BIOFILMS IN

MEDICINE
PSBT 302
UNIT 4
POINTS
• Formation of Biofilms- Stages
• Formation of Biofilms by various organisms
• Dental Plaques- formation and control/removal
• Biofilms associated with indwelling medical
devices
• Biofilms and Pathogenesis
• Factors favoring Biofilm formation
• Role of Biofilms in microbial community
• Removal of Biofilms
FORMATION
• BIOFILM- A thin but robust layer of mucilage
adhering to a solid surface and containing a
community of bacteria and other microorganisms.
• Organisms in Biofilms- Pseudomonas spp.
Streptococcus spp. Staphylococcus spp.
Enterococcus spp. Klebsiella, E. coli, etc.
Surface Conditioning

 Consist of loose collection

of organic soils which
include proteins and
carbohydrates

 These combine with

minerals in hard water

 Organics neutralize surface

charge that repels
bacteria.

 Conditioning begins within

seconds of exposure to
Attachment/Adhesion/Attractio
n
 Electrical charge builds on
surface which now becomes
increasingly attractive to
bacteria carrying opposite
charge

 Planktonic bacteria attach by

electrostatic attraction and
physical forces

 Electrostatic forces are weak

and reversible

 MOs easily removed or killed by

mild cleansers and sanitizers
Slime Formation
 Bacteria become firmly attached to the
surface and each other within 8-24 hrs.

 Do so by exuding a polysaccharide material

that entraps cells and debris within a glue
like matrix- Extracellular Polymer (EPS)

 EPS consist of charges and neutral

polysaccharide grps that cement cells to the
surface and acts as am ion exchange sys for
trapping nutrients

 Pioneer cells reproduce, daughter cells

produce their own exopolymers increasing
vol of ion exchange surface

 Thriving colony established, grows in

thickness

 Capable of blocking cleaners and sanitizers

Mature Biofilm- Detachment
SECONDARY
COLONIZERS
 Other type of
microbial cells.
 Metabolize wastes
from primary
colonizers.
MATURE BIOFILM
 Fully functional living tissue on pipe surface.
 Complex, metabolically co-operative community made up of diff
species reach living in a customized microniche
 Complex diffusion channels for transport of nutrient, O2, etc.
 Anaerobic layer may develop underneath aerobic biofilm
 May take several hrs. to several weeks to develop

DETACHMENT
 Continuous supply of cells that easily slough away and contaminate
other surfaces
DENTAL PLAQUE BIOFILMS
Dental Plaque
• Diverse microbial
community found http://www.intersurgical.com/info/oralca
on the tooth
surface embedded
in a matrix of
polymers of host
and microbial
origin

http://www.therabreath.com/dental-
 Structure
• Mushroom-shaped
microcolonies
• Independent
community
• Protected by
extracellular slime
layer
• Series of fluid
channels
• Communication is
done by chemical
signal
Jill S. Nield-Gehrig,
 Phases
of
Plaque
Formati
on

Jill S. Nield-Gehrig,
RDH, MA
 Pattern of
biofilm
Development

Jill S. Nield-Gehrig, RDH, MA

 Components of Plaque
• Found more than 500
bacterial strain
• Mostly found Streptococci
and Lactobacilli
1. rapidly metabolize
dietary sugars to acid
2. creates low pH
3. normal bacteria
(enamel) sensitive to low
pH
4. dissolve organic and
inorganic components of
teeth
http://www.dr-beaupere-
chirurgien-dentiste.fr/en/oral-
hygiene.php
CONTROL AND REMOVAL
• Antibiotic or antimicrobial therapy not an option-
doses need to be increased 1500 times which will
kill the patient before killing biofilm!!!
• Only viable method- Physical removal
• Frequent periodontal debridement of subgingival
surfaces by a dentist
BIOFILMS ASSOCIATED WITH
INDWELLING MEDICAL DEVICES
BIOFILMS ASSOCIATED WITH
INDWELLING MEDICAL DEVICES
• Medical devices are critical in modern-day medical practice .

• The use of medical device is the greatest exogenous predictor of

healthcare-associated infections

Common sites for occurrence of medical device associated

infections
• 95 % of urinary tract infections are associated with a urinary
catheter .

• 86 % of pneumonias are associated with mechanical ventilation .

• 87 % of the bloodstream infections are associated with an

intravascular device
CENTRAL VENOUS CATHETER-
ASSOCIATED INFECTIONS
 Why are central venous
catheters used ?
• It’s a catheter placed into a large
vein in the neck (internal jugular
vein ),chest (subclavial or axillary
vein)or groin (femur vein).
 Doctor recommends central
venous catheters :-
• Hemodialysis

• Its used to administer medication

or fluids, obtain blood tests and
measure central venous pressure
PATHOGENESIS
• Its is the most commonly
infected ,surgically
implanted device .

