Recent Trends in Biofilm Science and Technology

Recent Trends in Biofilm Science and Technology helps researchers working on fundamental aspects of biofilm formation and control conduct biofilm studies and interpret results. The book provides a remarkable amount of knowledge on the processes that regulate biofilm formation, the methods used, monitoring characterization and mathematical modeling, the problems/advantages caused by their presence in the food industry, environment and medical fields, and the current and emergent strategies for their control. Research on biofilms has progressed rapidly in the last decade due to the fact that biofilms have required the development of new analytical tools and new collaborations between biologists, engineers and mathematicians.

• Presents an overview of the process of biofilm formation and its implications
• Provides a clearer understanding of the role of biofilms in infections
• Creates a foundation for further research on novel control strategies
• Updates readers on the remarkable amount of knowledge on the processes that regulate biofilm formation

• Language: English
• Publisher: Academic Press
• Release date: Jun 4, 2020
• ISBN: 9780128194980

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Recent Trends in Biofilm Science and Technology

Editors

Manuel Simões, PhD

Assistant Professor, Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Department of Chemical Engineering, Faculty of Engineering, University of Porto (FEUP), Porto, Portugal

Anabel Borges, PhD

Junior Researcher, Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE), Department of Chemical Engineering, Faculty of Engineering, University of Porto (FEUP), Porto, Portugal

Lúcia Chaves Simões, PhD

Junior Researcher, Centre of Biological Engineering, University of Minho, Braga, Portugal

Table of Contents

Cover image

Title page

Copyright

Contributors

Preface

Acknowledgments

Chapter 1. Biofilm formation and resistance

1.1. Biofilm mode of growth

1.2. Biofilm formation is a multistep process

1.3. Biofilm-specific resistance to antimicrobials

1.4. Conclusions

Chapter 2. Nuclear magnetic resonance to study bacterial biofilms structure, formation, and resilience

2.1. Introduction

2.2. Biofilm formation and structure

2.3. The composition of extracellular polymeric substances and how it affects biofilm architecture

2.4. Applications of nuclear magnetic resonance spectroscopy to study biofilms

2.5. Nuclear magnetic resonance–based metabolomics approach to study biofilms

2.6. Conclusion

Chapter 3. Design and fabrication of biofilm reactors

3.1. Definition of a biofilm reactor

3.2. Design process

3.3. Implementing the design process: industrial surfaces biofilm reactor

3.4. Conclusions

Chapter 4. Oral biofilms

4.1. Defining the problematic: an introduction

4.2. The oral cavity and its microbiota

4.3. Dental plaque

4.4. Disease-associated oral biofilms

4.5. Non-oral infections associated with oral bacteria

4.6. Conclusions

Chapter 5. The role of filamentous fungi in drinking water biofilm formation

5.1. Drinking water concerns

5.2. Microbiology of drinking water distribution systems

5.3. Drinking water distribution systems maintenance

5.4. Bacterial and fungal interactions

Chapter 6. Microalgal and cyanobacterial biofilms

6.1. Microalgae and cyanobacteria

6.2. Applications of microalgae and cyanobacteria

6.3. Microalgal/cyanobacterial cultivation

6.4. Microalgal harvesting techniques

6.5. Factors affecting microalgal/cyanobacterial biofilms

6.6. The role of microalgal/cyanobacterial biofilms in wastewater treatment processes

6.7. Conclusions

Chapter 7. Biofilms in membrane systems for drinking water production

7.1. Introduction

7.2. Methods to evaluate biofilm growth potential of feedwater

7.3. Conventional biofouling control strategies

7.4. New control strategies

7.5. Future perspectives

Chapter 8. Biofilm fuel cells

8.1. Processes involved in the biofilm of a microbial fuel cell

8.2. Microbial fuel cell structures

8.3. Integration of main processes in a microbial fuel cell model

8.4. Dimensional electrodes

8.5. Conclusions

Chapter 9. Application of lactic acid bacteria and their metabolites against foodborne pathogenic bacterial biofilms

9.1. Introduction

9.2. Antibiofilm activities of lactic acid bacteria and their metabolites against foodborne bacterial pathogens

9.3. Conclusions

Chapter 10. Role of equipment design in biofilm prevention

10.1. Introduction

10.2. Simple equipment geometries

10.3. Complex equipment design

10.4. Material properties

10.5. Conclusion

Chapter 11. Biofilm control with enzymes

11.1. Biofilms and problems associated with their control

11.2. Biofilm structure and mechanisms of bacterial resistance

11.3. Emergent strategies of biofilm control and eradication

11.4. Antibiofilm enzymes

11.5. Conclusions

Chapter 12. The potential of phytochemical products in biofilm control

12.1. Antimicrobial properties of phytochemicals

12.2. Phytochemicals as biofilm-controlling agents

12.3. Conclusions

Chapter 13. Photoinactivation of biofilms

13.1. Photodynamic therapy

13.2. Photoinactivation of biofilms

13.3. Concluding remarks

Chapter 14. The potential of drug repurposing to face bacterial and fungal biofilm infections

