Microbial Biofilms, Second Edition

Many fungal and bacterial pathogens build biofilms, establishing resilient communities on a variety of clinical surfaces ( 1 , 2 ). Biofilm formation has become increasingly appreciated as one of the most common modes of growth. Medically, these adherent conglomerates of cells pose a serious obstacle for the treatment of infection. Compared to nonbiofilm, planktonic cells, they are extraordinarily tolerant to anti-infective therapies and resist killing by host defenses ( 3 ). Biofilm formation has been well described for Candida albicans, the most common fungal pathogen in the hospital setting ( 4 , 5 ). More recently, the majority of clinically encountered fungi have been shown to produce biofilms. This group includes filamentous fungi (Aspergillus, Fusarium, zygomycetes), Pneumocystis, and yeasts (Blastoschizomyces, Saccharomyces, Malassezia, Trichosporon, Cryptococcus, and numerous Candida spp.) ( Table 1 ) ( 6 – 16 ).

One of the most remarkable features of the evolutionary process is its capacity to construct. In billions of years a primordial soup of organic compounds evolved to the theater of life extant today. This ability to construct is best illustrated by a number of transitions that have occurred during the natural history of our planet, such as the evolution of the first prebiotic cells, eukaryotes, multicellularity, and eusociality ( 1 , 2 ). These transitions all bear a number of striking similarities ( 2 ). First, construction evolves through cooperation ( 3 – 6 ). That is, new organizational layers come about through the cooperative interaction of biological units that previously functioned independently. For example, organelles evolved from microbes that engaged in mutualistic interactions through endosymbiosis, and multicellularity evolved from cells that cooperate by sticking together, either via incomplete cell division or through aggregation ( 7 ). In addition to cooperation, a second aspect characterizes major evolutionary transitions: the division of labor ( 8 , 9 ). A precise definition of the division of labor will be given below, but one can loosely speak of division of labor when individuals—during their cooperative interactions—specialize in performing different “tasks.” Perhaps the most striking example comes from multicellular development. Multicellular organisms consist of many specialized cell types (e.g., muscle cells, neurons, epithelia, etc.). Despite being genetically identical, these cells have differentiated and thereby organized themselves in different physiological and morphological structures (e.g., organs) that together make up the individual.

The use of indwelling devices in current therapeutic practice is associated with hospital-acquired bloodstream and deep tissue infections ( 1 ). Transplantation medical procedures, immunosuppression, and prolonged intensive care unit stays have also increased the prevalence of nosocomial infections. Device-associated infections are commonly associated with the ability of bacteria and fungi to form biofilms, which are defined as communities of sessile organisms irreversibly associated with a surface, encased within a polysaccharide-rich extracellular matrix, and exhibiting enhanced resistance to antimicrobial drugs ( 2 – 5 ). Forming a biofilm provides the microbes protection from host immunity, environmental stresses due to contaminants, and nutritional depletion or imbalances, while being dangerous to human health due to biofilms’ inherent robustness and elevated resistance.

Aspergillus fumigatus is the most important airborne fungal pathogen in the world. The conidia are inhaled by the entire population and cause a wide range of diseases from simple rhinitis to fatal invasive aspergillosis (IA) in immunocompromised patients ( 1 ). The number of A. fumigatus chronic infections is constantly increasing in immunocompetent patients suffering from respiratory problems such as chronic obstructive pulmonary disease (22%), asthma (1 to 5%), and cystic fibrosis (5 to 10%), along with 15% allergic bronchopulmonary aspergillosis. Aspergillus is also the cause of lung and sinus aspergilloma and serious fungal keratitis infections ( 2 , 3 ).

Microorganisms in the natural environment typically live on or in close association with surfaces and predominantly exist as biofilms, surface attached microbial communities composed of cells and extracellular matrix ( 1 – 4 ). The exact compositions of biofilm matrices differ greatly between different microorganisms and growth conditions under which biofilms are formed but generally consist of exopolysaccharides, proteins, and nucleic acids. Proteinaceous components include cell surface adhesins, protein subunits of flagella and pili, secreted extracellular proteins, and proteins of outer membrane vesicles.

