The majority of staphylococci produce biofilm on medical devices, which is the main mechanism to infect humans. Staphylococcal biofilms attach to abiotic or biotic surfaces, forming aggregates and protecting themselves against the immune system and the antimicrobial compounds of the host. Few studies on biofilm formation mechanism in Staphylococcus epidermidis and other coagulase-negative staphylococci (CNS) have been performed; however, there is a great interest in studying and controlling biofilm formation of this genus. This chapter exhibits the state of the art on biofilm formation in S. epidermidis and other staphylococcal species. The main goal of this chapter is to recognize the importance of biofilm formation in Staphylococcus. The participating molecules in staphylococcal biofilm formation are described. Currently, biofilm producer strains of Staphylococcus and mainly CNS have been frequently isolated at hospitals, causing significant economic losses. This chapter includes promising solutions in order to prevent medical device-associated infections, as the development of medical devices possessing anti-biofilm materials or surfaces that act against the adhesion or viability of the microorganisms.
- Staphylococcus epidermidis
- Staphylococcus aureus
- medical devices
During the last years, the study of biofilms has become relevant due to their significance on many microbiology areas. In the health field, biofilms have been of great relevance because many pathogenic and non-pathogenic bacteria can produce biofilm as a part of its virulence mechanism and protection against the host. A biofilm is considered a complex microbial community (or communities) attached to a defined surface and embedded within a cell matrix. Regarding the surface, biofilms may be formed on a wide variety of chemical or biological surfaces. Regarding bacteria of the
I.1. Medical and epidemiological relevance of staphylococci biofilms
Staphylococci are commensal bacteria inhabiting the human skin and mucus. However, they have been identified as infection-causing agents associated to biofilms. Animal models of biofilm-associated infections using staphylococci have allowed to determine the importance of their biofilms as a virulence mechanism. Therefore, staphylococci, particularly
I.2. Experimental models to study biofilm formation
The clinical relevance of biofilm formation on foreign materials has been demonstrated using cell culture models, a
PIA-dependent biofilm formation also interferes with the host’s complement activation. Biofilm-positive wild-type bacteria pre-opsonized with normal human serum are more resistant to complement-mediated elimination than the corresponding biofilm-negative isogenic bacteria . It has been also shown that
II. Mechanisms and molecules participating in staphylococci biofilm formation
In this chapter, we will divide the study of the biofilm formation process in three phases. During primary attachment, bacteria adhere to the biotic or abiotic surface in order to colonize it, whereas on the accumulation phase, bacteria build a tridimensional multi-cell and multi-layer array. Then, staphylococci are able to disassemble biofilm structure in order to release those cells capable to colonize other sites on the surface.
II.1. Participating molecules on the biofilm primary attachment phase
An essential step performed during the primary attachment stage is the tight binding of bacteria to the foreign material (medical device). This bacterial tight binding leads to a successful establishment of a medical device-associated infection. Regarding
The interaction between
eDNA function during
II.2. Participating molecules during the biofilm accumulation phase
The main component during the accumulation phase is the expression of molecules possessing intercellular (cell-cell) adhesion properties leading to cell aggregation and to subsequent biofilm development having a multi-cell and multi-layer tridimensional structure. Based on the early electron microscopy studies, it has been shown that
The PIA structure was first described in biofilm-forming
Conversely, the first observation made through biochemical analysis on biofilm matrix extracts indicated the presence of oligosaccharide, proteins, and nucleic acids. The specific proteins that comprise a biofilm have been identified and characterized; one of them is the biofilm-associated protein Bap . The Bap is rarely found in invasive
II.3. Multifunction proteins during the biofilm accumulation phase
Protein factors contributing to the accumulation phase of staphylococci biofilm have features of multifunctional proteins. In
The Embp protein and its
The Aap protein is covalently bound to the cell wall and consists of an A domain and a B domain. The A domain has 584 amino acids and includes an export signal at its N-terminal, 16 amino acid repeats and a globular region of 212 amino acids with alpha-helical and beta-sheet contents. This 212-amino acid-long region is highly conserved between Aap and its
The importance of Aap for
Although the intercellular adhesion property of Aap was recognized, currently there is evidence supporting its significant role in the primary attachment phase as well. The binding of
II.4. Molecular mechanisms for mature biofilm disassembly
A primary biofilm disassembly mechanism used by
Extracellular protease production has been implicated on the disassembly of the mature biofilm. In
PSMs are peptides possessing surfactant features, which are produced by both
III. Regulation of biofilm formation in staphylococci
Biofilms are a lifestyle adopted by a wide variety of microorganisms that requires the consumption of an enormous amount of energy. Thus, it is expected that biofilm growth may be controlled by more regulatory mechanisms regarding planktonic growth. Some of the factors that impact on biofilm formation are mentioned in the following sections.
