Posts Tagged ‘Biofilms’

New Broad-Spectrum Peptide Antibiotic Targets Biofilms

Friday, May 23rd, 2014

Biofilm Biofilms are structured multicellular communities of microorganisms associated with surfaces. They have been widely studied, in part because they cause at least 65% of all human infections, being particularly prevalent in device-related infections, on body surfaces and in chronic infections. Biofilms represent a major health problem worldwide due to their resistance to host defence mechanisms and to conventional antimicrobials, which generally target free-swimming (planktonic) bacteria. So there is an urgent need to identify compounds that effectively clear biofilm-related infections.

A new report in PLoS Pathogens identifies a potent anti-biofilm peptide that works by blocking (p)ppGpp, an important signal in biofilm development. The peptide had at least three effects on biofilms, which might reflect the role of (p)ppGpp in cells. First when added prior to initiation of biofilms it prevented biofilm formation, second it specifically led to cell death in biofilms at concentrations that were not lethal for planktonic (free-swimming) cells, and third it promoted biofilm dispersal even in maturing (2-day old) biofilms. This anti-biofilm strategy represents a significant advance in the search for new agents that specifically target many bacterial species.


Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response. (2014) PLoS Pathog 10(5): e1004152. doi:10.1371/journal.ppat.1004152
Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.


What is it about amyloids?

Tuesday, December 10th, 2013

Curli Amyloids are insoluble fibrous protein aggregates with a beta sheet structure. They have had a lot of bad publicity because they are associated with diseases such as Alzheimer’s, Huntington’s, and the spongiform encephalopathies associated with prions. In these conditions, the deposits of amyloid protein within nerve cells are toxic and lead to the death of cells and consequent neurological symptoms. But recently, we have slowly become aware that amyloids are not necessarily “evil” and that “functional” amyloids contribute to normal cellular biology, being used as structural elements by lower organisms and involved in the production of proteins such as melanin.

So what is it about amyloids – why are they so widespread in living organisms? The amyloid fold which produces the characteristic beta sheet structure of all amyloids is a pre-programmed event, so amyloid polymers can self-assemble without any exogenous energy, inside or outside the cell from which they originate. Once made, amyloid polymers are resistant to harsh conditions that would denature most proteins – a pretty useful characteristic. A new article in PLOS Pathogens looks at the various roles of bacterial amyloids in host, polymicrobial, and environmental interactions and makes some interesting suggestions (Disease to Dirt: The Biology of Microbial Amyloids. (2013) PLoS Pathog 9(11): e1003740. doi:10.1371/journal.ppat.1003740).

Amyloid fibres are common as part of the extracellular matrix that holds biofilms together. It is easy to see how useful amyloid is in this environment. The fibres are produced extracelluarly and reinforce the complex protein and polysaccharide matrix of the biofil that protects the communities of cells it contains. Escherichia coli, Salmonella enterica serovar Typhimurium, Bacillus subtilis, Staphylococcus aureus, Mycobacterium tuberculosis, and many others, all produce extracellular amyloid fibers, as do between 5–40% of species isolated from natural biofilms found in seawater, sludge, and drinking water. Amyloids are very common then, but they do much more than acting as reinforcing rods in the biofilm matrix.

Curli are fibres composed of amyloid found in the complex extracellular matrix produced by many Enterobacteriaceae such as E. coli and Salmonella (Curli biogenesis and function. (2006) Annu Rev Microbiol. 60: 131-47). Curli are involved in adhesion to surfaces, cell aggregation, biofilm formation and mediate host cell adhesion and invasion, and they are potent inducers of the mammalian inflammatory response and plant hypersensitive immune response. This is where it gets interesting because in this context amyloid has moved from being mere structural reinforcement to what is now known as “functional amyloid”. In come cases curli act as adhesins, and in a few cases (such as Klebsiella pneumoniae), as toxins which can trigger apoptosis in human cells.

The reason amyloid has had such as bad press is because the tangled plaques of amyloid fibrils found in conditions such as Alzheimer’s diseases are clearly examples of amyloid formation at the wrong time, in the wrong place or with the wrong constituents. Mature amyloid fibrils do not appear to be inherently toxic if handled appropriately by the cell and essential components of many aspects of cell biology. Finding out more about the normal as opposed to the abnormal biology of amyloid will have great benefits in understanding bacterial growth and pathogenesis. This knowledge could lead to potential therapies or probiotics to designed beneficially exploit amyloid formation in the host and environment or to counteract bacterial cell invasion mechanisms.


