Bacterial biofilms are formed by communities that are embedded in a self-produced matrix of extracellular polymeric substances (EPS). Importantly, bacteria in biofilms exhibit a set of 'emergent properties' that differ substantially from free-living bacterial cells. In this Review, we consider the fundamental role of the biofilm matrix in establishing the emergent properties of biofilms, describing how the characteristic features of biofilms - such as social cooperation, resource capture and enhanced survival of exposure to antimicrobials - all rely on the structural and functional properties of the matrix. Finally, we highlight the value of an ecological perspective in the study of the emergent properties of biofilms, which enables an appreciation of the ecological success of biofilms as habitat formers and, more generally, as a bacterial lifestyle.

Biofilms: an emergent form of bacterial life.

Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

Pseudomonas aeruginosa is an opportunistic pathogen affecting immunocompromised patients. It is known as the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and as one of the leading causes of nosocomial infections. Due to a range of mechanisms for adaptation, survival and resistance to multiple classes of antibiotics, infections by P. aeruginosa strains can be life-threatening and it is emerging worldwide as public health threat. This review highlights the diversity of mechanisms by which P. aeruginosa promotes its survival and persistence in various environments and particularly at different stages of pathogenesis. We will review the importance and complexity of regulatory networks and genotypic-phenotypic variations known as adaptive radiation by which P. aeruginosa adjusts physiological processes for adaptation and survival in response to environmental cues and stresses. Accordingly, we will review the central regulatory role of quorum sensing and signaling systems by nucleotide-based second messengers resulting in different lifestyles of P. aeruginosa. Furthermore, various regulatory proteins will be discussed which form a plethora of controlling systems acting at transcriptional level for timely expression of genes enabling rapid responses to external stimuli and unfavorable conditions. Antibiotic resistance is a natural trait for P. aeruginosa and multiple mechanisms underlying different forms of antibiotic resistance will be discussed here. The importance of each mechanism in conferring resistance to various antipseudomonal antibiotics and their prevalence in clinical strains will be described. The underlying principles for acquiring resistance leading pan-drug resistant strains will be summarized. A future outlook emphasizes the need for collaborative international multidisciplinary efforts to translate current knowledge into strategies to prevent and treat P. aeruginosa infections while reducing the rate of antibiotic resistance and avoiding the spreading of resistant strains.

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Pseudomonas aeruginosa Lifestyle: A Paradigm for Adaptation, Survival, and Persistence.

Biofilm refers to the complex, sessile communities of microbes found either attached to a surface or buried firmly in an extracellular matrix as aggregates. The biofilm matrix surrounding bacteria makes them tolerant to harsh conditions and resistant to antibacterial treatments. Moreover, the biofilms are responsible for causing a broad range of chronic diseases and due to the emergence of antibiotic resistance in bacteria it has really become difficult to treat them with efficacy. Furthermore, the antibiotics available till date are ineffective for treating these biofilm related infections due to their higher values of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), which may result in in-vivo toxicity. Hence, it is critically important to design or screen anti-biofilm molecules that can effectively minimize and eradicate biofilm related infections. In the present article, we have highlighted the mechanism of biofilm formation with reference to different models and various methods used for biofilm detection. A major focus has been put on various anti-biofilm molecules discovered or tested till date which may include herbal active compounds, chelating agents, peptide antibiotics, lantibiotics and synthetic chemical compounds along with their structures, mechanism of action and their respective MICs, MBCs, minimum biofilm inhibitory concentrations (MBICs) as well as the half maximal inhibitory concentration (IC50) values available in the literature so far. Different mode of action of anti biofilm molecules addressed here are inhibition via interference in the quorum sensing pathways, adhesion mechanism, disruption of extracellular DNA, protein, lipopolysaccharides, exopolysaccharides and secondary messengers involved in various signaling pathways. From this study, we conclude that the molecules considered here might be used to treat biofilm-associated infections after significant structural modifications, thereby investigating its effective delivery in the host. It should also be ensured that minimum effective concentration of these molecules must be capable of eradicating biofilm infections with maximum potency without posing any adverse side effects on the host.

