class about biofilms and treatment of the same.

1. Dr. Rajendra Singh Lakhawat
Department Of Otorhinolaryngology,
SMS Medical College & Hospital, Jaipur, India

2.  Biofilms can be seen as a preliminary stage of
a multicellular life that provides the bacteria
with an important tolerance against
antibiotics and host defense.
 These are Self assembled microbial
communities characterized by cells that are
embedded in a matrix of self-produced
extracellular polymeric substances, mainly
polysaccharides, proteins and DNA.

3.  The variation in microbe phenotypes is wide
and several bacterial or fungal species and
strains may live in harmony in the same
biofilm with certain specialization between
them.
 Between the surface and deep layers of the
biofilm a gradient in oxygen level exists and
the deepest parts may be practically
anaerobic.

4.  In a thick biofilm mass a water channel
system, resembling a simple circulation,
provides distribution of oxygen and
nutrients.
 To be able to produce biofilm a cell-to-cell
communication called quorum sensing (QS)
is essential. The bacteria produce and
simultaneously react to these small QS
signaling molecules that promote a density
dependent gene regulation and bacterial
behavior.

5.  Through the QS the microorganisms can
sense when critical bacterial concentration
has been reached to be able to produce a
biofilm and suppress further multiplication.
 Multiplication is suppressed using different
signaling molecules.
 Many natural antibiotic molecules such as
erythromycin or rifampicin are originally part
of this communication system.

6.  These antibiotics orchestrate the growth of
the community by turning on or off the genes
of other microbes.
 Dispersal is a mechanism of biofilm bacterial
spreading in infected organ or human body.
 It happens either in planktonic form as more
or less programmed phenomenon or in
bacterial clusters by the shear forces.

7.  By the present knowledge more than 99% of
the bacteria in nature live in biofilms, and in
80% of the chronic diseases biofilms are
suggested to play an important role.
 Biofilm community provides a safe and
favorable habitat for different species against
the environmental challenges such as host
defense and antimicrobial agents.

8.  The volume of the extracellular matrix
restricts the penetration of antibiotics by
diluting and binding them before they can
reach all individual cells of the biofilm.
 Safety of the community may be provided in
unselfish all-for-few manner: if the
extracellular mass is not thick enough to stop
the attack, majority of the bacteria will be
sacrificed to produce more barrier biomass
which protects the sessile microbials in the
deep parts of the biofilm.

9.  Subpopulations of dormant, nondividing
microbial cells are highly resistant to
antibiotics.
 vast majority of the nature's biomass exists in
biofilms.

11.  A suspicion of a biofilm infection should
always rise when seen a chronic and recurrent
infection.
 A typical pitfall in clinical practice is caused
by cultivation of a planktonic bacteria
detached from a biofilm.
 The study may result with infectious agents
sensitive to antimicrobials and decent
minimal inhibitory concentration (MIC)
levels even though the biofilm itself most
probably is highly resistant.

12.  For diagnosing the biofilm etiology the
presence of biofilm growth must be
determined. As no specific markers exist, the
diagnosis is still based on direct microscopy.
 The most widely used methods are scanning
electron microscopy (SEM) and confocal
scanning laser microscopy (CSLM).

13. Scanning electron micrograph of a staphylococcal biofilm on
the inner surface of an indwelling medical device. Bar, 20 μm.

14.  The diagnosis is established when a
community of viable microbes covered with
extracellular matrix is visualized within or
upon the affected tissue or prosthesis.
 SEM provides a 3 D picture allowing
assessment of architecture and developmental
stage of the biofilm.
 It requires robust tissue preparation, which
may lead to artifacts and difficulties in
differentiating mucus, clots and biofilm. It
does not identify the pathogen.

15.  There are high chances of false positive
results in electron microscopy.
 Therefore, fluorescent in situ hybridization
(FISH) with CSLM is used in biofilm
identification.
 With this method a 3 D view of biofilms is
obtained and depending on hybridization
probes, its microbiological identity can be
revealed.

16.  BacLight Live/Dead stain, may be used for
biofilm detection together with CSLM.
 BacLight provides generic nucleic acid
labeling, that stains living cells green and
dead or damaged cells red. BacLight stain
with CSLM can be used for accurate biofilm
detection in clinical samples, but the exact
identity of pathogen is not provided.