III.1. Regulation of the factors participating on the primary attachment phase
III.2. Regulation of PIA synthesis
The regulation of PIA expression is probably the best-studied system among those regulation systems involved in staphylococci biofilm formation. Anaerobiosis significantly increases PIA expression . This constitutes an important finding for biofilm physiology, as the oxygen concentration would restrict biofilm formation at the oxygen-loaded arterial bloodstream. In an already established biofilm, PIA expression would be higher at the most deep biofilm sections because oxygen concentration significantly decreases. Conversely, it has been found that sub-inhibitory concentrations of specific antibiotics increase the transcription of the
Some overall regulators of
III.3. Regulation of the PSMs expression
It has been discussed that the
III.4. Biofilm regulation against host’s defenses and antibiotics
One of the advantages possessed by bacteria in the biofilm state is high resistance toward antibiotics and the host innate defense, such as AMPs and the phagocytosis performed by neutrophils. However, the molecular basis of this phenomenon has been recently investigated. Two of the main mechanisms contributing to biofilm resistance are: (1) keeping antibacterial substances from reaching their target, for example, by limited diffusion or repulsion and (2) biofilm’s specific physiology that limits the efficiency of antibiotics, mainly those targeting active cells, and it may include specific subpopulations of resistant cells (“persistent”).
Limited antibiotic diffusion provided by the biofilm is mainly due to the nature of the biofilm matrix. However, this limited diffusion is the resistance mechanism toward some antibiotics, such as ciprofloxacin in
Phagocytosis, mainly performed by neutrophils, is a major mechanism by which the innate immune system eliminates microorganisms invading the human body. Staphylococci in a biofilm are not readily subjected to phagocytosis by neutrophils. The responsible elements for this constraint are the PIA exopolysaccharide and the PGA exopolymer, and therefore they contribute to biofilm resistance toward the host’s innate defense mechanisms.
IV. Therapeutic strategies against biofilm formation in medical devices
Medical devices are widely used for diagnostic and therapeutic treatment in most medical specialties. Infection risk is a frequent complication linked to the permanent use of medical devices such as orthopedic or heart prostheses, vascular catheters, urinary catheters, and endotracheal tubes. A promising solution in order to prevent medical device-associated infections is to develop devices possessing materials or surfaces that act against microorganism adhesion or their viability. The first strategy was the use of biocides in coatings. A number of clinical assays have been conducted producing conflicting results. Some authors suggest that the extended use of biocide on the coating may lead to an increase of microbial resistance toward the microbiocide agent. The other strategy consists in the development of materials impeding bacterial adhesion.
IV.1. Biological strategies for biofilm treatment
The chemical diversity of the biofilm matrix, including protein material, eDNA, and polysaccharides, is susceptible to degradation by a wide variety of exogenously added enzymes. Some research groups have observed that proteinase K and trypsin may disperse the mature biofilm of
A current topic is the development of an antimicrobial coating interfering with quorum-sensing mechanisms. This has been observed for halogenated furanones synthesized by the red algae
IV.2. Anti-adhesive chemical strategies
IV.2.1. Hydrophobicity and surface charge
Bacterial adhesion depends on hydrophobocity of the cell and material constituting the surface. The self-autoassembled monolayers (SAMs) can modulate the exposure of their different residues on a surface and they are used in bacterial adhesion studies as models of surfaces with chemically controlled properties. SAMs with hydrophilic residues (OH, NH2) tend to decrease bacterial adhesion when compared to those with hydrophobic surfaces containing methyl groups (CH3) . Some hydrophilic linings, such as hydrogels or medical devices with chemically modified surfaces have been developed in order to restrict biofilm development. Some clinical studies have reported that urinary catheters lined with heparin can reduce
IV.2.2. Steric barriers
The chemical modifications of the surface may also consist on grafting long-chain polymers in order to form brush-type structures on it. The density of the chains provides a steric barrier that repels bacterial adhesion. The most widely studied polymers are derivatives of polyethylene oxides. In fact, residues of SAMs with ethylene glycol (4EG and 3EG) have lower bacterial adhesion in comparison to hydrophilic surfaces . Polymers with ester residues (CHO2-) or cyclic hydrocarbons (C4H-, C6H-) exhibit less bacterial attachment strength than materials containing ethylglycol or hydroxyl group fragments .
IV.2.3. Anti-adhesive strategies based on topographic modifications of the surface
In the theories of bacterial adhesion, the appearance of the surface of the material was not considered. The relief of a surface depends on the scale, that is, for bacterial adhesion, the submicron scale is used. The reliefs are divided into: i) areas with irregular or random traits defined as rough; ii) areas with organized features, often made by an engineering process, defined by the term surface topography.
One study showed that adherence of
IV.2.4. The influence of nanofeature physical structure on bacterial response
Nanofeatures may adopt different shapes: nanotubes, notches, channels or grooves, holes or pillars. There are few studies regarding the relationship between nanofeatures and bacterial adhesion. Ercan et al. compared
As mentioned above, the nanoscale level, size, and bacterial shape regarding nanofeature dimensions play a significant role. Bacterial features (adhesion, surface charge) are also important to the adhesion process. The surface of a nanostructure must be tested with several bacterial strains, as they exhibit different adhesion behaviors. For instance, a titanium surface nanostructured by femtosecond laser ablation and mimicking the superhydrophobic surface of the lotus leafs was not colonized after 18 hours by
Conversely, silver nanoparticles (AgNPs) are gaining interest for biomedical applications because of their features having a higher surface/mass ratio and a potent antibacterial activity. These AgNPs may be applied as monolayers at the surface of biomaterials. A study on glass surfaces modified with AgNPs was carried out and it was found that they possess a great stability in aqueous media, an extended Ag+ release without AgNPs detachment and a strong anti-biofilm activity against
This work was supported by a grant from the “CONACyT México” (No. 153268). JJR, SRM, MECD, and JCCD appreciate the COFAA and EDI-IPN fellowships; also that provided by SNI-CONACyT.
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