Biofilm is a complex community of microorganisms that attach to surfaces and produce an extracellular matrix. It forms in stages including initial attachment, adhesion, colonization and maturation. Biofilm provides microbes protection from environmental threats and antimicrobial agents. It is characterized by surface attachment, an extracellular matrix, structural heterogeneity and genetic diversity. Biofilm plays a role in various chronic infections like chronic rhinosinusitis, otitis media, mastoiditis and laryngitis. New therapies targeting biofilms include agents that neutralize, disperse or disrupt quorum sensing in biofilms.

1. BIOFILM

2. Biofilm
1. Introduction and definition
2. Ultrastructure
3. Characteristics
4. Development of biofilm
5. Protection
6. Resistance of microbes in biofilm
7. Benefits of biofilm to microbes
8. Applications
9. Conclusion

3. 1. Introduction and definition
Grossman - Biofilm is defined as a community of micro colonies of
microorganisms in an aqua solution that is surrounded by a Matrix of glycocalyx,
which also attaches the bacterial cells to a solid substratum
Ingle - biofilm is a mode of microbial growth where sessile cells are irreversibly
attached to a solid substratum and are embedded in a self-made Matrix of extra
cellular polymeric substance.

4. Must fulfil four basic criteria
1. Autopoiesis - ability to self organise
2. Homoeostasis
3. Synergy - more effective in association than in isolation
4. Communality - Respond to environmental changes as a unit rather than
single individuals

5. Biofilms are characterised by
- Surface attachment
- Extracellular matrix of polymeric substances
- Structural heterogenicity
- Genetic diversity
- Complex community interactions
Biofilms require
- Moisture
- Nutrient supply
- Surface

6. Sites include
- Natural materials above and below ground
- Metals and plastics
- Medical implant materials
- Plant and body tissue

8. 2. Ultrastructure of biofilm
- composed of microbial cells or cell cluster and glycocalyx like Matrix.
— 85% - Matrix ( polysaccharides, proteins, nucleic, acids, and
salts )
— 15% - bacterial cells
- viable, fully hydrated, biofilm appears as Tower or mushroom shaped structure other into two
substrate.
- Water channels regarded as circulatory system in a biofilm establish connection between
micro colonies and facilitates exchange of materials between bacterial cells and bulk fluid.

11. Matrix
- A Glycocalyx Matrix made up of extra cellular polymeric substance, surrounds
the micro colonies and anchors the bacterial cell to the substrate
Functions
1. Maintains the integrity of biofilms.
2. Prevents desiccation
3. Resists microbial agents
4. Create a nutritionally rich environment
5. Access a buffer and retains extra cellular enzymes

12. - bacterial cells within matrix produce beta-lactamase against antibiotics
- also produce catalases and superoxide dismutase against oxidising ions of a
phagocytes
- Elastase’s and cellulases produced by bacteria or concentrated in the Matrix
and produce tissue damage.

13. 3. Characteristics of biofilm
- Biofilm structure protects the residing bacteria from environmental threats.
- Structure of biofilm permits, trapping of nutrients and metabolic cooperativity
between Resident cells of the same species or different species
- Biofilm structure displays organised internal compartmentalisation
- Bacterial cells in a biofilm community may communicate and exchange genetic
material to acquire new traits.

14. 4. Development of biofilm
- bacteria can form biofilm on any surface that is bathed in a nutrition
containing fluid
- Three Major components involved in biofilm formation
a) bacterial cells
b) A solid surface
c) A fluid a medium

15. Stages of biofilm formation
- Stage 1 - Attachment
- Stage 2 - Adhesion
* phase 1 * phase 2 *phase 3
- Stage 3 - Secondary colonisation
- Stage 4 - Mature biofilm ( Detachment)

18. 5. Protection of biofilm bacteria
- Bacteria residing in a biofilm experiences certain degree of protection and homeostasis .
- Bacteria are capable of producing polysaccharides either as cell surfaces or extra cellular excretions
( EPS )
- EPS creates microniche
Protects from environmental stresses
Sequestration of metallic cations and toxins
Physically prevents diffusion of certain compounds by acting as ion exchanger

