Biofilms

Bacterial Biofilms: A general view
Blgo. Carlos A. Fernández Miñope
FernandezC4@gmail.com – Marzo, 2014
Segunda Especialidad en Microbiología Clínica
Hospital Regional Lambayeque
Universidad Nacional Pedro Ruiz Gallo

What are Biofilms?
• Biofilms are communities of surface-associated
microorganisms encased in a self-produced extracellular
matrix.
• Biofilm formation is a nearly universal bacterial trait,
and biofilms are found on almost all natural and
artificial surfaces
• Some examples of biofilms are the plaque that forms on
teeth and the slime that forms on surfaces in watery
areas.
Blgo.CarlosA.FernándezMiñope

Biofilms are everywhere
Biofilms appear early in the fossil record. There is evidence of biofilm formation
early in the fossil record, particularly in hydrothermal environments.

High-resolution scanning electron microscopy image of a community-
associated methicillin-resistant Staphylococcus aureus biofilm embedded
in extracellular matrix, formed on implanted catheter tubing during
experimental biofilm infection.
M. Otto, D. Dorward, E. Fischer and A. Mora,
National Institute of Allergy and Infectious
Diseases, USA.

Human Infections linked to biofilms

A simple view: B. subtilisbiofilm formation
Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nature
Reviews Microbiology, 11: 157-168 (2013).

Vlamakis, H., Chai, Y., Beauregard, P., Losick, R., Kolter, R. Sticking together: building a biofilm the Bacillus subtilis way. Nature

A closer look at biofilms Extracellular Matrix
In situ observations of biofilm structure using confocal laser
microscopy showed sessile bacteria growing in heterogeneous
matrix-enclosed microcolonies interspersed with open water
channels.

Thecomplexstructureof bacterialbiofilms
McDougald, D., Rice, S., Barraud, N., Steinberg, P., Kjelleberg, S. Should we stay or should we go: mechanisms and ecological
consequences for biofilm dispersal . Nature Reviews Microbiology, 10: 39-50 (2010).
ΔPf4

Oralbacterialcolonization
Kolenbrander, P.E., Palmer, R.J., Periasamy, S., Jakubovics, N.S. Oral multispecies biofilm development and the key role of cell–
cell distance. Nature Reviews Microbiology, 8: 471-481 (2010).

There is a strong relationship between the cell’s
environment and its physiology and structure.
Science News
Biofilms form Nutrient levels
decrease towards the
center
Variety of environmental
niches form
Biofilm formationand growth

• Quorum sensing and chemical signaling
• Microcolonies with extracellular matrix
• Network of channels
• Cells experience different environmental
conditions
• Multi-species
Biofilm formation and growth

Stages of Biofilm Formation
Bacteria switch from a free-floating (planktonic) state where
they function as individuals to a sessile state where they
function as communities.

Watnick, P., Kolter, R. Biofilm, City of microbes. J. Bacteriol. 182 (10): 2675-2679 (2000).
Biofilm formation in Vibrio cholerae

Candida albicans biofilmstructureinvitroandinvivo
Scanning electron micrograph (SEM)
of an in vitro Candida albicans
biofilm. The biofilm sample was
sliced to show three layers in a cross-
sectional view.
SEM of an in vivo C. albicans
biofilm from the rat catheter
model.
Images of J. Suhan (Carnegie Mellon University, Pittsburgh,
Pennsylvania, USA), and J. Nett and D. Andes (University of
Wisconsin–Madison, USA).

Singh, P.K., Schaefer, A.L., Parsek, M.R., Moningerk, T.O., Welsh,
M.J., Greenberg, E.P. Quorum-sensing signals indicate that cystic
fibrosis lungs are infected with bacterial biofilms. Nature 407: 762-
764 (2000).
In the microscopic examination of
sputum from patients colonizated
by P. aeruginosa reveled that they
are encased in a densely stained
matrix. This appearance is
consistent with previous
microscopic observations and
consistent with the hypothesis that
in CF sputum, P. aeruginosa exists
in biofilms.
a, Low magnification; b, high
magnification.