• The range of microbes

encountered in these
infections is wide
,including components of
the skin microflora ,some
from the environment and
some from enteric origin .

 Most commonly
found
microorganisms
are :-
• Staphylococcus aureus ,
Enterococci , Candida
albicans .
INFECTIONS AND SYMPTOMS
 Bacteremia –
Asymptomatic

 Types –
1) Transient
2) Intermittent
3) Continuous

 Septicemia –Associated
with multiplication of
microorganism .

• Symptoms – Fever ,chills,

tachycardia .
DIAGNOSIS AND PREVENTION
Diagnosis –
• Catheter tip culture

• Rapid diagnostic technique

• Scanning electro microscopy

 Prevention -
• Proper placement of CVC in an area where skin flora is less dense .

• Proper skin cleansing .

• Maintaing aseptic insertion technique during the actual insertion process .

• Clean hands with soap and water or an alcohol-based hand rub before changing
any bandages ,also known as dressings which ,cover the catheter area .
URINARY CATHETER-ASSOCIATED
INFECTIONS
Why are catheters used ?
• Urinary catheters are hollow ,flexible
tubes used to collect urine from the
bladder .

 Doctors recommend use of catheters

if :-

1) A person is unable to control while

urinating

2) During urine incontinence

3) A person is unable to empty

bladder when required
PATHOGENESIS
Sources of
microorganisms
• Endogenous – By rectal or
vaginal colonization .
• Exogenous – Usually via
contaminated hands of health
care personnel .
• During catheter insertion .
• Manipulation of the collecting
system.

• Most commonly found

organisms:-
Staphylococcus , Proteus mirabilis
,
E. coli , Klebsiella ,
Enterococcus , Streptococcus ,
Pseudomonas , Candida .
INFECTIONS AND SYMPTOMS
Bacteriuria – Develops in
about 25% of patients .Its
asymptomatic .

Urinary tract infections -

16-32 % of those who are
bacteriuric
• Fever ,chills , headache
,burning of urethra or
penis .

Candiduria – Develops in
about 25% of patients
 DIAGNOSIS AND PREVENTION
Diagnosis :-

• Urinalysis – Look for presence of white blood cells

• Urine microscopy –Looks for the presence of Red blood cells ,White
blood cells or bacteria .Urine culture is deemed positive if it shows
bacterial colony count of greater than or equal to 103 colony forming
units per ml

Prevention :-

• Insert catheters only for appropriate indications .

• Leave catheters in place only as long as needed .
• Ensure that only trained persons insert and maintain catheters .
• Insert catheters using aseptic technique and sterile equipment .
• Following aseptic insertions , maintain a closed drainage system
• Maintain unobstructed urine flow .
• Hand Hygiene
 ENDOTRACHEAL TUBE
ASSOCIATED INFECTIONS
Why is endotracheal tube
used ?
• Endotracheal tubes is a breathing tube .It
keeps airway open .Its placed through the
patient’s nose or mouth into his trachea
Doctor recommend use of
endotracheal tube :–
• To attach a ventilator if the patient is
unable to breathe on its own .

• To keep the patients trachea open .

• To allow the staff to remove mucus from

the patient’s lungs that the patient is
unable to cough up himself.
PATHOGENESIS
Sources of
microorganisms :-

• Bacteria from the biofilm lining of the

endotracheal tube might be scattered
into the lungs during ventilation gas
flow.
• Colonizing bacteria may arise from the
upper airway and ventilator tubing .

Most commonly found

microorganisms :-

• Staphylococcus aureus ,
Streptococcus pneumoniae , Klebsiella
pneumoniae ,
Pseudomonas aeruginosa .
INFECTIONS AND SYMPTOMS
Bacteria Tracheitis
infections caused in
children
• High fever

• Difficulty breathing

• A high-pitched sound when

breathing

• Ventilator-associated
pneumonia :-
• Fever

• Hypoxemia
 DIAGNOSIS AND PREVENTION
• Diagnosis
• Microbiologic study of sputum endotracheal aspirate specimen .

• Central line and pheripheral blood cultures obtained from

neonates .

• Chest radiograph is done .

• Prevention :-
• Prevention of aspiration .

• Prevention of contamination of equipments .

• Educate health care personnel about VAP.

• Perform regular oral care with an antiseptic solution .

