Download A life in slime – biofilms rule the world

A life in slime – biofilms rule the world
Author: Dr. Kristen Kerksiek
September 3, 2008
In 1905, Robert Koch (1843-1910) was awarded the Nobel prize for his identification of Mycobacterium tuberculosis as the causative agent of tuberculosis. More
than a century later, the methods Koch used to grow bacteria in culture, which
have enabled the identification of countless microorganisms as well as insight
into the way they "work", are little changed and still form the foundation of medical microbiology. It therefore seems ironic that the pure cultures that have
proven so valuable in the laboratory are virtually absent in nature: >99.9% of bacteria exist in heterogeneous communities called biofilms.
It wasn’t until the 1970s that scientists realized the ubiquitous nature of biofilms, which form when microorganisms
(bacteria, fungi, archaea, algae) attach to wet surfaces (living or nonliving) and encase themselves in a slimy extracellular polymer matrix. Biofilms are critical component
of food chains in rivers and streams – they prefer nutrientrich, flowing water – but can be found in even the most
hostile environments, from the scorching-hot, acidic pools
of Yellowstone to glaciers in the Antarctic. They can be
composed of single species but tend to be diverse: more
than 300 bacterial species have been identified in biofilms
that make up dental plaque. And while many acute infecA mature biofilm of P. aerugitious diseases are caused by free-floating (also known as
nosa grown for 5 days in a flow
cell system. Living cells appear
planktonic) bacteria, the National Institutes of Health
green and dead or inactive cells
(NIH) now estimates that upwards of 80% of infectious
appear red. © A. Dötsch / S.
Häußler, HZI
diseases in the developed world are caused by biofilms. Research in the 1980s and 1990s revealed that biofilms are not
just masses of microorganisms and the slime that connects
and protects them, they are elaborately organized and differentiated communities with an enhanced ability to resist environmental stresses.
The building of a biofilm: cities in slime
Humans and a wealth of other animal species live in communities, benefiting not only from
“safety in numbers”, but also from a structure that makes survival easier by sharing the
work…and the risk. Community living is advantageous not only for individuals but also for survival of the species. We are accustomed to thinking about bacteria in terms of free-floating entities, but they (and other microorganisms) have embraced the advantages of group living.
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The founding of a biofilm is humble: “pioneers” bind with
weak, reversible forces to a moist surface and, if they can
stay fixed to the substrate, attach more permanently by cell
adhesion. This attachment activates the transcription of different genes; in fact, the genes expressed by biofilm cells of
different species are more similar than genes expressed by
biofilm and planktonic cells of the same species. Some of
these biofilm-induced genes stimulate secretion of a sticky
extracellular matrix, which composes ~85% of a mature
biofilm. As the landscape becomes more diverse (and
sticky), other microorganisms are able to join. Microcolonies, the basic unit of a biofilm (analogous to tissues forming
an organ), form by recruitment of free-floating cells or cell
clumps as well as by aggregation of cells and cell division.
The microcolonies typically assume a mushroom or pillar
shape and are separated from one another by waterways that
serve to channel in nutrients and oxygen and remove wastes,
like miniature cities in slime.
The material surrounding the
abdomal pore’s orifice of a spider (Loxosceles reclusa) is
composed of an unidentified
bacterial biofilm. © J. H. Carr
CDCI
Formation of a biofilm follows this basic scheme irrespective of the environment and the participating microbial species. Dispersal of these microbial communities may be triggered by external factors (e.g. increased shear stress) or internal biofilm processes. Detachment can proceed
in different ways: biofilms often release cells in clumps but may also discharge planktonic cells
(“swarming/seeding”) or move across surfaces as an intact biofilm.
The establishment of biofilms is not random. Both the construction and the dispersion of such
structured multicellular communities is regulated by quorum sensing, cell-to-cell signaling that
controls population density-dependent gene expression; the microbes secrete small diffusible
signaling molecules (analogous to hormones) and can detect how many other microbes are in the
vicinity by the local concentration of the substance. They are also very responsive to external
factors, altering signaling based on environmental input. The “chemical vocabulary” differs depending on the species producing it, but bacteria also participate in cross-talk between species.