17.  In clinical practice BacLight/CSLM may be
more useful than FISH/CSLM with the
advantages of being rapid, simple, inter
observer reliable and covering all biofilm
forming bacteria.

18.  In a study Using FISH/CSLM with probes
against Staphylococcus aureus, Haemophilus
influenzae, Pseudomonas aeruginosa and
universal fungal element, detected biofilms in
77% of CRS patients. Of the detected biofilms
60% were polymicrobial, 54% had
Staphylococcus aureus biofilm, 36%
Haemophilus influenzae, 31% Pseudomonas
aeruginosa, and 26% fungal biofilm.

19.  Acute middle ear infection is the most
common bacterial disease in early childhood.
 The most common bacterial species cultured
from acute middle ear effusions are
Streptococcus pneumoniae, Haemophilus
influenzae and Moraxella catarrhalis.

20.  Effusions recovered from middle ears are
often bacteriologically sterile in cultures, but
often polymerase chain reaction-positive for
bacterial DNA. In addition, bacterial
messenger RNA has been found to be present
in middle ear effusions. This suggests the
existence of metabolically active bacteria in
middle ear fluids despite the inability to
culture them.

21.  All the most important bacterial species
causing acute middle ear infections are
known to form biofilms in vitro and in vivo.
 In glue ear, biofilms attach to mucus as well
as mucosa providing an inflammatory
stimulus leading to a middle ear effusion.

22.  Biofilm formation play an important role in
chronic external otitis as well.
 Removal of the bacterial biofilm has a high
correlation with a long-term clinical
remission.
 Mastoid mucosal biofilm has been found in
patients with chronic otitis media more often
than with control patients.

23. Confocal
scanning laser
microscopy
image of
bacterial
biofilm
formation in
Mastoid
mucosa. The
samples were
stained with
BacLight
Live/Dead
stain. The live
bacteria can be
seen stained
fluorescent
green and the
damaged
bacteria and
host cells red

24.  Bacterial biofilms are now acknowledged to
play an important role in the development of
chronic or recurrent otitis media, but the role
of biofilms in acute middle ear infections is
still understudied.

25.  The development of effective bacterial
vaccines might be the most effective way to
manage otitis media. By reducing the
colonization of bacteria and formation of
biofilms in the nasopharynx the vaccines
could reduce the possibility of retrograde
bacterial transition from nasopharynx into the
middle ear through eustachian tube and
minimize the risk of development of acute
otitis media and its sequelae.

26.  Adenoidectomy is considered to be beneficial
in children with CRS and chronic otitis media.
Recent studies identifying biofilms in adenoids
may support this clinical assumption.
Approximately 95% of the mucosal surface of
adenoids removed from children with CRS,
covered with biofilms.

27.  Confocal images of adenoidal tissue from
patients with otitis media exhibited the
presence of biofilms from multiple species
including S.aureus, H.influenzae, M. catarrhalis
and S. pneumoniae.
 In a study, 70.8% tonsil specimens removed
from patients with chronic or recurrent
tonsillitis, contained biofilms.

28.  Defective sinonasal mucociliary clearance
caused by epithelial inflammation is
suggested to be the common fundamental
pathophysiology of CRS.
 In many cases CRS has a clinical picture of a
biofilm disease.

29.  Antibiotics alleviate acute symptoms but fail
to eradicate biofilm nidus. Residual biofilm
periodically releases planktonic bacteria,
which cause clinical relapses. CRS is not a
classical bacterial infectious disease, although
bacteria admittedly play an important role in
exacerbation phases.

30.  Cryer and colleagues were the first to report
bacterial biofilms in the sinus mucosa of
patients with Pseudomonas aeruginosa
infection and CRS.
 Psaltis et al. (using BacLight/CSLM) found
biofilms in 44% of CRS patients, and none in
9 controls.
 Foreman et al. (FISH/CSLM) detected
biofilms in 72% of CRS patients and in 0/10
controls. S. aureus was the most common
biofilm-forming organism.

