Biofilm: Basic Principles,Pathophysiology,and Implications for Clinicians

Abstract

Background:

Biofilm is ubiquitous throughout nature including bacteria, fungi, protozoa-associated bacteriophages, and viruses. Whereas it is adaptive for certain organisms in a variety of environments, biofilm is important in understanding and treating clinically relevant infections, especially those involving temporarily or durably implanted devices.

Methods:

Review of pertinent English-language literature.

Results:

Important advances have been made in understanding biofilm structure and function that elucidate key events in biofilm-based infectious processes. Wounds, oral cavity, urinary tract, gastrointestinal tract, and device-associated biofilm-based infections dominate clinically relevant infections. Criteria have been articulated to detect and diagnose biofilm-associated infection but there are hurdles to overcome to treat effectively such infection. Native biofilm resistance mechanisms as well as incompletely effective human immune system responses impede successful infection resolution. Biofilm-appropriate education appears under-represented in standardized surgical education curriculum.

Conclusion:

Several potential methods of enabling primary prevention as well as treatment of biofilm-associated infection are on the near horizon. There is an opportunity to enhance surgical education regarding biofilm prevention, diagnosis, and therapy.

Biofilm is a naturally occurring accumulation of micro-organisms (bacteria, fungi, protozoa, and associated bacteriophages and other viruses) of either a single or multiple species, embedded in an extracellular polymeric matrix and adherent to a biologic or non-biologic surface. Bacteria in a biofilm differ profoundly from their planktonic (free-living) counterparts by growing in an enclosed, nutrient-sufficient ecosystem as a community of sessile organisms that exhibit an altered phenotype with respect to growth rate and gene transcription [1,2]. Such organized communities play crucial roles in human as well as non-human infections, and are relevant to all physicians who address patients with sepsis.

First discovered in dental plaque (including Van Leeuwenhoek's early description of “animalcules” in his own dental plaque), the extent of the role of biofilm in clinical infections, and especially in persistent and difficult-to-treat infections, has become clear [3]. Biofilm accounts for 65%–80% of microbial infections in human beings, as estimated by the U.S. Centers for Disease Control and Prevention (CDC) [4] and the National Institutes of Health (NIH) [5]. Accordingly, biofilm has clinically relevant implications for and a deleterious effect in patients with surgical infections. The present review describes the structure and function of biofilm and its components, explores biofilm in relation to clinical infections, delineates how biofilm imparts microbial resistance to antimicrobial agents, and highlights approaches to addressing biofilm-based infections, in addition to underscoring the need for increased teaching about biofilm in surgical education.

Structure and Function of Biofilm

Surface attachment and matrix formation

In general, the majority of clinically relevant surfaces that are ultimately a target site for bacterial attachment begin in a sterile state, exhibiting only naturally occurring macromolecules. For example, the human tissue reaction to biomedical devices includes the deposition of proteins or glycoproteins (plasma fibronectin, fibrinogen, albumin, and immunoglobulins) on surfaces or in cavities where they would not normally occur or would not occur at the concentrations that can be assayed. Initially, attachment of non-human cells to a biologic surface is reversible when bacteria remain in their planktonic form [6]. The attachment is either non-specific or involves specific receptor-to-ligand adhesion mechanisms. As bacteria adherent to the surface multiply, attachment strengthens and the bacteria begin to differentiate; bacteria attached firmly to the surface are termed sessile [7]. Within minutes of attachment, adherent cells up-regulate the secretion of a variety of inter-cellular signaling molecules that orchestrate community-wide phenotypic responses through a process termed “quorum sensing” [8]. Further maturation occurs through the consumption of soluble nutrients, up-regulation of virulence factors, secretion of extra-cellular polymers, and recruitment of other bacterial species or of mammalian cells (e.g., platelets). This organized process is both elegant and complex and describes the process of biofilm genesis.

