SOCIO-MICROBIOLOGY

BIOFILMS AND QUORUM SENSING IN

INFECTIOUS DISEASES

Dr.T.V.Rao MD

DR.T.V.RAO MD 1
 SOCIOMICROBIOLOGY
• Sociomicrobiology" is part
of the broader discipline
"Microbiology", the study of
organisms (bacteria, yeast,
molds, viruses and protists)
that cannot be observed
with the naked eye, but
having critical mechanisms
for propagation.

DR.T.V.RAO MD 2
 SOCIOMICROBIOLOGY

• The term "sociomicrobiology" was

introduced by Matt Parsek & Peter
Greenberg in 2005 (Trends in
Microbiology, 13:27-33) and refers to the
group behavior of micro-organisms, Two
topics that form the core of
sociomicrobiological research are microbial
biofilm formation and cell-cell
communication (quorum sensing).
DR.T.V.RAO MD 3
 INTRODUCTION
Quorum sensing is cell to cell signaling mechanism that enables
the bacteria to collectively control gene expression.

 This type of bacterial communication is achieved only at higher

cell densities.

 Bacteria release various types of molecules called as auto

inducers in the extracellular medium, these molecules are
mediators of quorum sensing.

 When concentration of these signaling molecules exceed a

particular threshold value, these molecules are internalized in the
4
cell and activate particular set of genes in all bacterial population,
DR.T.V.RAO MD
 SOCIOMICROBIOLOGY ADVANCES THE
UNDERSTANDING MICROBES
• The study of group
behavior in microbes
• • Debate over
environmental vs.
genetic determinates
• • Biofilms and quorum
sensing
• • Model for dissecting
social behavior at a
genetic level
DR.T.V.RAO MD 5
 BACTERIA ARE DYNAMIC CREATURES
• Bacteria are dynamic creatures • iosynthetic and regulatory prodigiosin
that are able to regulate their mutants of Serratia
metabolism and lifestyle in
response to a variety of
environmental cues. These cues
include changes in their
chemical, physical, and biological
surroundings. In recent decades,
microbiologists have come to
appreciate that bacteria are even
able to recognize changes in
their own population density. Cell
density-dependent regulation has
been termed "quorum
sensing."
DR.T.V.RAO MD 6
 MICROBES RUN IN OUR BODY AS
NORMAL FLORA
• Microbes run much of our body.
The human micro biome in our
gut, mouth, skin, and elsewhere,
harbors 3,000 kinds of bacteria
with 3 million distinct genes .
(Our own cells struggle by
on only 18,000 genes or
so.)…This biotech century
will be microbe enhanced
and maybe microbe
inspired….Confronting a
difficult problem we might
fruitfully ask, “What would a
microbe do?”
DR.T.V.RAO MD 7
MICROBES DO HAVE SOCIAL LIFE AND WELL ADOPTED
WHY DO WE WORK
ON IT?

• Models to understand biology

of sociality
• To develop new medicines to
treat devastating bacterial
infections
• Understanding bacteria

• Sociomicrobiology
• The “new” science of
• Tools for synthetic biology

DR.T.V.RAO MD 8
 GENETICS X ENVIRONMENT
• In the past decade,
significant debate
has surrounded the
relative contributions
of genetic
determinants versus
environmental
conditions to certain
types of human
behavior
DR.T.V.RAO MD 9
 WHAT IS A BIOFILM?
• A structured community of
bacterial cells enclosed in a
self-produced polymeric
matrix.
• -Biofilms are a protective
mode of growth that allows
survival in hostile
environments.
• -Bacteria in biofilms are
inherently resistant to
killing.

DR.T.V.RAO MD 10
 BIOFILMS ARE …….
• Biofilms are multicellular aggregates of bacteria and
yeast that congregate on surfaces.
• Biofilm may form on any surface exposed to biofilm-
forming bacteria and some amount of water.
• Biofilms are formed to protect the bacteria from host
defenses, antibiotics, and from harsh environmental
conditions.

DR.T.V.RAO MD 11
 BIOFILMS ARE COMMUNITIES OF
MICROORGANISMS
• Biofilms are communities of microorganisms that
develop on surfaces in most natural and artificial
environments. Biofilm maturation requires cell contact
with a surface and cell-cell adhesion counteracting the
shear forces of the environment. Biofilms are
characterized by a surface covered by a high number of
cells (a film) encased in a self-produced extra cellular
matrix, are highly heterogeneous environment, both at
structural, physiological and specific levels and biofilm
bacteria express still under-explored specific biological
properties such as a characteristic increased tolerance
to biocides
DR.T.V.RAO MD 12
 WHERE ARE BIOFILMS FOUND?
• Biofilms are found
almost everywhere in
nature, including
rivers, lakes, soil, water
pipes, and even inside
the human body
• Bacterial biofilms are
often a cause of
infections associated
with medical implants
such as catheters and
IV lines and other
medical devices.

DR.T.V.RAO MD 13
 BIOFILMS ARE CONCERN IN EVERY
ASPECT OF LIFE

DR.T.V.RAO MD 14
 THE DYNAMICS OF GROWING BIOFILM
• Quorum Sensing:
• What is it?
• How does it work?

