The physical characteristics of a biofilm allow for the development of antibiotic resistance through horizontal gene transfer events and inhibit the diffusion of various biocides into the center of the mature biofilms. This makes biofilms significantly more difficult to eradicate than planktonic organisms, which float freely within culture medium and have been most commonly studied in laboratory settings over the past century. In addition to presenting a physical barrier to disinfectants, microbes in a biofilm exhibit dramatic differences in gene expression
compared to their planktonic counterparts⁸ resulting in distinct phenotypes.

Biofilms form when planktonic bacteria are able to attach to a surface.⁹ Attached bacteria then produce an extracellular matrix typically consisting of polysaccharides, proteins, and DNA,¹⁰ which enables the bacteria to adhere irreversibly to the surface (Figure 67.1).⁹,¹¹,¹² These extracellular polysaccharides (EPS) are extensively hydrated, can impede diffusion of antimicrobials, and act as a physical barrier to host innate immune cells allowing the bacteria to cause persistent infections.⁹,¹³,¹⁴ The physiological generation of EPS is a critical feature of a biofilm and confers numerous advantages to the microorganisms residing in the biofilm community (Figure 67.2). One major benefit that the EPS layer provides to the community is resistance to desiccation because water stress is an environmental pressure that organisms frequently experience. The active production of EPS molecules makes biofilm bacteria significantly more resilient to desiccation than their planktonic counterparts (see Figure 67.2).¹⁶

Diffusion of reactive oxidants such as hydrogen peroxide produced by host immune cells, or similar molecules used in disinfection treatments, can be limited and slowed such that the oxidants are inactivated prior to penetrating the outer layer of the biofilm.¹⁷,¹⁸ Antimicrobials administered for the treatment of infections can also be
inactivated before they effectively penetrate a biofilm.¹⁹ This diffusion barrier can create an environment that promotes the development of random mutations with the potential of conferring greater antimicrobial resistance to some of the bacteria. Constant low-level exposure to antimicrobial agents in particular can promote increased resistance development within a biofilm. In addition, the physical characteristics and three-dimensional structure of biofilms prevent diffusion of various biocides, and this mode of growth is typically more resistant to biocides than planktonic cells.¹⁴,²⁰ The length of time that biofilms are permitted to grow and generate a more complex EPS enhances this resistance, as mature biofilms show increased tolerance to biocides such as glutaraldehyde.²¹ Biofilm-associated microbes experience environmental heterogeneity, meaning that different microniches within the biofilm have different concentrations of nutrients.¹⁵,²² At the center of a biofilm, the oxygen and other nutrient concentrations can be 30 times less than the concentrations at the surface.²³,²⁴ The conditions at the center of the biofilms can even be completely anaerobic in an otherwise aerobic environment.²⁴ This change in nutrient availability appears to cause cells near the center of the biofilm to exist in a slow-growing state.⁹,²⁵ Bacteria growing in this nutrient-restricted state are much less susceptible to antimicrobial agents because the mechanisms of many antimicrobials target the synthesis of nucleic acids and proteins in growing and dividing cells and minimally affect dormant cells that are not actively and rapidly producing these molecules.²⁶ The differences in metabolic states of the cells within a biofilm ensure that some bacteria will survive exposure to antimicrobials⁹ or other harsh environmental challenging conditions. Channels for water and nutrients are present throughout the entire structure of the biofilm, which help the microbes near the center receive necessary nutrients. Certain populations of planktonic bacteria, known as “swimmers,” can tunnel into the biofilm, creating another distinct system of pores that help supply nutrients and oxygen to nutrientdeficient areas.²⁴,²⁷

The frequency of mutation in biofilms is significantly higher than in planktonic organisms, leading to the development of antibiotic resistance and other genetic changes more rapidly in biofilms than in planktonic cells.¹³ Biofilmassociated bacteria exhibit what is termed a “hypermutable phenotype,” creating mutations in the DNA repair pathways leading to the enhanced expression of numerous mutant phenotypes within the community.¹³ This seems to be an evolutionary strategy for microbes to survive a variety of unique challenges by rapidly creating a diversity in their genotypes and phenotypes. Biofilm-associated bacteria are more resistant to various environmental stresses such
as desiccation, nutrient limitation, toxin actions, oxidative stresses, and heat shock through these mutations, and this hypermutable state can lead to increased antimicrobial resistance opportunities.¹³,²⁶,²⁸,²⁹,³⁰,³¹

