201 9 Biofilms: A Phenotypic Mechanism of Bacteria Conferring Tolerance Against 18 Stress and Antibiotics Anwar Alam, Ashutosh Kumar, Prajna Tripathi, Nasreen Z. Ehtesham, and Seyed E. Hasnain Abstract Biofilm, as a heterogenous congregation of microbial cells enclosed within a pellicle, has largely gained attention due to their historical importance in environ- ment as sludges, flocs, slimes, etc. Biofilms in medical research have been an active area of research in periodontics, in wounds, and in surgical implants. With the availability of whole genome sequences, it is now evident that the mechanisms that control biofilm formation have largely remained conserved during the course of evolution, pointing to the fact that biofilm formation is an Anwar Alam, Ashutosh Kumar and Prajna Tripathi have contributed equally to this chapter. A. Alam JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, Delhi, India Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, Delhi, India A. Kumar Department of Microbiology, Tripura University (A Central University), Agartala, Tripura, India P. Tripathi National Institute of Immunology, New Delhi, Delhi, India JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, Delhi, India N. Z. Ehtesham Inflammation Biology and Cell Signaling Laboratory, ICMR-National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, Delhi, India e-mail: [email protected] S. E. Hasnain (*) JH Institute of Molecular Medicine, Jamia Hamdard, New Delhi, Delhi, India Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India e-mail: [email protected] # Springer Nature Singapore Pte Ltd. 2019 315 S. E. Hasnain et al. (eds.), Mycobacterium tuberculosis: Molecular Infection Biology, Pathogenesis, Diagnostics and New Interventions, https://doi.org/10.1007/978-981-32-9413-4_18 316 A. Alam et al. integral part in the lifecycle of any unicellular organism. The ability to easily inter- switch between planktonic to a sessile life cycle is an important armor for these unicellular organisms to overcome stress. The matrix not only acts a physical barrier that protects the bacteria but also provides an ecological niche for close interaction and communication among these unicellular entities. This coordinated community-like behavior synchronizes metabolic upregulation or downregulation, both in time and space, and allows these microorganisms to achieve physiological proficiency in terms of ability to tolerate stress that might not be possible as a single cell. Research on biofilms from the perspective to explore the mechanisms of drug tolerance is now considered an apt model as compared to the use of planktonic microorganisms. The increasing use of medical implants further necessitates the need to accelerate research in anti-biofilm strategies for these medical devices. This chapter presents an overview of the mechanisms of biofilm formation and the various interventions for prevention of biofilms. Keywords Biofilm · Persister · Drug tolerance · Mycobacterium tuberculosis Abbreviations ABC ATP-binding cassette transporters CaCl2 Calcium chloride cAMP Cyclic adenosine monophosphate DNA Deoxyribonucleic acid FDA Food and Drug Administration GMP Guanosine monophosphate kHz Kilohertz MgSO4 Magnesium sulfate NaCl Sodium chloride RNA Ribonucleic acid 18.1 Introduction Microbial biofilm refers to assemblage of monospecies or poly-species microbial cells that is irreversibly attached with surface or with each other and is embedded within an extracellular matrix of polymeric substances. Cells within the microbial biofilms exhibit an altered phenotype in terms of growth rate or gene transcription and are therefore distinct as compared to the colonies of bacteria growing on agar plate macromolecular polymer matrix. Antony van Leeuwenhoek is credited with the first account of biofilm when he reported (1683–1708) the presence of aggregated microbes in the scurf of his teeth and tongue. Bacterial adhesion to surfaces is a common phenomenon that has routinely been observed in the environment. The term film has been used in marine microbiology 18 Biofilms: A Phenotypic Mechanism of Bacteria Conferring Tolerance Against... 