Collections of bacteria attached to a surface settle into aqueous systems, encased in a “glycocalyx” matrix that is composed principally of polysaccharides. The exact composition of the matrix varies according to the microbial composition of the bacterial collection and the host environment, but generally consists of polysaccharides, proteins, glycolipids, and bacterial deoxyribonucleic acid (DNA) [9]. Host-derived components are particularly important in clinically relevant biofilm, supporting the elaboration of an extracellular polymeric matrix (e.g., salivary proteins in dental biofilm, fibronectin on medical implants, and fibrin and platelets entwined in vegetations of endocarditis) [10]. Biofilm incorporates both single cells and microcolonies within its matrix [11]. Structurally heterogeneous building blocks of microbial cells develop into matrix-enclosed “towers” or “mushrooms,” with fabricated open water channels, inter-digitated between microcolonies, that serve as links. Living, fully hydrated biofilms are composed of approximately 15% cells and 85% matrix material by volume [12]. Phenotypically distinct populations of bacteria with metabolically active and inactive zones develop as a function of the prevailing gradients of nutrients, oxygen, or electron acceptors [13]. This architecture has been demonstrated in both Pseudomonas aeruginosa [14] and Staphylococcus aureus [15] biofilms, forming an outer oxygen-penetrated region and an inner nutrient-dense region. Although the foregoing examples may appear to differentiate biofilms into readily categorizable types, the reader should be aware that biofilm structure and organization are unique and perhaps best characterized in a fashion resembling that of snowflakes, which are similar but not identical to one another.

Dynamic process

Biofilm may form in environments with both high (rapidly flowing) and low (slowly flowing) fluid shear. Both smooth and rough surfaces are subject to bacterial adhesion in the laboratory and clinical environment [16]. Recent data derived from nanoscale technology-driven laboratory experimentation suggest that surface pore size and dynamics may influence protein deposition and bacterial adhesion [17]. In one study of Escherichia coli and S. aureus on nanostructured titania surfaces, increases in pore size above 20 nm decreased protein deposition, bacterial adhesion, and biofilm formation [17]. Biofilm structure is highly viscoelastic and behaves in a rubbery manner. Accordingly, tensile strength and resistance to mechanical breakage may be influenced by the stresses imposed by the environment in which a biofilm forms [18].

Although bacterial attachment occurs within minutes, there is significant variability in the rate and strength of attachment of different bacteria, and strongly attached microcolonies generally require 2–4 h for formation. Enhanced tolerance to antibiotics, antiseptic agents, and disinfectants may be noted within 6–12 h of attachment, at least in the laboratory environment, and attachment alone may enable some degree of resistance to antibacterial agents. Biofilm microcolony maturation is complete as early as 2–4 d after initial bacterial attachment, depending on the bacterial species forming the microcolony and the growth conditions for the colony [18]. In addition, with a laboratory-based flow-cell system of analysis, some mature biofilms, such as that of P. aeruginosa, recovers rapidly from mechanical disruption and reforms within 24 h [3]. Fully mature biofilms shed planktonic cells, microcolonies, and fragments of biofilm continuously into their surrounding environments, such as the human vascular tree, in which fragments may lodge in capillary beds and play a part in the hematogenous spread of infection [19].

Quorum sensing

The bacteria that form a biofilm regulate their cooperative activities and physiologic processes through a mechanism of communication termed “quorum sensing,” by releasing, sensing, and responding to small, diffusible signal molecules. This communication was first described in regard to the marine bioluminescent bacterium Vibrio fischeri with respect to their unique method of regulating group light production [20]. Research with this organism (also named Photobacterium fischeri) has revealed a density-dependent requirement for the production and accumulation of a minimum threshold concentration of “autoinducers,” the small, diffusible signal molecules required for quorum sensing functions [21]. Increased cell density favors chemical methods of signaling for communicating with the cellular community and facilitating social interactions [22].

Autoinducers diffuse through membranes, with their concentrations inside cells approximating their concentrations in the surrounding environment; at critical concentrations they bind to and activate receptors for gene expression [23]. Many classes of autoinductive molecules have been discovered, but most prevalent are N-acylhomoserine lactones (AHLs) from gram-negative bacteria, oligopeptides from gram-positive bacteria, and autoinducer-2 (AI-2) from both gram-negative and gram-positive bacteria [24]. The AHLs and oligopeptides are designed for intra-species signaling; remarkably, production of AI-2s has been detected in a large number of diverse bacteria, implying interspecies communication and social interaction among bacteria [25].

