Biofilm formation: mechanistic insights and therapeutic targets

Abstract

Biofilms are complex multicellular communities formed by bacteria, and their extracellular polymeric substances are observed as surface-attached or non-surface-attached aggregates. Many types of bacterial species found in living hosts or environments can form biofilms. These include pathogenic bacteria such as Pseudomonas, which can act as persistent infectious hosts and are responsible for a wide range of chronic diseases as well as the emergence of antibiotic resistance, thereby making them difficult to eliminate. Pseudomonas aeruginosa has emerged as a model organism for studying biofilm formation. In addition, other Pseudomonas utilize biofilm formation in plant colonization and environmental persistence. Biofilms are effective in aiding bacterial colonization, enhancing bacterial resistance to antimicrobial substances and host immune responses, and facilitating cell‒cell signalling exchanges between community bacteria. The lack of antibiotics targeting biofilms in the drug discovery process indicates the need to design new biofilm inhibitors as antimicrobial drugs using various strategies and targeting different stages of biofilm formation. Growing strategies that have been developed to combat biofilm formation include targeting bacterial enzymes, as well as those involved in the quorum sensing and adhesion pathways. In this review, with Pseudomonas as the primary subject of study, we review and discuss the mechanisms of bacterial biofilm formation and current therapeutic approaches, emphasizing the clinical issues associated with biofilm infections and focusing on current and emerging antibiotic biofilm strategies.

Keywords: Anti-biofilm drugs; Antibiofilm therapeutic strategy; Biofilm; Pseudomonas.

Fig. 1

Bacterial biofilm development produces a classic structure. The formation of biofilms consists of five stages: reversible attachment, irreversible attachment, maturation stage I, maturation stage II and dispersion. In the reversible attachment stage, bacteria attach to the matrix through the swing of flagella. In the irreversible attachment stage, the expression of the flagella gene is lost. Then, several cell clusters mature, and thick cell clusters embedded in the biofilm matrix enter mature stage I. In mature stage II, the bacterial clusters reach the maximum thickness, and microcolonies can be seen. When the biofilm is dispersed, the biofilm cycle will cycle again

Fig. 2

Abundant biofilm matrix molecules. In the matrix of biofilms, polysaccharides are important components, including alginate, fructan and other capsular polysaccharides, as well as Psl and other aggregation polysaccharides. Alginate is a highly molecular-weight acetylated polymer and is an anionic polysaccharide composed of β-1-4 glycosidic bond-linked α-α L-guluronic acid and β-D-mann β uronic acid. Levan is a high molecular weight b-2,6 polyfructose with extensive branching through the b-2,1 bond. Psl polysaccharides are neutral polysaccharides consisting of pentasaccharide repeating units composed of D-mannose, D-glucose, and L-rhamnose. In addition, there are a large number of proteins, enzymes, eDNA, lipids and other important components in the matrix

Fig. 3

Biofilm formation and dispersion. a In the mechanism of biofilm formation, the two-component system is usually composed of sensor kinases and response regulators. Quorum sensing includes AHL-based quorum sensing systems and PQS-based quorum sensing systems. Among them, LasR can positively regulate RhlR and PQS, while RhlR can negatively regulate PQS. LasR can regulate the activity of TpbB, reduce the level of c-di-GMP, and promote biofilm formation by activating PelD. RhlR controls the synthesis of rhamnolipid and maintains the channel in the mushroom-like structure together with eDNA released from the PQS system. The synthesis and degradation of c-di-GMP occurs through the opposite activity of diguanosine cyclase (DGC) with a GGDEF domain and phosphodiesterase (PDE) with an EAL or HD-GYP domain. DGC can promote the synthesis of c-di-GMP, PDE can reduce the level of c-di-GMP, and c-di-GMP will act on the four effectors Alg44, FleQ, PelD and FimX. The formation of biofilms is promoted by regulating the synthesis of Psl and CdrA and convulsive movement. In the sRNA pathway, sensor kinases include GacS, RetS, and LadS; GacS can phosphorylate GacA, and GacA can activate the transcription of rsmZ and rsmY, thereby regulating the synthesis of Psl and the corresponding movement. Sigma factors such as RpoS can positively regulate the expression of Psl to promote biofilm formation. b In the mechanism of biofilm dispersion, NO can activate BdlA, and then activated BdlA can recruit and activate RpdA and DipA, which can reduce the level of c-di-GMP. In addition, rhamnolipid and cis-2-decenoic acid can also reduce the level of c-di-GMP, which can increase the activity of the LapG homolog and LapD homolog, resulting in the release of LapA and CdrA and eventually leading to the dispersion of biofilms

Fig. 4

Mechanisms of drug action. Different antibiofilm drugs have different mechanisms of action. For example, ciprofloxacin works by interfering with bacterial DNA replication, while tetracycline, tobramycin and gentamicin interfere with translation, all of which specifically kill cells that are metabolically active at the top of the biofilm. Dfo-gallium is antibacterial by interfering with the iron metabolism of cells, while colistin, EDTA and SDS destroy the structure of the membrane by combining with the lipopolysaccharide terminal lipid outside the bacterial membrane, and these two kinds of drugs kill the cells in the deep biofilm