14.1. Introduction

14.2. Antimicrobial activity among drugs used for noninfectious human diseases

14.3. Drug repurposing—an alternative strategy against biofilm infections

14.4. Conclusions

Chapter 15. In silico development of quorum sensing inhibitors

15.1. Biofilms in health

15.2. Mechanisms of biofilm formation

15.3. Quorum sensing

15.4. In silico methods

15.5. Conclusions

Chapter 16. Challenges and perspectives in reactor scale modeling of biofilm processes

16.1. Introduction

16.2. Mathematical modeling of biofilm reactors

16.3. Modeling challenges and perspectives

16.4. Conclusion

Index

Copyright

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Contributors

Ana Cristina Abreu,     Department of Chemistry and Physics, Research Centre CIAIMBITAL, University of Almería, Almería, Spain

Mafalda Andrade,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Ana Cláudia Barros,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Thierry Bénézech,     Univ. Lille, CNRS, INRAE, ENSCL, UMET, Villeneuve d'Ascq, France

Anabela Borges,     LEPABE – Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Fernanda Borges,     Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales, United Kingdom

Szilard S. Bucs,     Water Desalination and Reuse Center (WDRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

Paula Aline Zanetti Campanerut-Sá,     Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil

Ana F.A. Chaves,     Faculty of Engineering, University of Porto, Porto, Portugal

João Vitor de Oliveira Silva,     Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil

Hermann J. Eberl,     Department of Mathematics and Statistics, University of Guelph, Guelph, ON, Canada

Christine Faille,     Univ. Lille, CNRS, INRAE, ENSCL, UMET, Villeneuve d'Ascq, France

Nadia M. Farhat,     Water Desalination and Reuse Center (WDRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

Ignacio Fernández,     Department of Chemistry and Physics, Research Centre CIAIMBITAL, University of Almería, Almería, Spain

Marcela N. Gatti,     Grupo de Control Automático y Sistemas, Facultad de Ingeniería. Universidad Nacional del Comahue, Neuquén, Argentina

Astrid Gędas,     Department of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

E. Giaouris,     Department of Food Science and Nutrition, School of the Environment, University of the Aegean, Ierou Lochou 10 and Makrygianni, Lemnos, Greece

D.M. Goeres,     Center for Biofilm Engineering, Montana State University, Bozeman, MT, United States

A.L. Gonçalves,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Nelson Lima,     Centre of Biological Engineering, University of Minho, Braga, Portugal

Rita P. Magalhães,     UCIBIO/REQUIMTE, BioSIM – Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Joana Malheiro

LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto, Portugal

Cardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, Wales, United Kingdom

Ana Meireles,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

André Melo,     LAQV/REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade do Porto Porto, Portugal

Luís Melo,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Filipe Mergulhão,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

M. Mettler,     Center for Biofilm Engineering, Montana State University, Bozeman, MT, United States

Jane Martha Graton Mikcha,     Department of Clinical Analysis and Biomedicine, State University of Maringá, Maringá, Paraná, Brazil

Ruben H. Milocco,     Grupo de Control Automático y Sistemas, Facultad de Ingeniería. Universidad Nacional del Comahue, Neuquén, Argentina

Isabel M. Oliveira,     LEPABE – Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Magdalena A. Olszewska,     Department of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

A.E. Parker

Center for Biofilm Engineering, Montana State University, Bozeman, MT, United States

Department of Mathematical Sciences, Montana State University, Bozeman, MT, United States

Russell Paterson,     Centre of Biological Engineering, University of Minho, Braga, Portugal

S. Pedersen,     BioSurface Technologies Corporation, Bozeman, MT, United States

Facundo Quiñones,     Grupo de Control Automático y Sistemas, Facultad de Ingeniería. Universidad Nacional del Comahue, Neuquén, Argentina

Marta Ribeiro,     LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Maria José Saavedra,     CITAB-Centre for Research and Technology of Agro-Environmental and Biological Sciences, Veterinary Science Department, University of Trás-os-Montes e Alto Douro, Vila Real, Portugal

Alex Fiori Silva,     Federal Institute of Paraná, Paranavaí, Paraná, Brazil

Lúcia Chaves Simões

Centre of Biological Engineering, University of Minho, Braga, Portugal

LEPABE — Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Manuel Simões,     LEPABE – Laboratory for Process Engineering Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal

Sérgio F. Sousa,     UCIBIO/REQUIMTE, BioSIM – Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