Breakthroughs in quorum sensing:
In a landmark study, Singh and researchers showed that P. aeruginosa in lungs of cystic fibrosis patients communicate like a biofilm: P. aeruginosa can secrete two forms of the signaling molecule, and while planktonic forms secrete primarily the long form, bacteria in
biofilms - and from patients - secrete the short form. (Nature 2000 407: 762–764)
Vibrio cholerae, the bacteria that cause cholera, form biofilms in the human intestine. Work
from the Bassler laboratory showed that cholera bacteria secrete a molecule called CAI-1,
and when enough has accumulated (i.e. the bacteria have multiplied enough), they release
toxins, which initiates release from the body. (Nature 2007 450: 883–886)
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The structural organization of the biofilm allows all “members” to receive the basics they need
to thrive. The variety of microenvironments within the biofilm provide niches for microorganisms with diverse phenotypes, strengthening the ability of the community to resist diverse environmental stresses as well as biocides, the immune system and antimicrobials.
The good and the bad…
Bacterial biofilms aren’t a new phenomenon; fossil records show their presence at least 3.25 billion years ago. Biofilms form wherever water is present and form an integral part of aqueous
ecosystems. They have been harnessed to our benefit in water treatment plants, where they help
remove pathogens and reduce the amount of organic matter in the water, and oil-and gas-eating
biofilms are invaluable in cleaning up contaminated soil and water.
However, biofilms can also be a nuisance…and in some cases, downright dangerous. They are
notorious for their pipe-clogging and -corroding properties, a costly irritation in industrial settings. And there is an increasing awareness that pathogenic bacteria also appreciate the benefits
of living in a community, with biofilms playing a prominent role in infectious disease.
Biofilms and disease
In wealthy, industrialized nations, improved sanitation and
hygiene standards as well as immunization and antibiotics
have dramatically reduced the spread of infectious diseases…but they are not gone. Estimates of the frequency of
infections caused by biofilms (bacterial and/or fungal) lie between 65% (Centers for Disease Control and Prevention/CDC) and 80% (NIH).
Biofilms are responsible for a significant portion of acute infections. A classic case is that of Legionnaire’s disease, an
acute respiratory infection resulting from aspiration of
clumps of Legionella biofilms detached from air and water
This scanning electron microheating/cooling systems. Many foodborne pathogens such as
graph (SEM) depicts a number
of Leptospira sp. bacteria atop a
E. coli 0157:H7, Listeria monocytogenes, Yersinia enteroco0.1. µm polycarbonate filter
litica, Salmonella spp. and Camphylobacter jejuni can form
either single-species or multi-species biofilms on food surfaces and equipment, and these are highly resistant to biocides, drying, heat, antibiotics, sanitizers and other stresses. With the realization that biofilms are more widespread than previously
thought, new research is revealing that many other pathogenic bacteria utilize biofilms to survive and infect. For example, biofilms of Cholera appear to play a central role in the dissemination of disease. And researchers at the Institut Pasteur recently found that species of Leptospira,
which cause the deadly emerging disease Leptospirosis, also readily form biofilms, which could
explain the long-term survival of the bacteria in water and in the kidneys of rats and other
mammalian carriers (Microbiology 2008 154: 1309–1317).
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An even greater number of chronic infections are associated
with biofilms. Persistent infections in the context of indwelling medical devices are clearly linked to biofilms. Many
pathogenic microorganisms grow readily on the materials
used for catheters, artificial joints, mechanical heart valves
and other devices. The microbial culprits are many of the
same responsible for a wide spectrum of nosocomial infections (see Perspectives, August 2008): coagulase-negative
staphylococci, Staphylococcus aureus, Enterococcus faecalis, Streptococcus spp., Escherichia coli, Klebsiella pneumoniae, Acinetobacter spp., Proteus mirabilis, P. aeruginsa
and Candida spp.. In many cases, it is impossible to eradicate the infection using antimicrobial agents, and the implant
has to be removed, which is expensive as well as dangerous
and traumatic for the patient.
