biofilm: a paradigm shift in microbiology

Studii și cercetări, Biology 17, Bistrița, p. 55-61

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BIOFILM: A PARADIGM SHIFT IN MICROBIOLOGY Anca FARKAS, Mihail DRǍGAN-BULARDA**

Abstract: A paradigm shift currently occurs in microbiology; the old misconception of free floating microbes is invalidated by a new knowledge pattern: over 99% of terrestrial microorganisms live in communities associated to surfaces, called biofilms. A biofilm represents a structured, usually plurispecific community of sessile populations, encased in a self-produced exopolymeric matrix, adherent to a living or inert interface. These forms of organization are ubiquitous and geomicrobiological evidences confirm their ability to persist over the evolutionary periods. Although observations upon bacterial accumulations are attested since the 17th century, their research gained momentum in the end of the 20th century, especially due to the impact in industry, medicine and environment. Biofilms may verify the hypothesis of multicellular organism’s evolution by unicellular population’s cooperation. Even the perception of science about the human body dramatically changes. Currently, the human organism is perceived as a biome, together with all its associated microorganisms, their genetic elements and established relationships. Fundamental differences between independent bacteria and sessile cells and the particularities in biofilm complexity and architecture generate the need for continuous development of new methods in their approach. Belonging to the community, from a microbial perspective is a benefit, materialized in increased chances of survival by perpetuating their resistance, protection against disinfectants, antibiotics or host’s immune system and enhanced virulence. From the human perspective, the biofouling prevention and control together with biofilms exploitation are targeted. Key words: biofilm, microbial communities, paradigm shift, microbial perspective versus human perspective.

Introduction Microorganisms and particularly bacteria display two behaviour types: the planktonic state, characterized by independent float or swimming into the liquid environment, and the attached state, with cells adherent to each other and to the surface, into a solid layer called biofilm. Biofilms are everywhere, from glaciers to hot acidic waters, on rivers rocks, on standing water surfaces, in water and air heaters and coolers, in pipes, on surfaces in food and other industries, on the hulls of ships, on humans and animals teeth, in digestive system and other hollow organs previously considered sterile, in implants, 

Compania de Apǎ Someş SA, Bd. 21 Decembrie 1989, nr. 79, 400604, Cluj-Napoca, Romania, E-mail: [email protected]. ** Universitatea Babeş-Bolyai, Facultatea de Biologie şi Geologie, Catedra de Biologie Experimentalǎ, str. Kogǎlniceanu, nr. 1, 400084, Cluj-Napoca, România.

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Anca FARKAS, Mihail DRǍGAN-BULARDA

hospital devices, at the surface and inside vegetation, in water and wastewater systems. Paleontological inventory showed that biofilms are robust fossils which can remain unchanged due to their peculiar chemical composition for up to 3.5 billion years (Westall et al., 2000). They are strong candidates for the most reliable and indisputable category of morphological biomarkers and represent a tool in the search for evidence of life in the oldest terrestrial sites and, by extension, in extraterrestrial materials (Toporski et al., 2003). Biofilm represents a structured community of sessile microorganisms characterized by surface attachment, self-produced exopolysaccharidic matrix, structural, functional and metabolic heterogeneity, capable of intercellular communication by quorum sensing and generally plurispecific composition. Organized in biofilms and protected by the matrix, microorganisms are able to produce substances that are not synthesized by individual cells, enhance their resistance, use chemical weapons in order to protect against disinfectants, antibiotics or host’s immune system and increase their virulence (Dreeszen, 2003; Flemming, 2008). When compared with planktonic state, biofilm displays superior characteristics due to specialization inside this emergent structure and to complex relationships established. The benefit of belonging to the community is materialized in increasing the chances of survival to its members; natural selection preserves a community whether its growth advantages are transferred to descendants (Costerton, 2007). Biofilms may verify the hypothesis of multicellular organism’s evolution by unicellular population’s cooperation (Shapiro, 1998), thus representing a paradigm shift in microbiology. Fundamental differences between independent bacteria and sessile cells together with particularities in biofilm complexity and architecture generate the need for continuous development of new methods in their approach. The well known and standardized laboratory techniques used by now in comprehensive plankton’s investigations are no longer sufficient. Conventional concepts in microbiology and genetics are overturned by cuttingedge fields of molecular biology implementing and exploration techniques improvement. History and stages in biofilm research Although their observation and empiric investigation started hundreds of years ago, researchers’ concerns in the biofilms’ subject were intensified in the last two decades. The evolution of science in biofilm research may be traced in the three stages described by physicist and philosopher Thomas Kuhn, as follows: prescience, normal science and revolutionary science. 1. Prescience stage lacks the central paradigm, but it presupposes it. The paradigm in Kuhn’s view describes a collection of beliefs shared by the scientific community, a knowledge pattern. Biofilm empiric investigation was determined since its origins by curiosity, hypotheses being issued starting from practical reasons. Since 1684 Anthony van Leeuwenhoek noted the broad