• Heterogeneous structures:

• How do these cells use polymer gel for locomotion?

• What are the mechanisms of pattern (structure) formation?

• Why is polymer gel so effective as a protective environment?

DR.T.V.RAO MD 15
 BIOFILMS ARE ADVANTAGEOUS TO
MICROORGANISMS
• Biofilms are important survival
mechanisms for bacterial cells.
According to in vitro studies, they
can avoid attack by host defenses. it
is difficult for phagocytic cells to
engulf bacteria in biofilms. Also,
biofilms are much more resistant
than planktonic cells to antimicrobial
agents. The bacteria within the
biofilm remain healthy, and the
biofilm can regrow. Repeated use of
antimicrobial agents on biofilms can
cause bacteria within the biofilm to
develop an increased resistance to
biocides.

DR.T.V.RAO MD 16
 BIOFILMS IN MEDICINE
• In medicine, biofilms spreading
along implanted tubes or wires
can lead to pernicious infections
in patients. Biofilms on floors and
counters can make sanitation
difficult in food preparation
areas.
• Dental plaque is a yellowish
biofilm that build up on the teeth.
If not removed regularly, it can
lead to dental caries.

DR.T.V.RAO MD 17
 WHY RESEARCH ON BIOFILMS?
• Due to the morphology of biofilms, bacteria
capable of forming them are highly resistant to
antibiotics, making treatment very difficult.
• In the US alone, one million nosocomial (hospital
acquired) infections each year are caused by
bacterial biofilms, leading to longer
hospitalization, surgery, and even death.

DR.T.V.RAO MD 18
 BIOFILMS AND INFECTIONS:
• Biofilms are responsible for Otitis Media, the most common acute ear
infection.
• Biofilms play a role in Bacterial Endocarditis (infection of the inner
surface of the heart and its valves).
• Biofilms form frequently in patients with Cystic Fibrosis (a chronic
disorder resulting in increased susceptibility to serious lung infections).
• Biofilms also play a role in Legionnaire's disease (an acute respiratory
infection resulting from the aspiration of clumps of Legionnella biofilms
detached from air and water heating/cooling and distribution systems).

DR.T.V.RAO MD 19
 QUORUM SENSING
• A process that enables
bacteria to communicate
using secreted signaling
molecules called auto
inducers
• This process enables a
population of bacteria to
regulate gene expression
collectively and therefore,
control behavior on a
community-wide scale.

DR.T.V.RAO MD
Henke and Bassler, 2004
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 Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis

Environmental Pseudomonas

Bacterial Adaptation Lung

Disease
• surface modifications • Alginate/mucoidy
• Increased PQS • Auxotrophy
(biofilm, virulence,
antibiotic resistance)
Innate Immune Selective Pressure

PA colonization-ASYMPTOMATIC Increased bacteria - SYMPTOMATIC

DR.T.V.RAO MD 21
 BEHAVIORS CONTROLLED BY
QUORUM SENSING
• Structuring of multicellular
communities
• Stress survival
• Production of
• Antibiotics
• Pigments
• Host tissue degrading
enzymes
 QUORUM SENSING CAN OCCUR …..
• Cell-cell
communication can
occur within and
between bacterial
species, and
between bacteria
and their eukaryotic
hosts.

DR.T.V.RAO MD 23
 QUORUM SENSING HELPS IN …
• Bacteria use Quorum
sensing to mastermind
behaviors including

• Mating
• Releasing toxins
• Causing disease
(virulence )

DR.T.V.RAO MD 24
HOW PATHOGENIC BACTERIA USE QUORUM
SENSING

The bacteria appear These changes culminate in an

relatively innocuous as they infection that can ambush and
quietly grow in number. overwhelm our immune system
defenses.

When their population reaches a certain

level, instant changes occur in their
• Behavior
• Appearance
• Metabolism

DR.T.V.RAO MD 25
 QUORUM SENSING SYSTEMS:
GRAM NEGATIVE BACTERIA
• Lux I/R systems
• Auto inducers: acylated homoserine lactone
• Lux I-type enzymes synthesize acylated homoserine lactone
(AHL) auto inducers by ligating a specific acyl moiety to the
homocysteine moiety of S-adenosylmethionine (SAM)
• LuxR-type proteins bind their cognate autoinducers and
control transcription of target genes.

DR.T.V.RAO MD 26
 QUORUM SENSING SYSTEMS:
GRAM POSITIVE BACTERIA
• Two-component systems involved
• Autoinducers: modified oligopeptides
• The signals are synthesized as precursor peptides, which are
subsequently processed and secreted
• Sensor histidine kinases detect the extracellular peptide
autoinducers, autophosphorylate and transmit sensory information
via phosphorylation of a response regulator
• Response regulator changes gene expression

DR.T.V.RAO MD 27
 THE CHAIN OF COMMAND IN BACTERIAL
COMMUNICATION
Signals from sRNAs turn Signals from LuxR
Small RNA molecules (sRNAs)

LuxR on and off control quorum-sensing

Qrr1

Qrr2 LuxR
Master Regulator
Protein
Qrr3

AI-2
Qrr4 AI-2 attaches to LuxR to
initiate cellular
Qrr = Quorum Regulatory RNA communication

DR.T.V.RAO MD 28
 QUORUM SENSING IN P. AERUGINOSA
Quorum Sensing: The ability of a bacterial colony to sense its
size and regulate its activity in response.