My Hummocks

Friday, April 19th, 2013

Bacterial surface adhesion The attachment of bacteria to solid surfaces is the first step in the formation of biofilms – communities of sessile microbes surrounded by a polymeric matrix. A growing appreciation of the ubiquity and importance of biofilms in medicine and industry has led to a proliferation of antiadhesion strategies that include chemical, biological, and physical approaches. Attempts to block biofilm formation must also take into account the rapid evolution of bacteria and their ability to resist many chemical assaults. By preventing adhesion in a manner that is nontoxic for the bacteria as well as for the application (e.g. medical implants), there is hope that we may reduce the costs associated with biofilm formation without imposing selective pressures for the development of antibiotic resistance. Physical strategies, particularly the use of rationally designed surface topographies, have gained attention recently as highly nonspecific methods for prevention of attachment without the use of antimicrobials.

Understanding how bacteria interact with surfaces that have roughness on the micrometer and submicrometer length scales comparable with the length scale of the bacteria themselves is critical to the development of antiadhesive materials. Such surfaces are also relevant for a deeper understanding of the native bacterial lifestyle, because most surfaces in nature are not atomically smooth. Indeed, the microvilli of the intestines are between 80 and 150 nm in diameter, creating considerable topographic constraints for enteric pathogens that might attempt to colonize the epithelium. Geometric considerations suggest that surfaces with roughness on the bacterial length scale provide less available surface area and fewer attachment points for rigid bacterial bodies than smooth surfaces. However, the simplistic view of bacteria as rigid rods or spheres ignores the presence of bacterial appendages, such as pili and flagella.

Previous work has demonstrated that type I pili and flagella are required for biofilm formation by Escherichia coli. Flagella have been suggested to aid in overcoming surface repulsive forces and, possibly, to aid in spreading of cells along a surface. Additionally, exopolysaccharides and surface antigens help to develop biofilm morphology. It is possible that such extracellular features also play important roles in allowing bacteria to adapt and adhere to bumpy surfaces. Using wild-type cells and deletions of several biofilm-associated genes, a recent paper in PNAS examines the role of surface appendages on adhesion to patterned substrates and show that flagella in particular appear to aid in attachment to surfaces inaccessible to the cell body, by reaching into crevices and masking surface topography. The results suggest that flagella may provide an structural function in biofilm formation additional to their role in motility.


Bacterial flagella explore microscale hummocks and hollows to increase adhesion. (2013) PNAS USA 110(14): 5624-5629. doi: 10.1073/pnas.1219662110
Biofilms, surface-bound communities of microbes, are economically and medically important due to their pathogenic and obstructive properties. Among the numerous strategies to prevent bacterial adhesion and subsequent biofilm formation, surface topography was recently proposed as a highly nonspecific method that does not rely on small-molecule antibacterial compounds, which promote resistance. Here, we provide a detailed investigation of how the introduction of submicrometer crevices to a surface affects attachment of Escherichia coli. These crevices reduce substrate surface area available to the cell body but increase overall surface area. We have found that, during the first 2 h, adhesion to topographic surfaces is significantly reduced compared with flat controls, but this behavior abruptly reverses to significantly increased adhesion at longer exposures. We show that this reversal coincides with bacterially induced wetting transitions and that flagellar filaments aid in adhesion to these wetted topographic surfaces. We demonstrate that flagella are able to reach into crevices, access additional surface area, and produce a dense, fibrous network. Mutants lacking flagella show comparatively reduced adhesion. By varying substrate crevice sizes, we determine the conditions under which having flagella is most advantageous for adhesion. These findings strongly indicate that, in addition to their role in swimming motility, flagella are involved in attachment and can furthermore act as structural elements, enabling bacteria to overcome unfavorable surface topographies. This work contributes insights for the future design of antifouling surfaces and for improved understanding of bacterial behavior in native, structured environments.



Monday, July 2nd, 2012

Biofilm formation Bacteria adhere to virtually all natural and synthetic surfaces. Although there are a number of different reasons as to why bacteria adhere to a surface, the summarizing answer is brief: “Adhesion to a surface is a survival mechanism for bacteria”. Nutrients in aqueous environments have the tendency to accumulate at surfaces, giving adhering bacteria a benefit over free floating, so-called planktonic ones. This is why mountain creeks may contain crystal clear, drinkable water, while stepping stones underneath the water may be covered with a slippery film of adhering microbes. In the oral cavity, adhesion to dental hard and soft tissues is life-saving to the organisms, because microbes that do not manage to adhere and remain planktonic in saliva are swallowed with an almost certain death in the gastrointestinal tract.

