Staphylococcal Biofilms: Challenges and Novel Therapeutic Perspectives

Abstract

Staphylococci, like Staphylococcus aureus and S. epidermidis, are common colonizers of the human microbiota. While being harmless in many cases, many virulence factors result in them being opportunistic pathogens and one of the major causes of hospital-acquired infections worldwide. One of these virulence factors is the ability to form biofilms-three-dimensional communities of microorganisms embedded in an extracellular polymeric matrix (EPS). The EPS is composed of polysaccharides, proteins and extracellular DNA, and is finely regulated in response to environmental conditions. This structured environment protects the embedded bacteria from the human immune system and decreases their susceptibility to antimicrobials, making infections caused by staphylococci particularly difficult to treat. With the rise of antibiotic-resistant staphylococci, together with difficulty in removing biofilms, there is a great need for new treatment strategies. The purpose of this review is to provide an overview of our current knowledge of the stages of biofilm development and what difficulties may arise when trying to eradicate staphylococcal biofilms. Furthermore, we look into promising targets and therapeutic methods, including bacteriocins and phage-derived antibiofilm approaches.

Keywords: S. aureus; antibiotics; bacteriocins; bacteriophages; biofilm; coagulase-negative staphylococci.

Figure 1

Stages and pathways involved in staphylococcal biofilm development. (A–C) During the attachment stage, an initial population of staphylococcal cells adhere onto a surface via (A) surface proteins, (B) teichoic acids and/or (C) eDNA released from cells, the latter due to cell lysis mediated by peptidoglycan hydrolases like AtlA/E and Sle1.(D–E) The maturation stage is mediated by intercellular adhesion, which allows the formation of multiple cell layers and the increase of the biofilm cell population. Intercellular adhesion is made via (D) a polysaccharide-dependent or (E) polysaccharide-independent pathway. The former involves homophilic or heterophilic protein-protein interactions by surface proteins (i) or amyloid fibers (ii). Certain surface proteins are processed by proteases to remove part of it, allowing the protein to polymerise into a fiber to reach a neighboring cell. The polysaccharide-dependent pathway is mediated by the icaADBC locus, which is responsible for the synthesis of β-1,6-N-acetylglucosaminoglycan (PIA/PNAG - grey hexagons). Then, PNAG chains are translocated to the outside of the cells, probably by IcaC, and deacetylated (red hexagons) by IcaB. (F,G) During the dispersion stage, cells are released from the biofilm. It has been suggested that the nuclease Nuc (F) participates in the degradation of eDNA during this stage. In addition, the accessory gene regulatory (agr) system (G) also plays a role during dispersion (please see the main text for details). (H,I) Examples of how the expression of selected genes affects S. aureus biofilms, imaged by confocal microscopy. Effects on S. aureus NCTC8325–4 biofilm formation when the genes tarA and tarO were depleted by using CRISPR interference (H), and on S. aureus JE by Δatl mutation (I). Biofilms were stained using a LIVE/DEAD kit. Green fluorescence indicates live cells, while red fluorescence indicates dead cells. Scale bar: 10 µm.

Figure 2

Model of anti-biofilm and bactericidal action of bacteriocins. (A) The structure of nisin in conjugation with lipid II is shown along with a representation of its dual mechanism of action. By interacting with lipid II molecules exposed on the cell surface, the bacteriocin inhibits the cell-wall biosynthesis and leads to pore formation in the bacterial cell membrane. (B) The structure of lysostaphin is shown as a representative example of staphylococcins. Its anti-biofilm action relies in part on the perturbation of wall teichoic acid (WTA), the function of which has been shown to be important in the process of biofilm formation. Epidermin-like bacteriocins (e.g., epidermin and gallidermin) follow a similar mode of action, and also lead to the downregulation of biofilm-promoting genes such as ica and atl. The protein structures were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDP) (http://www.rcsb.org).

Figure 3

Visualization of the effects of a bacteriocin-based formulation on MRSA biofilms.

Figure 4

Bacteriophage replication through the lytic cycle. (A) Phage-infected S. aureus biofilm in which different cells are undergoing consecutive stages of the lytic cycle, while others remain uninfected. (B) Virion-associated proteins with antibiofilm potential that participate in early stages of the lytic cycle (VAPGHs and exopolysaccharide depolymerases). (C) Role of endolysins in cell lysis at the end of the lytic cycle. WTA, wall teichoic acids.