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``Ironing out'' the Problem - Bacterial Biofilm Development Dr. Ehud Banin
The increase in bacterial antibiotic resistance is a major concern for clinicians and medical officials worldwide.
One of the modes by which bacteria enhance their resistance is to create biofilms.
Biofilms are surface-associated bacterial communities encased in an extracellular polymeric matrix.
The problem of bacterial biofilm formation on abiotic surfaces (estimated to cost many billions of dollars each year) is common to a wide range of both medical and industrial problems.
The prevalence of biofilm formation and the difficulties in biofilm removal, make biofilm prevention a major research challenge at the interface between microbiology and materials science.
Dr. Banin's research focuses on understanding the basic aspects of the signals and processes involved in biofilm development with a goal of finding new methods of treating biofilm-related infections.
A main interest in the laboratory is to uncover the role of iron in biofilm development.
Biofilm development is known to follow a series of complex but discrete and well regulated steps.
Adhesion initially involves reversible association with the surface. As this proceeds bacteria undergo irreversible attachment with the substrate through cell surface adhesions.
In later stages bacteria will start secreting a protective extracellular matrix and form microcolonies that develop into mature biofilms.
These structures protect the bacteria from host defenses and systemically administered antibiotics.
An important characteristic of microbial biofilms is their innate resistance to immune system- and antibiotic-killing.
This has made microbial biofilms a common and difficult-to-treat cause of medical infections.
It has recently been estimated that over 60% of the bacterial infections currently treated in hospitals are caused by bacterial biofilms.
The number of implant-associated infections approaches 1 million/yr in the US alone and their direct medical costs exceed $3 billion annually.
Thus, there is an urgent need to find novel approaches to eradicate biofilms.
Iron is an essential element for most living organisms. Recent work has shown that iron concentration serve as a signal for biofilm development.
By sequestering iron, sub-growth inhibitory concentrations of the mammalian iron chelator lactoferrin block the ability of P. aeruginosa biofilms to mature from thin layers of cells attached to a surface into large multicellular biofilm structures (Fig. 2).
Dr. Banin's work has shown that P. aeruginosa requires active iron transport to support normal biofilm development and P. aeruginosa could effectively be killed and dispersed by exposing them to a strong chelator.
The discovery that iron acts as a critical checkpoint in biofilm development provides us with an important tool to investigate biofilm physiology.
Dr. Banin is using iron as a valuable ``switch'' to intervene at defined points in the biofilm process, and he can now better understand both the role iron plays in mediating biofilm formation and gain significant knowledge of the basic processes required for successful biofilm development and maintenance.
The genetic and genomic approaches Dr. Banin is taking are expected to reveal genes that are directly involved in biofilm formation and dispersal as well as genes involved in iron-regulation and signaling.
Under normal iron concentrations, bacteria attach, multiply and develop into microcolonies that mature into structured biofilms.
In low iron, the cells show increased surface motility, they attach and multiply but daughter cells move away from the point of replication and thus do not form microcolonies and structured biofilms.
Another major theme in the laboratory is the search for novel antibiofilm agents.
Dr. Banin's preliminary findings suggest that by interfering with bacterial iron homeostasis we may be able to eradicate bacterial biofilms.
Based on this, Dr. Banin is currently testing novel desferrioxamine-metallo complexes.
Because P. aeruginosa posses two uptake systems for ferrioxamine (the iron loaded form of desferrioxamine), there is reason to predict there might be a synergistic effect of imposing iron limitation by directly delivering the toxic metal loaded in the DFO molecule to the cells via the ferrioxamine uptake systems (``Trojan horse'' approach) and by sequestering any free available iron by the siderophore.
Results show these complexes effectively block biofilm formation and can eradicate mature biofilms when combined with antibiotic treatment.
Dr. Banin had similar success in vivo using a P. aeruginosa eye infection (keratitis) animal model. Topical addition of DFO-complex plus gentamicin decreased both infiltrate and final scar size by about 50% compared to topical application of the antibiotic alone.
Another approach Dr. Banin is taking to try and develop novel antibiofilm coating is based on nanotechonology.
In collaborations with researchers in the Center for Advanced Materials and Nanotechnology at Bar-Ilan, Dr. Banin is utilizing novel surface nanofabrication techniques that allow us to change surface properties such as charge and topography as well as attach nanocrystals with antimicrobial activity inorder to create sterile abiotic surfaces.
The recent advancements in biological research tools provide Dr. Banin with the opportunity to begin and explore fundamental aspects of bacterial life-style such as the processes that lead to the development of biofilms.
At the same time there is an immediate necessity for discovery of novel antimicrobial agents as pathogenic bacteria rapidly gain resistance to existing antibiotics.