 22/072016

FACTORS FAVOURING
BIOFILM FORMATION
FACTORS FAVOURING BIOFILM
FORMATION
• Biofilms formed on wide variety of surfaces- living
tissues, indwelling medical devices, indus or
portable water sys piping or natural water sys piping

• Diff factors-
1. Substratum Effect
2. Conditioning film
3. Hydrodynamics
4. Characteristics of Aqueous Medium
5. Horizontal Gene Transfer
6. Quorum Sensing
SUBSTRATUM EFFECT
• Surface roughness increases- Extent of microbial
colonization increases.
• Max attachment- high surface free energy or
wettablity
• E.g. Stainless steel, glass more hydrophillic
• Show greater bacterial attachment
CONDITIONING FILM
• Chemical modification of surface
• Acquired Pellicle in Oral Cavity- consists of
albumin, lysozyme, glycoprotein,
phosphoproteins, lipids and gingival crevice fluid.
Aid in colonization of bacteria
• Host produced conditioning films- blood, saliva,
tears, urine, intravascular fluid and respiratory
secretions influence attachment
HYDRODYNAMICS
• Laminar and Turbulent flow
• Laminar flow- Patchy biofilms with rough cell
aggregates separated by interstitial voids
• Turbulent flow- Patchy but elongated biofilms
CHARACTERISTICS OF AQUEOUS
MEDIUM
• pH, nutrient levels, ionic strength, temperature
etc. play imp role in attachment
• Increase in conc of cations like sodium, calcium,
lanthanun, ferric ions etc. also influence
attachment
HORIZONTAL GENE TRANSFER
• Transfer of mobile genetic elements- conjugative
plasmids, transposons, bacteriophages
• Imp for evolution and genetic diversity
• Diff genes transcribed in planktonic and biofilm-
associated phases of bacterial life cycle- bacteria
express diff phenotypic characters
• Some genes expressed in response to specific
surface on which bacterium settles
• E.g. Vibrio spp have diff structural genes for
attachment to chitin and abiotic non-nutritive
surfaces- plastic and glass
QUORUM SENSING
• Cell-to-cell Signalling
• Communication carried out by bacterial products
that are able to difuse away from 1 cell into
another. Ideal method in diffusion limited envt
• Gram-negative bacteria- production and secretion of an
acyl homoserine lactones (AHL), which diffuse through the cell
wall, from the cell to the medium.

• Gram-positive bacteria- use secreted peptides as signal

compounds and a two-component regulatory system
(composed of a membrane-bound histidine kinase receptor
and an intracellular response regulator) to detect the peptide
and trigger the required changes in gene expression.
• Third examined form of quorum sensing
mechanism employes a family of related
molecules termed autoinducers-2.
• The cell-to-cell signaling mechanism in
Pseudomonas aeruginosa controls the expression
of the catalase and superoxide dismutase genes
and mediates biofilms resistance to hydrogen
peroxide.
• The ability of signal molecules to trap external
positive-charged compounds is similar to
antimicrobial action of glycocalyx matrix.
• Signal may alter distribution of specific bacterial
spp in the biofilm, alter protein expression on
neighboring cells, introduce new genetic trait in
neighboring cells and incorporate bacteria in
biofilm
ROLE OF BIOFILMS IN
MICROBIAL
COMMUNITIES
ROLE OF BIOFILMS IN
MICROBIAL COMMUNITIES
• Survival