Phenotypic differentiation

An individual bacterium within a species is often thought of as a clone of its brethren. This conceptual framework does not apply to biofilm. As a strategy to help individual cells within a biofilm withstand diverse environmental conditions, phenotypic differentiation produces functionally specialized cells. These cells diversify with targeted purposes within the biofilm community, including the expression of different receptor-ligands for surface adhesion, production of extracellular matrix polymers, metabolic regulation, and maintenance of the integrity or dissolution of the architecture of the biofilm [26]. The expression of different adhesins, their receptors, and exopolymeric components by individual cell types within a biofilm community contributes to the overall development of the biofilm by producing and maintaining biodiversity and ecosystem microbial homeostasis [27]. This differentiation produces both “cooperative” phenotypes that divide labor, form metabolic food chains, and create synergistic nutritional systems, and “competitive” phenotypes that produce peptide bacteriocins and cannibalistic toxins, and ultimately produce group-based genetic competence within a particular biofilm.

Cell-to-cell signaling is required for the differentiation of the common bacterium P. aeruginosa into complex multicellular structures, implying that this phenomenon is a required element for biofilm genesis [28]. In addition, secondary metabolites, such as antibiotics and pigments, may signal the initiation or inhibition of biofilm formation; in particular, biofilm inhibition may be driven by other organisms already resident in a target habitat [29].

Biofilm-associated Infection

A multitude of pathogens have been noted to form biofilms. This adaptive bacterial behavior ubiquitously affects human life in the natural, medical, and industrial settings. Biofilm-associated infections are difficult to diagnose because typical culture techniques (blood, urine, sputum, and abscess-cavity sampling) may not identify accurately a biofilm-associated infection [30]. Biofilm-based infections have been identified in virtually all tissue and organ systems of the human body, including oropharyngeal soft tissues, teeth and dental implants, the middle ear, eye, endobronchial and pulmonary parenchymal tissues, cardiac valves, the gastrointestinal tract, and the urogenital tract. Biofilm-based infections also form readily on and around a foreign body, including urinary tract prostheses, peritoneal dialysis catheters, venous access catheters, and other devices that indwell for long periods, tunneled and non-tunneled catheters for hemodialysis, implanted cardiac devices and prostheses (e.g., pacemakers, cardiac valves, ventricular assist devices), vascular endoprostheses (synthetic vascular grafts and stents), orthopedic internal-fixation devices, sutures, endotracheal tubes (oral, nasal, and trans-tracheal), and ventilator tubing [31]. Clearly, biofilm-based infections are widespread in diverse patient populations both in and out of the ICU. The reader should note that the microbiology laboratory uses planktonic bacteria for identifying pathogens, and as a result, the data derived from the laboratory may have little relevance to biofilm-associated infection.

Wounds

Chronic wounds represent common soft tissue entities that are managed frequently by surgeons; examples of such entities are diabetic foot ulcers, venous ulcers of the lower extremities, pressure ulcers, and open abdominal wounds. Any surgery-related wound is subject to chronic infection, and failure of wound healing has been attributed to an imbalance of destructive and healing processes that often hinge on microbial colonization and infection. Unsurprisingly, biofilms have been shown to be among the factors responsible for chronic wound infection [32]. Despite common culture results showing aerobic gram-positive cocci on the surface of wounds, it is now known that multiple organisms can reside within a biofilm in deeper aspects of wounds. Pseudomonas aeruginosa embedded in the deep layers of a wound biofilm can be missed entirely with the use of a standard swab culture technique [33]. Accordingly, the role of antimicrobial agents in the management of chronic wounds is less clear than in acute wounds because a biofilm may prevent antibiotic agents from reaching their target. Choice of an antibiotic on the basis of a wound swab culture, coverage may yield woefully inadequate antimicrobial coverage. Ultimately, regular debridement with deep tissue culture, in addition to targeted antimicrobial therapy, remains the most efficient tactic for addressing biofilms in chronic wounds [34].