P. Sturman,     Center for Biofilm Engineering, Montana State University, Bozeman, MT, United States

Tatiana F. Vieira,     UCIBIO/REQUIMTE, BioSIM – Departamento de Biomedicina, Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Johannes S. Vrouwenvelder,     Water Desalination and Reuse Center (WDRC), Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

Matthew J. Wade,     School of Engineering, Newcastle University, Newcastle-upon-Tyne, United Kingdom

D.K. Walker,     Center for Biofilm Engineering, Montana State University, Bozeman, MT, United States

B. Warwood,     BioSurface Technologies Corporation, Bozeman, MT, United States

Preface

The ability of microorganisms to adhere on surfaces and form biofilms is a privilege. These aggregates of cells enclosed in a matrix of extracellular polymeric substances show a physiology that is distinctly different from that of the same cells in planktonic state. Biofilm growth is arguably the most relevant growth state for the majority of microorganisms, particularly bacteria. Its complexity relative to planktonic growth means that we still have a poor understanding of how microorganisms behave in such a complex structure. Biofilms are as versatile as they are ubiquitous. Intentional and unintentional biofilms concern a broad range of areas, comprising special attention in the industrial/environmental and biomedical areas. As consequence, research on biofilm science and technology is an evolving research area.

This book contributes with new insights regarding the biofilm mode of life, giving the readers a significant content focusing the recent advances on multidisciplinary biofilm research. The book is strategically outlined with data on biofilm formation by diverse microorganisms—bacteria, microalgae, and filamentous fungi. Top-notch methods for biofilm analysis and characterization are described in terms of analytical chemistry and mathematical modeling. Advanced strategies for biofilm control are detailed in several chapters as well as the in silico analysis for the development of biofilm-targeting molecules. Biofilms are further conveniently described for their biotechnological potential, particularly for wastewater treatment and for bioenergy production as biofilm fuel cells.

Acknowledgments

This work was financially supported by Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy (LEPABE)—funded by national funds through the FCT/MCTES (PIDDAC); projects PTDC/BII-BTI/30219/2017 - POCI-01-0145-FEDER-030219, POCI-01–0145-FEDER-028397, POCI-01–0145-FEDER-033298, and POCI-01–0145-FEDER-035234 funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through the FCT/MCTES. FCT under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Sabbatical grant SFRH/BSAB/150379/2019.

Chapter 1

Biofilm formation and resistance

Astrid Gędas, and Magdalena A. Olszewska     Department of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

Abstract

To increase the chance of survival, microorganisms form biofilms enclosed in self-produced extracellular polymeric substances (EPS). Biofilm formation is a complex process that depends on many factors, such as environmental conditions, specific strain attributes, and material surface properties. Biofilms are also responsible for the increased microbial resistance to antimicrobial agents, especially to antibiotics. This effect is reinforced by, e.g., persisters that—as a subpopulation of cells with slowed down metabolism—escape antibiotic treatment. This and many other biofilm-related resistance mechanisms are therefore important to be continuously studied to overcome serious problems biofilms caused in particular in the medicine field. In this chapter, we focus on the factors including molecular aspects that play a key role in both the biofilm formation process and the biofilm-based resistance to antimicrobials that bring new knowledge and insight in this regard.

Keywords

Biofilm formation process; Biofilm-based antibiotic resistance; Biofilms

1.1. Biofilm mode of growth

Overall, microorganisms exist in two modes of growth: a unicellular, in which single, free-living cells (planktonic) prevail in the microbial population, and a multicellular one, in which the cells bind to each other (cohesion) and to the substratum (adhesion) (Bjarnsholt, 2013). In nature, microbes barely occur as planktonic cells but instead exist as communities of sessile cells that grow as biofilms (Berlanga and Guerrero, 2016; Rabin et al., 2015; Donlan, 2002). Biofilms can be defined as aggregated microbial communities surrounded by a matrix of self-produced extracellular polymeric substances (EPS), which develop on a wide variety of inert or organic surfaces (Armbruster and Parsek, 2018; Kim and Lee, 2016).

Biofilm development depends upon different factors, including those associated with environmental conditions, specific strain attributes, and material surface properties (Chmielewski and Frank, 2003). In fact, biofilm formation is usually enhanced in harsh environmental conditions, such as nutrient-deficient or toxic media (Rendueles and Ghigo, 2015). Besides, microbes within the biofilm can coordinate their behavior for promoting growth and producing EPS (Moradali et al., 2017). The ability to form biofilms seems to be universal among microorganisms. Microbial communities exhibiting this ability may be composed not only of single species but of multiple species as well (O'Toole et al., 2000; López et al., 2010). In most biofilms, however, microorganisms account for less than 10% of the biofilm dry mass, whereas the EPS may account for over 90% (Flemming and Wingender, 2010; Tsagkari and Sloan, 2018). Indeed, this self-produced matrix is responsible for the cohesion and adhesion of cells, but more importantly, for the development of a microenvironment that allows the microbes for cell–cell interaction and communication and serves as a reservoir of metabolic substances, nutrients, and energy for biofilm inhabitants (Flemming and Wingender, 2010).