 Protection from Environment

 Nutrient availability
 Acquisition of New Genetic Trait
 Penetration of Antimicrobial Agent
Protection from Environment
• EPS provides certain degree of shelter and
homeostasis to bacteria in biofilm
• EPS Matrix- can physically prevent access of
certain microbial agents by acting as an anion
exchanger
• Restricts diffusion of compounds from
surroundings mainly hydrophilic antibiotics e.g.
positively charged aminoglycosides
• Sequester metal ions, cations and toxins
• Protection from environmental stresses- pH shift,
UV radiation, osmotic shock and dessication
Nutrient availability
• Water channel- enhance nutrient availability, removal of
toxins
• Inter-spp substrate exchange and removal & distribution
of metabolic products
• E.g. degradation of complex org matter into methane and
CO2 during anaerobic digestion requires interaction of
atleast 3 spp of bacteria
• Fermentative bact initiate catabolism producing acids &
alc, utilized as substrate by Acetogenic bact,
Methanogens convert acetate, CO2 and H2 to CH4
• Biofilm provides ideal envt for syntrophism- symbiosis
where metabolically distinct bact depend on each other to
utilize substrates and energy requirement
Acquisition of New Genetic
Trait
• Horizontal gene transfer
• Transcription of algC gene (alginate production)
increased 4-fold in biofilm cells. P. aeruginosa cells
mucoid due to alginate.
• Alginate production increases, flagella production
decreases- regulated by sigma factors
• To become effective member of biofilm
community, bact must diff into biofilm-associated
cells by repressing flagella production
(destabilization) and increase EPS production
• Inherent Resistance to antimicrobial agents,
antibiotics, disinfectants or germicides
Penetration of Antimicrobial
Agent
• EPS- diffusion barrier
• Influences rate of transport of mol from interior to
exterior of biofilm and vice versa
• Biofilm mode of growth is slow and restricted.
Advantages-
1. All energy is used by bact in making EPS that will protect
microbial community
2. Bact will remain in dormant stages. Most antibiotics are
inactive towards dormant bacteria
 (1) Antimicrobial agents may fail to penetrate beyond the surface layers of the
biofilm. (2) Antimicrobial agents may be trapped and destroyed by enzymes in
the biofilm matrix. (3) Altered growth rate inside the biofilm. (4) Expression of
biofilm-specific resistance genes (e.g., efflux pumps). (5) Stress response to
hostile environmental conditions.
1.The biofilm matrix (glycocalyx)
may act as diffusion barrier
• Slime or capsule may provide the forces responsible for
cohesion and adhesion to the solid surfaces .
• It may cause alterations in the gaining access of
antibacterial molecules to its targets located inside the cells.
2.Differentiation into persister
cells
• Bacterial biofilms include persisters, cells that neither grow nor
die during exposure to bactericidal agents, thus exhibit multidrug
tolerance
• differentiate into dormant cells, are able to survive extreme
antibiotic treatment
• The increased resistance to killing of biofilm is due to high level
of persisters produced by stationary phase bacteria inside biofilm
.
3.Increased production of
oxidative stress
• Oxidative stress is caused by an imbalance between the
production of oxidants, such as the free radicals, peroxide
and nitric oxide, with the levels of antioxidant defenses.
• Oxidative stress is considered to cause enhanced mutability
in biofilms.

4.Efflux pumps
• Efflux pumps can affect both intrinsic and acquired resistance to
antimicrobial agents by applying the energy to limit the
cytoplasmic compound concentration to subtoxic level.
• Exposure the bacterial biofilms to insufficient dose of antibiotics,
such as tetracycline and chloramphenicol, and to xenobiotics,
such as salicylate and chlorinated phenols, induces the
expression of multi-drug resistance operons and efflux pumps.
BIOFILMS AND
PATHOGENESIS
ENDOCARDITIS
• An infection of the heart valves or the heart’s
inner lining (endocardium)

• Types of Endocarditis-
 Native Valve Endocarditis

 Prosthetic Valve Endocarditis

Native Valve Endocarditis
• Cause
 Interaction b/w vascular endothelium- of mitral,
aortic, tricuspid and pulmonic valves of heart and
microbes circulating in blood stream

• Causative Agents
 Streptococcus, Staphylococcus, Pneumococci,
Candida, Aspergillus spp and some Gram-ve bact.
 Enter into bloodstream via pharynx, GI tract and
genitourinary tract
• Mechanism
 Injury- damaged endothelium. Development of
Non-Bacterial Thrombotic Endocarditis (NBTE-
accumulation of platelets, fibrin and occasionally
RBCs)
 Fibronectin secreted by endothelial cells, platelets,
and fibroblast have been identified in thrombotic
lesions of heart valve in response to vascular
injury
 Fibronectin can simultaneously bind to fibrin,
collagen, human cell and bacteria
 Many bacterial spp have fibronectin receptors-
Staphylococcus and Streptococcus spp.
 Prosthetic Valve Endocarditis
• Patients in whom endocarditis occurred within 60
days of the original prosthetic valve replacement
were defined as having "early" endocarditis

• Patients in whom endocarditis occurred 60 days or

more after insertion of the prosthetic valve were
defined as having 'late" endocarditis

• The risk is greatest during the initial three months

after surgery
SOURCE OF INFECTION
 Early endocarditis
 Late endocarditis
 Contaminated intravenous
 urinary infection
catheter
 Postoperative pneumonia
 genitourinary
infection
 Postoperative wound infection
 Dental infection

Organisms Causing Prosthetic

Valve Endocarditis
 Early endocarditis - Staphylococcus aureus, Klebsiella,
Pseudomonas aeruginosa, candida species, Diptheroid

