Paradigm of Infectious Disease
Bacteria in biofilms tend to be more difficult to culture and
more resistant to control strategies (antibiotics and biocides) and host
defenses than when grown planktonically in the laboratory. Their resilience
has been related to physiology and protection by the EPS slime matrix that
they produce. These phenomena may explain seemingly conflicting features of
the disease when signs and symptoms are otherwise consistent with
- Chronic in Nature
- Culture Negative
- Poor Response to Antibiotics
- Potential for Metastasis
The more we study biofilms the more we found out about their structural
complexity and development. This schematic was drawn by Peg Dirckx from the
Center for Biofilm Engineering to incorporate various biofilm behaviors and
concepts based largely on observations from confocal and time-lapse
microscopy. An interactive version can be found at http://www.erc.montana.edu/MultiCellStrat/default.html.
A version of this schematic has also been published in Nature Reviews
Microbiology 2, 95-108 (2004). Click on the image for a larger version.
Confocal image (left) of a Mycobacterium
fortuitum biofilm illustrating key biofilm characteristics. The individual
bacterial cells are stained green with a nucleic acid stain which stains the
DNA. The surrounding EPS matrix is stained red with a lipophilic stain,
giving both locational and compositional information. The cells and EPS are
organized into a secondary structure of cell clusters separated by water
channels and voids (black areas). M.
fortuitum is an opportunistic bacterium that inhabits water and
can form biofilms. It can cause cervical lymphadenitis and otomastoiditis in
children as well as cutaneous, wound, ocular and catheter-related infections.
Image provided by J. Janzen & L. Hall-Stoodley. A scanning electron
microscopy (SEM) image of a M. fortuitum
biofilm is shown for comparison (right). Single cells (blue arrow), branching
cells (red arrow) and putative EPS (green arrow) are indicated. The SEM has
higher resolution but requires dehydration which can result in artifacts. The
combination of microscopic techniques enhances interpretation of
What are Biofilms?
Biofilms are difficult to define because of their
diversity, both in terms of the micro-organisms that inhabit them and the
environmentsin which they are found. One working definition is:
Biofilms are communities of micro-organisms encased
within an extracellular polymeric slime (EPS) matrix living on surfaces.
Biofilm microbiology is often contrasted with
What are Planktonic Cells?
Planktonic or free-floating microorganisms come in a
number of forms:
Planktonic cultures are cultures that are
"forced" to grow primarily as single cells under constant mixing
in conventional chemostat or batch shake flask type cultures.
Microbial flocs have many of the same characteristics
as biofilms but are not attached to a solid surface. Flocs are suspended
aggregates of micro-organisms within an EPS matrix that GREW in liquid
Detached biofilm particles can be single cells or
clumps of cells that GREW on a surface in a biofilm and subsequently
detached due to either a biological or environmental stimuli. In an
infectious disease context "biofilm emboli" may be a useful term
for detached biofilm.
At the CGS we are particularly interested in the dynamic nature of
biofilms and how these composite microbial communities change over time,
moving, shedding and re-growing adherent colonies.
The spatio-temporal aspect of biofilm development underlies several key
research questions that remain to be elucidated such as:
- Why are biofilms less
susceptible to antimicrobial treatments than free-floating bacterial
- How do biofilm organisms respond
to surfaces genetically and phenotypically?
- How does biofilm development
proceed over time?
- How do hydrodynamics and shear
affect biofilm development, structure and detachment?
- How does detachment of the
biofilm play a role in overall biofilm development?
These questions require state-of-the-art microscopic imaging capabilities
in order to examine the spatio-temporal relationships between biofilm
structure and function. Both confocal microscopy and real-time imaging are
essential components of these investigations that allow us to use fluorescent
molecular reporter probes to follow gene regulation during biofilm
Biofilms and Infection
In industrial systems cells on surfaces (biofilms) may be difficult to
culture, hard to kill and persist for long periods of time (years) resulting
in problems of chronic contamination. Similar criteria are now increasingly
being used to flag a putative biofilm etiology for chronic illnesses in which
signs and symptoms suggest infection, but cultures may be negative and the
illness does not respond to antibiotic treatment. The Biofilm Paradigm may
explain these contravening data. Initial links between biofilms and
persistent infection were made in the context of indwelling devices such as
catheters and implants. For an online CDC review see Biofilms and
Device-Associated Infections by Rodney M. Donlan. More recently the Biofilm
Paradigm has been increasingly applied to explain infection on native tissue.
These diseases include periodontitis (teeth and gums), tonsillitis (tonsils),
Cystic Fibrosis respiratory illness (lungs) otitis media (middle ear),
osteomyelitis (bone) and urinary tract infections (bladder).