Examples: P. aeruginosa
P. Aeruginosa
•Major cause of hospital infection in the US.
•Major cause of deaths in intubated CF patients, and IV fed patients.

P. Aeruginosa in planktonic (non-colonized) form are non-toxic, but

as a biofilm, they are highly toxic and well protected by the polymer
gel in which they reside. However, they do not become toxic or begin
to form polymer gel until the colony is of sufficient size to overwhelm
the immune system. Before this, they cannot be detected by
the immune system.
DR.T.V.RAO MD 29
 QUORUM SENSING IN P. AERUGINOSA

Planktonic

Loosely Bound EPS secreting

DR.T.V.RAO MD 30
 “WALL SENSING” IN P. AERUGINOSA

Wall Sensing: The ability of bacteria to differentiate in response to

Contact with a wall (the substratum).

Planktonic Loosely Bound EPS secreting

DR.T.V.RAO MD 31
 INTRASPECIES VS. INTERSPECIES
COMMUNICATIONS
• AHL type autoinducers are
for intraspecies
communications
• AI-2 and its synthase,
LuxS, are widespread,
existing in many bacterial
phyla. AI-2 is suggested to
serve as an interspecies
bacterial communication
signal.

DR.T.V.RAO MD 32
 How quorum sensing works?
Signaling compounds, auto inducers
AI synthases (luxI gene products)
cell density indicators
- non-essential aa, acyl homoserine lactones
lactone ring part - binding to a receptor site
acyl chain tail – determining the species specificity
- oligopeptides
- diketopiperazines
- quinolone
- furanones

Recognition systems
LuxR transcriptional regulator
specific binding sites for AHL and DNA (sensor/transducer)

Genetic basis
regulatory circuit involving both regulatory genes
accumulation of AHL - activating gene transcription
 LABORATORY MADE MOLECULES HAVE
SOLUTIONS TO COUNTER QUORUM SENSING
Autoinducer 2 may hold the key to
disrupting quorum-sensing.
AI-2 contains the
element boron

AI-2 and similar boron-

containing molecules made
in the laboratory could
serve as decoys to subvert
virulence and other
quorum-sensing behaviors

DR.T.V.RAO MD 34
 The three general classes of quorum-sensing systems
Class Autoinducer Strain
QS1 AI1
P. aeruginisa
V. fisheri
E. carotovora
A. tumefaciens
Y. enterocolitica

QS2 AI2
E. coli O157:H7
V. harveyi
V. cholerae
V. vulnificus
S. Typhimurium

G(+) PAI
Modified
oligopeptides B. subtilis
Processin S. aureus
g and S S. pneumoniae
secreation H
A
K
S. epidermidis
T A L. lactis
AD
P
P
R
 COMMUNICATION BETWEEN
PROKARYOTES AND EUKARYOTES
• Chemical communication extends to the eukaryotic
hosts with which bacteria engage in pathogenic and
symbiotic relationships.
• P. aeruginosa AHLs enter eukaryotic cells and stimulate
production of chemokine interleukin 8 (IL-8), which in
turn induces the NF-kB transcription factor.
• These responses cause recruitment of neutrophils to
the lung, in which they contribute to pulmonary
inflammation and tissue deterioration.

DR.T.V.RAO MD 36
 NEED FOR INHIBITION OF
QUORUM SENSING
 Inhibition of quorum sensing has been proved to be very potent method
for bacterial virulence inhibition.

 Several QS inhibitors molecules has been discovered.

 QS inhibitors have been synthesized and have been isolated from several
natural extracts such as garlic extract.

 QS inhibitors have shown to be potent virulence inhibitor both in in-vitro

and in-vivo, using infection animal models.

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WHAT IS THE NEED FOR QUORUM
SENSING INHIBITORS ?

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 STRATEGIES FOR QUORUM
SENSING INHIBITION
3 strategies can be applied

Targeting signal Targeting AHL signal Targeting the signal

generation dissemination receptor
Signal precursor Signal precursor Signal precursor
X
Signal Signal Signal
X
X
Signal receptor Signal receptor Signal receptor
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 FURTHER RESEARCH IS NEEDED IN
SOCIOMICROBIOLOGY ON ….
• Further studies are needed on quorum
sensing
• regulated gene expression
• – Onset of QS
• – Affects on Onset
• • Architecture and physical flow
• • Functional consequences on biofilm
• community
• • Role in mixed species systems
• • Effects of signal consumption

DR.T.V.RAO MD 40
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DR.T.V.RAO MD 41
 • Created by Dr.T.V.Rao MD for ‘ e’ learning
resources for Medical Microbiologists in the
Developing world
• Email.
• doctortvrao@gmail.com

DR.T.V.RAO MD 42