BIOFILM: A PARADIGM SHIFT IN MICROBIOLOGY

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accumulation of microorganisms in dental plaque, his statement to the Royal Society of London is considered the first scientific report on biofilms: “The number of these animalcules in the scurf of a man’s teeth are so many that I believe they exceed the number of men in a kingdom” (Sauer et al. 2007). In the mid-nineteenth century Robert Koch and Louis Pasteur have initiated microbes’ research methods by their cultivation on solid growth media, thus isolating the first pure bacterial cultures. Biofilms have been observed by researchers all over the world: Henrici (1933) developed a microbial fishing technique placing microscope slides into an aquarium and explained biofilm formation. Heukelekian and Heller (1940) observed “the bottle effect”, Zobell (1943), the pioneer in microbial induced corrosion research, introduced the terms “attached films” and “sessile bacteria” as a result of his studies upon the marine bacteria adherent to surfaces (Donlan, 2002; Lappin-Scott, 1999). Marshall et al. (1971) and Characklis (1973) described the reversible and irreversible surface attachment and microbial slimes resistance to disinfectants. Numerous studies upon bacterial sessile populations have been initialized, including the fascinating social behaviour of bacteria. In their microbiological investigations in mountain streams, Geesey et al. (1978), Costerton et al. (1978) realized the huge bacterial density in biofilms on rocks (>1x106 cells/cm2) compared to planktonic microorganisms occurrence (8-10 cells/ml). 2. Normal science stage begins with biofilms definition and characterization, introduced in 1978 by J. W. Costerton, the pathfinder in biofilm research, who explained the beneficial organization of these ecological micro niches from a microbial perspective. Costerton’s career, started in the 1950’s expanded in the 1980’s, when was realized that biofilms represent the natural mode of microbial growth, the free, planktonic mode being artificial and constituting only a step in biofilm development, when dispersion occurs. During the 1990’s a narrow scientific community in biofilms research explored the attached microbial communities in a multitude of habitats, mostly the ones with relevant negative impacts from a human perspective in industry, environment and medicine. Practical imperativeness of problems solving in such branches promote the investigation of biofouling: surfaces undesirable colonization by microorganisms and their accumulation together with the associated products. Epidemic outbreaks in the early twentieth century inducted a comprehensive approach of biofilms in medicine, launching the hypothesis that severe and recurrent infections may be caused by inoffensive microorganisms with environmental origin, which acquired resistance to antibiotics, disinfectants and innate host’s immune system mechanisms. Biofilms were identified as the source of infection, visualized or isolated from medical devices, implants and compromised tissues. Since 1990 to 1996 the structural, functional and metabolic heterogeneity of those emergent structures was discovered, as well as the intercellular communication by quorum sensing and kin selection evolutional mechanisms inside of constitutive populations.

