinos E., Esteve I., Guerrero R. Comparison between Direct Methods for Determination of Microbial Cell-Volume—Electron-Microscopy and Electronic Particle Sizing. Appl. Environ. Microb. 1983;45:1651–1658. PubMed PMC

Webster P., Wu S., Webster S., Rich K., McDonald K. Ultrastructural preservation of biofilms formed by non-typeable Hemophilus influenzae. Method Enzymol. 2004;1:165–182. doi: 10.1017/S1479050504001425. DOI

Graham L.L., Beveridge T.J. Effect of Chemical Fixatives on Accurate Preservation of Escherichia-Coli and Bacillus-Subtilis Structure in Cells Prepared by Freeze-Substitution. J. Bacteriol. 1990;172:2150–2159. doi: 10.1128/jb.172.4.2150-2159.1990. PubMed DOI PMC

Hayat M.A. Principles and Techniques of Scanning Electron Microscopy. Biological Applications, Volume 1. Van Nostrand Reinhold Company; New York, NY, USA: 1974.

Wu Y., Liang J., Rensing K., Chou T.M., Libera M. Extracellular Matrix Reorganization during Cryo Preparation for Scanning Electron Microscope Imaging of Staphylococcus aureus Biofilms. Microsc. Microanal. 2014;20:1348–1355. doi: 10.1017/S143192761401277X. PubMed DOI

Fassel T.A., Edmiston C.E. Ruthenium red and the bacterial glycocalyx. Biotech. Histochem. 1999;74:194–212. doi: 10.3109/10520299909047974. PubMed DOI

Reese S., Guggenheim B. A novel TEM contrasting technique for extracellular polysaccharides in in vitro biofilrns. Microsc. Res. Tech. 2007;70:816–822. doi: 10.1002/jemt.20471. PubMed DOI

Galway M.E., Heckman J.W., Jr., Hyde G.J., Fowke L.C. Advances in High-Pressure and Plunge-Freeze Fixation. Methods Cell Biol. 1995;49:3–19. PubMed

Wang A.B., Lin C.H., Chen C.C. The critical temperature of dry impact for tiny droplet impinging on a heated surface. Phys. Fluids. 2000;12:1622–1625. doi: 10.1063/1.870413. DOI

Dahl R., Staehelin L.A. High-pressure freezing for the preservation of biological structure: Theory and practice. J. Electron Microsc. Tech. 1989;13:165–174. doi: 10.1002/jemt.1060130305. PubMed DOI

Moor H. Theory and Practice of High Pressure Freezing. In: Steinbrecht R., Zierold K., editors. Cryotechniques in Biological Electron Microscopy. Springer; Berlin/Heidelberg, Germany: 1987. pp. 175–191.

Studer D., Michel M., Muller M. High-Pressure Freezing Comes of Age. Scanning Microsc. 1989:253–269. PubMed

Shimoni E., Muller M. On optimizing high-pressure freezing: From heat transfer theory to a new microbiopsy device. J. Microsc. 1998;192:236–247. doi: 10.1046/j.1365-2818.1998.00389.x. PubMed DOI

Samek O., Mlynarikova K., Bernatova S., Jezek J., Krzyzanek V., Siler M., Zemanek P., Ruzicka F., Hola V., Mahelova M. Candida parapsilosis Biofilm Identification by Raman Spectroscopy. Int. J. Mol. Sci. 2014;15:23924–23935. doi: 10.3390/ijms151223924. PubMed DOI PMC

Rebrosova K., Siler M., Samek O., Ruzicka F., Bernatova S., Jezek J., Zemanek P., Hola V. Differentiation between Staphylococcus aureus and Staphylococcus epidermidis strains using Raman spectroscopy. Future Microbiol. 2017;12:881–890. doi: 10.2217/fmb-2016-0224. PubMed DOI

Notingher I., Hench L.L. Raman microspectroscopy: A noninvasive tool for studies of individual living cells in vitro. Expert Rev. Med. Devices. 2006;3:215–234. doi: 10.1586/17434440.3.2.215. PubMed DOI

Mc F.J. The nephelometer: An instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J. Am. Med. Assoc. 1907;49:1176–1178.

Maquelin K., Kirschner C., Choo-Smith L.P., Ngo-Thi N.A., van Vreeswijk T., Stammler M., Endtz H.P., Bruining H.A., Naumann D., Puppels G.J. Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures. J. Clin. Microbiol. 2003;41:324–329. doi: 10.1128/JCM.41.1.324-329.2003. PubMed DOI PMC

De Gelder J., De Gussem K., Vandenabeele P., Vancanneyt M., De Vos P., Moens L. Methods for extracting biochemical information from bacterial Raman spectra: Focus on a group of structurally similar biomolecules—Fatty acids. Anal. Chim. Acta. 2007;603:167–175. doi: 10.1016/j.aca.2007.09.049. PubMed DOI

Tuma R. Raman spectroscopy of proteins: From peptides to large assemblies. J. Raman Spectrosc. 2005;36:307–319. doi: 10.1002/jrs.1323. DOI

Notingher I. Raman Spectroscopy cell-based Biosensors. Sensors. 2007;7:1343–1358. doi: 10.3390/s7081343. DOI

Neugebauer U., Schmid U., Baumann K., Ziebuhr W., Kozitskaya S., Holzgrabe U., Schmitt M., Popp J. The influence of fluoroquinolone drugs on the bacterial growth of S-epidermidis utilizing the unique potential of vibrational spectroscopy. J. Phys. Chem. A. 2007;111:2898–2906. doi: 10.1021/jp0678397. PubMed DOI

De Gelder J., De Gussem K., Vandenabeele P., Moens L. Reference database of Raman spectra of biological molecules. J. Raman Spectrosc. 2007;38:1133–1147. doi: 10.1002/jrs.1734. DOI