Oral cavity and respiratory tract

Biofilm formation in the oral cavity is well described. There is substantial evidence linking oral disease and systemic conditions including cardiovascular disease, diabetes mellitus, preterm or low birth weights, and rheumatoid arthritis with the pathogens in oral biofilm [35]. In addition, oral biofilm plays a considerable role in lower respiratory tract infections. Oral biofilms of Pseudomonas aeruginosa are strongly implicated in the recurrent pneumonia and tracheobronchitis of patients with cystic fibrosis [36].

Endotracheal tube-related biofilm formation impedes the successful treatment of ventilator-associated pneumonia (VAP). Oral decontamination, such as with the use of chlorhexidine-based oral hygiene, in conjunction with use of the VAP prevention bundle, is effective in reducing the incidence of VAP and the duration of mechanical ventilation in patients in the surgical ICU [37]. However, the role of such decontamination in preventing or disrupting biofilm formation and biofilm-associated pneumonia remains unclear. One study found that oral decolonization with chlorhexidine reduced the absolute number of S. aureus organisms but not other respiratory pathogens [38]. Silver coating of endotracheal tubes has been shown to reduce significantly the incidence of VAP, as well as to delay the time to its occurrence, but may be too costly for routine application [39].

Gastrointestinal tract

Bacterial biofilm formation is related to infection of the gastrointestinal (GI) tract and inflammatory conditions of the tract. Helicobacter pylori can form biofilm despite residing in the harsh acidic environment of the stomach [40]. Difficult-to-treat cases of H. pylori infection have been associated with biofilm formation and resistance to therapy; pretreatment with N-acetylcysteine (before triple therapy) improves treatment results in refractory cases of refractory H. pylori infection and is believed to be related in part to biofilm disruption [41]. Salmonella species have been shown to cause biofilm-related cholecystitis [42]. Colon inflammation has also been connected to biofilm, and may provide an explanation for certain “auto-immune” inflammatory conditions [43]. The vast range and abundance of gastrointestinal flora leads to a multitude of possibilities by which biofilm may affect susceptible patients. Imbalances between resident species of the bacterial flora that follow an empiric or therapeutic course of antimicrobial agents may be all that is needed to render a patient susceptible to a biofilm-related infection.

Urinary tract

Urinary catheters are readily subject to biofilm formation and biofilm is strongly related to persistent or recurrent urinary tract infections (UTIs) in patients with chronic indwelling catheters [44]. Escherichia coli is the most commonly implicated biofilm-forming pathogen associated with genitourinary infection [45]. Biofilms may persist on bladder epithelium despite the removal of indwelling or temporary catheters, may resist penetration by a host of antibiotics, and may explain the persistent nature of UTIs despite presumed adequate antimicrobial therapy [46].

Device associated

Among the most important and clinically relevant examples of biofilm-associated infections are those occurring on indwelling devices. Central venous catheters are responsible for the highest proportion of bloodstream infections in hospitalized patients [47]. A staggering 28% of central venous catheter-related infections in ICU patients are associated with sepsis [48]. After the insertion of a central venous catheter, the intravascular portion of the device is coated rapidly by a thrombin layer that is rich in host-derived proteins. This sheath promotes surface adherence of both blood-borne microbes and those introduced iatrogenically during catheter insertion [49]. Even after catheter removal, the biofilm remains in the tract left by the device, rendering the patient susceptible to chronic seeding by biofilm bacteria if the cather site is reaccessed, and may increase the risk of hematogenous bacterial spread as sessile biofilm bacteria are released in their planktonic state.

Diagnosis or detection

Robert Koch is credited with developing criteria to establish a causal relationship between a microbial pathogen and human disease. Many of Koch's postulates have been deemed irrelevant in specific disease states. Even Koch himself abandoned the exclusivity of the postulates when he discovered asymptomatic carriers of cholera. Relatedly, biofilm-forming bacteria and related infections also violate many of these postulates.

Hall-Stoodley and others [30] have recently published proposed diagnostic guidelines that may serve as criteria for determining a biofilm-associated infection. The proposed criteria include the following elements:

1. Microbiologic evidence of a localized chronic or foreign body-associated infection.

2. Microscopic evidence of aggregated microorganisms.

3. A medical history of a biofilm-predisposing condition (e.g., implanted medical device, cystic fibrosis, infective endocarditis, chronic otitis media).

