Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms.

https://tandfonline.com/doi/pdf/10.1080/1040841X.2016.1208146

Critical review on biofilm methods

Electroactive microorganisms markedly affect many environments in which they establish outer-surface electrical contacts with other cells and minerals or reduce soluble extracellular redox-active molecules such as flavins and humic substances. A growing body of research emphasizes their broad phylogenetic diversity and shows that these microorganisms have key roles in multiple biogeochemical cycles, as well as the microbiome of the gut, anaerobic waste digesters and metal corrosion. Diverse bacteria and archaea have independently evolved cytochrome-based strategies for electron exchange between the outer cell surface and the cell interior, but cytochrome-free mechanisms are also prevalent. Electrically conductive protein filaments, soluble electron shuttles and non-biological conductive materials can substantially extend the electronic reach of microorganisms beyond the surface of the cell. The growing appreciation of the diversity of electroactive microorganisms and their unique electronic capabilities is leading to a broad range of applications.

Electromicrobiology: the ecophysiology of phylogenetically diverse electroactive microorganisms

Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.

https://www.mdpi.com/2079-6382/10/1/3/pdf

Biofilms as Promoters of Bacterial Antibiotic Resistance and Tolerance.

“Slime” played a brief and spectacular role in the 19th century founded by the theory of primordial slime by Ernst Haeckel. However, that substance was never found and eventually abandoned. Further scientific attention slowly began in the 1930s referring to slime as a microbial product and then was inspired by “How bacteria stick” by Costerton et al. in 1978, and the matrix material was considered to be polysaccharides. Later, it turned out that proteins, nucleic acids and lipids were major other constituents of the extracellular polymeric substances (EPS), an acronym which was highly discussed. The role of the EPS matrix turns out to be fundamental for biofilms, in terms of keeping cells in proximity and allowing for extended interaction, resource capture, mechanical strength and other properties, which emerge from the life of biofilm organisms, including enhanced tolerance to antimicrobials and other stress. The EPS components are extremely complex and dynamic and fulfil many functional roles, turning biofilms into the most ubiquitous and successful form of life on Earth.

https://www.mdpi.com/2076-2607/4/4/41/pdf

EPS—Then and Now

One of the key elements in the establishment and maintenance of the biofilm structure and properties is the extracellular matrix. The extracellular matrix is composed of water and extracellular polymeric substances (EPS): primarily polysaccharides, proteins and DNA. Characterization of the matrix requires component identification, as well as determination of the relative concentration of EPS constituents, including their physicochemical properties and descriptions of their interactions. Several types of experimental approaches with varying degrees of destructiveness can be utilized for this characterization. The analysis of biofilm by infrared spectroscopy gives information about the chemical content of the matrix and the proportions of different EPS. The sensitivity of a biofilm to hydrolytic enzymes targeting different EPS gives insight into the composition of the matrix and the involvement of matrix components in the integrity of the structure. Using both chemical and physical treatments, extraction and purification of EPS from the biofilm also provides a means of determining matrix composition. Purified and/or artificial EPS can be used to obtain artificial matrices and to study their properties. Using examples from the literature, this review will illustrate selected technologies useful in the study of EPS that provide a better understanding of the structure-function relationships in extracellular matrix, and thus the structure-function relationships of the biofilm phenotype.

Extracellular polymeric substances, a key element in understanding biofilm phenotype.

Summary
A hallmark of bacterial biofilms is a self-produced extracellular matrix of exopolysaccharide, extracellular DNA (eDNA) and proteins that hold bacterial cells together in the community. However, interactions among matrix components and how the interactions contribute to the formation of matrix remain unclear. Here, we show the physical interaction between exopolysaccharide Psl and eDNA, the two key biofilm matrix components of the opportunistic pathogen Pseudomonas aeruginosa. The interaction allows the two components to combine to form a web of eDNA–Psl fibres, which resembles a biofilm skeleton in the centre of pellicles to give bacteria structural support and capability against agents targeted on one matrix component. The web of eDNA–Psl fibres was also found in flow-cell biofilms at microcolonies initiation stage. The colocalization of eDNA or Psl fibres with bacterial cell membrane stain suggests that fibre-like eDNA is likely derived from the lysis of dead bacteria in biofilms. Psl can interact with DNA from diverse sources, suggesting that P. aeruginosa has the ability to use DNA of other organisms (such as human neutrophils and other bacterial species) to form its own communities, which might increase the survival of P. aeruginosa in multispecies biofilms or within a human host.