A biofilm containing the rodshaped bacteria, Alcaligenes xylosoxidans in association with
fibrinlike material on a catheter’s
surface. © J. H. Carr CDC
It has taken longer to make a clear connection of biofilms to
persistent infections in native tissue. However, in the last years there has been increasing evidence that biofilms, either as single species or mixtures of bacterial and/or fungal species, are
responsible for a number of chronic infections. Some of these (e.g. otitis media, prostatitis) are
infections in which negative cultures are common despite clear clinical symptoms. Biofilm
growth seems to make the microorganisms less conspicuous to the immune system and/or the
immune system less effective in fighting them, and many biofilms are highly resistant to antimicrobials. The persistent nature of some infections may also be caused by periodic release of
biofilm fragments, spreading the infection to other locations within the body. Biofilm infections
tend to be moderate in virulence, serving the interest of the infecting pathogens by increasing the
longevity of the host.
Biofilms and chronic infections:
In many cases, biofilms cannot be proven to be the cause of disease according to Koch’s
postulates, but very strong associations have been determined for some chronic infections.
Otitis media (middle ear infections): Analysis of biopsy specimens as well as animal models indicate that bacterial biofilms on the eardrum are the main cause of both recurrent
and chronic childhood ear infections.
Cystic Fibrosis: Pseudomonas aeruginosa repeatedly infects most CF patients and becomes a chronic infection (forming biofilms) in >80% of adult patients, accelerating lung
damage.
Periodontal disease: Dental plaque is a biofilm that is responsible for dental caries, gingivitis and periodontitis. Dental biofilm in periodontitis seems to be associated with a variety of systemic diseases (e.g. cardiovascular and respiratory diseases, diabetes mellitus)
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Chronic bacterial prostatitis: Electron microscopy of biopsy specimens collected from
men with prostatitis clearly showed bacterial biofilms.
Native valve endocarditis: Fibronectin secreted at the site of damaged vascular endothelium (typically valves) enables bacteria or fungi in the bloodstream to adhere, initiating
biofilm formation.
Sinusitis: Biofilms are found on tissues from 80% of patients undergoing surgery for sinusitis.
Biofilms have also been shown to be associated with chronic corneal infections (connected
to contact lens use), diabetic wounds, tonsillitis, osteomyelitis, urinary tract infections, biliary tract infections, infectious kidney stones…the list keeps growing.
It’s not that Koch had it all wrong. Many pathogens that
cause acute infection are planktonic, and the culture methods
he developed for free-floating bacteria are incredibly valuable for medical diagnoses and microbiological studies, including the development of antibiotics. However, when diseases are caused by biofilms, traditional culture methods and
the antimicrobial agents developed using them are often ineffective.
Antimicrobial resistance
Robert Koch
Microorganisms in biofilms are different from their planktonic counterparts: they express distinct genes, they exhibit large metabolic heterogeneity depending on their position within the biofilm, and their close contact may facilitate genetic transfer. The extreme resistance of biofilms to antibiotics – up to 1000-fold more resistant than
planktonic bacteria of the same strain – has caught us off-guard but should perhaps come as no
surprise: antibiotics are tested on planktonic bacteria, not on biofilms.
One way biofilms may resist treatment with antibiotics is by preventing their entry. However,
the structural heterogeneity of biofilms seems to be more important in resistance than the physical barrier of biofilm exopolymers: the presence of niches with low levels of oxygen and nutrients results in microbial populations with low metabolic activity, and in a dormant state, bacteria
are relatively resistant to antibiotics. During antibiotic treatment, metabolically active bacteria in
the outer layers of the biofilm may be killed, but a few “persisters” can initiate regrowth of the
biofilm when treatment is discontinued; the sacrifice of individuals on the outer wall of the
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biofilm, which shields microbes in the interior, enables the survival of the community.
