Bacterial biofilms and healthcare-associated infections

Biofilms are survival mechanisms implemented by bacteria in order to adapt to hostile extracellular environments.¹ The National Institutes of Health report that 80% of all chronic infections are due to biofilms.² Bacterial biofilms are known to increase antimicrobial resistance and improve microbial survival, probably by evasion of phagocytosis and other innate, as well as adaptive, immune responses: In this capacity, they contribute to persistence of infection.^2,3 Biofilms are the cause of many chronic infections of the upper respiratory tract as well as native valve endocarditis, otitis media, periodontitis, and chronic prostitis.⁴ Many medical devices have been associated with biofilm growth and, in this way, can increase the incidence of healthcare-associated infections (HAIs).⁴ Current treatment plans include aggressive antibiotic treatment and surgical debridement to remove infectious agents.⁴

Biofilms are communities of microorganisms that remain adherent to solid surfaces in an aqueous environment.⁵ These microbes produce a network of extracellular carbohydrates, proteins, and nucleic acids known as extracellular polymeric matrix which acts as a protective shield for the biofilm-associated bacteria. A biofilm community creates an environment suitable for the exchange of genetic material between microbes within the biofilm architectural structure and conjugation (plasmid transfer) occurs at higher rates within biofilms than in free-floating (planktonic) species. The increased level of conjugation occurring in biofilms may be the result of protection of the microbes from shear forces brought on by fluid flow in the extracellular environment. Close cell-to-cell contact created by biofilms can enhance conjugation to efficiently spread bacterial resistance to antimicrobials via plasmid exchange.⁴

Biofilms are characterized by their heterogeneity or makeup of mixed bacterial species.⁴ Biofilms composed of a single microbial strain intrinsically form thinner biofilms, while thicker biofilms form due to one species stabilizing the other species in mixed-culture biofilms. Biofilms are capable of forming on a variety of surfaces including living tissue, indwelling medical devices, industrial water pipes, and natural aquatic systems. When on surfaces, colonization increases as the surface roughness increases. This is because shear forces of the aqueous external environment are diminished, and the surface area is characteristically higher on rougher platforms. Thus, surface area exerts a strong influence on the rate and extent of biofilm attachment.⁴

Biofilm formation is a process characterized by five stages:

Stage One is initiated by changing environmental signals of pH, temperature, osmolality, and nutrient or iron concentration.⁶ During this stage, microbes attach to a surface, be it a medical device or living tissue. This initial attachment is reversible, characterized by repeated attachment and detachment from the surface.

Stage Two involves irreversible binding to the solid surface and is characterized by microbes remaining adherent to surfaces even under shear forces present in the microenvironment. During this stage, biofilm growth and aggregation occurs, and microbe motility decreases due to formation of the extracellular polymeric matrix.

Stage Three is the first phase of maturation and causes a layering of biofilm to a thickness >10 um. It is at this stage that development of biofilm architecture starts, a process that is dynamic due to constantly changing external and internal environments.⁴ During maturation, the biofilm produces three distinct layers of organisms that are important for survival, function, and architectural integrity. The inner layer is the attachment layer and contains the oldest bacterial cells. These bacteria support the biofilm structure by remaining attached to the adherent surface, are the least metabolically active, and contain the foundation of genetic inheritance for the biofilm community.⁶ The middle layer contains bacteria with down-regulated metabolic activity that are arranged in close proximity to each other, which allows them to exchange nutrients and genetic information (e.g., for antimicrobic resistance). Most of the cell-to-cell communication and genetic interaction occurs in this layer of the biofilm. The outer layer is composed of the protective matrix and active bacteria behaving in a manner similar to individual planktonic (non-biofilm-associated) bacteria.

Stage Four is a second maturation level and is attained when the biofilm reaches its ultimate thickness of >100 um.