Bacterial adhesion is generally recognized as the first step in biofilm formation, and for the human host, the ability of a bacterium to adhere is a definite virulence factor, especially in immunocompromised patients and in the growing number of elderly patients relying on biomaterials implants and devices for the restoration of function after surgery, trauma, or wear. Well-known examples of biomaterials implants are dental implants, vascular grafts, and prosthetic hips and knee joints. Bacterial adhesion is a virulence factor, because it stimulates the organism to produce extracellular polymeric substances, such as polysaccharides, proteins, nucleic acids, and lipids, through which they embed themselves in a protective matrix. This protective matrix provides mechanical stability to a biofilm and constitutes the main difference between planktonic bacteria and bacteria adhering to a surface in a so-called biofilm mode of growth. Bacteria organized in biofilms are at least ten to 1,000 times more resistant to antibiotics than bacteria in a planktonic state and can cope much better with unfavorable external conditions in the host immune system than their planktonic counterparts. Not surprisingly, the fate of an infection associated with a biomaterials implant is mostly removal and replacement of the implant, at high costs to the health care system and great inconvenience to the patient.

How Do Bacteria Know They Are on a Surface and Regulate Their Response to an Adhering State? (2012) PLoS Pathog 8(1): e1002440. doi:10.1371/journal.ppat.1002440

Fungal Biofilms

Wednesday, April 25th, 2012

Biofilms are a principal form of microbial growth and are critical to development of clinical infection. They are responsible for a broad spectrum of microbial infections in the human host. Many medically important fungi produce biofilms, including Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, and Pneumocystis. This review emphasizes common features among fungal biofilms and points toward genes and pathways that may have conserved roles.


Fungal Biofilms. (2012) PLoS Pathog 8(4): e1002585. doi:10.1371/journal.ppat.1002585


Matrix-producing cannibals

Friday, November 18th, 2011

A tense bacterial standoff Cell-cell interactions are common to all living systems. Bacteria are no exception, and numerous mechanisms that use secreted products as signaling molecules are known. Among these, the so-called “quorum sensing” systems are perhaps the best studied. In quorum sensing, all bacterial cells within a population produce secreted molecules. Only when population densities are high is there a response to these compounds, thus allowing the bacteria to coordinate their behavior. However, it is clear that there is much more to bacterial cell–cell interactions than simply counting numbers and coordinating behavior. Secreted molecules also play key roles in microbial development so that different cell fates can arise and coexist within a single-species population. In addition, in settings where multiple species coexist, their interactions often are mediated through extracellular compounds. Development in one microbe can be influenced by small molecules secreted by other species.

Almost all quorum sensing studies have been performed under laboratory conditions – far removed from how bacteria actually live. What happens in the “wild” when rival bacterial gangs contest the same territory?


Interspecies interactions that result in Bacillus subtilis forming biofilms are mediated mainly by members of its own genus. PNAS USA November 10 2011. doi: 10.1073/pnas.11036301
Many different systems of bacterial interactions have been described. However, relatively few studies have explored how interactions between different microorganisms might influence bacterial development. To explore such interspecies interactions, we focused on Bacillus subtilis, which characteristically develops into matrix-producing cannibals before entering sporulation. We investigated whether organisms from the natural environment of B. subtilis – the soil – were able to alter the development of B. subtilis. To test this possibility, we developed a coculture microcolony screen in which we used fluorescent reporters to identify soil bacteria able to induce matrix production in B. subtilis. Most of the bacteria that influence matrix production in B. subtilis are members of the genus Bacillus, suggesting that such interactions may be predominantly with close relatives. The interactions we observed were mediated via two different mechanisms. One resulted in increased expression of matrix genes via the activation of a sensor histidine kinase, KinD. The second was kinase independent and conceivably functions by altering the relative subpopulations of B. subtilis cell types by preferentially killing noncannibals. These two mechanisms were grouped according to the inducing strain’s relatedness to B. subtilis. Our results suggest that bacteria preferentially alter their development in response to secreted molecules from closely related bacteria and do so using mechanisms that depend on the phylogenetic relatedness of the interacting bacteria.

A Tale of Two Biofilms

Monday, September 19th, 2011

Candida albicans One of the great advances in medical technology has unwittingly spawned a serious threat to public health. Implanted medical devices, from cardiac stents to artificial hip joints, are commonly infected with biofilms, complex microbial communities that can prove remarkably resistant to host defenses and treatment. It appears, however, that biofilms, even those arising from the same microbe species, may harbor innate differences in their response to treatments like antifungal agents. Understanding how these microbe colonies might produce structures that appear similar but have very different physical properties and functions is a critical step in figuring out how to overcome antimicrobial resistance.