This highly hydrated EPS matrix is mainly composed of polysaccharides (PSs), but it can also consist of proteins, lipids, extracellular DNA (eDNA), and other biopolymers (Das et al., 2016; Kim and Lee, 2016; Kostakioti et al., 2013 ). The PSs synthesized by microbial cells differ significantly in their composition and thus in their chemical and physical properties (Limoli et al., 2015). For instance, in gram-negative bacteria, some PSs are neutral or polyanionic and hence rendered more anionic, whereas in gram-negative bacteria, the EPS show mostly cationic nature due to teichoic acid and certain quantities of proteins (Donlan, 2002; Vu et al., 2009). Enzymes may also play an important role in a biofilm life cycle, i.e., they can break down EPS polymers and provide carbon and energy during starvation or cause biofilm degradation during detachment and dispersal (Rabin et al., 2015). Several studies have shown the significance of enzymes particularly in releasing cells from biofilms to start a new biofilm life cycle (Petrova and Sauer, 2016). In biofilms, e.g., these formed by Vibrio cholerae and Pseudomonas aeruginosa, nonenzymatic proteins, such as lectins, which take part in formation and stabilization of the matrix, can also be found (Fong and Yildiz, 2015). Moreover, a crucial role in biofilm formation has recently been demonstrated for eDNAs which, among others, take part in cell adhesion. In the case of Staphylococcus aureus, eDNA is responsible for matrix structure and enables cell–cell as well as cell–surface interactions (Boles and Horswill, 2011). eDNA is also essential in cell-to-cell connection and in Pseudomonas biofilm stabilization especially at the initial stages of biofilm development, when the amount of EPS components is small (Kostakioti et al., 2013).

Not only composition but also the quantity of EPS changes depending on the type of microorganisms, the age of the biofilm, and current environmental conditions (Donlan, 2002). Importantly, EPS form the structure and architecture of the biofilm and protect cells from adverse and disruptive environmental conditions (Vu et al., 2009). Several studies have shown that biofilms can increase microbial resistance to dehydration (Roy et al., 2018), UV radiation (Kirmusaoğlu, 2016), extreme temperature and pH (Achinas et al., 2019; Kirmusaoğlu, 2016 ), high salinity (Kirmusaoğlu, 2016), nutrients deficiency (Roy et al., 2018; Chmielewski and Frank, 2003), various sanitizers (Lu et al., 2019), antibiotics (Lu et al., 2019; Kirmusaoğlu, 2016 ), etc. Therefore, biofilm formation is of serious and ongoing concern in a wide range of fields from industrial processes such as food processing, to health-related fields such as medicine and dentistry (Van Houdt and Michiels, 2010). Aside from economic and health problems, biofilms play a positive role and can arouse commercial interest in the immobilization technology (Nie et al., 2016; Żur et al., 2016 ).

1.2. Biofilm formation is a multistep process

Biofilm formation is a dynamic process that can be divided into several steps, i.e., initial attachment, irreversible attachment, early development of biofilm architecture, maturation, and dispersion and which involves a highly coordinated cascade of gene expression (Reen, 2019) (Table 1.1).