All biofilms found in the natural environment are polymicrobial (ie, composed of multiple microbial genera and species) because this provides numerous advantages to the microbes.³² Multiple species living together exhibit an increased rate of horizontal gene transfer (movement of genes from one bacterium to another), and this, in conjunction with the hypermutable phenotype, allows bacteria living within the biofilm to more efficiently deal with environmental stresses than any single species in planktonic form.³³ Certain types of bacteria are able to promote the growth of other bacterial species when in proximity, such as in coculture. For example, the presence of Fusobacterium nucleatum in a culture allows Porphyromonas gingivalis to grow under microaerophilic conditions that would otherwise be toxic to monocultures of P gingivalis.³⁴ The process of symbiotic nitrification is another example of metabolite cooperation in biofilms. In this phenomenon, specific bacteria from the genus Nitrospira oxidize ammonium to nitrite, which then is oxidized by other members of this genus to nitrate.³⁵ These observations provide clear evidence for the reasons why bacteria aggregate and form polymicrobial communities that exhibit social interactions such as competition, cooperation, and synergism.

Just like in higher organisms, communication among individual cells is a critical aspect of microbial life in a complex community. All the processes described earlier rely on the exchange of information between microorganisms within a biofilm. Bacteria in biofilms communicate with one another via various signaling molecules in a process initially termed quorum sensing. Waters and Bassler³⁶ comprehensively review the quorum sensing concept. Disruption of quorum sensing pathways may represent a powerful alternative to traditional antimicrobial treatments. For example, a recent study on aquatic, biofilm-forming species of Vibrio demonstrated that intermediates of polyhydroxy butyrates effectively inhibit biofilm formation in a strain-specific fashion by disrupting communication between cells.³⁷ Such unique strategies may lead to novel mechanisms of inhibiting and killing microbes.

Biofilms are a significant issue in any system that relies on the movement of water or other nutrient-laden liquids through a pipeline, tube, or planar structure. The shear forces in these systems create environments that are conducive toward biofilm development and growth.³¹ Biofilms and their associated structures can act as a reservoir for pathogens in various medical devices that require water or other liquids, such as washer-disinfectors, and may result in relatively mild consequences such as water discoloration or, in catastrophic cases, can result in large outbreaks of infectious disease.³⁸,³⁹,⁴⁰ Biofilms present a substantial problem in the water reservoirs and associated liquid delivery systems in surgical and medical equipment such as those used in eye surgery facilities.³⁸ One particularly notable pathogen known to be capable of biofilm formation in air conditioner and domestic water supply cooling towers is Legionella pneumophila.⁴⁰ This particular bacterium can cause serious outbreaks of pneumonia infections or a condition known as legionellosis or Legionnaires’ disease. Biofilms of Legionella within contaminated cooling towers are one of the earliest reported and major sources of infection in such outbreaks.⁴⁰ These outbreaks are particularly severe in immunocompromised populations.⁴¹ New methods to detect bacteria growing in these systems include DNA amplification and next generation DNA sequencing, which have brought biofilms to the attention of researchers particularly in the field of water contamination.⁴⁰,⁴²

Up to 60% of clinical infections treated with antibiotics are estimated to have involved biofilms.⁸ Biofilm-associated infections can slowly lead to chronic tissue damage caused by the continual production or overproduction of proinflammatory molecules by frustrated host immune cells such as neutrophils (see Figure 67.1). Inflammationinduced tissue damage occurs in this fashion in conditions such as cystic fibrosis,⁴³ periodontal disease,⁴⁴ and a number of other chronic infectious diseases. In vivo experiments have shown neutrophil migration within biofilms. In some cases, the EPS matrix incorporates damaged and/or dead host immune cells, likely strengthening the barrier to other host immune cells (Figure 67.3).³ Quiescent biofilms act as nidi of future infection in a host; however, certain toxins (such as endotoxin; see chapter 66) and other bacterial products can be released periodically from mature biofilms that can result in immune responses and inflammation at near and distant locations. Biofilms growing in an environment such as the water reservoirs of medical washing-disinfection equipment can cause postsurgical aseptic inflammation in patients through the release of heat-resistant endotoxins. Such aseptic endotoxin-mediated inflammation events have been implicated in cases of diffuse lamellar keratitis (DLK) in patients undergoing corneal LASIK surgery³⁸ as well as in outbreaks of toxic anterior segment syndrome (TASS).⁴⁵ A combination of mechanical abrasion and biocide application was required to dramatically reduce the incidence of DLK³⁸ and TASS caused by these biofilmcolonized surfaces.⁴⁵