/pdf/the-exopolysaccharide-psl-edna-interaction-enables-the-4dnw0vv3re.pdf

The exopolysaccharide Psl-eDNA interaction enables the formation of a biofilm skeleton in Pseudomonas aeruginosa.

Bioelectrical nanowires as ecomaterials have great potential on environmental applications. A wide range of bacteria can express type IV pili (T4P), which are long protein fibers assembled from PilA. The T4P of Geobacter sulfurreducens are well known as "microbial nanowires," yet T4P of Pseudomonas aeruginosa (PaT4P) was believed to be poorly conductive. P. aeruginosa is an aerobic and electrochemically active bacterium. Its T4P have been known to be responsible for surface attachment, twitching motility and biofilm formation. Here, we show that PaT4P can be highly conductive while assembled by a truncated P. aeruginosa PilA (PaPilA) containing only N-terminus 61 amino acids. Furthermore, increasing the number of aromatic amino acids in the PaPilA1-61 significantly enhances the conductivity of pili and the bioelectricity output of P. aeruginosa in microbial fuel cell system, suggesting a potential application of PaT4P as a conductive nanomaterial. The N-terminal region of PilA from diverse eubacteria is highly conserved, implying a general way to synthesize highly conductive microbial nanowires and to increase the bioelectricity output of microbial fuel cell.

Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa.

Antibiotic resistance or tolerance of pathogens has become one of the global public crises. Finding new drug targets may open up a way of infection control. Phenazine pyocyanin (PYO) is an important virulence factor produced by the pathogen Pseudomonas aeruginosa. Here we show that a multidrug efflux pump repressor, MexL, acts as a transcriptional activator to enhance phenazines production via binding with a conserved DNA motif within the promoters of phenazines biosynthesis genes. Moreover, PYO functions as a self-regulating ligand of MexL for restricting its own production and the mexL knockout attenuates the virulence and antibiotics tolerance of P. aeruginosa. Based on the structure of MexL we resolve, we find two antimicrobials that can interact with MexL to reduce the PYO production and virulence of P. aeruginosa. Our in vivo studies suggest that the antimicrobials combination by using MexL-antagonists to reduce bacterial virulence and enhance the efficacy of common antibiotics can be an effective way to combat P. aeruginosa infection. Finding new drug targets may open up a way to combat microbial infections. In this work, authors present the molecular basis for the dual-function regulator MexL as a target to control the virulence and pathogenesis of opportunistic pathogen Pseudomonas aeruginosa.

Dual-function regulator MexL as a target to control phenazines production and pathogenesis of Pseudomonas aeruginosa

Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs. In this study, a high-throughput transposon-insertion site sequencing (Tn-seq) approach was employed to identify novel P. aeruginosa biofilm genetic determinants. When analyzing the novel biofilm regulatory genes, we found that the cell division factor ZapE (PA4438) controls the P. aeruginosa pqs quorum sensing system. The ∆zapE mutant lost fitness against the wild-type PAO1 strain in biofilms and its production of 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) had been reduced. Further biochemical analysis showed that ZapE interacts with PqsH, which encodes the synthase that converts 2-heptyl-4-quinolone (HHQ) to PQS. In addition, site-directed mutagenesis of the ATPase active site of ZapE (K72A) abolished the positive regulation of ZapE on PQS signaling. As ZapE is highly conserved among the Pseudomonas group, our study suggests that it is a potential drug target for the control of Pseudomonas infections. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/mlf2.12059

Cell division factor ZapE regulates
 Pseudomonas aeruginosa
 biofilm formation by impacting the
 pqs
 quorum sensing system

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The exopolysaccharide Psl-eDNA interaction enables the formation of a biofilm skeleton in Pseudomonas aeruginosa.

Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa.

Dual-function regulator MexL as a target to control phenazines production and pathogenesis of Pseudomonas aeruginosa

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system