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BIOFILMS IN INFECTION
UPDATED
DR.T.V.RAO MD
Dr.T.V.Rao MD 1
BEGINNING OF MICROBES
• Bacteria first appeared on
earth about 3.6 billion years
ago, long before the
appearance of Homo
s...
BIO FILM CONSTITUTES
• A biofilm is an aggregate of
microorganisms in which cells
are stuck to each other and/or
to a surf...
BIOFILM IS A COMPLEX SUBSTANCE.
• A biofilm is a complex
aggregation of
microorganisms growing on
a solid substrate. Biofi...
BIOFILMS FOUND IN NATURE EVERYWHERE
WHERE IS THERE IS MOISTURE
• More properly known as
biofilm, slime cities thrive
where...
BIOFILM SUPPORTS THE
BACTERIAL GROWTH
• Biofilm are a common mode of bacterial growth in
nature and their presence has an ...
FORMATION OF BIOFILMS
• Biofilms may form on
living or non-living
surfaces, and
represent a prevalent
mode of microbial
li...
BIOFILMS INCREASES ANTIBIOTIC
RESISTANCE
With
microorganisms
are highly
resistant to
antimicrobial
treatment and are
tenac...
MECHANISMS OF BIOFILM FORMATION
• Formation of a biofilm
begins with the attachment
of free-floating
microorganisms to a
s...
FACTORS INFLUENCING RATE AND
EXTENT OF BIOFILM FORMATION
• Indwelling medical device when contaminated
with microorganisms...
TECHNOLOGY UNDERSTANDS
BIOFILMS BETTER…
• Technological progress in
microscopy, molecular
genetics and genome
analysis has...
STEPS IN BIOFILM DEVELOPMENT
• Biofilm development can be
divided into several key
steps including attachment,
micro colon...
STAGES OF BIOFILM DEVELOPMENT.
Dr.T.V.Rao MD 13
STEPS IN BIOFILM FORMATION
Dr.T.V.Rao MD 14
BACTERIA ASSOCIATED WITH
BIOFILMS DIFFER• Bacteria living in a biofilm can
have significantly different
properties from fr...
BIOFILMS MAJOR CAUSE OF
NOSOCOMIAL INFECTIONS
• Microbial biofilms,
which often are
formed by
antimicrobial-
resistant org...
BIOFILMS A GREAT THREAT TO IMPLANTS
• A significant number of people
are affected by biofilm
infections which develop on
m...
Dr.T.V.Rao MD 18
BIOFILMS A GROWING CONCERN
IN MODERN MEDICINE
• Prosthetic device infections,
such as those involving artificial
heart val...
DENTAL PLAQUE
• Dental plaque is a
yellowish biofilm
that build up on
the teeth. If not
removed regularly,
it can lead to
...
DENTAL PLAQUES
• The formation of
dental plaque bio
films includes a
series of steps that
begins with the initial
coloniza...
FORMATION OF DENTAL BIOFILMS
• Additionally, through
the growth process of
the plaque bio film, the
microbial composition
...
CELL-CELL SIGNALLING (EX. QUORUM SENSING), AND
COMMUNICATION WITH DIFFERENT BACTERIA
ENHANCE BIOFILM FORMATION
Dr.T.V.Rao ...
Dr.T.V.Rao MD 24
BIOFILMS EVERYWHERE
• They're everywhere: on your
shower curtain, on medical
devices implanted in
patients, on rocks in ri...
CDC – ON BIOFILMS
• Biofilms form on the surface of
catheter lines and contact lenses. They
grow on pacemakers, heart valv...
Dr.T.V.Rao MD 27
BIOFILMS INTERFERE IN ANTIBIOTIC
THERAPY
• Bacteria growing in a biofilm
are highly resistant to
antibiotics, up to 1,000
...
BIOFILM AND ANTIBIOTIC RESISTANCE
• A key property of bio films is
that individual
microorganisms are bound
together by a ...
BACTERIAL RESISTANCE AND
BIOFILMS
• Another area of great importance
from a public health perspective is
the role of biofi...
BIOFILMS IN CYSTIC FIBROSIS
• Biofilms are involved
in numerous
diseases. In cystic
fibrosis patients have
Pseudomonas
inf...
