Bacterial Community

Biofilm and Quorum sensing control

5-3-2010
Biofilm-
the real ecosystem
 Biofilms are the predominant
phenotypes in natural and
pathogenic ecosystems
 Pure culture planktonic growth
is rarely how bacteria exist in
nature !!!
 Microbial biofilms (2004) , ASM
Press, edited by Mahmoud
Ghannoum and George A.
O’Toole
Biofilm composition
 Sessile cells- immobilize on solid
surface
 complex attached and

aggregated communities
(hydrodynamics)- open water channels
 collective and heterogenous
behavior (structural and metabolic
heterogeneity) Costerton and Stewart, 2001,
 periodicity of cell attachment Scientific American
patterns

Nature rev Microbiology (2008) Feb

A complex developmental process
5. dispersion
4. maturation II
3. Maturation I
1. Initial
attachment
2. Irreversible
attachment

PLoS Biology (2007) Nov

 Biofilm development- Pseudomonads
WT
 Planktonic cells attach to
surface Microcolony
aggregation to recruit
planktonic cells Mushroom-
shaped macrocolony PilA mutant
Detachment
Cellular chain formation in
Escherichia coli biofilms

Microbiology 2009 April

Environmental and genetic
requirements for M. tuberculosis biofilms


Mol Microbiol 2008

EPS (slime) matrix is the determinant
 Extracellular polymeric substance
(including extracellular polysaccharides)
 the key factor of biofilm structure
 Provides the slimy matrix
 Protective layer

 Supply of carbon and energy source

 comprise proteins, humic acids, and DNA in

addition to carbohydrates
 EPS regulation
in Vibrio cholerae biofilm
Flagella and fimbriae are
necessary for motility
and initial adherence to a
solid surface

MotX
Synthesis of an EPS
occurs after
attachment to solid
at least 3 signaling pathways known
surfaces and
for EPS production-
microcolony formation
- quorum sensing pathway, flagellum-
dependent pathway, phase variation
pathway
J. Bacteriol. (2004) 186:4835
An intricate network of Bacilli biofilm

EPS- polysaccharide
synthesis

EPS protein
2CS-RR

Mol Microbiol (2008)

 Cross-talk in Escherichia coli biofilm
formation
 Signals leading to
activation of stress
responses often lead
to biofilm formation
which, in turn, can
trigger induction of
stress response
mechanisms
 The regulatory
systems of a tight and
complex interplay
include TCS, RpoS
and cyclic di-GMP
Res Microbiol 2009
Correlation between virulence factors
and in vitro biofilm formation
 Among 30 VFs-associated genes analyzed,
mannose-resistant haemagglutination was the
only phenotypically expressed in the strong
biofilm group and papC and papG alleles,
sfa/focDE, focG, hlyA and cnf1 were more
common (p<0.05) in strong biofilm producers.

Microbiol Pathogenesis 2008

Escaping from the biofilm- detachment
 Reenter into planktonic population
 Biological factors
 Nutrient limitation
 lyase activity detected only in older biofilm

 production of biosurfactants
 Pseudomonas rhamnolipid
 Physical mechanisms
 Disrupt the hydrodynamic conditions
 viscosity, pH, or electrical properties
 Increase of external shear forces
 Decrease of the internal strength
 Monospecies biofilms

P. aeruginosa- different B. subtilis- diverse aerial structures

differentiated phenotypes
FEMS Rev (2009) accepted Oct 24 2008
Heterogenous biofilm
 Subpopulation interaction
 expression of the iron-siderophore pyoverdin in one
subpopulation  necessary for development of
another subpopulation which does not itself express
the pyoverdin syntheses genes
 QS-controlled DNA-release in one subpopulation 
necessary for development of another subpopulation
which does not itself express the QS genes

Molecular Microbiology (2009)

Natural biofilms- mixed species

G- Pseudomonas putida
G- Green algae R- Acinetobacter sp.