The Enterobacteriaceae bacterial family includes a variety of intestinal symbionts as well as notable pathogens such as Salmonella enterica, Serratia marcescens, Klebsiella, and Yersinia pestis ( 1 ). Also included among the Enterobacteriaceae family is the most well-documented bacterial species on Earth, E. coli. E. coli is a fascinatingly diverse bug, featuring a cadre of different strains that have adapted to diverse environmental conditions and lifestyles. While the typical E. coli genome contains roughly 4,800 genes, only approximately 1,700 are shared by every E. coli strain ( 2 ). In total, there are over 15,000 genes that make up the E. coli pangenome ( 2 , 3 ). The genomic plasticity of various E. coli isolates provides E. coli the ability to proliferate and survive in an array of environments ( 4 , 5 ).

Historically, microbial organisms have been grown in pure liquid cultures as free-floating “planktonic” cells, promoting the general theory of the unicellular lifestyle. However, in the late 1970s, Costerton et al. ( 1 ) demonstrated that groups of bacteria were embedded in a highly hydrated polysaccharide matrix that mediated adhesion to solid aquatic surfaces. Several years later, the same research team called these cellular communities “biofilms,” defined as a functionally heterogeneous aggregate of microcolonies or single cells encased in a matrix of self-produced extracellular polymeric molecules that could adhere either to organic, abiotic surfaces or to each other ( 2 ). Microbial biofilms can develop into highly organized structures containing channels in which water, nutrients, and metabolic waste can be transported. Adhesion to substrates or surfaces induces expression of a large number of genes, while cell aggregates in different regions in a biofilm exhibit different gene expression profiles that regulate biofilm development and maturation processes ( 3 , 4 ). A large amount of research since the 1980s has brought to light the theory that most, if not all, bacteria and fungi can form biofilms as a survival mechanism in hostile environments, providing protection from biotic and abiotic stresses ( 5 – 8 ). Prime candidates for cell attachment and biofilm growth are surfaces exposed to or containing moisture and some nutrients. Natural or man-made substrates for cell attachment and biofilm growth include river stones, oil and gas installations, ship hulls, water pipes, food-processing surfaces, contaminated surgical instruments, indwelling medical devices, human teeth, and infected wounds ( 9 – 11 ). In this chapter, we will present an overview of the life cycle of biofilms and their diversity, detection methods for biofilm development, and host immune responses to pathogens. We will then focus on current concepts in bacterial and fungal biofilm immune evasion mechanisms.

The rapid development of new sequencing technologies and the use of metagenomics revealed the great diversity of microbial life and enabled the emergence of a new perspective on population dynamics. Moreover, it has highlighted the central role of social interactions in ecological and evolutionary processes. Microbes living in multispecies communities are prevalent in nature and have been shown to extensively cooperate and compete. Both intra- and interspecies interactions are instrumental in major geochemical cycles and are important in human health and homeostasis (e.g., the human microbiome has been associated with several diseases) and in industrial and clinical settings ( 2 ). Few studies have addressed the role of individual species within mixed communities ( 3 ), and they generally focus on cooperative interactions and increased benefits of community life ( 4 , 5 ). However, recent work pointed out that most interactions are competitive rather than cooperative, suggesting that adaptation is more likely achieved by competitive success ( 6 ). A further degree of complexity in understanding multispecies interactions and dynamics is brought by increasing evidence suggesting that phenomena occurring in complex communities cannot be predicted by the observation of single-species communities ( 7 ).

Signaling pathways are required for bacterial biofilm formation and antimicrobial resistance. Among them, quorum sensing (QS) and c-di-GMP signaling are the best characterized. QS is a widely distributed intercellular signaling mechanism by which microorganisms regulate gene expression in response to small diffusible signaling molecules ( 1 ). Bacteria have developed oligopeptides, N-acyl homoserine lactones (HSLs), and autoinducer-2 as signal molecules ( 1 ). When the QS signal molecules reach a local threshold concentration, they can interact with specific receptors and impact the expression of hundreds of genes. Many of the QS-regulated genes (motility, biosurfacant synthesis, EPS synthesis) are required for the biofilm formation and antibiotic resistance of various bacterial species ( 2 ).