ENDOCARDITIS AND BIOFILMS
• Microorganisms may attach and develop
biofilms on components of mechanical
heart valves and su...
EYE AND BIOFILMS
• The presence of bacterial
biofilms has been
demonstrated on many
medical devices including
intravenous ...
BIOFILMS AND CONTACT LENSES
• Bacterial biofilm
formation on contact
lenses and contact lens
storage cases may be a
risk f...
URINARY CATHETERS AND
BIOFILMS
• Urinary catheters are tubular latex or silicone devices,
which when inserted may readily ...
BIOFILMS AND INDWELLING
MEDICAL DEVICES
• Biofilms on indwelling medical devices may be
composed of gram-positive or gram-...
INDWELLING CATHETERS AND
BIOFILMS*• Central venous catheters, the reference method
for quantification of biofilms on cathe...
INDWELLING CATHETERS AND
BIOFILMS*
• In addition, this method cannot detect more
than 1,000 colony-forming units (CFU) per...
ANTIBIOTIC THERAPY ALONE MAY NOT CURE
?
• Antimicrobial agents are
administered during valve
replacement and whenever
the ...
BIOFILMS A CONCERN IN
ANTIMICROBIAL THERAPY
• Microbial biofilms may pose a public health
problem for persons requiring in...
BIOFILMS NEED HIGHER
CONCENTRATION OF ANTIBIOTICS
• Biofilms are remarkably difficult
to treat with antimicrobials.
Antimi...
BIOFILMS HELP GENE TRANSFER
• Biofilms increase the
opportunity for gene
transfer
between/among
bacteria.. Gene transfer
c...
BIOFILMS –QUORUM SENSING
• Certain species of bacteria
communicate with each
other within the biofilm. As
their density in...
QUORUM SENSING HELPS THE
SURVIVAL OF PATHOGENS
Dr.T.V.Rao MD 44
BIOFILMS CONTRIBUTE FOR NEW
PHENOTYPES
• Bacteria express new, and
sometimes more virulent
phenotypes when growing
within ...
BIOFILMS PROTECTS FROM IMMUNE
RESPONSES
• Bacteria embedded within
biofilms are resistant to
both immunological and
non- s...
BIOFILMS – PROTECTS FROM
PHAGOCYTOSIS• Phagocytes are unable to
effectively engulf a
bacterium growing within
a complex po...
CURRENT OBJECTIVES ON
BIOFILM RESEARCH
• o Development of improved imaging of biofilms in
situ; o
Development of improved ...
CURRENT OBJECTIVES ON BIOFILM
RESEARCH
• o Further characterization of biofilm-specific gene
expression;
o Studies of the ...
CURRENT OBJECTIVES ON BIOFILM
RESEARCH
• o Studies of pathogenic
mechanisms of microbes
growing in biofilms;
o Elucidation...
CURRENT OBJECTIVES ON
BIOFILM RESEARCH
• In studies of infectious lung disease in cystic
fibrosis; o Studies
on the potent...
SEARCHING FOR ALTERNATIVES –
TISSUE ENGINEERING
• Role of biofilms in multiple pathologies and the
difficulty in resolving...
EMERGING METHODS FOR CURE
• Several researchers are
finding solutions for the
cure of Biofilms , yet it is
experimental, w...
• Programme Created by Dr.T.V.Rao MD for
Medical and Microbiology Professionals for
Global education on developments in
Sc...
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Biofilms in infection

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BIO FILMS IN INFECTION
UPDATED

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  • Sir, kindly shed more light on the bio-film formation inside the Reverse- osmosis water supply pipe-lines to the dialysis machines,the recirculation of the water with the continuous motion,the use of bleach,formalin or heat clean to decontaminate or decolonizing them are available.Any better suggestion to solve these problem with better methods?