In P. aeruginosa, differentiation is cued by one of two quorum-
sensing signals N-(3-oxododecanoyl)-L-homoserine lactone
(3OC12-HSL) and N-butyryl-LHSL (C4-HSL). The signal required
for microcolony differentiation in biofilms is 3OC12-HSL).
Singh, P.K., Schaefer, A.L., Parsek, M.R., Moningerk, T.O., Welsh, M.J., Greenberg,
E.P. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with
bacterial biofilms. Nature 407: 762-764 (2000).
C4-HSL as a fraction of the total C4-HSL plus 3OC12-
HSL. From left to right, P. aeruginosa in early
logarithmic (A600 = 0.5), mid-logarithmic (A600 = 1),
late logarithmic (A600 = 2), stationary (A600 = 4) and
late stationary phase (A600 = 6).
C4-HSL 1 C4-HSL 3 C4-HSL 66
3OC12-HSL 3 3OC12-HSL 1 3OC12-HSL 1
A/(A+B) = 0.25 A/(A+B) = 0.75 A/(A+B) = 0.99
Incremento de 3OC12-HSL Incremento de C4-HSL
C4-HSL 1 C4-HSL 3 C4-HSL 66
3OC12-HSL 3 3OC12-HSL 1 3OC12-HSL 1
A/(A+B) = 0.25 A/(A+B) = 0.75 A/(A+B) = 0.99
Incremento de 3OC12-HSL Incremento de C4-HSL
QUORUM
SENSING

Singh, P.K., Schaefer, A.L., Parsek, M.R., Moningerk, T.O., Welsh, M.J., Greenberg, E.P. Quorum-sensing signals indicate that cystic
fibrosis lungs are infected with bacterial biofilms. Nature 407: 762-764 (2000).
The findings are
consistent with
the hypothesis
that
P. aeruginosa in
CF lungs exist as
a biofilm.
QUORUM
SENSING

Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S., Greenberg, E.P. Gene expression in
Pseudomonas aeruginosa biofilms. Nature 413: 860-864 (2001).
Genes for synthesis of pili and flagella are repressed in
biofilms. Pili and flagella are reported to be involved in the
initial steps (attachment and microcolony formation) of
development of P. aeruginosa biofilms. These appendages
may not be required for maintenance of a mature biofilm.
Once development has proceeded through these steps, pili
and flagella are no longer required.
Diferential gene expression

Coordinatedcell-deathis necessaryto
maintainthe biofilm
A confocal image (400× magnification) showing a mixed population of live (green) and dead (red) Staphylococcus aureus cells within
a 3-day-old biofilm. Image by Ethan Mann, University of Nebraska Medical Center, Nebraska, USA.
The cidABC and lrgAB
operons of S. aureus regulate
cell lysis during the
stationary phase of growth
and in response to antibiotic
exposure. There are
homologues of cid/lrg genes
present in a wide range of
Gram-positive and Gram-
negative bacteria.

Moving of bacteria in biofilms

Activebiofilmdispersalandvariantformation
McDougald, D., Rice, S., Barraud, N., Steinberg, P., Kjelleberg, S. Should we stay or should we go: mechanisms and ecological
consequences for biofilm dispersal . Nature Reviews Microbiology, 10: 39-50 (2010).
Each biofilm population becomes a mutator

Hall-Stoodley, L., Costerton, J.W., Stoodley, P. Bacterial
biofilms: from the natural environment to infectious
diseases. Nature Reviews Microbiology, 2: 95-108 (2004).
Bacterial endocarditis shows
how microorganisms on the
skin or in the oral cavity that
transiently enter the
bloodstream can colonize
abnormal or implanted valves,
or altered endothelial surfaces
in the heart.

Hall-Stoodley, L., Costerton, J.W., Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature

ProgressionofStaphylococcusaureusfroman
infectedorcontaminatedsourcethroughthe
bloodstreamtoametastatictarget
Thwaites, G.E., Gant, V. Are bloodstream leukocytes Trojan Horses for the metastasis of Staphylococcus aureus? Nature Reviews
Microbiology, 9: 215-222 (2011).