These two paths converge as our improved knowledge on bacterial physiology and resistance can assist in developing novel therapeutic approaches.
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This begs the question, "Has anyone really proven that Bart or Haemobart really has a biofilm or is that just theoretical at this point?"
I listened to Dr Fry from the radio interview and got the impression that he believes in the biofilm theory, but in my opinion it is just a theory at this point.
Hubby had iron shots at one point for at least 6 months and currently has a low RBC and is most likely anemic -- hard to say that either supplemental iron or iron deficiency has made a difference in how he has responded to treatment.
And if Marnie is right then supposedly extra iron is toxic to Lyme (couldn't prove this by hubby) but the iron feeds the Bart and presumably Babesia as well.
Bea Seibert
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sparkle7
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I'm not a scientist or know much about this but from what I've read it seems that the biofilms may be different with each individual bacteria.
Just an educated guess...
If someone knows - please post.
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posted
Here is one recent paper on biofilms. I will post another after this. My impression is that biofilm formation may not always be the same. If iron is not essential to Bb, then adding iron or subtracting it will presumably not change a biofilm containing Bb.
However, as you can see in the second abstract, the partners in a biofilm change into an association with characteristics that are different from the individual components.
Biofilms have long been known to comprise architecturally complex communities of sessile bacterial cells, but what switches off bacterial motility within biofilms has been a mystery. Now, a new study published in Science reveals that in Bacillus subtilis biofilms, motility is switched off through the activity of a protein that acts as a clutch and disables the flagellar motor.
In B. subtilis, motility and biofilm formation are oppositely regulated by the master regulator SinR. Previous work had shown that SinR is a direct negative regulator of the eps operon, which is responsible for the production of the extracellular matrix that is essential for biofilm formation, but precisely why sinR mutants are non-motile was unknown.
Kris Blair and colleagues began by looking at flagellar distribution and function in sinR mutants using a fluorescently labelled flagellar filament protein. They found that in sinR mutants, flagella are present but non-functional.
To investigate in more detail how SinR regulates flagellar function, Blair et al. looked for suppressor mutations that restored motility in sinR mutants. Of the 18 suppressors that were isolated, 9 mapped to the epsE gene, and introduction of wild-type epsE could complement the inhibition of motility.
The authors therefore concluded that sinR mutants are non-motile because epsE is derepressed. Further work revealed that EpsE is sufficient for motility inhibition and that this inhibition involves stopping flagellar rotation.
So what is the target for EpsE? Again, the authors turned to suppressor mutations, but this time looked for suppressors that rescued motility in epsE mutants.
All of the suppressors that were isolated mapped to fliG, which encodes a component of the flagellar motor that is responsible for transmitting torque to the rotary motor through the MotA/B proton channel. The subcellular localization patterns of EpsE were studied in different mutant backgrounds using a fluorescent EpsE fusion protein, and the results confirmed that EpsE interacts with FliG in vivo and that this interaction is responsible for motility arrest.
Finally, by tethering B. subtilis cells to a surface by a single flagellum and observing the rotation of the cell body in the presence and absence of EpsE, the authors discovered that EpsE acts as a clutch that disengages the flagellar rotor from the power source, the MotA/B proton channel.
So, the motility of B. subtilis cells within biofilms is shut down through the use of a molecular clutch, EpsE. The authors conclude that clutch control is a "simple, rapid and potentially reversible" way to switch off motility.
ORIGINAL RESEARCH PAPER
Blair, K. M. et al. A molecular clutch disables flagella in the Bacillus subtilis biofilm. Science 320, 1636-1638 (2008)
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Nature 445, 533-536 (1 February 2007) | doi:10.1038/nature05514; Received 13
September 2006; Accepted 8 December 2006
Evolution of species interactions in a biofilm community
Susse Kirkelund Hansen1, Paul B. Rainey2, Janus A. J. Haagensen1 and S�ren Molin1
1. Infection Microbiology Group, BioCentrum-DTU, The Technical University
of Denmark, Building 301, DK-2800 Lyngby, Denmark 2. School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
Biofilms are spatially structured communities of microbes whose function is dependent on a complex web of symbiotic interactions1, 2. Localized interactions within these assemblages are predicted to affect the coexistence of the component species3, 4, 5, community structure6 and function7, 8, 9, 10, but there have been few explicit empirical analyses of the evolution of interactions11. Here we show, with the use of a two-species community, that selection in a spatially structured environment leads to the
evolution of an exploitative interaction. Simple mutations in the genome of one species caused it to adapt to the presence of the other, forming an intimate and specialized association. The derived community was more stable and more productive than the ancestral community.