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Transcript of "Biofilms in infection "

  1. 1. BIOFILMS IN INFECTION UPDATED DR.T.V.RAO MD Dr.T.V.Rao MD 1
  2. 2. BEGINNING OF MICROBES • Bacteria first appeared on earth about 3.6 billion years ago, long before the appearance of Homo sapiens around 100,000 years ago.. Van Leeuwenhoek was the first person to visualize, graphically illustrate, and label "animalcules" (bacteria) that he found in plaque scraped from his own teeth. Dr.T.V.Rao MD 2
  3. 3. BIO FILM CONSTITUTES • A biofilm is an aggregate of microorganisms in which cells are stuck to each other and/or to a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS). Biofilm EPS, which is also referred to as "slime," is a polymeric jumble of DNA, proteins and polysaccharides. Dr.T.V.Rao MD 3
  4. 4. BIOFILM IS A COMPLEX SUBSTANCE. • A biofilm is a complex aggregation of microorganisms growing on a solid substrate. Biofilms are characterized by structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances. Dr.T.V.Rao MD 4
  5. 5. BIOFILMS FOUND IN NATURE EVERYWHERE WHERE IS THERE IS MOISTURE • More properly known as biofilm, slime cities thrive wherever there is water - in the kitchen, on contact lenses, in the gut linings of animals. When the urban sprawl is extensive, bio films can be seen with the naked eye, coating the inside of water pipes or dangling slippery and green from plumbing.“ (Coghlan 1996) Dr.T.V.Rao MD 5
  6. 6. BIOFILM SUPPORTS THE BACTERIAL GROWTH • Biofilm are a common mode of bacterial growth in nature and their presence has an enormous impact on many aspects of our lives, such as sewage treatment, corrosion of materials, food contamination during processing, pipe collapse, plant-microorganisms interaction in the biosphere, the formation of dental plaque, the development of chronic infections in live tissue (mastitis, Otitis, pneumonia, urinary infections, osteomyelitis) or problems related to medical implants. Dr.T.V.Rao MD 6
  7. 7. FORMATION OF BIOFILMS • Biofilms may form on living or non-living surfaces, and represent a prevalent mode of microbial life in natural, industrial and hospital settings Dr.T.V.Rao MD 7
  8. 8. BIOFILMS INCREASES ANTIBIOTIC RESISTANCE With microorganisms are highly resistant to antimicrobial treatment and are tenaciously bound to the surface Dr.T.V.Rao MD 8
  9. 9. MECHANISMS OF BIOFILM FORMATION • Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible van der Waals forces. If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures such as pili Dr.T.V.Rao MD 9
  10. 10. FACTORS INFLUENCING RATE AND EXTENT OF BIOFILM FORMATION • Indwelling medical device when contaminated with microorganisms, several variables determine whether a biofilm develops. First the microorganisms must adhere to the exposed surfaces of the device long enough to become irreversibly attached. The rate of cell attachment depends on the number and types of cells in the liquid to which the device is exposed, the flow rate of liquid through the device, and the physicochemical characteristics of the surfaceDr.T.V.Rao MD 10
  11. 11. TECHNOLOGY UNDERSTANDS BIOFILMS BETTER… • Technological progress in microscopy, molecular genetics and genome analysis has significantly advanced our understanding of the structural and molecular aspects of biofilms, especially of extensively studied model organisms such as Pseudomonas aeruginosa. Dr.T.V.Rao MD 11
  12. 12. STEPS IN BIOFILM DEVELOPMENT • Biofilm development can be divided into several key steps including attachment, micro colony formation, biofilm maturation and dispersion; and in each step bacteria may recruit different components and molecules including flagella, type IV pili, DNA and exo polysaccharides. Dr.T.V.Rao MD 12
  13. 13. STAGES OF BIOFILM DEVELOPMENT. Dr.T.V.Rao MD 13
  14. 14. STEPS IN BIOFILM FORMATION Dr.T.V.Rao MD 14
  15. 15. BACTERIA ASSOCIATED WITH BIOFILMS DIFFER• Bacteria living in a biofilm can have significantly different properties from free-floating bacteria, as the dense and protected environment of the film allows them to cooperate and interact in various ways. One benefit of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and the outer layer of cells protect the interior of the community. Dr.T.V.Rao MD 15