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3. Revolutionary science involves the paradigm shift in microbiology: more than 99% of the microorganisms on Terra live in complex communities associated to surfaces (Costerton et al., 1987), the old misconception of free floating unicellular organisms being invalidated by innovative techniques implementation. In the beginning, most observations and approaches in order to characterize biofilms are based on electron microscopy, especially scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and also on conventional cultivation techniques. The new research tools in microbiology, genetics and molecular biology promoted the transition from a descriptive, qualitative study to the quantitative investigation. Since the comprehensive research of planktonic prokaryotes has led to the development of standardized detection methods in microbiology, a new stage of attached microorganisms approach is required, since it was shown that they develop a totally different type of behaviour. Techniques of investigation in this case are still emerging, but quantum leaps in research have been registered in the past 20 years. A great support in biofilms understanding has been offered by major research tools such as confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), mass spectrometry, genetic sequencing, metagenomics and so on. The new challenge is represented by the viable but non cultivable state of microorganisms, enhanced in sessile bacteria. Biofilm is currently percept as a microbiome defined by the microbial communities’ totality, their genetic elements and interactions with the environment. This distinct phenotype expression is determined by the regulation of genes involved in adhesion, aggregation, response to nutrients limitations, disinfectants, antibiotics and heavy metal resistance. Is estimated that approximately 40% of sessile bacteria genome is up or down regulated, compared with genetic expression of planktonic cells in the logarithmic growth state (Prakash et al., 2003). Even scientists’ perception about the human body dramatically changed; until now considered as a whole, unified life entity, capable of growth, metabolism and reproduction, focusing on gene expression and on morphological, structural and functional ensembles: cells, tissues, organs, systems and organism. Acknowledging the role of microorganisms associated to the human body in its functions accomplishment and mostly their regulatory mechanisms led to the new paradigm: human body represents a biome. If associated microorganisms are removed, the human body cannot survive. Researchers are intensively studying the microbial communities on the skin surface and inside the human body in relation to different diseases and immunity (Ventura et al., 2009). Recent studies indicate a certain correlation between the vaginal flora composition in pregnant women and preterm birth. It was realized that a specific bacterial structure is present in the digestive system of diabetic patients, or in those who are suffering from obesity (Cani et al, 2008; Schwiertz et al., 2009), the complex role of microorganisms and the


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causes that lead to dominance of some species over others being largely unknown. These perspectives generated the Human Microbiome Project initialization by the USA National Institutes of Health, which “aims to characterize the microbial communities found at several different sites on the human body, including nasal passages, oral cavities, skin, gastrointestinal tract and urogenital tract and to analyze the role of these microbes in human health and disease”. A realistic approach of biofilms involves investigations of structure, specific composition, populations’ distribution and microbial activities within the biomass beyond a simple identification of the component species, by applying more or less invasive methods. The measurements performed on biofilms target their components and properties, structure and architecture as well as processes and interactions within the ecological micro niche. Suggestive became the approaches of Watnick and Kolter: “Biofilm, city of microbes”, Orent’s: “Slime and the city” and Hentzer’s et al.: “Quorum sensing in biofilms: gossip in slime city”. The foundation of specialized centres in USA, Germany, United Kingdom, Denmark, Australia, Singapore so on conducted the intensively biofilms research all over the world in the XXI century. Recent findings demonstrate that microorganisms are essential in almost all processes taking place on our planet (Maloy and Schaechter, 2006). The biofilm concept represents a revolution in microbiology and the approach of the attached microbial consortia brings new research perspectives and insights. Cell differentiation, specialization and social behaviour of individuals have launched the hypothesis that biofilms are primitive multicellular systems (Webb, 2007). Microbiology has become an integrative science, with practical applications in public health, industry and environmental protection. This new interdisciplinary field involves a complex research work of microbiologists, ecologists, evolutionists, geneticians, chemists, physicists, medical doctors and engineers from all around the world, which continuously bring fundamental contributions in biofilms understanding, preventing, controlling and exploitation. Rezumat. Vechea concepţie în microbiologie potrivit căreia microorganismele plutesc liber este înlocuită în prezent cu o nouǎ paradigmă, un nou model de cunoaştere: peste 99% din microorganisme trăiesc organizate în comunităţi asociate suprafeţelor, sub formǎ de biofilme. Biofilmul reprezintă o comunitate structurată de microorganisme sesile, care-şi dezvoltă o matrice polimerică aderentă la o interfaţă vie sau inertă. Aceste forme de organizare sunt ubicuitare, iar evidenţe geomicrobiologice le atestă capacitatea de a persista de-a lungul perioadelor evolutive. Deşi observaţii asupra acumulǎrilor bacteriene la suprafeţe sunt atestate încǎ din secolul XXVII, cercetarea lor a cǎpǎtat amploare abia în secolul XX, special datoritǎ impactului în industrie,

