BIOFILMS

BIOFILMS

‘Bio’ means ‘living’ and ‘film’ means ‘coating.’ So in the simplest sense, Biofilms is nothing but a coating of living organisms.

·        Historical aspects:-

*  In 17^th century, Anton Von Leeuwenhook invented microscope and he saw aggregates of microorganisms on the scrapings of his own dental plaques.So this is considered to be the first description of a biofilm

*Then later on, in 1978 Bill Costerton coined the term ‘Biofilm’

A microorganism can exist in two forms,either as a free coating one,called as  plankton or as an aggregate which is attached to a surface,called as a biofilm. So, something which can exist as a free being is aggregating itself with others to form a Biofilm.What is that for?

·        How does forming a BF help an organism?

Biofilm form(BF) ation is actually a defence mechanism. Biofilms are found everywhere, be it damp soil,aquatic plants,oreven our body,in wounds,implants etc.

It offers six advantages to the microbes:-

(1)    BF protects it from host defences

(2)    BF is sometimes referred to as “cities of microbes” because it is like a community with very good co-operation and very good communication with each other. It provides structural stability.

(3)    It adds to the virulence

(4)    The gene expression of an individual microbe and that of a BF is entirely different. The genotype of BF has got more survival advantage.

(5)    BF has increased resistance to antimicrobials.

(6)    As a group, they can breakdown more nutrients and have better nourishment.

 

·        How is a biofilm produced?It has six stages.

1^st –Stage of Attachment i.e,the organism attaches to a biotic/abiotic surface

2^nd –Stage of microcolony formation.This microcolony is called the basic structural unit of a Biofilm.

*  How is this microcolony formed?

The microbes will produce EPS i.e.,Extracellular Polymeric Substance. This EPS is the hallmark of BF formation.EPS consists of polysaccharides,lipids,proteins and extracellular DNA.So, biofilm is an aggregate of microbes,embedded in this EPS,also called glycocalyx.

* What is the need of this EPS?

(1)       It will help in attachment to surfaces

(2)       It protects from neutrophilic penetration

(3)       It masks phagocytic detection of opsonins

(4)       It sequestrates host antibodies and complement factors

(5)       It facilitates communication among them ,through biochemical signals

(6)       It protects from antibiotics    

3^rd –Stage of Quorum Sensing (QS). QS means, when the microbes reach a critical number they will produce certain signalling molecules, called auto inducers.These molecules act as communication circuits between the organisms

Eg:-

Gram negative bacteria secrete acylated homoserine lactones

 Gram positive bacteria secrete oligopeptides

4^{th -}Stage of Maturation i.e,they will divide and grow in mass

5^th-Stage of Development i.e,cell division stops but they will increase in size.By this time,it has become a fully established biofilm.

6^th –Stage of Dispersal i.e, the BF disperses and spreads to form new colonies.This is brought about by enzymes like dispersin B, deoxyribonucleases etc which are produced by the BF itself.

Thus,through these 6 stages,a biofilm is produced.Now,once it is formed,what next?It will either remain dormant or it will become active.

* If it is dormant,we call it as ’persistors’.Cell metabolism is shut down in these persistors. So,antibiotics are not going to work.

*  If it is active,it will produce abscess,lumps,nodules,sinuses,granulomas,or even systemic infections.

 

·        Why should a clinician need to be aware of biofilms?

First reason is because BF makes our conventional antimicrobial treatment useless. Organisms in a BF are drug resistant most of the times.Also,one important reason for making some diseases c/c or persistent is this BF.And moreover even if we are aware of it and we send for a culture,it will come as negative.We need special techniques to detect them, like electron microscopy, epifluorescent microscopy,peptide nucleic acid fluorescence insitu hybridization,cryoscanning,confocal laser scanning microscopy,denaturating gradient gel electrophoresis and 16s rRNA sequencing.If it is a single bacterium,we can easily see it with light microscopy.But when it is forming a biofilm,it can’t be seen with a light microscope because,during the processing, glycocalyx will collapse and also,the phenotype of a BF is so much different from that of individual planktons.