317 to distinguish sessile microorganisms from the planktonic forms. The “bottle effect” observed for marine microorganisms pointed that bacterial growth and activity increased significantly due to attachment of these organisms on the surface. Initial investigation about the composition of biofilm using “ruthenium red” dye followed by fixation with osmium tetroxide showed that biofilms consist of polysaccharides. The medical rele- vance of biofilm emanated from the studies carried out on sputum samples of patients suffering from lung infection due to cystic fibrosis (CF) caused by Pseudomonas aeruginosa.Thefirst image of medical biofilms, published in 1977, was obtained from lung biopsies and showed aggregated bacteria surrounded by “glycocalyx.” The term glycocalyx was soon replaced with “biofilms” to emphasize the in vivo sessile growth of colonized microorganisms that were considered to cause antimicrobial resis- tance as compared to planktonic microorganisms (Costerton et al. 1987). The focus of research that was initially directed toward testing medicaments on planktonic forms of microorganisms soon diverged as it was observed that organisms in the biofilms showed altered physiology and gene expression. Nearly 80% of the hospital-borne infections is attributed to biofilm-forming microorganism and hence studies on biofilm-forming microorganism have taken the front seat of all major research on drugs (Kumar et al. 2017). The major thrust in biofilm research has been due to the advent of instrumentation such as scanning electron microscopy (SEM), confocal laser scanning microscopy, Robbins device biofilm sampler, etc.; novel techniques such as that have complemented standard microbiological culture techniques for biofilm characterization. Bacteria ubiquitously form biofilm and the mechanism of adhesion is highly conserved across different species of bacteria during the course of evolution. Biofilm formation is an inherent and distinct phenomena among bacteria surviving in the environment, where adhesion and protection is of higher importance and hence a sessile life cycle is favored. The evolution of nonpathogenic environmental bacteria to patho- genic strains has led to shedding of the biofilm machinery and enhancement of motility machinery required for pathogenicity-invasion of host cells and escape from immune cells. This is evident from the observation that subculturing of planktonic laboratory strains of bacterium leads to shunting off of the genetic machinery involved in expres- sion of cellular components required for adhesion, thereby rendering these cells inca- pable to form biofilms. In the presence of selective pressure such as surfactants and antibiotics, genetic machinery essential for formation of protective surface glycocalyx is triggered. Cells with surface-bound glycocalyx continue to survive while planktonic cells lacking protective surface structures fail to survive. The interaction of bacteria with the environment, within and outside the community, and the outcome of the response toward drugs are predominantly influenced by the phenotype as well. In drug discovery strategies, biofilm model could provide more relevant data that may address core questions as compared to the use of planktonic laboratory strains. 18.2 Biofilm Formation by Bacteria Bacterial species are known to inhabit a variety of ecosystems and persist even under extremely adverse conditions. The survival mechanisms of some pathogenic microbes can be clearly credited to their ability to form biofilms. In addition to 318 A. Alam et al. acting as a physical barrier against shear forces and antimicrobial agents generated by the host system, biofilms also provide a protective niche for the survival of bacteria (Jefferson 2004). Bacteria use their ability to form biofilms as a defense strategy against host-mediated assaults like perturbations in pH, generation of reactive oxygen intermediates, nutrient deprivation, attack by phagocytic cells, etc. Biofilms are considered as the product of evolution against adverse conditions. The fact that certain stress regulators and chaperones significantly contribute to the phenomenon of biofilm formation indicates that a major motivation behind biofilm formation is defense. Stress response mediators such as σB, RpoS, GroEL, DnaK, and DnaJ have been implicated in microbial biofilm formation. In addition to stress, there are other factors as well that trigger the formation of such a sessile microbial community. Pathogenic and commensal bacteria also use biofilm development as a means to colonize and persist in environments that seem propitious within the host and thus flourish well with time. Bacterial species such as Staphylococcus, Strepto- coccus, Pseudomonas, Vibrio cholerae, etc., display elaborate biofilm development under conditions that offer readily utilizable carbon sources in abundance suggesting that this may also act as a trigger factor. Biofilms formed at high shear locations, such as blood or saliva stream, are highly viscous and display remarkable tensile strength, thus conferring tremendous survival advantage to the constituent microbial cells. Moreover, microbes also reap other benefits from this interactive community such as division of metabolic labor. Close proximity of microorganisms within the biofilm