Our results show that evolution in a spatially structured environment can stabilize interactions between species, provoke marked changes in their symbiotic nature and affect community function.
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posted
Here is an outfit working on the problem of biofilms:
The Centers for Disease Control and Prevention estimate that 65 percent of human infections involve biofilms. Biofilm infections are less susceptible to antibiotics and contribute to the increased occurrence of resistant strains of bacteria.
"It is now abundantly clear that many chronic infections are established and persist because the bacteria involved form biofilms that resistant host defenses and conventional antimicrobial agents," said Professor Bill Costerton, Director of the Center of Biofilm Engineering. "The most effective potential therapeutic strategy that emerges from this new biofilm concept is the use of chemical signals and signal inhibitors to control or reverse biofilm formation. Biofilm control signals are used by aquatic plants to control microbial fouling on their photosynthetic surfaces. Natural compounds these plants use have great potential in the effective control of biofilm formation (and chronic bacterial infection) in patients, in both medical and dental contexts."
Confocal scanning laser microscope micrographs are shown of mixed biofilms containing Acinetobacter sp. C6 (red) and ancestral Pseudomonas putida (green) (a), and Acinetobacter sp. C6 (red) and a rough variant of P. putida (green) (b). Figure reproduced from Nature 445, 533-536 (2007).
Biofilm communities are not simply surface-adherent mixtures of bacterial species, rather they are dynamic and structurally complex systems. As biofilms might be the default mode of bacterial life, understanding how they assemble, function and evolve is fundamentally important. Now, publishing in Nature, Paul Rainey, Sّren Molin and colleagues have used a deceptively simple approach to reveal how bacterial species can interact and evolve to form a stable biofilm.
When Acinetobacter sp. (strain C6) is grown with benzyl alcohol as a sole carbon source it secretes benzoate. Pseudomonas putida (strain KT2440) cannot grow on benzyl alcohol, but it can catabolize benzoate. So P. putida can thrive when benzyl alcohol is the only carbon source supplied, but only if Acinetobacter is also present. Rainey and colleagues exploited this simple metabolic partnership to examine biofilm development.
Initial experiments revealed that when these species were grown together on a surface, they were able to utilize a drastically reduced concentration of benzyl alcohol compared with that required when co-cultured in liquid suspension. This is because local interactions -- particularly the development of chemical gradients -- are possible when bacteria grow in a spatially structured environment such as the glass surface used in this study. In well-mixed liquid cultures, however, neighbours constantly change and opportunites for local interactions are reduced.
Microscopic inspection revealed that Acinetobacter colonies (see figure, panel a) were initially surrounded by groups of P. putida colonies. Within 5 days a more intimate association developed, with a mantle of P. putida coating Acinetobacter colonies (see figure, panel b). Growing samples from these biofilms on agar plates revealed that they all contained a stable rough-colony variant of the P. putida strain (the wild-type strain is smooth).
The genetic basis of this switch was pinned down to mutations in a gene encoding a key lipopolysaccharide biosynthetic enzyme. The rough variant only evolved when the ancestral P. putida strain was grown on a surface in the presence of Acinetobacter. After reinoculation of biofilm chambers with the rough variant of P. putida and Acinetobacter the intimately associated biofilm, which took five days to form previously, formed in just one day, showing that the evolutionary adaptation promoted biofim formation. Further experiments confirmed that the evolved rough variant was fitter than the ancestral wild-type strain, but only when it was grown in a biofilm with Acinetobacter.
These experiments beautifully clarify the first steps in the formation of a mixed-species biofilm. Because the evolution of the rough variant only took place in a mixed culture and in a spatially structured environment, this research shows that spatial structure is intrinsically linked to the evolution of new functions in biofilm communities.
1.
Hansen, S. K., Rainey, P. B., Haagensen, J. A. J. & Molin, S. Evolution of species interactions in a biofilm community. Nature 445, 533-536 (2007)
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ByronSBell 2007
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This topic is a "key" to getting well or staying sick. If you can't get rid of the biofilm, you cant get rid of the infections.
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sparkle7
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The idea is that the biofilms are different depending on the bacteria. So, Bb would have a different chemical make-up than some other bacteria. It would require different solutions based on the types of bacteria...
I sort of imagine them being like a city. From what I've read - they can incorporate more than one bacteria & they can communicate with each other to trade genetic materials to benefit the colony.
This is why it's so hard to bust them up. After they detatch - they can start new colonies using the gained genetic materials to make them resistant to abx, etc.
If this is correct, some researchers are looking for ways to stop the communication (called quorem sensing).
They seem to behave alot better than most human communities do. Maybe we should learn a lesson from them?
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