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medicinǎ şi mediul înconjurǎtor. Biofilmele ar putea verifica ipoteza apariţiei organismelor pluricelulare prin cooperarea între populaţii de celule individuale, reprezentând astfel o nouǎ paradigmǎ în microbiologie. Chiar percepţia despre organismul uman s-a schimbat, fiind perceput ca un biom, împreunǎ cu totalitatea microorganismelor asociate, a elementelor genetice ale acestora şi relaţiilor stabilite. Diferenţele fundamentale între bacteriile planctonice independente şi cele sesile ataşate, împreunǎ cu particularitǎţile de arhitecturǎ şi complexitate a biofilmului genereazǎ nevoia continuǎ a unor noi modalitǎţi de abordare a acestor structuri emergente. Avantajele apartenenţei la comunitate din perspectiva microbianǎ se concretizeazǎ în creşterea şanselor de supravieţuire, perpetuarea rezistenţei la antibiotice, dezinfectanţi, metale grele şi întǎrirea virulentă a bacteriilor patogene; din perspectiva umanǎ se urmǎreşte prevenirea formǎrii, controlul şi exploatarea biofilmelor. REFERENCES: CANI, P.D., DELZENNE, N.M., AMAR, J. and BURCELIN, R., 2008, Role of gut microflora in the development of obesity and insulin resistance following high-fat diet feeding, Pathol. Biol., 56, 305-309. CHARACKLIS, W.G., 1973, Attached microbial growths – II. Frictional resistance due to microbial slimes. Water Res., 7, 1249-1258. COSTERTON, J.W., GEESEY, G.G. and CHENG K.J., 1978, How bacteria stick, Sci. Am., 238, 86-95. COSTERTON, J.W., CHENG, K.J., GEESEY, G.G., LADD, T.I., NICKEL, J.C., DASGUPTA, M. and MARRIE, T.J., 1987, Bacterial biofilms in nature and disease, Ann. Rev. Microbiol., 41, 435-464. COSTERTON, J.W., 2007. The biofilm primer, Springer-Verlag Berlin Heidelberg. DONLAN, R.M., 2002, Biofilms: microbial life on surfaces, Emerg. Infect. Dis., 8(9), 881-890. DREESZEN, P.H., 2003, Biofilm, Edstrom Industries Inc., USA. FLEMMING, H.C., 2008, Why microorganisms live in biofilms and the problem of biofouling. Biofouling, 3-12. GEESEY G.G., MUTCH, R. and COSTERTON, J.W., 1978, Sessile bacteria: An important component of the microbial population in small mountain streams, Limnol. Oceanogr., 23(6), 1214-1223. HAUKELEKIAN, H. and HELLER, A., 1940, Relation between food concentrate and surface for bacterial growth, J. Bacteriol., 40, 547-558. HENRICI, A.T., 1933, Studies of freshwater bacteria: I. A direct microscopic technique, J. Bacteriol., 25, 277-287. HENTZER, M., GIVSKOV, M. and EBERL, L., 2004, Quorum sensing in biofilms: gossip in slime city? In: O’TOOLE, G. and GHANNOUM, M.A., Microbial Biofilms, ASM Press, USA, 118-140. KUHN, T.S., 1999, The structure of scientific revolutions (in Romanian), Humanitas Publishing, Bucharest. LAPPIN-SCOTT, H.M., 1999, Claude E. Zobell – his life and contributions to biofilm microbiology, Proceedings of the 8th International Symposium on Microbial Ecology, Halifax, Canada.


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