19. 6. Resistance of microbes in biofilm to antimicrobials
- Biofilms offer an inherent resistance to anti microbial agents such as
antibiotics, disinfectants or germicides
- Mechanism responsible for resistance
Resistance associated with extra cellular polymeric matrix
Resistance associated with growth rate and nutrient availability
Resistance associated with adoption of resistance phenotype

21. 7. Benefits of biofilm to microbes
- Helps the bacteria to survive in unfavourable environmental and a nutritional
conditions
- Resistance to antimicrobial agents
- Increase in local concentration of nutrients
- Opportunity of genetic material exchange
- Ability to communicate between bacterial population of same and different
species
- Produce growth factors across species boundaries

22. 8. Applications

24. Chronic rhinosinusitis
- CRS involves a complex interplay of infectious, inflammatory, and host
factors.
- Biofilms are the preferred state of bacterial existence.
- Several novel therapies directed against biofilms are in use or in
development.
- However, the efficacy of these agents and their potential integration into the
armamentarium of strategies directed against CRS will largely be reliant on
better establishing the role of biofilms in the pathogenesis of chronic
rhinosinusitis.

25. The existing evidence strongly supports the role of biofilms in the pathogenesis of
CRS. Several anti-biofilm therapies have been investigated for use in CRS and
these are at variable stages of development.
Generally, these strategies:
1) neutralize biofilm microbes;
2) disperse existing biofilms; or
3) disrupt quorum sensing.

26. 1. Neutralise biofilm microbes
- Macrolides have been shown to, potentially, harbor an anti-biofilm effect
through their inhibition mechanism
- Study with roxithromycin
- topical mupirocin could reduce S. aureus biofilm mass by more than 90%,
while topical ciprofloxacin and vancomycin were both largely ineffective

27. randomized controlled clinical trial of 25 patients after FESS
- Patients were randomized to one month of twice daily nasal saline rinses
with 0.05% mupirocin or one month of twice daily nasal saline rinses with oral
Augmentin.
- At one month, negative cultures were noted in 89% of patients in the
mupirocin group compared to 0% in the placebo group.
- Furthermore, re-assessment of the mupirocin treated cohort at 2–6 months
post-treatment demonstrated that 83.3% of participants developed positive
cultures for S. aureus.
- The implication of this finding is that the observed clinical benefits of
mupirocin therapy are, generally, short-lived.
- Indeed, subsequent studies demonstrate that mupirocin rinses have a
microbiologic failure rate of 75% over time.

28. There have been efforts to develop novel non-antibiotic antimicrobial agents. One
such example, N,N-dichloro-2,2-dimethyltaurine (NVC-422), is a synthetic and
stable form of N,N-dichlorotaurine
Manuka honey is a natural product of New Zealand whose main active ingredient,
methylglyoxal (MGO), has a high phenol content which is known to be
bactericidal.
Manuka honey has demonstrated significant bactericidal effect against planktonic
and biofilm forms of S. aureus and P. aeruginosa, eliminating 82% of methicillin-
sensitive S. aureus, 63% of MRSA biofilms, and 91% of P. aeruginosa biofilms

29. Studies conducted in sheep models of CRS revealed statistically significant
reductions in biofilm biomass compared to saline flushes
Corticosteroids have been shown to enhance some functions of the mucosal
innate immune system including increased production of complement and acute
phase proteins.
A recent study found that high concentrations of fluticasone (400 μg/200 μL),
budesonide (750–2000 μg/200 μL) and mometasone (200–400 μg/200 μL) directly
reduced biofilm biomass by up to 99% in vitro

30. 2. Dispersion of existing biofilms
- Another tactic for biofilm eradication is the use of surfactants to disrupt biofilm
integrity
- A subsequent randomized controlled trial by Farag et al98 involved 44
patients with CRS randomized to receive post-FESS baby shampoo rinses or
hypertonic saline rinses. Both treatment arms demonstrated similar
improvements in symptoms and olfactory thresholds
- Citric acid/Zwitterionic surfactant (CAZS) is a novel surfactant consisting of
citric acid, which chelates calcium in the calcium ion bridges integral to biofilm
structural integrity

31. 3. Interruption of quorum sensing
- In the context of Pseudomonas biofilms, macrolide antibiotics have been
shown to reduce the expression of quorum sensing systems.
- Recently, paraoxonases (PONs) have been shown to play an important role
against biofilm formation.