Bacteria then proceed to Stage Five, which is also known as the dispersal stage. Biofilm-associated bacteria transfer to new sites by forming projections that break off (disperse) from the community and colonize at new tissue sites.⁶ Specifically, shear forces present in the microenvironment are high enough to cause detachment of a portion of the biofilm and formation of projections called streamers that result in colonization of dispersed biofilm components at new locations. Biofilm dispersion is a means of effective spread of infection from one site to another site (metastasis of infection). The rate of biofilm dispersal increases with increasing biofilm thickness and external shear force. Detachment provides a mechanism for bacteria to migrate from heavily colonized areas depleted of nutrients to environments more supportive for growth.⁴

During biofilm formation, cooperative communication — known as quorum sensing — occurs to coordinate biofilm efforts during different stages of development. For example, quorum sensing can enhance initial attachment (Stage One effect) or cause detachment (Stage Five effect) and metastasis of infection.⁴ Quorum-sensing monitors increasing population density and coordinates changes in gene expression and biofilm community behavior. Accordingly, quorum-sensing molecule number increases with a correlated increase in population density of bacteria. Quorum sensing involves a chemical communication system of ligand-receptor interactions that initiates intracellular signal pathways via a second messenger pathway.² These chemical messengers promote cooperative gene expression as a tool to enhance microbial survival by allowing bacteria in the biofilm to adapt physiologically in response to changing environmental stressors. These quorum-sensing signaling molecules are known as autoinducers and vary, depending upon the bacterial composition of the biofilm. Several categories of autoinducers have been identified: acylated homoserine lactones found in Gram-negative bacteria, autoinducing peptides found in Gram-positive bacteria, and autoinducer-2 compounds found in both Gram-negative and -positive bacteria or in mixed species biofilms.

A treatment strategy has been hypothesized that blocking quorum-sensing molecular interactions with bacterial receptors or intracellular signal blockers causes biofilms to switch from being antimicrobic-resistant to antimicrobic-susceptible by inhibition of interspecies communication. Approaches for drug development have included blockade of autoinducer signal receptors; interception and degradation of autoinducer signaling molecules; and competitive inhibition of signaling. Plants and bacteria have been identified that secrete chemicals which block quorum sensing. For example, red algae secrete halogenated furanones and enones that act as antagonists of acylated homoserine lactone signaling molecules present in biofilms. A competitive mechanism used by bacteria known as intraspecies interference has been identified in which bacteria secrete compounds that block autoinducer effects, thereby allowing for the advantage gain of competitive colonization of a suitable environment.²

There is direct evidence that synergistic treatment of infection with autoinducer antagonist, appropriate antimicrobic; and quorum-sensing inhibitor (e.g., RNA III-inhibiting protein) causes disruption of quorum sensing in staphylococcal biofilms and resultant antibacterial effect.² Synergistic drug therapies are useful and frequently necessary to diminish mechanisms by which biofilms establish and maintain resistance to antimicrobic treatment. As a result, synergistic drug therapies that include the use of traditional antibiotics appear to be a valuable tool in the effective treatment of chronic biofilm infections.

Current research has improved our understanding of the transition from free-floating (planktonic) to biofilm-associated (sessile) bacteria. It is now known that when an organism assumes biofilm growth behavior, most of its metabolic, chemical, and physical properties are altered.⁵ For example, the growth rate of bacteria in planktonic form is usually quite different from the rate of the same bacteria in sessile form.⁷ Due to vast changes in physical and chemical properties, biofilm-associated bacteria have drastically altered responses to stresses in the environment (e.g., antibiotics, host immune responses) compared to planktonic bacteria.

Biofilms are known for their high level of antimicrobic resistance and resilience to host defense mechanisms. Little is known of the immune response to biofilm infections; however, mucosal surfaces have a defense mechanism that inhibits initial attachment of bacteria to a surface.⁶ Prompt killing of these bacteria may account for the resistance of mucosal surfaces to biofilm formation. The caveat must be remembered, however, that if a biofilm matrix does form, it causes enhanced bacterial resistance to protective host defenses.³ For example, biofilms are able to evade phagocytosis, a component of the innate immune response, by preventing biofilm engulfment by phagocytes (e.g., macrophages, neutrophils) which is a normal and protective response against bacteria in the planktonic form.⁷