The first step in the process can be either active or passive, depending on cell adherence mechanism (Dufour et al., 2012; O'Toole et al., 2000; Srey et al., 2013). Adhesive surface structures, such as fimbria and pili, are promotive to the active adhesion. Flagella facilitate bacteria transfer to a specific attachment site and together with pili help overcoming the repulsive barriers encountered by the cell when it approaches the surface (Berne et al., 2015; Chmielewski and Frank, 2003). However, they can also play an important role in surface sensing by which microbes sense and respond to contact with the surface (O'Toole and Wong, 2016). In the case of a biofilm-forming bacterium P. aeruginosa, flagellar mechanosensing is regulated by a secondary messenger, bis-(3́–5́)-cyclic dimeric guanosine monophosphate (c-di-GMP), which affects the activity of a regulator of flagellar gene expression, FleQ. Normally, FleQ suppresses the expression of pel gene, which is responsible for the synthesis of biofilm exopolysaccharides. When c-di-GMP binds FleQ, the inhibition of pel gene expression decreases, thus promoting biofilm formation (Belas, 2014; O'Toole and Wong, 2016). This small cytoplasmic signaling molecule—c-di-GMP—is a key regulator of the motility-to-biofilm transition for many other bacteria, e.g., Escherichia coli, Vibrio cholerae, and Salmonella enterica serovar Typhimurium (Guttenplan and Kearns, 2013; Valentini and Filloux, 2016). Importantly, several genes encoding adhesins associated with biofilm formation were identified within the gram-positive and nonmotile bacteria like Enterococcus, such as SagA, Acm (Enterococcus faecium), and Ace (Enterococcus faecalis) that facilitate adherence to eukaryotic extracellular matrix components (Kostakioti et al., 2013). Moreover, a new study has identified bacterial membrane vesicles (MVs) produced by E. faecium and suggested that MV-associated proteins are involved in virulence and antimicrobial resistance including biofilm-promoting proteins and extracellular matrix–binding proteins (Wagner et al., 2018). Indeed, at the beginning of biofilm development, adhesins play a major role in anchoring to the surface (Esteban et al., 2014). Biofilm formation by Staphylococcus epidermidis and S. aureus requires also other surface proteins, such as biofilm-associated protein (Bap) and the accumulation-associated protein (Aap) (Conrady et al., 2008). Fibronectin is also associated with enhancing cell adhesion. There are two types of fibronectin synthesized by S. aureus—fibronectin-binding protein A (FnBPA) and fibronectin-binding protein B (FnBPB), which differ by ligands. A group of adhesins that bind collagen (CAN) may be distinguished as well (Esteban et al., 2014). In turn, Cairns et al. (2013) showed that the gram-positive model organism Bacillus subtilis flagellum also acts as a mechanosensor upon contact with the surface and that the inhibition of flagellar rotation involving the flagellar stator gene, motB, results in the activation of the DegS-DegU two-component signal transduction system with sensory kinase that controls biofilm formation. Moreover, a recent study has shown that c-di-GMP levels are higher in matrix producers than motile cells and differ between sporulating and competent cell types and thus might also play an important role during B. subtilis biofilm formation (Weiss et al., 2019).

Table 1.1

In turn, the passive adhesion is facilitated by gravity, diffusion, and fluid dynamics (Chmielewski and Frank, 2003). Initial attachment is a reversible stage due to the loose binding with the abiotic or biotic surface, where van der Waals and electrostatic forces are involved, and at this stage, cells might still detach and return to the planktonic mode (Armbruster and Parsek, 2018). However, after 5–30   s of weak interaction, when cells lie flat on the surface forming a monolayer and resist to physically dislodge, the attachment becomes irreversible (Chmielewski and Frank, 2003). Importantly, material properties affect bacterial adhesion. Surface charge plays an important role in cell–surface interaction. Most bacterial cells are negatively charged; therefore the adhesion may be disrupted by a negative charge of the surface, whereas an opposite effect can be observed with the positively charged surface (Song et al., 2015). Moreover, surface charge may influence bacterial motility and biofilm cells morphology. A study by Rzhepishevska et al. (2013) showed that P. aeruginosa developed a mushroom-shaped structure on negatively charged poly(3-sulfopropylmethacrylate) (SPM) and zwitterionic poly(2-(methacryloyloxy)ethyl)dimethyl-3-sulfoproyl) ammonium hydroxide) (MEDSAH), whereas a flat biofilm on positively charged surfaces. Additionally, high levels of c-di-GMP were found in the mushroom structure, which may suggest that—through physiological adaptation—bacteria can attach even to the negatively charged surfaces. A more recent study has indicated, however, that Pseudomonas putida NBRC 100650 easily adhered and formed biofilm on the hydrophobic polyvinylidenefluoride (PVDF) surface. In contrast, biofilm was rarely observed on the hydrophilic polyvinyl alcohol (PVA) surface (Saeki et al., 2016). Surface energy, roughness, and topography are also significant factors in cell attachment (Achinas et al., 2019; Song et al., 2015). Nonetheless, irreversible attachment is characterized by permanent bonding such as dipole–dipole interaction, hydrogen bonds, or ionic covalent bonding, and thus is hard to break (Chmielewski and Frank, 2003; Srey et al., 2013).