32. Otitis media with effusion
Bio-film research, has begun to mount indicating that otitis niedia with effusion may more reflect a
chronic inflammatory state and that Eustachian tube dysfunction plays a secondary role.
traditional swab cultures of the fluid within the middle-ear space are usually negative, as the
bionln1-aggregated bacteria are not free-floating in the fluid but are sequestered onto the surface
of the lining mucosa.
Several studies have supported the biofil111 hypothesis of the etiology of otitis 111edia with
effusion. In 2001, Post demonstrated biofiln1s in a chinchilla model by injecting J-f. influenzae into
the 111iddle-ear space.
Scanning electron n1icroscopy {SEM) was used to exan1ine the n1iddle-ear 111ucosa and
den1onstrated bio-films.
He also used SEM to document the presence of biofilms within tympanoston1y tubes.

33. Mastoid biofilm in chronic otitis media
Background: Biofilm formation has been found in several chronic airway infections. COM
is associated with chronic, recalcitrant infection of the mastoid mucosa, and surgery often
is required.
Methods: COM patients were divided into 2 groups: one with chronic suppurative otitis
media (CSOM) and one with cholesteatoma presence. All COM patients had mastoid
involvement in a preoperative computed tomographic scan. The control group consisted
of patients undergoing cochlear implantation, with no previous history of chronic otitis
media. Mastoid mucosa samples were harvested during mastoidectomy. The samples
were studied with multiplex-polymerase chain reaction and with CSLM using BacLight
Live/Dead stain. Routine bacterial culture was performed in selected cases.

34. Results: A total of 29 COM patients underwent mastoidectomy. Mastoid mucosal
biofilm formation could be found in 19 (66%) of these patients. In the control
group, there were 11 cases of cochlear implantation, and 1 patient (9%) presented
mastoid mucosal biofilm. In the cholesteatoma group, there were 17 patients, of
which, 14 (82%) presented biofilm, whereas in the CSOM group, 5 (42%) of 12
patients presented biofilm. The correlation between COM and biofilm was
statistically significant (Fisher's exact test, p = 0.003), as was the correlation
between cholesteatoma and biofilm, in comparison with the CSOM group (Fisher's
exact test, p = 0.046).
Conclusion: Mastoid mucosal biofilm could be seen in patients with COM with or
without cholesteatoma. The role of mastoid biofilm in the development of
cholesteatoma should be studied further.

37. Biofilm formation on tracheostomy tube
Acinetobacter baumannii and Klebsiella pneumoniae were the commonest biofilm
forming organisms
Amikacin, Gentamicin and Ciprofloxacin were most sensitive drugs.
Multi drug resistant organisms were also commonly found, stressing the need for
sensitivity-based antibiotics.

38. Biofilm formation in chronic laryngitis
Direct detection of biofilm in laryngeal biopsy specimens from patients with chronic
laryngitis supports the hypothesis that chronic laryngitis may be biofilm related.
Biofilm was found in 62% of the cases of chronic laryngitis.

46. 9. Conclusion
- The stages of biofilm formation follows basically the same model in various
micro organisms, the biofilm architecture and molecular mechanisms involved
in biofilm formation appear to differ
- Information on the genetic regulation of oral biofilm formation is still lacking.
- A better understanding of these processes is necessary to the development
of novel strategies for oral disease prevention and control based on
interference of two-component signal transduction.

47. Latest Journals on biofilm
1. The biofilm matrix - multitasking in a shared space ( 2023 )
2. Microfluidics for biofilm studies ( 2023 )
3. Biofilm formation of pathogenic bacteria isolated from aquatic animals
4. Biofilm formation and inhibition mediated by bacterial quorum sensing
5. The biofilm life cycle- expending Da conceptual model of biofilm formation
6. Biofilm and wound healing : from bench to which side
7. Biofilm formation and control of food borne pathogenic bacteria.
8. Combating Boli microbial biofilm: recent approaches
9. Efflux pumps and microbial biofilm formation
10.Dispersing biofilm myths.

48. Thank you