https://www.tandfonline.com/doi/pdf/10.1080/21505594.2017.1313372

Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action

Biofilms are a predominant form of growth for bacteria in the environment and in the clinic. Critical for biofilm development are adherence, proliferation, and dispersion phases. Each of these stages includes reinforcement by, or modulation of, the extracellular matrix. Pseudomonas aeruginosa has been a model organism for the study of biofilm formation. Additionally, other Pseudomonas species utilize biofilm formation during plant colonization and environmental persistence. Pseudomonads produce several biofilm matrix molecules, including polysaccharides, nucleic acids, and proteins. Accessory matrix components shown to aid biofilm formation and adaptability under varying conditions are also produced by pseudomonads. Adaptation facilitated by biofilm formation allows for selection of genetic variants with unique and distinguishable colony morphology. Examples include rugose small-colony variants and wrinkly spreaders (WS), which over produce Psl/Pel or cellulose, respectively, and mucoid bacteria that over produce alginate. The well-documented emergence of these variants suggests that pseudomonads take advantage of matrix-building subpopulations conferring specific benefits for the entire population. This review will focus on various polysaccharides as well as additional Pseudomonas biofilm matrix components. Discussions will center on structure–function relationships, regulation, and the role of individual matrix molecules in niche biology.

Pseudomonas biofilm matrix composition and niche biology.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

https://www.pnas.org/content/pnas/112/36/11353.full.pdf

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix

Background: Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the ‘‘scaffolding’’ polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings: In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for

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Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

In order to investigate the mechanism of benzo[a]pyrene uptake by a filamentous fungus Fusarium solani, a biochemical characterization of its concentrated culture filtrate has been conducted. The preparation contained approximately (w/w): 50% of total carbohydrate, 6.5% of uronic acid and 6% protein, as determined by colorimetric tests. Gel filtration and anion-exchange chromatographic profiles indicated that the main product of the culture filtrate was a glycoprotein, which contained mannose, glucose and galactose in an approximate molar ratio of 1.5: 0.8: 1. The polysaccharide fraction of the culture filtrate was prepared by treatment with proteinase K, followed by gel-filtration chromatography. Its chemical structure was studied by methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear Magnetic Resonance spectroscopy (NMR). The major carbohydrate was a polymer of β-(1 → 6)-linked galactofuranose units fully branched at positions O-2 by single residues of α-glucopyranose. The Fusarium concentrated culture filtrate increased 4-fold the BaP solubilization in comparison with its aqueous solubility and suggested that the carbohydrate present in this filtrate should probably be involved in this enhancement. Our findings point out the potential role of fungal glycoproteins in PAH microbial bioavaibility, an important step for PAH biodegradation.

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Preliminary Characterizations of a Carbohydrate from the Concentrated Culture Filtrate from Fusarium solani and Its Role in Benzo[a]Pyrene Solubilization

Biofilm formation is an important virulence factor of Staphylococcus epidermidis. However, little is known about the mechanisms of staphylococcal biofilm dispersal. In the present study, we investigated biofilm dispersal of the model biofilm-forming strain S. epidermidis RP62A under oligotrophic stress conditions. We found that oligotrophic stress led to rapid dispersal of pre-formed biofilms and concomitant changes in the composition of the extracellular matrix, including a decrease in poly-N-acetylglucosamine polysaccharide and an increase in proteins. Our results suggest that modifications in biofilm integrity caused by compositional changes in the biofilm matrix can induce biofilm dispersal.

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Charlène Coulon

Papers

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix

Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

Preliminary Characterizations of a Carbohydrate from the Concentrated Culture Filtrate from Fusarium solani and Its Role in Benzo[a]Pyrene Solubilization

Stress-Induced Dispersal of Staphylococcus epidermidis Biofilm Is Due to Compositional Changes in Its Biofilm Matrix