 Late endocarditis – Streptococcus, gram negative rods,

micrococcus
Mechanism
 Host proteins are exposed in areas of endothelial
disruption at sites of contact with prosthetic
devices
 microbial surface components responsible for
attachment to molecular molecules allow
Staphylococcus aureus to bind to host proteins that
coat the surface of prosthetic devices
 S. aureus is able to bind to fibrinogen, fibronectin,
and collagen, as well as to gene regulators that
control the expression of these adhesins.
 Infections of prosthetic heart valves usually
originate when fibrin and thrombi occur at the site
of the suture line and/or annulus, allowing
microorganisms to subsequently adhere
Common Microorganisms and
Treatment Regimens
OTITIS MEDIA
• Chronic ear infection
• Involves inflammation of mucoperiosteal lining
(Eustachian tube, tympanic cavity, mastoid
antrum and mastoid air cell)

• Causative Agents
 S. pneumoniae, H. influnzae, Moraxella
catarrhalis, S. epidermidis, P. aeruginosa etc
• Types

• Acute OM (AOM)- defined by the presence of fever,

irritability, pulling at the ears and tympanic membrane
changes

• OM with effusion (OME)- diagnosed when fluid is

visible in the middle ear in the absence of symptoms
and in the absence of recent episodes of AOM

• Chronic suppurative OM- persistent ear infection

that results in tearing or perforation of the eardrum

• Adhesive OM- occurs when a thin retracted ear drum

becomes sucked into the middle ear space and stuck
• Treatment
 Low conc of antibiotic is present in middle ear
fluid due to limited penetration of antibiotic

 Powerful antibiotics- Amoxyxillin, Cefaclor,

Erythromycin, and Clarithromycin are used
CYSTIC FIBROSIS
• Chronic disease of lower respiratory tract
• inherited, autosomal recessive disease affecting
exocrine gland function and usually manifests itself
at birth or early childhood as a gastrointestinal or
pulmonary disorder
• Patients with CF have various recurrent and chronic
lung infections from early life, and most will
eventually acquire chronic P. aeruginosa lung
infection

• Causative agents
 P. aeruginosa, S. aureus, H. influenzae, S.
pneumoniae, Burkholderia cepacia
• Mechanism
 Many species of bacteria produce extracellular
polymers that may facilitate non-specific adhesion
to surfaces and provide the framework for biofilm
formation

 The major reason why P. aeruginosa persists in the

lungs appears to be due to its ability to produce
alginate-containing microcolonies

 These bacterial populations adapt to the highly

compartmentalized and anatomically deteriorating
lung environment of CF patients, as well as to
challenges by the body’s immune defences and
antibiotic therapies
 Alginate has been shown to inhibit phagocytosis
and biofilm bacteria appear to stimulate a lesser
oxidative burst response by neutrophils than free-
swimming bacteria

 Alginate, together with the host’s mucus, may

reduce the action of some antibiotics and provide
increased protection from toxic oxygen radicals
• Treatment
 Possibilities for successful treatment of CF may
ultimately hinge on early antimicrobial treatment
to prevent or delay chronic infection with P.
aeruginosa

 Early treatment with oral Ciprofloxacin and

Colistin may postpone chronic infection for several
years
CHRONIC BACTERIAL PROSTITIS
• Prostate- small gland located directly below the
bladder in men
• Infection of prostate gland due to MOs ascended
from urethra or by reflux of infected urine into
prostatic ducts emptying into posterior urethra
• results in swelling, inflammation, and frequent
urinary tract infections (UTIs)

• Causative Agents
 E. coli, P. aeruginosa and spp of Klebsiella,
Proteus, Serratia, Bacteroides etc.
• Mechanism
 Ascending urethral infection
 Reflux of infected urine into prostatic ducts
 Migration of rectal bacteria via direct extension or
lymphogenous spread
 Hematogenous infection
• Treatment
• Prostatitis treatments vary depending on the
underlying cause. They can include:
 Antibiotics. This is the most commonly
prescribed treatment for prostatitis.