4. Recurrent or recrudescent infection (particularly if the same organism is responsible at multiple time points).

5. A documented history of antibiotic failure or persistent infection despite appropriate selection of an antibiotic agent and duration of therapy.

6. Evidence of local or systemic signs and symptoms of infection that resolve with antibiotic therapy, only to recur after therapy is terminated.

7. Evidence of a specific immune response to an identified microorganism, such as antibodies to specific pathogens (e.g., to alginate or other P. aeruginosa antigens in cystic fibrosis patients).

We caution that more research is needed to validate these criteria, including better methods for ascertaining the presence of a biofilm in a suspected infection. Nonetheless, the elaborated criteria make sense physiologically and appear to match known elements and aspects of biofilm-based infection. However, the guidelines do not inform the practitioner about how to address a biofilm-based infection.

Difficulty in Treating Biofilm-Associated Infections

It is problematic for the treating physician that microorganisms within a biofilm tend to be far more difficult than ordinary to eradicate with antimicrobial agents, and to be particularly difficult for the host immune system to recognize and respond to in an appropriate manner. Although mechanisms that underpin antimicrobial resistance have been elaborated in great detail, new mechanisms that are unique to biofilm continue to be discovered. It is to be hoped that understanding those mechanisms will improve the ability of novel tactics to enhance antimicrobial efficacy in addressing biofilm-based infections.

Resistance of biofilm to antimicrobial agents

Multiple mechanisms for biofilm-related resistance have been identified or hypothesized. Three main mechanisms have been elaborated, but are incomplete with regard to encompassing all of the observed resistance patterns currently ascribed to biofilms. These three general mechanisms are as follow:

1. Slow or incomplete penetration of antimicrobial agents through the biofilm matrix [50]. Electrostatic repulsion as well as the creation of a protected environment that is hydrophobic (despite the embedded water channels) helps to prevent polar and charged antibiotics from reaching the inner regions of a biofilm community. Although this appears to be a primary mechanism, it does not account fully for antibiotic resistance, as in the case of daptomycin, which has been shown to be able to penetrate S. epidermidis biofilm rapidly [51].

2. The physiologic response of organisms to the heterogeneous environment of biofilms and the phenotypically differentiatial effect on susceptibility to antimicrobial therapy [52]. Biofilms increase the opportunity for gene transfer between bacteria [53]. This is important because bacteria resistant to antimicrobial agents or chemical biocides can transfer the genes for resistance to neighboring, receptive bacteria. Such promiscuous gene transfer can convert a previously avirulent commensal organism into a highly virulent pathogen [54].

3. The development of subpopulations of dormant and metabolically less active “persister” bacteria, reducing the efficacy of bacteriostatic antimicrobial agents [55]. Persister cell populations, which are often non-dividing and multi-drug-resistant, are genetically identical to the rest of the population of a bacterial pathogen, but have been shown to express antitoxin systems that effectively block the target of a substance toxic to these populations, such as the binding site for an antimicrobial drug [56].

Regardless of the mechanism, microorganisms growing as biofilms are significantly less susceptible to antibiotics [57]. Alarmingly, exposure to antibiotics has even been shown to induce biofilm formation as a specific and defensive reaction by specific bacteria [58].

Immune-system response

The inflammation and tissue destruction of chronic infections is well chronicled. Biofilms have gotten increasing attention for promoting and sustaining chronic inflammation, illuminating dynamic and reciprocating interactions between the organisms in biofilms and human immune effector cells. It had been believed that immune responses were triggered primarily by antigens on the outer surface of a biofilm, with the matrix serving as a mechanical barrier to immune cells and antibodies and other proteins [59]. Studies of the interactions between biofilms and immune cells have discredited this intuitively attractive concept, as leukocytes appear to penetrate easily the biofilm of S. aureus [60]. It is unclear whether this leukocyte capability is limited to S. aureus biofilm or is a general capability of the organisms that create biofilms; it remains unclear whether all lines of white blood cells have this functionality as well, or whether it is neutrophil-specific. Furthermore, phagocytosis alone is ineffective against biofilm-residing bacteria because bacteria resident in biofilms evidence a detachment response that releases a cloud of bacteria from the biofilm to envelop attracted and homing neutrophils and obscure their targets [61]. Paradoxically, the release of neutrophil-generated polymers enhances the density and integrity of an established biofilm matrix [62]. These examples illustrate the manipulation of immune responses by the bacteria in biofilms and the effect of immune cells on the formation of biofilm, as well as the effect of biofilm on bacterial virulence.