Following this stage, cells start to multiply and form microcolonies and simultaneously produce a polymer matrix (Taraszkiewicz et al., 2013). For many gram-negative bacteria, this stage is characterized by high levels of c-di-GMP because it is associated with intensified production of a biofilm matrix (Armbruster and Parsek, 2018). From now on, bacteria display a coordinated behavior through cell–cell communication commonly known as quorum sensing (QS), which is important for controlling, e.g., sporulation, competence, antibiotic production, virulence factor secretion, and biofilm formation (Rutherford and Bassler, 2012). For this purpose, they secrete autoinducers (AIs), which accumulate in the environment, as the bacterial population density increases and alters gene expression (Reen, 2019). For biofilm development, the genes involved in biofilm formation and maturation are activated, as population density reaches a critical value. There are three groups of signaling molecules in bacteria: acyl homoserine lactones (AHLs) that are specific for gram-negative bacteria, peptides called autoinducing peptides (AIPs) that are specific for gram-positive bacteria, and a universal signaling molecule called autoinducer-2 (AI-2) (Taraszkiewicz et al., 2013). When concentration of AHLs is high, the bonding with cytoplasmic receptors occurs that further governs the gene expression. Gram-negative bacteria use the LuxI/LuxR system, in which AIs are synthesized by LuxI-type enzymes and detected by LuxR-type transcriptional regulators (Bassler, 2002). There are many gram-negative bacteria identified to use LuxI/LuxR to control a wide range of biological behaviors, including those encoding virulence factors and antibiotics biosynthesis, plasmid transfer, bioluminescence, and biofilm formation (Ziemichód and Skotarczak, 2017). Each species produces a specific AHL or a combination of AHL, and the members of the same species respond only to the signal molecule (Li and Tian, 2012). When AIP concentration is high, it binds to a two-component sensor kinase receptor transmitting signal and consequently activating the expression of genes, e.g., an accessory gene regulator (Agr) and RNAIII (Lu et al., 2019; Taraszkiewicz et al., 2013). For example, the agr global regulator, besides the transition between planktonic and sessile modes, controls cell detachment, contributing to survival via cell dispersal and colonization of new niches by S. aureus (Grande et al., 2014). A recent understanding of the molecular mechanisms underlying agr QS was reviewed by Tan et al. (2018). Agr operon controls Agr system and consists of AgrA, AgrB, AgrC, and AgrD. When cell density is high, AIPs bind to the membrane-located AgrC, which further activates AgrA and consequently promotes the target gene expression. There are two promoters. One of them, P3, may also activate RNAIII expression, which prompts the upregulation of exo-toxins and exo-enzymes (Lu et al., 2019; Otto, 2013). Ciulla et al. (2018) described a potential method for the treatment of S. aureus biofilm infections with using RNA III-inhibiting peptide (RIP). This peptide is capable of blocking the QS mechanisms by competing with RNA III-activating peptide, and it leads to the inhibition of S. aureus pathogenesis. In turn, AI-2 is produced by a range of gram-positive and gram-negative bacteria and detected by a variety of them, thus allowing for intra- and interspecies communication, including development of dental plaque (Rickard et al., 2006), and affecting gut microbiota composition (Thompson et al., 2015). It has recently been shown that AI-2 produced by E. faecalis biofilms attracts E. coli cells, resulting in enhanced aggregation and microcolony formation by E. coli and in increased stress resistance of both species (Laganenka and Sourjik, 2017).

Once the first layer of the biofilm is developed, biofilm often grows to a mushroom- or tower-shaped structure, as observed for P. aeruginosa, S. aureus (Rabin et al., 2015), or honeycomb-like organizational structures of Listeria monocytogenes biofilms (Guilbaud et al., 2015) (Fig. 1.1). Biofilm structure development depends on many factors, including changes in the amount of nutrients or atmospheric pressure (Reen, 2019). At this stage, a monolayer biofilm, where there are cell–surface interactions only, transforms into a multilayer biofilm, where bacteria are attached to both the surface and other biofilm cells (Karatan and Watnick, 2009). Along with maturation, an increasingly complex biofilm structure is being formed together with water channels, which distribute nutrients and wastes (Lu et al., 2019). The arrangement of bacteria in the structure is not accidental and determined by their metabolism and tolerance to oxygen. For instance, anerobic bacteria are located in deeper layers, thus avoiding exposure to oxygen (Rabin et al., 2015). This is also associated with different cell growth rates—fast growers are usually located on the periphery and slow growers in deeper layers, which indicates that the periphery of biofilm microcolonies is compositionally and mechanically dynamic (Chmielewski and Frank, 2003).

Figure 1.1 3D projections (A–D) from confocal laser scanning microscope (CLSM) images of the biofilm formed by Listeria monocytogenes serotype 1/2a showing the honeycomb-like morphotype (obtained with the Zeiss Zen software—authors' own contribution). The biofilm was grown in an eight-well chamber slide system in TSB at 37   °C and stained with the Live/Dead BacLight.

