Periphery Of Biofilm Research Articles

Biofilm dispersal patterns revealed using far-red fluorogenic probes.

Bacteria frequently colonize niches by forming multicellular communities called biofilms. To explore new territories, cells exit biofilms through an active process called dispersal. Biofilm dispersal is essential for bacteria to spread between infection sites, yet how the process is executed at the single-cell level remains mysterious. Here, we characterize dispersal at unprecedented resolution for the global pathogen Vibrio cholerae. To do so, we first developed a far-red cell-labeling strategy that overcomes pitfalls of fluorescent protein-based approaches. We reveal that dispersal initiates at the biofilm periphery and ~25% of cells never disperse. We define novel micro-scale patterns that occur during dispersal, including biofilm compression and the formation of dynamic channels. These patterns are attenuated in mutants that reduce overall dispersal or that increase dispersal at the cost of homogenizing local mechanical properties. Collectively, our findings provide fundamental insights into the mechanisms of biofilm dispersal, advancing our understanding of how pathogens disseminate.

Systematic microscopical analysis reveals obligate synergy between extracellular matrix components during Bacillus subtilis colony biofilm development

Single-species bacterial colony biofilms often present recurring morphologies that are thought to be of benefit to the population of cells within and are known to be dependent on the self-produced extracellular matrix. However, much remains unknown in terms of the developmental process at the single cell level. Here, we design and implement systematic time-lapse imaging and quantitative analyses of the growth of Bacillus subtilis colony biofilms. We follow the development from the initial deposition of founding cells through to the formation of large-scale complex structures. Using the model biofilm strain NCIB 3610, we examine the movement dynamics of the growing biomass and compare them with those displayed by a suite of otherwise isogenic matrix-mutant strains. Correspondingly, we assess the impact of an incomplete matrix on biofilm morphologies and sessile growth rate. Our results indicate that radial expansion of colony biofilms results from the division of bacteria at the biofilm periphery rather than being driven by swelling due to fluid intake. Moreover, we show that lack of exopolysaccharide production has a negative impact on cell division rate, and the extracellular matrix components act synergistically to give the biomass the structural strength to produce aerial protrusions and agar substrate-deforming ability.

Neutralization of ionic interactions by dextran-based single-chain nanoparticles improves tobramycin diffusion into a mature biofilm

The extracellular matrix protects biofilm cells by reducing diffusion of antimicrobials. Tobramycin is an antibiotic used extensively to treat P. aeruginosa biofilms, but it is sequestered in the biofilm periphery by the extracellular negative charge matrix and loses its efficacy significantly. Dispersal of the biofilm extracellular matrix with enzymes such as DNase I is another promising therapy that enhances antibiotic diffusion into the biofilm. Here, we combine the charge neutralization of tobramycin provided by dextran-based single-chain polymer nanoparticles (SCPNs) together with DNase I to break the biofilm matrix. Our study demonstrates that the SCPNs improve the activity of tobramycin and DNase I by neutralizing the ionic interactions that keep this antibiotic in the biofilm periphery. Moreover, the detailed effects and interactions of nanoformulations with extracellular matrix components were revealed through time-lapse imaging of the P. aeruginosa biofilms by laser scanning confocal microscopy with specific labeling of the different biofilm components.

Effects of noise and harmonic excitation on the growth of Bacillus subtilis biofilm

We study the dynamic growth behaviors of Bacillus subtilis biofilms by using a stochastic delay differential equation similar to the Mackey-Glass equation. We observe a wealth of dynamic behavior from a mathematical perspective. Firstly, the change of the time delay (i.e. the time that the growth state of the periphery to affect the stress of the interior cells needs and the time required for the stress signal coming from the interior to reach the peripheral cells) and the external excitation (i.e. the change in temperature) frequency will influence the size of Bacillus subtilis biofilm and the collective growth of the cell community. We also found the resonance phenomena in mathematics, which is of great help in understanding the behavior of stress levels in the biofilm periphery. Secondly, the direction of the Hopf bifurcation and the stability of the bifurcating periodic solutions are derived by using the normal form theory and the center manifold reduction. Numerical simulations are carried out to ensure theoretical results. Finally, the effects of noise on stress levels in the biofilm periphery are analyzed and reveal more complex dynamics of the system.

The Consequences of Being in an Infectious Biofilm: Microenvironmental Conditions Governing Antibiotic Tolerance.

The main driver behind biofilm research is the desire to understand the mechanisms governing the antibiotic tolerance of biofilm-growing bacteria found in chronic bacterial infections. Rather than genetic traits, several physical and chemical traits of the biofilm have been shown to be attributable to antibiotic tolerance. During infection, bacteria in biofilms exhibit slow growth and a low metabolic state due to O2 limitation imposed by intense O2 consumption of polymorphonuclear leukocytes or metabolically active bacteria in the biofilm periphery. Due to variable O2 availability throughout the infection, pathogen growth can involve aerobic, microaerobic and anaerobic metabolism. This has serious implications for the antibiotic treatment of infections (e.g., in chronic wounds or in the chronic lung infection of cystic fibrosis patients), as antibiotics are usually optimized for aerobic, fast-growing bacteria. This review summarizes knowledge about the links between the microenvironment of biofilms in chronic infections and their tolerance against antibiotics.

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

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.

Metabolic co-dependence gives rise to collective oscillations within biofilms.

Cells that reside within a community can cooperate and also compete with each other for resources. It remains unclear how these opposing interactions are resolved at the population level. Here we investigated such an internal conflict within a microbial biofilm community: Cells in the biofilm periphery not only protect interior cells from external attack, but also starve them through nutrient consumption. We discovered that this conflict between protection and starvation is resolved through emergence of long-range metabolic codependence between peripheral and interior cells. As a result, biofilm growth halts periodically, increasing nutrient availability for the sheltered interior cells. We show that this collective oscillation in biofilm growth benefits the community in the event of a chemical attack. These findings indicate that oscillations support population-level conflict resolution by coordinating competing metabolic demands in space and time, suggesting new strategies to control biofilm growth.

The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin

Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.

MRT Letter: Spatial distribution of vancomycin‐induced damage in Staphylococcus epidermidis biofilm: An electron microscopic study

This study was planned to elucidate the efficacy of antibiotics on Staphylococcus epidermidis and Staphylococcus aureus biofilms by scanning electron microscopy (SEM). Biofilms of S. epidermidis ATCC 35984 and S. aureus ATCC 29213 were grown on black, polycarbonate membranes placed on tryptic soy agar plates for 48 h at 37 degrees C, and then exposed to vancomycin or amikacin or ciprofloxacin at clinically achievable levels for 24 h at 37 degrees C. The morphology of antibiotic-treated and untreated biofilms was elucidated by SEM. SEM analysis indicated a differential affection of S. epidermidis ATCC 35984 in the center and periphery of biofilm upon treatment with vancomycin. The center of biofilm revealed damaged cells with sparse distribution, smaller size, and irregular shape, whereas cells in the periphery were unaffected. This differential distribution of susceptibility within S. epidermidis ATCC 35984 biofilms was specific for vancomycin only and was not observed on exposure to amikacin or ciprofloxacin. No such response was found in S.aureus ATCC 29213 biofilms. Thus, our study suggests a spatial distribution of vancomycin-induced damage in S. epidermidis biofilms. To our knowledge, this is the first report that indicates a differential affection of S. epidermidis in the center and periphery of biofilm upon treatment with vancomycin. Studies on the factors controlling this differential distribution could provide valuable insights into the mechanisms of antimicrobial resistance in S. epidermidis biofilms.