  16. 16. BIOFILMS MAJOR CAUSE OF NOSOCOMIAL INFECTIONS • Microbial biofilms, which often are formed by antimicrobial- resistant organisms, are responsible for 65% of infections treated in the developed world.Dr.T.V.Rao MD 16
  17. 17. BIOFILMS A GREAT THREAT TO IMPLANTS • A significant number of people are affected by biofilm infections which develop on medical devices implanted in the body such as catheters (tubes used to conduct fluids in or out of the body), artificial joints, and mechanical heart valves. When implanted material becomes colonized by microorganisms, a slow developing but persistent infection results. Dr.T.V.Rao MD 17
  18. 18. Dr.T.V.Rao MD 18
  19. 19. BIOFILMS A GROWING CONCERN IN MODERN MEDICINE • Prosthetic device infections, such as those involving artificial heart valves, intravascular catheters, or prosthetic joints, are prime examples of biofilm- associated infections. With the increasing use of such devices in the modern practice of medicine, the prevalence of these infections is expected to increase. Dr.T.V.Rao MD 19
  20. 20. DENTAL PLAQUE • 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 20
  21. 21. DENTAL PLAQUES • The formation of dental plaque bio films includes a series of steps that begins with the initial colonization of the pellicle and ends with the complex formation of a mature bio film.Dr.T.V.Rao MD 21
  22. 22. FORMATION OF DENTAL BIOFILMS • Additionally, through the growth process of the plaque bio film, the microbial composition changes from one that is primarily gram-positive and streptococcus-rich to a structure filled with gram-negative anaerobes in its more mature state.Dr.T.V.Rao MD 22
  23. 23. CELL-CELL SIGNALLING (EX. QUORUM SENSING), AND COMMUNICATION WITH DIFFERENT BACTERIA ENHANCE BIOFILM FORMATION Dr.T.V.Rao MD 23
  24. 24. Dr.T.V.Rao MD 24
  25. 25. BIOFILMS EVERYWHERE • They're everywhere: on your shower curtain, on medical devices implanted in patients, on rocks in rivers and streams, and in your nose. While the sheer number of different organisms a biofilm may contain makes it a challenge to study, Dr.T.V.Rao MD 25
  26. 26. CDC – ON BIOFILMS • Biofilms form on the surface of catheter lines and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints and other surgical implants. The CDC (Centers for Disease Control) estimate that over 65% of Nosocomial (hospital-acquired) infections are caused by biofilms.Dr.T.V.Rao MD 26
  27. 27. Dr.T.V.Rao MD 27
  28. 28. BIOFILMS INTERFERE IN ANTIBIOTIC THERAPY • Bacteria growing in a biofilm are highly resistant to antibiotics, up to 1,000 times more resistant than the same bacteria not growing in a biofilm. Standard antibiotic therapy is often useless and the only recourse may be to remove the contaminated implant. Dr.T.V.Rao MD 28
  29. 29. BIOFILM AND ANTIBIOTIC RESISTANCE • A key property of bio films is that individual microorganisms are bound together by a polymeric substance excreted by the microorganisms.. This protective encapsulation is believed to play a role in some antibiotic-resistant infection. Dr.T.V.Rao MD 29
  30. 30. BACTERIAL RESISTANCE AND BIOFILMS • Another area of great importance from a public health perspective is the role of biofilms in antimicrobial- drug resistance. Bacteria within biofilms are intrinsically more resistant to antimicrobial agents than plank tonic cells because of the diminished rates of mass transport of antimicrobial molecules to the biofilm associated cells or because biofilm cells differ physiologically from plank tonic cells Dr.T.V.Rao MD 30
  31. 31. BIOFILMS IN CYSTIC FIBROSIS • Biofilms are involved in numerous diseases. In cystic fibrosis patients have Pseudomonas infections that often result in antibiotic resistant biofilms. Dr.T.V.Rao MD 31
  32. 32. ENDOCARDITIS AND BIOFILMS • Microorganisms may attach and develop biofilms on components of mechanical heart valves and surrounding tissues of the heart, leading to a condition known as prosthetic valve endocarditis. The primary organisms responsible for this condition are S. epidermidis, S. aureus, Streptococcus spp., gram-negative bacilli, diphtheroids, enterococci, and Candida spp. These organisms may originate from the skin, other indwelling devices such as central venous catheters, or dental work. Dr.T.V.Rao MD 32