Environmental stress or nutrient deficit leads to the last and critical stage in the biofilm life cycle, wherein cells leave the biofilm and return to the planktonic state or settle in a new niche (Dufour et al., 2012). In general, there are three main ways of bacterial cells removal, i.e., desorption, detachment, and dispersion. Desorption means a direct transfer of microorganisms to the bulk liquid and is observed throughout the biofilm development process (Petrova and Sauer, 2016). Detachment may be categorized into four mechanisms: abrasion, grazing, erosion, and sloughing. Abrasion means detachment caused by collisions with particles from the bulk liquid. Feeding activity of eukaryotic organisms leads to the release of a biofilm, called grazing. Peripheral cells in the structure are constantly exposed to fluid shear, which results in the continuous removal of biofilm portions. This kind of detachment is called erosion. In contrast, sloughing is a rapid removal of intact pieces or entire biofilm mass. Besides shear forces, the chemical or enzymatic degradation is also involved in this process (Kim and Lee, 2016; Petrova and Sauer, 2016). Enzymes are produced by community inhabitants, which break down polysaccharides holding the biofilm together, thereby actively releasing bacteria for the colonization of new niches in a process called dispersion. For instance, P. aeruginosa produces alginate lyase, E. coli uses N-acetyl-heparosan lyase, and Streptococcus equi uses hyaluronidase to break down a biofilm matrix (Kregiel and Antolak, 2016). Another example of a detachment agent can be a hydrolase called dispersin B (DspB), which is produced by Aggregatibacter actinomycetemcomitans. It catalyzes the hydrolysis of poly-N-acetyl-D-glucosamines (PGAs) and, as a result, causes the biofilm to lose its structural integrity (Ragunath et al., 2015). DspB effectively inhibits biofilm formation by both gram-positive and gram-negative bacteria, for instance, by S. epidermidis and Actinobacillus pleuropneumoniae (Kaplan et al., 2004). For this reason, Ghalsasi and Sourjik (2016) proposed using engineered E. coli to produce dispersin B as a targeting system to biofilm removal. The last way of cell release from biofilm is dispersion, wherein the sensing of certain signals is involved. The regulatory network is crucial in physiological changes, which facilitate bacteria release. Dispersion can be divided into two classes by the source of signals. In native dispersion, signaling molecules are self-synthesized, whereas changes in the external environment are the cues for environmentally induced dispersion (Petrova and Sauer, 2016).

1.3. Biofilm-specific resistance to antimicrobials

To survive, microbes have developed cell protection or resistance mechanisms against the harsh environmental conditions (Možina et al., 2013). Along with the transformation of planktonic cells into a sessile form, biofilm resistance becomes a more complex phenomenon with a range of genes that showed increased expression in biofilms (Table 1.2). Biofilm displays therefore unique properties, making the community inhabitants even 1000   times more resistant than the planktonic cells (Chadha, 2014). Due to this fact, human infections related to biofilms are often difficult or impossible to eradicate and turn into serious chronic conditions (Aswathanarayan and Vittal, 2013). A group of bacteria comprising P. aeruginosa, E. faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter species is characterized by tolerance and resistance to antimicrobial agents and referred to as ESKAPE pathogens (Santajit and Indrawattana, 2016). P. aeruginosa is especially important owing to its capability to form biofilms, where the bacteria embed themselves in the EPS and escape from antibiotic treatment (Ciofu and Tolker-Nielsen, 2019). Aside from the healthcare, antimicrobial resistance via attachment of cells to the surface has serious consequences also in the petroleum industry, drinking water distribution system, paper industry, metalworking industry, and food-processing industry (Singh et al., 2017). However, because biofilm-related health issues generate significant global health and economic costs, the link between biofilm and antibiotic resistance deserves a profound interest. Several studies have revealed certain antibiotics can induce biofilm formation, as recently reviewed by Ciofu and Tolker-Nielsen (2019) or Song et al. (2016). Other researchers have focused on the association between biofilm production and multidrug resistance (MDR) and demonstrated antibiotic resistance to be higher among biofilm producers than nonproducers, e.g., P. aeruginosa or A. baumanii (Gurung et al., 2013). Zeighami et al. (2019) have recently revealed the high frequency of biofilm producers among extensively drug-resistant (XDR) A. baumannii clinical isolates, with a high prevalence of biofilm-related genes of csuE and pgaB. Moreover, although Qi et al. (2016) found that MDR A. baumannii isolates were weaker biofilm producers than the non-MDR isolates, the enhancement in resistance occurred irrespective of the quantity of biofilm produced.

Different species in the community are able to cooperate with each other rather than compete, which is also unique to biofilms. These biofilm aggregates could easily give rise to a horizontal gene transfer, thus spreading resistance genes (Bordi and de Bentzmann, 2011). Haaber et al. (2017) suggests that mobile genetic elements (MGEs), which carry antibiotic resistance genes (ARGs) in S. aureus that forms biofilm on medical implants, are transferred by conjugation or bacteriophage transduction, with the latter being potentially higher in biofilms compared with the planktonic cells. In turn, gene operons are involved in biofilm-associated tolerance and resistance. E. coli uses a four-gene operon, pgaABCD, which encodes the exopolysaccharide, poly-N-acetylglucosamine, and plays an important role in biofilm formation and resistance. Besides E. coli, poly-N-acetyl-glucosamine was also described in S. aureus, S. epidermidis, and Actinobacillus species. Cerca and Jefferson (2008) revealed that the expression of pga and polysaccharide synthesis was induced in E. coli by several chemicals, i.e., ethanol, NaCl, and glucose, and biofilm formation was also induced by one of them (glucose). Besides, many of the studies on biofilm-induced resistance have been done with P. aeruginosa, as reviewed by, e.g., Olsen (2015), where biofilm-specific resistance genes and operons were identified. It was, for instance, suggested that glucose polymers encoded by the ndvB present in P. aeruginosa prevent antibiotics from reaching their ribosomal target sites by isolating them in the periplasm and by the activation of ethanol oxidation genes (Bordi and de Bentzmann, 2011; Olsen, 2015).