Research at the Center for Genomic Sciences has revealed that chronic
otitis media with effusion (OME), once thought to be a sterile inflammatory
process, may in fact be a biofilm disease. Similar combinatorial techniques
of confocal microscopy and genotyping are being used to investigate other
Mucosal biofilm formation on middle-ear
mucosa in the chinchilla model of otitis media. Ehrlich GD, Veeh R, Wang X,
Costerton JW, Hayes JD, Hu FZ, Daigle BJ, Ehrlich MD, Post JC. JAMA. 2002 Apr
Confocal images of Haemophilus influenza cells and biofilm (green) associated
with host cells (nuclei stained red) 24 hours (A) and 10 days (B) post
Controlling the Growth of Biofilms
An active area of research is in the development of materials and surfaces
that resist or prevent biofilm formation. In industry this can mean reduced
operating costs and reductions in product spoilage, while in medicine the
prevention of biofilms on implants and medical equipment may result in
reductions in patient morbidity and mortality.
Pseudomonas aeruginosa PAO1
(pMF230) biofilms grown on untreated polyurethane (left panel) and polyurethane
with incorporated usnic acid (right panel) in flow
cells. Polyurethane is used to make catheters and usnic acid is a natural
"anti-biofilm" antibiotic produced by lichens, a biofilm system
composed of fungi, algae and/or cyanobacteria. In Staphylococcus aureus, a Gram positive human pathogen,
biofilm formation was inhibited. Intriguingly, in P. aeruginosa biofilm formation was not inhibited but the
structure was changed. This is interesting since usnic acid has structural
similarities with the homoserine lactones, a family of cell signaling
molecules involved in the maturation of biofilm architecture. This work was
done in collaboration with Gianfranco Donelli and Iolonda Francolini from
University of Rome "La Sapienza". Individual bacterial cells have
been genetically engineered to produce GFP for live visualization. The strain
was constructed and gifted by Mike Franklin, Dept. Microbiology, MSU-Bozeman.
Francolini, I., Norris,P.M., Piozzi, A., Donelli, G. and Stoodley, P.
2004. Usnic acid as a natural inhibitor of biofilm formation on polymer
surfaces. Antimicrob. Agents. Chemother.
Streptococcus pneumoniae, Biofilm Formation and Ear Infection
Streptococcus pneumoniae is a significant human pathogen which can cause a variety of infections. In addition to pneumonia this bacterium can cause sinusitis, otitis media (infection of the middle ear) ear infection), meningitis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess. (http://en.wikipedia.org/wiki/Streptococcus_pneumoniae). Recently it has been shown that S. pneumoniae can form biofilms (Allergrucci et al. 2006) and such biofilms are present in the middle ear of children suffering from repeated episodes of otitis media (Hall-Stoodley, et al. 2006). Since biofilms are resistant to many antibiotics we suspect that biofilm formation may explain why infections by S. pneumoniae and other organisms may be so difficult to treat in some people.
This movie shows the dynamic motility of S. pneumoniae (BS72) biofilm "towers" grown in vitro for six days and examined in situ in real time by confocal microscopy. The movie shows sections from top to bottom through the tower. In the movie the detachment of cells and clusters of cells into the surrounding fluid can be seen. Detachment of bacteria from biofilms has been well documented in other types of bacteria and suggests that in the presence of effusion in the ear, individual cells and clusters of pneumococci could readily detach from biofilm aggregates and propagate to other sites. Therefore, biofilm development may also increase the risk of recurrent OM and other acute exacerbations, due to repeated cycles of shedding of pneumococci from biofilm sources in the nasopharynx.
S. pneumoniae Type 23 biofilm with a large mushroom structure protruding from the surface. The extracellular polymeric slime (EPS) matrix was stained green with lectins. Bacteria were stained red with the nucleic acid stain Syto59. Co-localization of these stains appears yellow. Scale = microns.
Model showing progression of pneumococcal biofilm starting with the attachment of cells to a surface, the growth of those bacteria into towers, and the detachment of the tops of the towers which can then reattach elsewhere.
Allegrucci M, Hu FZ, Shen K, Hayes J, Ehrlich GD, Post JC, Sauer K. 2006. Phenotypic characterization of Streptococcus pneumoniae biofilm development. J Bacteriol. 188(7):2325-35.
Hall-Stoodley, L., Hu, F.Z., Gieseke, A., Nistico, L., Nguyen, D., Hayes, J., Forbes, M., Greenberg, D.P., Dice, B., Burrows A., Stoodley, P., Post, J.C., Ehrlich G.D., and Kerschner, J. 2006. Direct detection of bacterial biofilms on the middle-ear mucosa of children with chronic otitis media [Clinical investigation]
Scanning electron micrograph of a QS
BHL mutant Pseudomonas aeruginosa PANO67 biofilm streamer grown under
turbulent flow. Image colored by Kathy Lange, CBE, MSU-Bozeman.