Biofilm

Dr Jyoti
 Intro
• Biofilms 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, provides
the bacteria with an important tolerance
against antibiotics and host defense through
several mechanisms.
• Between the surface and deep layers of the
biofilm a gradient in oxygen level exists and
the deepest parts may be practically
anaerobic.
• In a thick biofilm mass a water channel system,
resembling a simple circulation, provides
distribution of oxygen and nutrients, however,
in some niches oxygen level may be low
enough for anaerobic bacteria to survive.
• The microbes are more active in the surface,
however in the deep layer their metabolic
activity and cell division are decreased and
microbes may be totally inactive in a dormant
sessile state out of reach of the antimicrobial
treatment.
• 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.
 Formation of a biofilm
• Initially -a stage of reversible attachment of the
planctonic (free-living) form of the organism to a
surface.
• As the biofilm develops, this attachment becomes
irreversible and the bacterial community becomes
covered in an extracellular matrix slime.
• Within this matrix the individual cells communicate
with each other via quorum sensing, which allows
regulation of gene expression throughout the colony.
• A fully mature biofilm is able to release planktonic
organisms back into the environment. Biofilms
may involve more than one bacterial species.
• 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.
• A mature biofilm can stay in a sessile form for long
periods of time.
• When suitable conditions arise, the biofilm activates
and bacteria may also seed to other locations to
form new biofilms.
• 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.
 Diagnostics
• A suspicion of a biofilm infection should
always rise when seeing a chronic and
recurrent infection.
• As no specific markers exist, the diagnosis is
still based on direct microscopy.
• Most widely used are scanning electron
microscopy (SEM) and confocal scanning
laser microscopy (CSLM).
• Fluorescent in situhybridization (FISH) with
CSLM- CSLM is used to detect the biofilms and
FISH to identify the pathogens.
• BacLight Live/Dead stain (Molecular Probes,
Leiden, The Netherlands) with CSLM.
• BacLight provides generic nucleic acid labeling,
that stains living cells green (Syto 9) and dead
or damaged cells red (propidium iodide).
• Basically the diagnosis is established when a
community of viable microbes covered with
extracellular matrix is visualized within or
upon the affected tissue or prosthesis.
 Rhinology
• CRS - Antibiotics alleviate acute symptoms but
fail to eradicate biofilm nidus. Residual biofilm
periodically releases planktonic bacteria,
which cause clinical relapses.
• Cryer and colleagues were the first to report
bacterial biofilms in the sinus mucosa of
patients with Pseudomonas aeruginosa
infection and CRS.
 Adenotonsillar disease
• Biofilms can be found both in adenoids and, in
the crypts of tonsils.
• Typically, these biofilms consist of both gram-
positive and gram-negative bacteria.
 Otology
• Using extended cultures and confocal
microscopy live bacteria have been
demonstrated in more than 90% of middle ear
effusions in children with glue ear and/ or
recurrent acute otitis media, suggesting that
bacteria and biofilms play an important role in
the pathogenesis of this condition.
• Chronic otitis externa, COM, cholesteatoma.
 Biomaterial Infections
• 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.
• Each implant can be colonized either by
exogenous or endogenous route.
• Tympanostomy tube (TT), materials ranging from
silicone to titanium, ossicular chain prostheses,
voice prosthesis and tracheostomy tubes.
• 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.
 Biofilms in medicine

• Dental plaque
• Otitis media
• Chronic rhinosinusitis
• Colonization of the lung in chronic respiratory disease,
e.g. cystic fibrosis
• Colonization of chronic wounds
• Osteomyelitis
• Intestinal microbiome
• Endocarditis
• Device-related infections
 Treatment
• Treatment of infections involving a biofilm are
complicated by both the presence of the
extracellular slime, which makes penetration
of antimicrobials difficult, and the fact that
the bacteria themselves will exhibit reduced
metabolic activity and divide less frequently
than as their free-living planktonic forms.
• Successful treatment of infections involving a
biofilm may require mechanical debridement
of the biofilm and if a device is involved, this
may ultimately require removal.
• Development of improved device surfaces that
do not allow attachment of the organisms to
initiate biofilm formation is a focus of research
that may in the future prevent device-related
infections.
 Future Therapeutic Options For Biofilm
Treatment
• 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.
• These results highlight the difficulty of biofilm
eradication using antibiotics. Systemic
treatment may require intolerable high
concentrations or may not be effective at all.
Also, surgical removal of all affected mucosa is
seldom possible.
• Basically three different strategies for biofilm
eradication with topical treatments exist.
• Firstly, traditional antimicrobial treatment is
aimed against causative organism.
• Second strategy is based on physical force to
achieve biofilm detachment from mucosal
surface by, e.g. surfactants, irrigation and
surgery.
• Third strategy aims at 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).
• 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.
• QSIs such as plant furanones have been found
in nature.
• In theory, QSIs increase biofilms susceptibility
to antibiotics and phagocytosis.
• 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.
• Mupirocin is widely used clinically to eradicate
Staphylococcus aureus from nasal vestibule,
and it has been shown to have activity against
Staphylococcus aureus biofilms.
• Other topical antimicrobial agents with some
effect on CRS biofilms demonstrated include
gallium nitrate, manuka honey, moxifloxacin,
tobramycin and antimicrobial peptides.
Manuka (Leptospermum scoparium) honey

• From New Zealand is the most therapeutically

potent honey. It has been shown to be active
against a broad spectrum of gram-positive and
gram-negative bacteria.
• The principal active ingredient responsible for
the antibacterial property in Manuka honey is
methylglyoxal (MGO).MGO is present in
Manuka honey at a concentration of up to 100-
fold that of conventional honey.
• In an in vitro study by Alandejani et al., Manuka
honey at a concentration of 33% v/v was shown to
be effective in eradicating methicillin-susceptible
Staphylococcus aureus (MSSA), methicillin-
resistant Staphylococcus aureus (MRSA) and
Pseudomonas aeruginosa (PA) biofilms.
• In fact, conditions that rapidly induced antibiotic
resistance did not cause bacterial resistance to
honey.
• A 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.
• Introduction of Esp-secreting Staphylococcus
epidermidis to nasal cavities of volunteers,
who were Staphylococcus aureus carriers,
inhibited Staphylococcus aureus colonization.
• 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.
• In case of a mature biofilm infection, a long-
term suppressive antibiotherapy may be useful.
• Systemic long-term low-dose macrolide
therapy inhibits bacterial virulence and possibly
biofilm formation, which might make it useful
adjuvant therapy in chronic infections.
 Conclusion
• Current evidence suggests bacterial and fungal
biofilms a role in several infections in ENT.
• 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.