Biofilms form on a wide variety of medical devices, often resulting in chronic infections that are difficult to treat. Infections surrounding medical implants are a serious therapeutic challenge. The economic effect of such biofilm-associated infections is staggering, with estimates of up to 40 billion US dollars annually in the United States alone.⁴⁶ The impact of biofilms in implant infections is reviewed by Costerton et al in 2005.⁴⁷

Biofilms are associated with infections on implanted artificial heart valves, orthopedic joint prostheses, catheters, stents, dental implants, contact lenses, and numerous other artificial substrates placed in or adjacent to host tissue.⁴⁸,⁴⁹,⁵⁰,⁵¹,⁵² These implant-associated biofilms often result in chronic infections that require multiple treatments and, in severe cases, result in the removal of the afflicted implant.⁹,⁵⁰,⁵³

Suture material is at high risk of biofilm development from skin-based microorganisms, and this is a significant cause of surgical site infection (SSI)⁵³ and surgical wound dehiscence. These types of infections are a heavy burden on the medical industry and can account for substantial proportions of the total hospital budget.⁵⁴ In a large portion of these infections (up to 30%), clinicians are unable to culture any viable species from the wounds and this may be due to the biofilm mode of growth itself⁵⁵ or the involvement of microorganisms that are challenging to culture such as mycobacteria.⁵⁶ Similarly, SSIs may
involve bacteria that are slow to grow or are not typically scrutinized by medical diagnostic laboratories. Some researchers hypothesize that the slow-growing phenotype makes it more challenging to identify clinically relevant biofilm-associated bacteria because traditional clinical diagnostic approaches do not allow enough time for visible growth on culture plates.⁵⁵ Biofilms composed of nontuberculous mycobacteria have also been implicated as causative agents of postsurgical infections, and these are very difficult to treat because many typical antibiotics or antimycobacterial drugs do not eradicate the bacteria.⁵⁶

Using confocal laser scanning microscopy (CLSM) and fluorescent in situ hybridization (FISH) on samples from 15 patients with SSIs associated with biofilm growth on permanent, multifilament sutures, investigators determined that the biofilms on the sutures were all polymicrobial in nature.⁵³ These biofilms were composed of several microbial taxa, with Staphylococcus aureus (and in particular methicillin-resistant S aureus) being the most prevalent. An earlier case study demonstrated that a polymicrobial biofilm attached to sutures caused a persistent surgical wound infection in a patient.⁵⁷ In these particular cases, which are likely to be more common than the literature suggests, treatments using antibiotics were not successful, and the sutures needed to be removed to eliminate the chronic infections.⁵³,⁵⁷ These studies illustrate several complicating issues with biofilm-associated chronic infections. Fortunately, removal of sutures is relatively simple. In other types of biofilm-associated infection, this is not a trivial task (eg, hip and knee joint prostheses or artificial heart valves). As described earlier, treatments with antimicrobials often fail due to lack of diffusion of the antimicrobial into biofilm, the increased incidence of gene transfer (that can convey resistance) within the biofilm as well as the presence of “persister” or metabolically dormant microbial cells in biofilms. The result is increased time spent in health care facilities, making patients even more susceptible to subsequent infections and most particularly when the individual is immunocompromised.

The generation of multiple, equivalent, and uniform biofilms for high-throughput in vitro studies is a significant challenge that microbiologists face. The major issue is reproducibility. There remains controversy among research groups regarding which method of biofilm generation in the laboratory provides the most accurate and realistic representation of biofilms and therefore several different techniques have become widely employed by groups around the globe. The use of a variety of methods creates challenges when reproducing previously published work. Therefore, standardization of experimental design, and perhaps even methods, is crucial in this important field of microbiological research. The following section briefly describes some of the common methods for biofilm generation in the laboratory, highlighting the advantages and disadvantages of each technique.