31.  Singhal et al.4 3 (FISH/CSLM) detected
biofilms in 77% of CRS patients. Their FISH
probes covered Staphylococcus aureus,
Haemophilus influenza, Pseudomonas
aeruginosa and universal fungal element. Of
the detected biofilms 60% were polymicrobial
and 54% had Staphylococcus aureus biofilms.

32.  It seems convincing, that biofilms are present
in a majority of CRS patients, and that
biofilms are not present in normal sinonasal
mucosa.
 Biofilms might be accountable, but a direct
evidence of biofilms being a causative factor
in CRS is still lacking.

33.  Psaltis et al. studied retrospectively the
postoperative outcome of 40 patients who had
undergone endoscopic sinus surgery (ESS) for
CRS. Bacterial biofilms were detected in 50%
of patients using CSLM.
 Patients with biofilms had worse
preoperative radiological scores and more
postoperative symptoms and mucosal
inflammation. In addition to biofilms, fungus
presence at the time of surgery was the only
other factor related to unfavorable outcome.

34.  The patients with Staphylococcus aureus
biofilms progressed especially poorly with
symptom scores and quality-of-life outcomes.

35.  The pathogenesis of implant-associated
biofilm infection involves interactions
between the microorganism, the implant and
host.
 The interaction of the microorganism with the
device results first in bacterial adhesion and
then in formation of a biofilm. Interaction of
host defense mechanisms with the implant
results in activated and exhausted phagocytes.

36.  Coagulase-negative staphylococci, S. aureus,
beta-hemolytic streptococci and
enterobacteriaceae are the most important
microorganisms in all types of implant-
associated infections.
 Common to all of these bacteria is their
ability to persist as biofilm

37.  Adherence of different bacteria to implant
surface involves rapid attachment mediated
by nonspecific factors, such as surface
roughness, tension, hydrophobicity and
electrostatic forces, or by specific adherins.
 Bacterial adherence to tympanostomy tube
(TT) materials has been studied widely.

38.  It is hypothesized that the biofilm
colonization on the TTs causes recurrent
suppuration.
 In vitro studies have demonstrated that inert
TT materials and smooth surface preparations
can inhibit the adsorption of key bacterial
binding proteins, such as fibronectin, and the
development of P. aeruginosa and S. aureus
biofilms.

39. Extruded titanium
tympanostomy tube
surface covered with
biofilm (a);
Human serum albumin
pre-treated titanium tube
with less biofilm adhesion
(b)

40.  Coating of TTs has been used to change
biomaterial surface properties. In vitro results
with albumin- and phosphorylcholine-coated
TTs are encouraging.
 The effect of reducing surface imperfections
and increasing surface antimicrobial activity
may be reduced if middle ear mucus, blood,
or cellular debris fouls the TT surfaces
because adherent debris may serve as
microbial binding sites

41.  Biofilms can also be found on ossicular chain
prostheses in patients who undergone
ossicular chain reconstruction.

42.  Antonelli et al. and Pawlowski et al. detected
biofilm formation on cochlear implants
removed from patients after intractable
infection.
 The infection was caused by S. aureus.

43.  Due to the close contact of the implant with
the middle ear mucosa and because the
electrode array is positioned in the
perilymphatic space via cochleostomy.
 There is a potential risk of bacterial transferal
along the electrode array into the cochlea.

44.  Cochlear implant material can provide a
surface for bacterial biofilm formation.
 Impressions can provide an environment
conducive to biofilm establishment and
growth, ultimately necessitating device
removal, with loss of implant function

45.  Biofilm infections also occur in laryngeal
mucosa.
 Thus, it is not surprising that biofilm
formation can cause problems in endotracheal
devices such as voice prosthesis and
tracheostomy tubes and can cause a need for
their frequent replacement.

46.  Biofilms were present on tracheostomy tubes
in greater than 90% of tracheostomy tubes
collected as early as 7 days after insertion in
both the inpatients and outpatients.

47. Confocal
scanning laser
microscopy
image of
bacterial
biofilm
formation
in laryngeal
mucosa. The
samples were
stained with
BacLight
Live/Dead
stain

48.  Throughout the history of medicine surgery
of the chronically infected focus has been in a
key role when treating chronic resistant
infections.
 Removal of contaminated implants and the
infected tissue as well as aeration of the
infection focus all limit the possibilities of a
biofilm infections.