 Alpha blockers. These medications help relax

the bladder neck and the muscle fibers where
your prostate joins your bladder. This treatment
may lessen symptoms, such as painful urination.
PERIODONTITIS
• Involve supporting tissues of teeth, gums
(gingiva) and periodontal tissues (gingiva,
alveolar bone and periodontal ligament)
• May lead to exfoliation (falling out) of teeth

• Causative Agents
 Porphyromonas gingivalis, Fusabacterium
nucleatum, Peptostreptococcus micros,
Eubacterioum timidum, E. brachy, P. anaerobicans
• Mechanism
• Dental plaque biofilm formation

• Treatment
• Physical removal
 Formation of
Biofilms by
various organisms
• Staphylococcus aureus
• Staphylococcus epidermidis
• Helicobacter pyroli
• Pseudomonas aeruginosa
• Escherichia coli
Staphylococcus aureus
• Staphylococcus aureus is a Gram-positive, coccal
ubiquitous bacterial species
• Positive for catalase and nitrate reduction
• Usually found in nose, respiratory tract or skin
• It can produce a multilayered biofilm embedded
within a glycocalyx or slime layer.
• the solid component of the glycocalyx is
composed of teichoic acids and staphylococcal
and host proteins such as PIA.
• Attachment
 S. aureus express MSCRAMMs
 MSCRAMMs have a common structure - an
exposed binding domain, a cell-wall spanning
domain and a domain that is responsible for the
covalent or non-covalent attachment.
 Covalent attachment is catalyzed by a family of
enzymes called sortases .
 Autolysins demonstrate non-covalent attachment.
• Maturation
 The maturation phase of biofilm formation is
characterized by
1) intercellular aggregation
• PIA dependent
• PIA independent

2) biofilm structuring forces that lead to the typical

3-dimensional appearance of mature biofilms
• Detachment
• Several factors may contribute to detachment:
1) mechanical forces
2) cessation of the production of biofilm building
material
3) others detachment factors, such as enzymes
that destroy the matrix, or surfactants.
• control biofilm thickness and expansion.
Mechanisms in biofilm regulation
Staphylococcus epidermidis
• Staphylococcus epidermidis is a gram positive bacteria
• Commensal organism
• It belongs to coagulase negative species
Biofilm formation
• The genomes of two S. epidermidis isolates,
ATCC12228 (~2.5 Mb) and RP62A (also known
as ATCC35984; ~2.6 Mb), have been fully
sequenced
• 3 steps:
1. Adhesion :
• MSCRAMMs (microbial surface components recognizing
adhesive matrix molecules) binds to serum proteins like
fibrinogen and fibronectin
2. Intercellular aggregation and accumulation:
• Exopolysaccharide polysachharide intercellular adhesin
(PIA) is a facilitator of biofilm formation.
 Synthesized by the gene products of the ica operon,
icaA, icaD, icaB, and icaC and has icaR regulator .
 PIA increases the persistence of infections and leads to
a decreased efficacy of antibiotic-induced bactericidal
activity
• Accumulation-associated protein (Aap):
• Helps in accumulation and intercellular adhesion
• Forms polymeric fibrils on the surface of cells
• It is proteinuous factor for biofilm formation
• Requies zinc for fibril formation
• The extracellular matrix-binding protein
(Embp) :
 of 1MDa and binds to fibronectin
Toxin- Phenly soulble modulins
(PSMs)
• PSMs have shown to lyse neutrphills and erythrocytes
• PSMs consists of 3 peptide: PSMα, PSMβ, PSMδ
• PSMβ is not cytolytic
• Poly-γ-DL-glutamic acid that protects S.
epidermidis against high salt concentrations in addition to
mediating resistance to antimicrobial peptides and
phagocytosis
Regulation
• Most extracellular and surface-attached virulence
factors are regulated by the agr (accessory gene
regulator) locus
Pseudomonas aeruginosa
Attachment
 Transport: diffusive, convective, and active flagellum-driven
transport.

 Attachment: flagella, type IV pili, Cup fimbria, extracellular DNA

(eDNA), and Psl polysaccharide.

 After P. aeruginosa has attached to a surface:

 Either detaches,
 remains attached at the position of attachment,
 or moves along the surface by means of type IV pili or flagella.

• Gene sadB: regulation of the frequency by which P. aeruginosa cells

attach to and detach from surfaces
Motility
 Surface-associated motility is an integrated part of P. aeruginosa biofilm
formation.

 Pattern of motility depends on the prevailing conditions.

 Glucose as a carbon source: the attached bacteria differentiate initially into a

nonmotile subpopulation and a motile subpopulation.
 Citrate is used as a carbon source: reduces the level of iron available to the
bacteria hence entire population is motile.
 Rhamnolipid is necessary for initial microcolony formation in
• P. aeruginosa biofilms.

 Low intracellular levels of c-di-GMP promote motility, whereas high

intracellular cdi- GMP levels induce the formation of cell–cell interconnecting
matrix components and promote microcolony formation.
Matrix formation
 The extracellular polymeric substance (EPS) matrix serves as the
• ‘house for biofilm cells’.