B- Cyanobacteria
P- EPS
FEMS Rev (2009)
Microbial interactions
in mixed species biofilms
 Commensalism/ competition in biofilms
 Most natural biofilm communities harbor more
than one species
Biofilm of
Pseudomonas
aeruginosa,
P. fluorescens, and
Klebsiella pneumoniae
Chronic periodontitis
 Porphyromonas gingivalis is a major etiologic agent
 a late colonizer of subgingival dental plaque, a

complex and dynamic polymicrobial biofilm

 adhere to primary plaque colonising species,
particularly Streptococcus spp. such as Streptococcus
gordonii
 formation of a bi-species biofilm with P. gingivalis attached to
S. gordonii bound to a salivary pellicle
 adheres to later colonising Gram-negative bacteria
including Fusobacterium nucleatum and Treponema
denticola
Staphylococcal biofilms
 Staphylococcal infections
 biofilm formation is a
critical factor in association
of implanted medical
devices (80 % form biofilm)
 tend to be chronic and
less acute (endocarditis)
PLoS Biology (2007) Nov
Biofilm development in S. epidermidis


Nature reviews | Microbiology | AUGUST 2009

Antimicrobial resistance in biofilms
 Depletion of the antimicrobial agent
 slow penetration of the agents
 slow or no growth (persisters)
 adaptive stress responses
 specific gene expression?
 the susceptibility is influenced by biofilm
thickness, age, areal cell density, antimicrobial
dose concentration, and species composition,
and genotype
 Persisters
 Persisters are
cells that remained
at stationary phase
and did not
respond to the
external cues until
they switch back to
normal growth

PNAS
(2008) April
Ciprofloxacin causes persister formation by
inducing the TisB toxin in Escherichia coli

 PLoS Biol 2010.

8(2):
e1000317.doi:10.137
1/journal.pbio.100031
7
Medically important bacterial-fungal
interactions


Nature Reviews Microbiology | published online 29 March 2010


Blocking biofilm formation
 Phage treatment (highly specific)
 Prophages in planktonic phase lytic phages in
biofilm  Promote biofilm detachment
 Anti-adhesive proteins (AEM 2008 April)
 Anti-Quorum sensing (QS) control
 Dispersion inducers
 make the infections vulnerable to traditional

antibiotics or even to normal immune response

PLoS Biology (2008) Jan

 Dispersing biofilms with engineered
enzymatic bacteriophage
 DspB- a dispersin to
degrade the polymer
-1,6-N-acetyl-D
glucosamine which
is a crucial adhesin
for the biofilm
formation

PNAS (2007) 104:11197-

 Biofilm detection
 Enhancing ATP
by enzymatic 104 CFU

pyrophosphate
regeneration

103 CFU

Analytical Biochemistry 399 (2010) 168–173

Model of cholera transmission
 Vibrio cholerae exist in water mostly as clumps
of cells (CVEC or VBNC), which resist cultivation
by standard techniques but revive into fully
virulent (Biofilm) form in the intestinal milieu
 CVEC- Conditionally viable environmental cells
 VBNC- Viable but nonculturable cells (VBNC)

PNAS Early Edition 2010

 Quorum sensing-regulated biofilms
 in vivo-formed biofilms
 CVEC (environmental
water)
 loss of QS  break
down biofilms 
interfere with CVEC
formation

PNAS Early Edition 2010

Quorum Sensing (QS)
 A density-dependent control
 At low bacterial density synthesis of
signaling autoinducer diffusion of the small
molecule accumulation of the molecules 
activate specific regulator  synchronous
activation of a certain phenotypes including
biofilm formation, toxin production, extra-
cellular polysaccharides production, virulence
factor production, and motility
Micro Mol Biol Rev (2006) 70: 859-
A prototype of quorum sensing
 1970, Photobacterium (Vibrio) fischeri
 The bacteria do not luminescence until they
reach a high population density
 Molecular messenger: autoinducer (AI)
 N-acyl homoserine lactone (AHL or HSL, AI1)