Research indicates that aqueous environmental conditions of nutrient type and concentration, pH, ionic strength, and temperature play roles in the rate of biofilm attachment to surfaces. For example, increased concentrations of cations — such as sodium, calcium, lanthanum, and ferric iron — affect initial attachment of Pseudomonas fluorescens to glass surfaces.⁴ These ions reduce repulsive forces between negative-charged bacteria and negative-charged host surfaces, thereby increasing the likelihood of initial contact of biofilm-producing bacteria with living tissue. Another environmental factor that can increase number of attached bacterial cells is increased nutrient concentration.⁴ Increasing concentration of sodium chloride causes increased biofilm formation in Listeria monocytogenes and increasing glucose concentration from 1% to 10% causes 97% of the strains to yield higher biofilm content.⁸

Similarly, in methicillin-resistant and methicillin-susceptible Staphylococcus aureus, thehigher the glucose concentration, the more the bacteria increased their biofilm content.¹ The excess carbon provided by the increased number of sugar molecules promotes biofilm matrix synthesis in the outside layer of the biofilm.⁴ Stresses (e.g., starvation, presence of toxic metabolites, and low pH environment) cause the biofilm to enhance its defenses by producing more matrix on the surface layer of the biofilm.⁸ Bacterial attachment is enhanced on surfaces that are rough or coated with bacteria and/or in environments with increased temperature, cations, or nutrients.

When these conditions are established, the site is known as an “enhanced surface” for biofilm formation.⁴ A synergistic effect of these environmental conditions (e.g., temperature, salt, and/or glucose concentration) is enhanced biofilm development.⁸ It has been reported that environmental conditions may cause different responses in biofilm development for different strains of bacteria and inherent factors exhibited by each bacterial strain affects biofilm formation.⁸ This variation in biofilm development among different bacterial species may be due to different combinations of regulatory genes or different levels of gene expression that play roles in the phases of biofilm formation.¹

Enhanced understanding of biofilms and environments that support their growth will contribute to (a) improved techniques for growth and isolation of bacteria causing biofim-associated infections in patients; (b) development of experimental designs focused on understanding biofilm behavior in the clinical and research setting; (c) elucidation of in vivo methods to identify effective treatment strategies for patients with biofilm-associated infections; (d) identification of means by which biofilm metastasis is preventable; and (e) design of mechanisms by which biofilm colonization of medical devices is preventable. Similarly, future experimentation needs to focus on (a) factors that influence the environment in which bacteria develop biofilms, (b) proteins, ions, and nutritional products needed for biofilm attachment; (c) environmental alterations that have inhibitory effects on biofilm formation; (d) in vivo studies to understand biofilm development; and (e) in vitro and in vivo studies to determine optimal conditions for prevention of bacterial attachment to indwelling medical devices.

To conclude, biofilms are efficient and highly effective strategies for bacterial survival. They allow microbes to survive in hostile immune and antimicrobic environments. Regulatory genes are shared among biofilm community members, and gene expression determines how rapidly and dense the biofilms form. Most biofilms are heterogeneous (i.e., contain multiple microbial species) and, therefore, produce larger biofilms than do single-species communities, probably by an increased stabilization effect of the multispecies constituency.⁴

Case Study. The following case has a successful outcome to a biofilm infection.⁹ A 56-year-old diabetic male developed progressive necrosis of the forefoot, beginning with a small painful wound of the second toe. Major limb amputation was recommended because of extensive tissue loss and necrosis; however, the patient refused amputation. A biofilm-based wound-care treatment plan was developed and implemented. Lactoferrin (that impairs irreversible bacterial attachment)⁹ and RNA-III-inhibiting peptide (that blocks expression of specific quorum-sensing molecules)⁹ were added to the antimicrobic treatment strategy. Extensive healing followed and extent of healing correlated with addition of antibiofilm agents (lactoferrin, RNA-III-inhibiting peptides) to the patient’s standard wound-care regimen.

Bradley A. Krivit, BA,
works within the Investigative and Medical Science Program in the Department of Clinical Laboratory Science, with Rita M. Heuertz, PhD, MT(ASCP), a professor in the Departments of Clinical Laboratory Science, Internal Medicine, Molecular Microbiology and Immunology at the Doisy College of Health Sciences and School of Medicine in Saint Louis University in St. Louis, MO.

References

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