Table 1.2

In biofilm environment, cells are confronted with different physical, chemical, or biological stress conditions, and they often cope with them via activation of a general stress response, in which a master regulator, RpoS, is involved that controls a specific physiological response to a specific stress and enhanced resistance of biofilm cells to antimicrobial agents (Možina et al., 2013). This largely falls into innate biofilm resistance mechanisms, where limited diffusion of an antibiotic agent through biofilm matrix, decreased oxygen and nutrient availability accompanied by altered metabolic activity, formation of persisters, and other specific molecules are considered (Ciofu and Tolker-Nielsen, 2019). Besides the innate resistance, induced resistance has also been recognized, where direct exposure to antimicrobial agents triggers a specific response. In the case of P. aeruginosa biofilm, overproduction of β-lactamase occurs as a result of β-lactam antibiotic exposure, causing the hydrolysis of this molecule (Aswathanarayan and Vittal, 2013). However, it is worth keeping in mind that the resistance to antibiotics in biofilms is due to both the innate and induced mechanisms. It has been shown that QS affects the tolerance of P. aeruginosa biofilms to tobramycin, kanamycin, and hydrogen peroxide (Olsen, 2015). It might be related with QS participation in eDNA production, which inhibits penetration of some antibiotics into the biofilm's structure. Under extreme conditions, the bacterial survival is strongly sustained by efflux pumps. There are single or multicomponent systems to get rid of toxins and waste products, and these pumps are widely involved in antibiotic resistance, even that related with MDR (Možina et al., 2013). The complete deletion of PA1874-1877 genes encoding the efflux pump in P. aeruginosa PA14 results in its increased sensitivity to tobramycin, gentamicin, and ciprofloxacin, in particular when this mutant is grown in a biofilm (Zhang and Mah, 2008). Thus, the efflux pump is more highly expressed in biofilm cells than in planktonic counterparts, suggesting that these genes play an important role for biofilm resistance to antibiotics. In addition, Zhang and Mah (2008) discovered that a mutant strain combining the ndvB mutation with PA1874-1877 gene deletion was more resistant than a single mutant, which shows the interaction of two different mechanisms of antibiotic resistance. P. aeruginosa has also a MexXY pump, which is a significant determinant of the resistance of aminoglycosides (such as amikacin, tobramycin). Besides MexXY pump, AcrD of E. coli, AmrAB-OprA of Burkholderia pseudomallei, and AdeABC of A. baumannii are aminoglycoside efflux pumps as well (Morita at al., 2012). In the case of K. pneumoniae, AcrAB and OqxAb efflux pumps play an important role in nitrofurantoin resistance (Xu et al., 2019). Mycobacterium tuberculosis resistance is also caused by the efflux pumps system. Among others, there is TetV causing resistance to tetracycline and Tap   (Rv1258c) conferring low resistance to aminoglycosides and tetracycline (Kanji et al., 2019).

Certainly, the so-called biofilm cell heterogeneity has an important impact on increased resistance to antimicrobial agents (Singh et al., 2017). A subpopulation called persister cells is frequently found in the biofilm; although it represents a relatively small part of the population, its frequency in biofilm is still higher than in the planktonic communities (Stewart, 2002). The metabolism of persisters is slowed down, perhaps even in the sporelike state. By this inhibition of metabolism, they are not antibiotic targets because they are inactive (Mah, 2012). A recent study has suggested the presence of a long-retention effect or memory effect in the persister cell state of not only gram-negative bacteria (E. coli, Acinetobacter, Salmonella) but of gram-positive bacteria as well (Staphylococcus, Bacillus) (Miyaue et al., 2018). In particular, it was revealed that E. coli forms more persister cells in biofilm culture than in the liquid culture and that these persisters can be sustained in higher numbers than those from liquid culture for up to 4   weeks   at 37°C in a fresh, antibiotic-containing medium, even after withdrawal from the biofilm culture. Hence, this memory effect may be caused by molecular signal inscribed into the persister cells during biofilm culture. Importantly, the number of persisters formed depends on the growth phase and is higher in