In humans, Candida albicans can cause problems like oral thrush and yeast infections. Far more serious is its increasing tendency to colonize catheters, heart valves, and other medical devices, where it serves as a seeding source for potentially deadly bloodstream infections. The finding that C. albicans can form two different types of biofilm is interesting not only because it is an example of how similar structures can have very different functions, but also because it provides information about how signaling pathways may evolve by modifying preexisting signaling modules for entirely new purposes. This phenomenon may be widespread, extending beyond slimy fungal biofilms to a variety of organisms. On a practical note, when researchers are designing new methods to discourage fungal biofilms it will be useful for them to keep in mind that there are two types of biofilms being formed by C. albicans.


A Tale of Two Biofilms. 2011 PLoS Biol 9(8): e1001119. doi:10.1371/journal.pbio.1001119


Can viruses form biofilms?

Friday, May 6th, 2011

HTLV Infection It is commonly accepted that the spread of most viruses occurs via the diffusion of ‘cell-free’ viral particles. In support of this, infectious viruses have been found in biological fluids and aerosols, and could be propagated in vitro using virus stocks produced from infected cell-culture supernatants. This mode of viral dissemination requires high numbers of stable viral particles released by the infected cell into the extracellular environment, such as the host bodily fluids. ‘Free’ viral particles were associated with a variety of components, such as lipids or proteins, which might reinforce virus envelop integrity and prevent envelope glycoprotein shedding. However, for other viruses, few viral particles are released, or they are poorly infectious when separated from infected cells. In such cases, virus propagation largely requires the presence of infected cells, suggesting that cell contacts mediate viral spread. This type of dissemination is mainly reported for enveloped viruses, such as some herpes viruses and some retroviruses, particularly deltaretroviruses such as the human T-cell leukemia virus type 1 (HTLV-1). Many aspects of this mode of virus dissemination are largely unknown, such as (i) the nature and the mechanism for forming cellular junctions that mediate cell–cell virus spread, and (ii) the nature of the infectious material transferred. Both of these factors depend on the virus and the type of infected cells.

Of note, the spread of two human retroviruses that infect leukocytes, HIV-1 and HTLV-1, occurs between mobile cells forming dynamic contacts with other cells. Both retroviruses cause severe chronic viral infections. Their transmission between individuals occurs through sexual contact and blood transfusion, and vertically from mother to child, including through breastfeeding. In addition to HTLV-1 dissemination through division of infected cells carrying viral genomes, transmission of HTLV-1 viral particles is known to occur mainly through cell contact in vitro and in vivo, with the exception of dendritic cells, which can be infected by cell-free viral particles. By contrast, HIV-1 spread can occur by both diffusion and direct cell–cell transfer. Nevertheless, compelling evidence indicates that direct spread through cell contact is the most efficient mode of HIV-1 dissemination in vitro, and might play a crucial role in vivo:

Can viruses form biofilms? Trends Microbiol. Mar 31 2011
The recent finding that the human T-cell leukemia virus type 1 (HTLV-1) encases itself in a carbohydrate-rich adhesive extracellular ‘cocoon’, which enables its efficient and protected transfer between cells, unveiled a new infectious entity and a novel mechanism of viral transmission. These HTLV-1 structures are observed at the surface of T cells from HTLV-1-infected patients and are reminiscent of bacterial biofilms. The virus controls the synthesis of the matrix, which surrounds the virions and attaches them to the T cell surface. We propose that, similar to bacterial biofilms, viral biofilms could represent ‘viral communities’ with enhanced infectious capacity and improved spread compared with ‘free’ viral particles, and might constitute a key reservoir for chronic infections.

Social interactions in a unicellular world

Wednesday, January 12th, 2011

Social interactions in a unicellular world Traditionally, research in bacterial communication and co-operation has been performed at the molecular level and less attention has been paid to evolutionary and ecological factors which govern such actions. But Steve Diggle and Roman Popat ask in this article in Microbiology Today, what is the relevance of social evolution for microbes?

Most of us at some point have had the rather unpleasant experience of putting our fingers down a plug-hole only to pull up a slimy goo. As microbiologists are now well aware, this is a large mass of microbial cells that we commonly refer to as a biofilm, and such microbial communities can be found almost everywhere. They are the pioneers of rocky shores which help enable seaweed to settle on rocks. They cause problems in industrial settings such as contamination of beer lines. Clinically, they are of huge importance, contributing to infection, colonization of medical devices and antibiotic resistance. Biofilms consist of numerous cells, often belonging to a number of diverse species surrounded by a complex exopolysaccharide matrix. They remind us that bacterial cells interact with each other and inevitably lead social lives. Recently, there has been a growing interest in studying aspects of sociality using bacteria or other microbial study systems.

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