Análisis de resultados

3.5.1

Bacterial adhesion to surfaces

Wild natural bacteria are necessary to study adhesion of pathogens on the surface of biomaterials as lab-adapted strains do not reproduce conditions inside the body. The DLVO theory constituted by van Loodsrecht et al. in 1990 is based on the comparison of bacterial cells with smooth colloid particles that interact with a surface as a result of an electrostatic attraction (99). However several studies showed that this theory is of limited application in terms of bacterial adhesion as the bacterial cells are not smooth-surfaced particles. The surface is rather covered by hydrophobic exopolysaccharide and sometimes a highly structured protein shell. There are different mechanisms of adherence ranging from utilization of flagella or pili to the point of producing windrows of cells (58). In general, pathogens show reversible and irreversible patterns of adherence (70), meaning that bacterial cells are able to adhere to the surface as well as to detach and leave the area again before attaching irreversibly and initiating the process of biofilm formation. Physical properties of the biomaterial surface seem to play an inferior role for adherence procedure as several studies have demonstrated. Pathogens seem to adhere equally well to hydrolphilic and hydrophobic, and to rough as well as to smooth surfaces (68, 91) even in systems with very a high shear flow rate (14). Scientists are still searching for new solutions.

Introduction and Objectives of the Thesis _____________________________________________________________________________

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Figure 2.Interaction between pathogens, tissue and implant (37).

3.5.2

Biofilm formation

The irreversible attachment of bacterial cells at a biomaterial surface is accompanied by a genetic switch which implicates a phenotypic change, in some cases up to 70 % (84). The phenotype of planktonic cells of Pseudomonas aeruginosa for instance, shows a higher agreement with planktonic cells of other species in the same genus than their biofilm counterparts (85). The production of exopolysaccharides by up-regulated genes is thereby one of the first activities of the bacterial cell with a differing phenotype (84, 93). These exopolysaccharides attach the cell irreversibly to the biomaterial surface and form the matrix of the biofilm. Binary fission or further attachment increases the number of phenotypically modified bacterial cells on the biomaterial surface resulting in a community of matrix and bacterial colonies of varying shape. These entities are separated by open water channels and water moves through these complex microcolonies in a convective pattern. A balance between multiplication and detachment of planktonic cells adjusts. Approximately one week after the initiation of colonization, a stable community structure has formed and can remain so for years. Observed resistance to antibiotics or to uptake by phagocytes can also be attributed to the upregulation of genes, as for example the gene fmt C is responsible for antibiotic resistance of staphylococcal species. The clinical consequence is that antibiotics can be used for treating acute infection occurrence caused by planktonic cells, but not for biofilm clearance. Biofilm-covered devices therefore have to be removed in many cases by surgery.

The complex structure and organization of these bacterial microcolonies lead to the conclusion that pathogens within these entities are able to communicate with each other via signals like

hormones or pheromones. The assumption of this so-called quorum sensing was first confirmed in 1998 by Davis et al. who demonstrated that biofim formation in Pseudomonas aeruginosa is controlled by an acylated homoserine lactone (AHL) quorum sensing signal. This AHL signal is only one of many signals which control the organization of pathogens in biofilm communities.

Figure 3.Staphylococcus aureus accumulation A) without B) with slime production (41).

3.5.3

Strategies of biofilm prevention

The most common way to prevent biofilm formation on medical devices is the incorporation of antibiotics into surface material in order to kill incoming planktonic cells before they can adhere and start biofilm formation. After complicated and longer operations, in particular, device- related infections can occur because skin and environmental bacteria have enough time to adhere and initiate biofilm formation. An antibiotic-modified surface is therefore a helpful and established strategy. The problem is that after the initial drug release, which guarantees effective protection against planktonic bacteria for a short period of time, the delivered sublethal antibiotic concentrations after months or years as well as the application of highly specialized drugs like ciprofloxacin can cause resistant strains. A switch to multitarget antiseptics like chlorhexidine has improved the situation, however, most are not approved yet for systemic use in humans. Another method of resolution is the development of intelligent coatings mimicking skin consisting of a self-assembled surface layer that contains molecules loaded into an underlying plastic. The advantage is the delivery of antimicrobial drugs in controlled impulses by ultrasonic waves post-operatively or any time preliminary signs of device-related infection show up. Other studies show that the application of ultrasonic energy itself (71, 77) or weak direct current fields (19) are promising strategies to render pathogens susceptible to antimicrobial agents.

Introduction and Objectives of the Thesis _____________________________________________________________________________

17 A more focused method to prevent biofilm formation is the utilization of biofilm signal blockers, for instance the RNA III-inhibiting peptide (RIP) analogue blocking the target of RNA III- activating protein (TRAP) receptor in gram-positives or brominated furanones targeted on the acylated homoserine lactone (AHL) system of gram-negative bacteria (6). This method is based on the idea to inhibit the genetic switch between planktonic cells and their biofilm counterparts and is derived from observations of plants that protect themselves from biofilm colonization by the use of signal blockers (92). This lock of pathogens in the planktonic phenotype makes them susceptible to conventional antibiotic therapy.

The new development from Westaim Biomedical Inc. for burn bandages uses a galvanic combination between silver and copper along with a simultaneous release system of silver and copper ions. This may have an effect on pathogen adhesion, biofilm formation and susceptibility to antibiotics. The synthesis of different concepts may be the best way for the development of new antimicrobial devices or so-called antifouling materials.