Perna G., Lastella M., Lasalvia M., Mezzenga E., Capozzi V. Raman spectroscopy and atomic force microscopy study of cellular damage in human keratinocytes treated with HgCl2. J. Mol. Struct. 2007;834:182–187. doi: 10.1016/j.molstruc.2006.12.014. DOI

Pyrgiotakis G., Bhowmick T.K., Finton K., Suresh A.K., Kane S.G., Bellare J.R., Moudgil B.M. Cell (A549)-particle (Jasada Bhasma) interactions using Raman spectroscopy. Biopolymers. 2008;89:555–564. doi: 10.1002/bip.20947. PubMed DOI

Pilat Z., Bernatova S., Jezek J., Kirchhoff J., Tannert A., Neugebauer U., Samek O., Zemanek P. Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress. Sensors. 2018;18:1623. doi: 10.3390/s18051623. PubMed DOI PMC

Rebrosova K., Siler M., Samek O., Ruzicka F., Bernatova S., Hola V., Jezek J., Zemanek P., Sokolova J., Petras P. Rapid identification of staphylococci by Raman spectroscopy. Sci. Rep. 2017;7:14846. doi: 10.1038/s41598-017-13940-w. PubMed DOI PMC

Samek O., Obruca S., Siler M., Sedlacek P., Benesova P., Kucera D., Marova I., Jezek J., Bernatova S., Zemanek P. Quantitative Raman Spectroscopy Analysis of Polyhydroxyalkanoates Produced by Cupriavidus necator H16. Sensors. 2016;16:1808. doi: 10.3390/s16111808. PubMed DOI PMC

Ruzicka F., Hola V., Votava M., Tejkalova R. Importance of biofilm in Candida parapsilosis and evaluation of its susceptibility to antifungal agents by colorimetric method. Folia Microbiol. 2007;52:209–214. doi: 10.1007/BF02931300. PubMed DOI

Azeredo J., Azevedo N.F., Briandet R., Cerca N., Coenye T., Costa A.R., Desvaux M., Di Bonaventura G., Hebraud M., Jaglic Z., et al. Critical review on biofilm methods. Crit. Rev. Microbiol. 2017;43:313–351. doi: 10.1080/1040841X.2016.1208146. PubMed DOI

Haque F., Alfatah M., Ganesan K., Bhattacharyya M.S. Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth. Sci. Rep. 2016;6:23575. doi: 10.1038/srep23575. PubMed DOI PMC

Ludecke C., Jandt K.D., Siegismund D., Kujau M.J., Zang E., Rettenmayr M., Bossert J., Roth M. Reproducible Biofilm Cultivation of Chemostat-Grown Escherichia coli and Investigation of Bacterial Adhesion on Biomaterials Using a Non-Constant-Depth Film Fermenter. PLoS ONE. 2014;9:e84837. doi: 10.1371/journal.pone.0084837. PubMed DOI PMC

Bray D.F., Bagu J., Koegler P. Comparison of Hexamethyldisilazane (Hmds), Peldri-Ii, and Critical-Point Drying Methods for Scanning Electron-Microscopy of Biological Specimens. Microsc. Res. Tech. 1993;26:489–495. doi: 10.1002/jemt.1070260603. PubMed DOI

Hazrin-Chong N.H., Manefield M. An alternative SEM drying method using hexamethyldisilazane (HMDS) for microbial cell attachment studies on sub-bituminous coal. J. Microbiol. Methods. 2012;90:96–99. doi: 10.1016/j.mimet.2012.04.014. PubMed DOI

Osumi M., Konomi M., Sugawara T., Takagi T., Baba M. High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: Ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy. J. Electron Microsc. 2006;55:75–88. doi: 10.1093/jmicro/dfl014. PubMed DOI

Psenicka M., Tesarova M., Tesitel J., Nebesarova J. Size determination of Acipenser ruthenus spermatozoa in different types of electron microscopy. Micron. 2010;41:455–460. doi: 10.1016/j.micron.2010.02.004. PubMed DOI

Brandt N.N., Brovko O.O., Chikishev A.Y., Paraschuk O.D. Optimization of the rolling-circle filter for Raman background subtraction. Appl. Spectrosc. 2006;60:288–293. doi: 10.1366/000370206776342553. PubMed DOI

Wold S., Esbensen K., Geladi P. Principal Component Analysis. Chemom. Intell. Lab. 1987;2:37–52. doi: 10.1016/0169-7439(87)80084-9. DOI

Kaech A., Ziegler U. High-Pressure Freezing: Current State and Future Prospects. Methods Mol. Biol. 2014;1117:151–171. PubMed

Kaech A., Woelfel M. Evaluation of Different Freezing Methods for Monolayer Cell Cultures. Microsc. Microanal. 2007;13:240–241. doi: 10.1017/S1431927607071978. DOI

Donlan R.M. Biofilms: Microbial life on surfaces. Emerg. Infect. Dis. 2002;8:881–890. doi: 10.3201/eid0809.020063. PubMed DOI PMC

Mahapatra S., Banerjee D. Fungal exopolysaccharide: Production, composition and applications. Microbiol. Insights. 2013;6:1–16. doi: 10.4137/MBI.S10957. PubMed DOI PMC

Maira-Litran T., Kropec A., Abeygunawardana C., Joyce J., Mark G., Goldmann D.A., Pier G.B. Immunochemical properties of the staphylococcal poly-N-acetylglucosamine surface polysaccharide. Infect. Immunity. 2002;70:4433–4440. doi: 10.1128/IAI.70.8.4433-4440.2002. PubMed DOI PMC

Raman Spectroscopy-A Novel Method for Identification and Characterization of Microbes on a Single-Cell Level in Clinical Settings