 Role of matrix:
• attachment, cell-to-cell interconnection, interactions between subpopulations,
tolerance, and exchange of genetic material.

 The mannose-rich Psl polysaccharide, encoded by the psl cluster, is highly

conserved in many P. aeruginosa strains.

 Alginate mainly produced by P. aeruginosa in chronic infections of the lungs of

CF patients.

 eDNA was recognized as one of the major matrix components of bacterial

biofilms.
Quorum sensing in P.
aeruginosa
 Mediated through three interconnected systems:
 the Las system : senses 3-oxo-C12-homoserine lactone
 the Rhl system : senses C4- homoserine lactone
 the Pqs system : senses 2-heptyl-3-hydroxy-4-quinolone
referred to as PQS

 Quorum sensing is necessary for the formation of the

cap portion of the mushroom-shaped structures in P.
aeruginosa biofilms.
Helicobacter pyroli
• Helicobacter pylori (H. pylori ) is a Gram negative
pathogen that selectively colonizes the human
gastric epithelium.
• Over 50% of the world population is infected with
H. pylori.
• This species is defined as a urease, catalase and
oxidase positive.
• Spiraled microaerophilic bacterium, mobile by
means of 3-5 polar flagella.
• Labile at pH 3.0
Mechanism of biofilm
formation
• The adherence of H. pylori to gastric mucosa
• BabA adhesin binds to Lewis b antigen
• Gastric epithelial cells are protected by a mucous
layer composed mainly by MUC5AC which
contains a domain rich in glycans (including the
Lewis b antigen), the target for H. pylori BabA
adhesin
• The MUC6 mucin synthesized in the deep mucosa
and secreted by glandular mucosal cells works as
a natural antibiotic by inhibiting H. pylori growth,
avoiding the colonization of the deep gastric
mucosa layer by the pathogen.
Genes responsible for bioflim
formation
1) luxS gene :- Autoinducer 2 (AI-2) of QS system
2) flaA gene :- type I and type IV pili and surface
Adhesins
3) lpxD gene:- cell envelop protein UDP-3-0-(3-
hydroxymyristoyl) glucosamine N-
acyltransferase
4) ureA gene :- H. pylori urease enzyme (ureA) is
essential for pH regulation
5) omp18 gene :- Bacterial outer membrane
proteins (OMPs) are important for ion transport,
osmotic stability, bacterial virulence and
adherence
References
• The In Vivo Dynamics Of Streptococcus Spp., Actinomyces Naeslundii, Fusobacterium Nucleatum And
Veillonella Spp. In Dental Plaque Biofilm As Analysed By Five-Colour Multiplex Fluorescence In Situ
Hybridization Ali Al-Ahmad, Axel Wunder, Thorsten Mathias Aauschill, Marie Follo, Gabriele Braun, Elmar
Hellwig And Nicole Birgit Arweiler Journal Of Medical Microbiology (2007), 56, 681–687
• Nield-Gehrig Js And Willmann De. Foundations Of Periodontics For The Dental Hygienist. Philadelphia:
Lippincott Williams & Wilkins 2003:67-73
• Dental Plaque As A Biofilm And A Microbial Community Implications For Health And Disease Philip D Marsh;
Bmc Oral Health 2006, 6(Suppl 1):S14 Doi:10.1186/1472-6831-6-S1-S14
• Dental Plaque Biofilms: Communities, Conflict And Control Philip D. Marsh, Annette Moter & Deirdre A. Devine
Periodontology 2000, Vol. 55, 2011, 16–35
• Vonezawa H, Osaki , Hanawa T, Kurata S, Ochiai K, Et Al. (2013) Impact Of Helicobacter Pylori Biofilm
Formation On Clarithromycin Susceptibility And Generation Of Resistance Mutations. Plos One 8(9): E73301.
Doi:10.1371/Journal.Pone.0073301
• Carolina Herrera, Carlos González Et Al. (2014) Biofilm And Helicobacter Pylori: From Environment To Human
Host. World J Gastroenterol 20(19): 5632-5638. Doi: 10.3748/Wjg.V20.I19.5632
• Alba E. Vega, Fabio A. Persia, Et Al. (2012) Helicobacter Pylori Biofilm Formation And Gene Expression On