  33. 33. EYE AND BIOFILMS • The presence of bacterial biofilms has been demonstrated on many medical devices including intravenous catheters, as well as materials relevant to the eye such as contact lenses, scleral buckles, suture material, and intraocular lenses. Many ocular infections often occur when such prosthetic devices come in contact with or are implanted in the eye.Dr.T.V.Rao MD 33
  34. 34. BIOFILMS AND CONTACT LENSES • Bacterial biofilm formation on contact lenses and contact lens storage cases may be a risk factor in contact lens-associated corneal infections. Studies have shown that contamination of lens cases by bacteria, fungi, and amoebae is common with 20% to 80% of lens wearers having a contaminated lens case. Dr.T.V.Rao MD 34
  35. 35. URINARY CATHETERS AND BIOFILMS • Urinary catheters are tubular latex or silicone devices, which when inserted may readily acquire biofilms on the inner or outer surfaces. The organisms commonly contaminating these devices and developing biofilms are S. epidermis's, Enterococcus faecalis, E. coli, Proteus mirabilis, P. aeruginosa, K. pneumoniae, and other gram-negative organisms. The longer the urinary catheter remains in place, the greater the tendency of these organisms to develop biofilms and result in urinary tract infections. Dr.T.V.Rao MD 35
  36. 36. BIOFILMS AND INDWELLING MEDICAL DEVICES • Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative bacteria or yeasts. Bacteria commonly isolated from these devices include the gram-positive Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus viridians; and the gram-negative Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. Dr.T.V.Rao MD 36
  37. 37. INDWELLING CATHETERS AND BIOFILMS*• Central venous catheters, the reference method for quantification of biofilms on catheter tips is the roll-plate technique, in which the tip of the catheter is removed and rolled over the surface of a nonselective medium. Quantification of the biofilm depends on the number of organisms recovered by contact with the agar surface. Biofilm-associated cells on the inner lumen of the device are not detected with this method, which has low diagnostic sensitivity and low predictive value for catheter-related bacteraemia.Dr.T.V.Rao MD 37
  38. 38. INDWELLING CATHETERS AND BIOFILMS* • In addition, this method cannot detect more than 1,000 colony-forming units (CFU) per tip. A method that used sonication plus vortexing as a means of quantifying biofilms on catheter tips showed that a level of 104 CFU per tip is predictive of catheter-related septicaemia * Biofilms and Device-Associated Infections Rodney M. Donlan Centers for Disease Control and Prevention Atlanta, Georgia, USA Dr.T.V.Rao MD 38
  39. 39. ANTIBIOTIC THERAPY ALONE MAY NOT CURE ? • Antimicrobial agents are administered during valve replacement and whenever the patient has dental work to prevent initial attachment by killing all microorganisms introduced into the bloodstream. As with biofilms on other indwelling devices, relatively few patients can be cured of a biofilm infection by antibiotic therapy alone Dr.T.V.Rao MD 39
  40. 40. BIOFILMS A CONCERN IN ANTIMICROBIAL THERAPY • Microbial biofilms may pose a public health problem for persons requiring indwelling medical devices. The microorganisms in biofilms are difficult or impossible to treat with antimicrobial agents; detachment from the device may result in infection. Although medical devices may differ widely in design and use characteristics, specific factors determine susceptibility of a device to microbial contamination and biofilm formation. Dr.T.V.Rao MD 40
  41. 41. BIOFILMS NEED HIGHER CONCENTRATION OF ANTIBIOTICS • Biofilms are remarkably difficult to treat with antimicrobials. Antimicrobials may be readily inactivated or fail to penetrate into the biofilm. In addition, bacteria within biofilms have increased (up to 1000-fold higher) resistance to antimicrobial compounds, even though these same bacteria are sensitive to these agents if grown under plank tonic conditions. Dr.T.V.Rao MD 41
  42. 42. BIOFILMS HELP GENE TRANSFER • Biofilms increase the opportunity for gene transfer between/among bacteria.. Gene transfer can convert a previous a virulent commensals organism into a highly virulent pathogen. Dr.T.V.Rao MD 42
