microbiology, wound infection, soft tissue infection

1. Dr.T.V.Rao MD
SOCIO-MICROBIOLOGY
BIOFILMS AND QUORUM SENSING IN
INFECTIOUS DISEASES
DR.T.V.RAO MD 1

2. • 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.
SOCIOMICROBIOLOGY
DR.T.V.RAO MD 2

3. 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

4. 4
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
cell and activate particular set of genes in all bacterial population,
DR.T.V.RAO MD

5. • 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
SOCIOMICROBIOLOGY ADVANCES THE
UNDERSTANDING MICROBES
DR.T.V.RAO MD 5

6. BACTERIA ARE DYNAMIC CREATURES
• Bacteria are dynamic creatures
that are able to regulate their
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."
• iosynthetic and regulatory prodigiosin
mutants of Serratia
DR.T.V.RAO MD 6

7. • 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?”
MICROBES RUN IN OUR BODY AS
NORMAL FLORA
DR.T.V.RAO MD 7

8. • 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
MICROBES DO HAVE SOCIAL LIFE AND WELL ADOPTED
WHY DO WE WORK ON IT?
DR.T.V.RAO MD 8

9. • In the past decade,
significant debate
has surrounded the
relative contributions
of genetic
determinants versus
environmental
conditions to certain
types of human
behavior
GENETICS X ENVIRONMENT
DR.T.V.RAO MD 9

10. • 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.
WHAT IS A BIOFILM?
DR.T.V.RAO MD 10

11. 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

12. 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

13. • 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.
WHERE ARE BIOFILMS FOUND?
DR.T.V.RAO MD 13

14. BIOFILMS ARE CONCERN IN EVERY
ASPECT OF LIFE
DR.T.V.RAO MD 14

15. 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

16. • 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.
BIOFILMS ARE ADVANTAGEOUS TO
MICROORGANISMS
DR.T.V.RAO MD 16

17. • 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.
BIOFILMS IN MEDICINE
DR.T.V.RAO MD 17

18. 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

19. 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

20. • 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.
QUORUM SENSING
Henke and Bassler, 2004
DR.T.V.RAO MD 20

21. Environmental Pseudomonas
PA colonization-ASYMPTOMATIC
Model of Chronic Pseudomonas aeruginosa
Infection in Cystic Fibrosis
Bacterial Adaptation
• Alginate/mucoidy
• Auxotrophy
Increased bacteria - SYMPTOMATIC
Lung
Disease
• surface modifications
• Increased PQS
(biofilm, virulence,
antibiotic resistance)
Innate Immune Selective Pressure
DR.T.V.RAO MD 21

22. • Structuring of
multicellular communities
• Stress survival
• Production of
• Antibiotics
• Pigments
• Host tissue degrading
enzymes
BEHAVIORS CONTROLLED BY
QUORUM SENSING

23. • Cell-cell
communication can
occur within and
between bacterial
species, and
between bacteria
and their eukaryotic
hosts.
QUORUM SENSING CAN OCCUR …..
DR.T.V.RAO MD 23

24. • Bacteria use Quorum
sensing to mastermind
behaviors including
• Mating
• Releasing toxins
• Causing disease
(virulence )
QUORUM SENSING HELPS IN …
DR.T.V.RAO MD 24

25. HOW PATHOGENIC BACTERIA USE QUORUM
SENSING
These changes culminate in an
infection that can ambush and
overwhelm our immune system
defenses.
The bacteria appear
relatively innocuous as they
quietly grow in number.
When their population reaches a certain
level, instant changes occur in their
• Behavior
• Appearance
• Metabolism
DR.T.V.RAO MD 25

26. 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

27. 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

28. THE CHAIN OF COMMAND IN BACTERIAL
COMMUNICATION
LuxR
Master Regulator
Protein
Qrr1
Qrr2
Qrr3
Qrr4
Small
RNA
molecules
(sRNAs)
Qrr = Quorum Regulatory RNA
Signals from sRNAs turn
LuxR on and off
Signals from LuxR
control quorum-sensing
AI-2
AI-2 attaches to LuxR to
initiate cellular
communication
LuxR
Master Regulator
Protein
Qrr1
Qrr2
Qrr3
Qrr4
Small
RNA
molecules
(sRNAs)
Qrr = Quorum Regulatory RNA
Signals from sRNAs turn
LuxR on and off
Signals from LuxR
control quorum-sensing
AI-2
AI-2 attaches to LuxR to
initiate cellular
communication
DR.T.V.RAO MD 28

29. QUORUM SENSING IN P. AERUGINOSA
P. Aeruginosa
•Major cause of hospital infection in the US.
•Major cause of deaths in intubated CF patients, and IV fed patients.
Quorum Sensing: The ability of a bacterial colony to sense its
size and regulate its activity in response.
Examples: P. aeruginosa
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

30. QUORUM SENSING IN P. AERUGINOSA
Planktonic
Loosely Bound EPS secreting
DR.T.V.RAO MD 30

31. “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

32. • 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.
INTRASPECIES VS. INTERSPECIES
COMMUNICATIONS
DR.T.V.RAO MD 32

33. 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

34. LABORATORY MADE MOLECULES HAVE
SOLUTIONS TO COUNTER 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
Autoinducer 2 may hold the key to
disrupting quorum-sensing.
DR.T.V.RAO MD 34

35. The three general classes of quorum-sensing systems
Modified
oligopeptides
Processin
g and
secreation
S
H
K
A
R
A
T
P
AD
P
Class Autoinducer Strain
P. aeruginisa
V. fisheri
E. carotovora
A. tumefaciens
Y. enterocolitica
E. coli O157:H7
V. harveyi
V. cholerae
V. vulnificus
S. Typhimurium
B. subtilis
S. aureus
S. pneumoniae
S. epidermidis
L. lactis
QS1
G(+)
QS2
AI1
PAI
AI2

36. 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

37. 37
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.

38. 38
WHAT IS THE NEED FOR QUORUM
SENSING INHIBITORS ?

39. 39
STRATEGIES FOR QUORUM
SENSING INHIBITION
3 strategies can be applied
Targeting AHL signal
dissemination
Targeting the signal
receptor
Targeting signal
generation
Signal precursor
Signal
Signal receptor
Signal precursor Signal precursor
Signal Signal
Signal receptor Signal receptor
X
X
X

40. • 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
FURTHER RESEARCH IS NEEDED IN
SOCIOMICROBIOLOGY ON ….
DR.T.V.RAO MD 40

41. VISIT ME FOR MORE TOPICS OF INTEREST IN
INFECTIOUS DISEASES
DR.T.V.RAO MD 41

42. • 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