Abiotic Surfaces Using A Cyanobacterial Extract. Journal Of Life Sciences 6 (2012) 1317-1327
• Staphylococcus Aureus Biofilms Properties, Regulation And Roles In Human Disease Archer Et Al Virulence 2:5,
1-15; September/October 2011.
• How Staphylococcus Aureus Biofilms Develop Their Characteristic Structure Periasamy Et Al Pnas | January
24, 2012 | Vol. 109 | No. 4 | 1283
• Staphylococcal Biofilms Michael Otto Curr Top Microbiol Iimmunol. 2008; 322: 207–228
• Michael Otto. Molecular Basis Of Staphylococcus Epidermidis Infections. Semin Immunopathol. 2012 March ;
34(2): 201-214
• James P. O’gara And Hilary Humphreys. Staphylococcus Epidermidis Biofilms: Importance And Implications. J.
Med. Microbiol.- Vol. 50 (2001), 582-587
• Shrout Jd, Chopp Dl, Just Cl, Hentzer M, Givskov M & Parsek Mr (2006) The Impact Of Quorum Sensing And
Swarming Motility On Pseudomonas Aeruginosa Biofilm Formation Is Nutritionally Conditional. Mol Microbiol
62: 1264–1277
• Klausen M, Heydorn A, Ragas P, Lambertsen L, Aaes-Jorgensen A, Molin S & Tolker-Nielsen T (2003b) Biofilm
Formation By Pseudomonas Aeruginosa Wild Type, Flagella And Type Iv Pili Mutants. Mol Microbiol 48: 1511–
• Yang L, Nilsson M, Gjermansen M, Givskov M & Tolker-Nielsen T (2009) Pyoverdine And Pqs Mediated
Subpopulation Interactions Involved In Pseudomonas Aeruginosa Biofilm Formation. Mol Microbiol 74:
1380–1392.
• Ryder C, Byrdm&Wozniak Dj (2007) Role Of Polysaccharides In Pseudomonas Aeruginosa Biofilm
Development. Curr Opin Microbiol 10: 644–648.
• Purevdorj B, Costerton Jw & Stoodley P (2002) Influence Of Hydrodynamics And Cell Signaling On
The Structure And Behavior Of Pseudomonas Aeruginosa Biofilms. Appl Environ Microb 68: 4457–
4464
• Trapnell Bc Et.Al For Inhalation (Fti): Efficacy Results Of A Phase 2 Placebo-Controlled Trial In Patients
With Cystic Fibrosis And Pseudomonasaeruginosa [Abstract]. Pediatr Pulmonol 2010, 45: 302
• Herrmann G Et.Al : Colistin-Tobramycin Combinations Are Superior To Monotherapy Concerning
Killing Of Biofilm Pseudomonas Aeruginosa. J Infect Dis 2010, 202: 1585-1592.
• C.Desrousseaux,V.Sautou,S.Descamps,O.Traove,October 2013,Modification Of The Surface Of Medical
Device To Prevent Adhesion And Bioflim Formation ,Doi:10:1016/Jhin.2013.06.015,Volume 85,Issue
2,Pages 87-93 .
• Infections Disease In Critical Care Book (Second Edition ) By Jordi Rello,Marin Kollef ,Emili Diaz .
• Rodney M. Donlan ,March –April 2001,Biofilms And Device-Associated Infections,Vol.7 ,No .2
• Li Zhang,John Gowardman And Claire M.Richard ,The Impact Of Microbial Attachment On
Intravascular Catheter- Related Infections .
• Leonard.A.Merme, Barry M. Farrr ,Robert J.Sherertz ,Issam I.Raad (2011)Guideline For Management Of
Intravascular Catheter Related Infection Volume 32,Issue 9,Pages 1249-1272
• Propionibacterium Acnes Prosthetic Valve Endocarditis With Abscess Formation: A Case Report Mario
Kurz1, Beat A Kaufmann, Larry M Baddour,Andreas ,F Widmer Bmc Infectious Diseases-Journal
2014, 14:105
• Biofilms: Survival Mechanisms Of Clinically Relevant Microorganisms Rodney M. Donlan, J. William
Costerton Clinical Microbiology Reviews-Journal Vol.15 No.2 ,Apr. 2002
• Prosthetic Valve Endocarditis Analysis Of 38 Cases William E. Dismukes, Adolf W. Karchmer,Mortimer
J. Buckley, W. Gerald Austen, Morton N. Swartz Circulation, 1973;48:365-377
• Mechanisms Determining Bacterial Biofilm Resistance To Antimicrobial Factors .Abee, T., Kovács,
Á.T., Kuipers, O.P. & Van Der Veen, S. (2011). Biofilm Formation And Dispersal In Gram-Positive
Bacteria. Current Opinion In Biotechnology, Vol. 22, Pp. 172-179, Issn 0958-1669.