Persister variants (as opposed to mutation or spread of a resistance gene) seem to be the major
factor in resistance of biofilms to antibiotics, as the bacterial survivors remain susceptible to antibiotics. That is, they remain susceptible if they ever were in the first place. In the hospital environment, where antibiotic resistance is a widespread problem, the development of biofilms complicates treatment even more. For example, infections related to medical devices are caused primarily by Staphylococcus epidermis, which is commonly multidrug resistant even when in
planktonic form. In addition to figuring out which antibiotic might be effective against such resistant pathogen, physicians have to consider the innate antibiotic resistance of biofilms. New
research is hoping to turn the tide by targeting the biofilms themselves.
Opportunities for treatment
Biofilms are difficult to work with in the lab, but with the realization of the importance of
biofilms in infectious disease, there have been many advances in research. The recognition that
biofilm formation is a genetically programmed developmental process was a huge step forward,
opening the possibility for directed development of new chemotherapeutic agents.
There are a variety of strategies in the search for effective treatments for biofilm infections, including prevention of bacterial cell adhesion, reduction of polysaccharide production and disruption of cell-to-cell communication. In particular, the discovery of quorum sensing communication has generated a lot of hope that compounds can be developed to interfere with biofilm formation or induce biofilm dispersion.
Recent research that gives hope…
Biofilms of Staphylococcus aureus use autoinducing peptides (AIPs) to communicate by
quorum sensing. When Horswill and colleagues synthesized AIPs and added them to
established S. aureus biofilms, the biofilms rapidly dispersed, and the released bacteria
regained susceptibility to antibiotics. (PLoS Pathogens 2008 4: e1000052)
It’s difficult to treat biofilms. Why not prevent their growth on medical devices entirely?
The Van Vliet group at MIT found that S. epidermidis and E. coli adhere preferentially to
stiffer polymers, giving hope that smart selection of implant materials can help fight biofilm
formation. (Biomacromolecules 2008 9: 1571–1578)
The slimy extracellular matrix of biofilms protects microorganisms from a host of enemies
including the immune system, antimicrobials and most bacteriophages, the viruses that
infect bacteria. Lu and Collins engineered bacteriophages to secrete slime-dissolving
enzymes, enabling their access into the biofilm interior where they continue to destroy the
biofilm by overproducing enzymes and then kill the bacteria. (Proc. Natl. Acad. Sci. USA
2007 104: 11197–11202)
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Biofilms are everywhere, forming an integral part of the world around us. Unfortunately they are
common uninvited guests within us as well. Biofilms present special challenges in infectious
diseases, but recent research indicates we are gaining ground. Perhaps we may even be able to
utilize biofilms in fighting infectious diseases in the near future: phagocytes are a critical
component of our immune defenses, but they seem to be powerless against biofilms. Similarly,
marine biofilms successfully defend themselves against amoebae. But it’s not all defense.
Research from the laboratory of Dr. Carson Matz at the Helmholz Center for Infection Research
in Braunschweig shows that marine biofilms are also offensive, producing chemicals that can
kill their protozoal enemies (read more). There is hope that the toxins they produce will also be
effective against human protozoal diseases such as malaria, Chagas’ disease and sleeping
sickness. (PLoS ONE 2008 3: e2744)
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Further information:
ƒ http://www.erc.montana.edu/ for general information (Biofilm basics) and updates on recent
research BiofilmsOnline online journal)
ƒ Lewis K. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. (2007) 5:48–
56.
ƒ Hall-Stoodley L, Costerton JW, Stoodley P. Bacterial Biofilms: from the natural environment
to infectious diseases. Nat. Rev. Microbiol. (2004) 2: 95–108.
ƒ Costerton W, Veeh R, Shirtliff M, Pasmore M, Post C, Ehrlich G. The application of biofilm
science to the study and control of chronic bacterial infections. J. Clin. Invest. (2003) 112:
1466–1477.
ƒ Stoodley P, Sauer K, Davies DG, Costerton JW. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. (2002) 56: 187–209.
ƒ Donlan RM, Costerton JW. Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. Clin. Microbiol. Rev. (2002) 15: 167–193.
ƒ Pace JL, Rupp ME, Finch RG. Biofilms, Infection and Antimicrobial Therapy. (2006) CRC
Press. (Can be partially viewed on books.google.com)
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