Future Directions

The most challenging aspect of controlling biofilm-associated infections is the prompt and accurate detection of the establishment of a biofilm and cataloging of the bacterial species embedded in it. Given that many bacteria employ quorum-sensing mechanisms in controlling virulence, pathogenicity, and biofilm formation, quorum sensing constitutes a new target for the development of antibacterial agents, with widespread potential application. Strategies for disrupting or directing quorum sensing have been proposed, including inhibition of signal generation, interference with signal dissemination, blocking of signal receptors, and inhibition of the endogenous signaling response system [27]. The substances known as quorum-sensing inhibitors increase the susceptibility of bacterial biofilms to existing antibiotics both in vitro and in vivo, thereby increasing the success of antibiotic therapy against biofilm-based infections [63].

Additional approaches may include the development of molecular-based diagnostic approaches (polymerase chain reaction, fluorescence in-situ hybridization, and confocal laser scanning microscopy, DNA microarray analysis, and immunohistochemical techniques) that support quantitative assessment of the bacterial load in biofilm-based infections. Imaging-based diagnostic approaches may be particularly helpful in that biofilm spatial organization is virtually impossible to examine in clinical specimens, which usually have a complicated and convoluted surface geometry. Biofilm-specific stains for gram stain-like analysis and biofilm-specific biomarkers, such as bacteria-specific antibodies, may enable more rapid diagnosis to differentiate biofilm-based infection from infection unrelated to biofilm. Additionally, designing biomedical devices to incorporate anti-biofilm defenses, such as chemical coatings to disrupt bacterial metabolism or enhance immune function, offers the promise of retarding biofilm-based infections of durable medical devices. Lastly, naturally occurring agents such as a mixture of D-amino acids manufactured by Bacillis subtilis, that can disrupt the adherence and internal structure of both P. aeruginosa and S. aureus biofilm, offer the potential to enhance the efficacy of existing antibiotic agents in biofilm-based infections [64].

Biofilm in Surgical Education

Biofilm is complex and likely to be incompletely explored in surgical education at the residency level. It is probable that biofilm-specific education is better explored at the level of fellowship in surgical critical care or acute care surgery, but specific data on how many programs address biofilm in a formal fashion is absent. It is hoped that the Surgical Council in Resident Education (SCORE) curriculum will embrace incorporating targeted biofilm education elements. At present, biofilm is not specifically mentioned in the curriculum outline, but biofilm may be found under nosocomial infections, biomaterials in hernia repair, post-operative pneumonia and VAP, vascular infections, surgical infections, vascular and peritoneal access, and mechanical circulatory support sections [65]. However, as crucial as biofilm may be in human infection, there is no single element of the SCORE curriculum element to inform the trainee about the details reviewed in this paper. Organizations such as the Surgical Infection Society may be particularly useful in helping to update and maintain the educational elements needed for enhancing resident and fellow education in surgical infections and the role of biofilm in those infections.

Conclusions

Biofilm is a common adaptive response of many if not all microorganisms in nature. Biofilm formation is complex and dynamic. The use of unique communication via quorum sensing allows elaborate communication between various microbes in a biofilm as well as between microbes in the biofilm and the host immune system. The formation of biofilm in clinical situations has the ability to create a chronic infection or persistent immune response in the host, leading to exacerbation of symptoms and destruction of host tissues in virtually every organ system. The need for better detection and diagnosis of biofilm-associated infections is apparent. Physicians should be aware of this element of infectious disease because the understanding and manipulation of biofilms will remain at the forefront of evolving treatments for many surgical infections and conditions.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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