Multiplicity of phenotypicstatesin biofilms
Stewart, P.S., Franklin, M.J., Physiological heterogeneity in biofilms. Nature Reviews Microbiology, 6: 199-210 (2008).

• High diversity
• Less parallelism
• Each biofilm population
becomes a mutator

Scanning electron micrograph of an untreated biofilm of S. epidermidis (a) and an
identical biofilm exposed to vancomycin and rifampin for 72 h at concentrations
exceeding the MIC and MBC for the organism (b). Despite obvious changes in the
treated biofilm, viable organisms were recovered for which the MIC and MBC of
both agents were unaltered. Photo: American Society for Microbiology.
Drugresistancein Biofilms

Time-and dose-dependentkilling of
biofilms by metals
Harrison, J.J., Ceri, H., Turner, R.J., Multimetal resistance and tolerance in microbial biofilms. Nature Reviews Microbiology, 5:
928-938 (2007).
a) In vivo b) In vitro

Drug resistance
in Biofilms
mediatedby
persistercells
Lewis, Kim. Persister cells, dormancy and
infectious disease. Nature Reviews
Microbiology, 5: 48-56 (2007).

Formationof persistercells
Lewis, Kim. Persister cells, dormancy and infectious disease. Nature Reviews Microbiology, 5: 48-56 (2007).
The treatment of a population with an antibiotic results in cell
death, leaving only persister cells or resistant mutants alive

Multiresistant Bacteria
• Horizontal gene transfer is promoted in biofilms;
therefore, it is possible that antibiotic resistance
spreads more easily than in planktonic populations

Existence in a biofilm induces moderate levels of resistance to all
antimicrobial treatments. This could afford cells in a biofilm the
opportunity to respond to an antibiotic by inducing genes more specific to
that antibiotic.
We compared biofilms exposed to tobramycin with untreated biofilms.
Consistent with our hypothesis, 20 genes were differentially
expressed in tobramycin-treated biofilms: 14 were
activated and 6 were repressed by tobramycin (seven times the minimum
inhibitory concentration for planktonic cells). Of these 20 genes, 12
were classified as genes coding for hypothetical
proteins of unknown function. As expected, treatment with
tobramycin, which causes errors in protein synthesis, seemed to induce a
stress response, with activation of dnaK and groES genes, for example.
Tobramycin strongly induced several genes coding for hypothetical
proteins.
Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S., Greenberg, E.P. Gene expression in Pseudomonas
aeruginosa biofilms. Nature 413: 860-864 (2001).

Whiteley, M., Bangera, M.G., Bumgarner, R.E., Parsek, M.R., Teitzel, G.M., Lory, S., Greenberg, E.P. Gene expression in Pseudomonas
aeruginosa biofilms. Nature 413: 860-864 (2001).
Survival of cells in wild-type and rpoS-mutant biofilms after treatment with the antibiotic
tobramycin. Open symbols, planktonic cultures; filled symbols, biofilms; squares, parent
strain PAO1; triangles, rpoS mutant. At tobramycin concentrations above 5 mg ml -1 there
were 2 viable cells ml -1 in planktonic cultures.

Hall-Stoodley, L., Costerton, J.W., Stoodley, P. Bacterial biofilms: from the natural environment to infectious diseases. Nature
Taken together, the data indicate that the ability to form
biofilms is an ancient and integral characteristic of
prokaryotes. In the context of EVOLUTION AND ADAPTATION
it is likely that biofilms provided homeostasis in the face of the
fluctuating and harsh conditions of the primitive earth
(extreme temperatures, pH and exposure to ultraviolet (UV)
light.
Biofilms are not new

Conceptualization of biofilm development and dynamic behaviours. Biofilms are
structurally and dynamically complex biological systems
Hall-Stoodley, L., Costerton, J.W., Stoodley, P. Bacterial biofilms: from the natural
environment to infectious diseases. Nature Reviews Microbiology, 2: 95-108 (2004).

Biofilms

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