49.  Using in vitro biofilm formation assay a study
have shown, that minimal biofilm eradication
concentration (MBEC) of different antibiotics is 4
to 500 fold higher for Staphylococcus aureus
biofilms than MIC of the same bacteria in
planktonic form.
 Planktonic Pseudomonas aeruginosa were
sensitive to enrofloxacin, erythromycin, and
oxytetracycline, but biofilms were sensitive only
to enrofloxacin.

50.  These results highlight the difficulty of
biofilm eradication using antibiotics.
 Systemic treatment may require intolerable
high concentrations or may not be effective at
all.
 Surgical removal of all affected mucosa is
seldom possible.

51.  With local application much higher effective
drug concentrations may be reached with less
systemic side effects.
 Much emphasis has been out to develop local
treatments in biofilm infections recently.

52.  Three different strategies for biofilm
eradication with topical treatments exist.
1. Traditional antimicrobial treatment is aimed
against causative organism.
2. Physical force to achieve biofilm detachment
from mucosal surface by, e.g. surfactants,
irrigation and surgery.

53. 3. Freeing bacteria from biofilm matrix
(dispersal) either by degrading the matrix or
by promoting a bacterial phenotype shift
from sessile to planktonic form with quorum
sensing inhibitors (QSI).

54.  Biofilm matrix degrading enzymes such as
alginate lyases have been proposed as a treatment
in cystic fibrosis. However, these biofilm specific
treatments are still more a theory than practice.
 There are in vitro studies on QSI that prevent the
bacteria to turn to thebio film mode. QSIs such as
plant furanones have been found in nature.
 QSIs increase biofilms susceptibility to
antibiotics and phagocytosis.

55.  Antibiotic treatment could also be combined
with biofilm matrix dissolving compounds
such as alginase which specifically dissolves
Pseudomonas aeruginosa biofilm and makes the
bacteria more susceptible to antibiotics.
 Some unspecific chemical surfactants could
also be used to disrupt biofilm integrity and
break the links between biofilm matrix and
mucosal surface.

56.  Some positive effects have been reported
with baby shampoo, citric acid/ switterionic
surfactant, and furosemide, convincing
clinical results are lacking.
 Combination of a sinonasal surfactant
SinuSurf™ was shown to improve the
effectiveness of a lower concentration of
topical antibiotics in biofilm mass and
viability in vitro.

57.  Combinations of local antibiotics and surfactants
together with mechanical biofilm removal by
surgery and/or irrigation will serve as logical
basis for future therapies.
 Various antimicrobial agents have been tested for
the topical treatment of biofilms. Mupirocin is
widely used clinically to eradicate
Staphylococcus aureus from nasal vestibule, and it
has been shown to have activity against
Staphylococcus aureus biofilms as well

58.  Other topical antimicrobial agents with some
effect on CRS biofilms demonstrated include
gallium nitrate, manuka honey, moxifloxacin,
tobramycin and antimicrobial peptides.
 Serine protease Esp, secreted by a subset of
commensal bacteria Staphylococcus
epidermidis, inhibits biofilm formation by
Staphylococcus aureus in vitro by a novel
mechanism of bacterial interference.

59.  Preventive measures have a high value.
Biofilms can be prevented by vaccinations
against organisms that tend to cause chronic,
recalcitrant infections such as pneumococci.
 Antibiotic prophylaxis or early aggressive
antibiotic therapy can also be seen as a
biofilm prevention.

60.  In case of a mature biofilm infection, a long-
term suppressive antibiotherapy such as used
in prevention of otitis media may be useful.
 It is known that systemic long-term low-dose
macrolide therapy inhibits bacterial virulence
and possibly biofilm formation, which might
make it useful adjuvant therapy in chronic
infections.

61.  Whether biofilms are the cause or the
consequence is still unclear, but their
presence seems to correlate with more severe
disease and unfavorable postoperative
outcome.
 Open access of topical treatments to infection
focuses offers the possibility of developing
new therapeutic strategies.

62.  Biofilms have been found in archaic fossils so
there is nothing new in biofilms except that a
few decades ago we did not know about
them.
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