 LuxI: AHL synthase

 LuxR: regulator
 LuxAB: luciferase
Molecular mechanics of AHL-mediated QS
Micro Mol Biol Rev (2006) 70: 859-
The QSs in Vibrio cholerae
 3 autoinducers: CAI, AI-2 (LuxSfuranosyl
borate diester ), and AI-3
 Accumulation of the autoinducers modulates the
2CS LuxL/LuxO
 Quorum regulatory RNA (Qrr)
 V. cholerae 4 Qrrs and V. harveyi 5 Qrrs
 At low cell density, expression of qrr genes is
activated by LuxO-P

Curr Opin Microbiol (2007) 10:1-10

QS and multifunctional signal
molecules in Pseudomonas aeruginosa
A regulatory network

3-oxo-C12-HSL C4-HSL

At least 5 to 20% of its genes and

proteins are directly or indirectly
subject to QS regulation

Current Opinion in Microbiology 2009, 12:1–10

QS in chronic cystic fibrosis
Pseudomonas aeruginosa infections


FEMS Minireview 2009

QS in Gram positive bacteria
An octapeptide autoinducer  Streptococcus mutans
the cause of dental caries
 21 aa peptide

pheromone (ComC) to
initiate QS for genetic
HK competence, biofilm
formation, stress
RR
responses, and
bacterocin production
J Bacteriol (2007) 189:1441-

Trends in Microbiology 2009

Bacillus subtilis QS
Phr derived pentapeptides
 The QS is mediated by
peptides that act as
phosphatase regulators
( 8 Phr) of receptor
aspartyl phosphatases
(11 Raps)
 Several Phr signals are

integrated in sporulation
phosphorelay signal
transduction

PNAS April 21, 2009 vol. 106 no. 16 6459–6464

 Interplay between QS (MvfR regulon) and
iron in virulence of Pseudomonas aeruginosa

 iron conditions
in vivo as a
signal indicating 4-hydroxy-2-alkylquinolines
a hostile
environment 
expression of
virulence or
fitness-related
genes PLoS Pathog 6(3): April 2010
Eukaryotes deal with bacterial QS
 Marine red alga Delisea pulcbra compounds
prevented fouling by mimicking AHL signals and
interfering with bacterial QS (1995)- QSI
 The algal furanones bind specifically to AHL receptor
proteins and enhance their rate of proteolytic
degradation
 The furanones also inhibit QS-regulated gene
expression in enteric bacteria such as E. coli and
Salmonella, which use AI-2 related signal molecules
 Biofouling: thick buildups of bacteria, algae, and
invertebrates on marine surface
ASM News (2005)71:129-
Micro Mol Biol Rev (2006) 70: 859-
 Plants produce AHL mimics
 In contrast to the purely inhibitory effects of
Delisea furanones, most of the AHL mimics
produced by higher plants stimulate receptor-
mediated gene expression in reporter strain
 By prematurely eliciting virulence gene expression in
pathogens, thus alerting host defenses before the
bacterium develops sufficient numbers to mount an
effective attack
Applications-
 Modulate cellular immune response
 30 M of 3-oxo-C12-HSL stimulate human
epithelial cells to produce IL-8
 QS blockers for therapeutic or industrial use
 Better toothpaste against Streptococcus mutants
 Anti-activation structural homolog (PNAS 2007 Oct)
 > 100 QS-related patents are pending
Intelligent implants to battle biofilm
 Self-diagnosing, self-treating, self-monitoring
artificial joints
 ASM News (2004):127-133

Garth D. Ehrlich, Fen Ze Hu, Qiao Lin, J. William Costerton, and

J. Christopher Post
Garth D. Ehrlich is Executive Director, Fen Ze Hu is Director of Operations,
and J. Christopher Post is Medical Director at the Center for Genomic
Sciences, Allegheny Singer Research Institute, Pittsburgh, Pa., and Drexel
University College of Medicine, Philadelphia, Pa.; Qiao Lin is Assistant
Professor at Carnegie Mellon University, Pittsburgh, Pa.; and J. William
Costerton is Director of the Center for Biofilm Engineering, Montana
State University, Bozeman.