  43. 43. BIOFILMS –QUORUM SENSING • Certain species of bacteria communicate with each other within the biofilm. As their density increases, the organisms secrete low molecular weight molecules that signal when the population has reached a critical threshold. This process, called quorum sensing, is responsible for the expression of virulence factors. Dr.T.V.Rao MD 43
  44. 44. QUORUM SENSING HELPS THE SURVIVAL OF PATHOGENS Dr.T.V.Rao MD 44
  45. 45. BIOFILMS CONTRIBUTE FOR NEW PHENOTYPES • Bacteria express new, and sometimes more virulent phenotypes when growing within a biofilm. Such phenotypes may not have been detected in the past because the organisms were grown on rich nutrient media under plank tonic conditions. The growth conditions are quite different particularly in the depths of biofilms, Dr.T.V.Rao MD 45
  46. 46. BIOFILMS PROTECTS FROM IMMUNE RESPONSES • Bacteria embedded within biofilms are resistant to both immunological and non- specific defence mechanisms of the body. Contact with a solid surface triggers the expression of a panel of bacterial enzymes which catalyze the formation of sticky polysaccharides that promote colonization and protection. Dr.T.V.Rao MD 46
  47. 47. BIOFILMS – PROTECTS FROM PHAGOCYTOSIS• Phagocytes are unable to effectively engulf a bacterium growing within a complex polysaccharide matrix attached to a solid surface. This causes the phagocyte to release large amounts of pro- inflammatory enzymes and cytokines, leading to inflammation and destruction of nearby tissues. Dr.T.V.Rao MD 47
  48. 48. CURRENT OBJECTIVES ON BIOFILM RESEARCH • o Development of improved imaging of biofilms in situ; o Development of improved clinically relevant in vitro and in vivo models of biofilms under specific in vivo conditions such as flow rate, nutrient content, and temperature; o Development of better probes (genetic, metabolic, and immunological) for real- time analysis; o Studies of quorum sensing/signaling molecules; Dr.T.V.Rao MD 48
  49. 49. CURRENT OBJECTIVES ON BIOFILM RESEARCH • o Further characterization of biofilm-specific gene expression; o Studies of the exchange of genetic material within biofilms; o Studies of organic contaminants on substrata, and their influence on biofilm structure; o Development of novel approaches to control pathogenic bacteria by, for example, devising strategies to favour growth of non-pathogenic microorganisms in biofilm communities; Dr.T.V.Rao MD 49
  50. 50. CURRENT OBJECTIVES ON BIOFILM RESEARCH • o Studies of pathogenic mechanisms of microbes growing in biofilms; o Elucidation of mechanisms of resistance of biofilms to antimicrobial agents; o Studies of host immune responses, both innate and adaptive to biofilms; Dr.T.V.Rao MD 50
  51. 51. CURRENT OBJECTIVES ON BIOFILM RESEARCH • In studies of infectious lung disease in cystic fibrosis; o Studies on the potential of diagnostic procedures such as Bronchoalveloar lavage and bronchoscopy to disturb local biofilm flora and inoculate distant locations; o Development of mathematical models and computer simulations of biofilms; o Development of the methodology for the prevention and control of biofilms from catheters, water unit lines, and other clinically important solid surfaces;.Dr.T.V.Rao MD 51
  52. 52. SEARCHING FOR ALTERNATIVES – TISSUE ENGINEERING • Role of biofilms in multiple pathologies and the difficulty in resolving these pathologies speaks to the importance of developing means of replacing or enhancing the therapies already in use. The use of synthetic materials in the body ranges from catheters to mesh to stents to heart valves and beyond. Until the development of viable and practical tissue engineering, then number and types of applications in which synthetic materials are used will continue to increase.Dr.T.V.Rao MD 52
  53. 53. EMERGING METHODS FOR CURE • Several researchers are finding solutions for the cure of Biofilms , yet it is experimental, with advances in molecular biology better model treatments can be identified to reduce the problem of Biofilm interference in Antibiotic therapy. Dr.T.V.Rao MD 53
  54. 54. • Programme Created by Dr.T.V.Rao MD for Medical and Microbiology Professionals for Global education on developments in Science • Email • doctortvrao@gmail.com Dr.T.V.Rao MD 54
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