MIC of antimicrobial agents is 10 -1000 fold greater when it is in BF state than in planktonic state.

* How is a BF helping an organism to develop antimicrobial resistance?

(1)    EPS act as a physical barrier to antibiotic penetration

(2)    Altered growth and decreased metabolism of organisms in biofilms

(3)    Increased chance of mutation in biofilms

(4)    Increased gene transfers as they are very close to each other

(5)    Phenotype switching

(6)    Persisters or spore like forms

(7)    Increased efflux pumps

(8)    Production of enzymes like beta lactamases

 

·        Pathological conditions associated with biofilms:-It is said that 80% of all human infections are associated with biofilms

*  Non dermatological conditions- cystic fibrosis, endocarditis,catheter infections, gingivitis, intrauterine devices infections, dental caries and periodontitis, UTI,coating of contact lenses, infection of joint prosthesis and heart valves,etc

 

*  Dermatological conditions-Chronic wounds, Atopic dermatitis, Hidradenitis suppurativa, Candidiasis,Onychomosis,Acne vulgaris,Dermal filler granuloma, Miliaria, Impetigo, Pemphigus foliaceus , Rosacea, Bacterial vaginosis, Buruli ulcer

Let us see these dermatologically relevant aspects of biofilms

1)Chronic wounds

Chronic,especially open wounds provide an ideal environment for microbial proliferation. BF may be formed as early as after 8 hours in a wound.Biofilms delays wound healing because:-

*  it delays epithelialisation

*  it hinders development of granulation tissue

* it induce apoptosis.

BFs are seen in 60% of  chronic wounds and 6% of acute wounds.The main culprits forming BFs in chronic wounds are staphylococcus aureus, pseudomonas aeruginosa, enterococcus and anerobes.Usually it is polymicrobial and if it is so,the pathogenicity will also be more due to the interspecies interactions.

So how to manage BFs in c/c wounds?

*  Repeated wound debridement/desloughing to remove the non-viable tissue. Because,it will reduce the surface area of the fertile environment for BFs. Also, when we debride, it will disrupt the BF structure,resulting in a period of reassembly during which they are more susceptible to our treatment.

*  Addition of lactoferrin and xylitol to hydrogel dressings.Both are synergistic especially to staphylococcus aureus and pseudomonas aeruginosa.MOA is that lactoferrin inhibit bacterial adhesion and reduce BF mass.While xylitol disrups glycocalyx formation.

*  Silver nitrate reduces BF viability.

*  Ammonium enhanced methane bandages

*  antimicrobialbluelight especiallyagainst acinetobacter baumanii and pseudomonas

*   5%Tea tree oil

*  PRP i.e. platelet rich plasma. 7-38 % of S. aurues BF mass can be reduced by it.

*  Others-Low frequency ultrasound,near infrared diode lasers, photodynamic treatment,ultrasonic assisted wound debridement etc.

*  Future treatment options under research- anti biofilm targetting QS

(2)Hidradenitis suppurativa

BFs are detected in hair follicular infundibulum and sinus tracts, because, there is abundant keratinous debri there,which provide a nidus for microbes. So it is suggested that combining medical treatment with early surgical excision/simple deroofing helps in better management of H. suppurativa.

(3)Atopic dermatitis

S. aureus biofilms are very important in its pathogenesis.BF induced occulsion of sweat ducts is an important cause for inflammation and pruritis in AD. Some studies have shown 0.02%farnesol and 5% xylitol is very useful in eradicating S.aureus BFs.Emollients also prevent BF formation in AD

(4)Candidiasis

C. albicansis notorious to cause BFs. Oral thrush with its characteristic white plaque is due to BF formation. BCR-1 transcription factor is involved in regulating BF formation in C. albicans . This is the reason why candida is protected from neutrophils and immune mechanism .Also, in a person using artificial dentures, chronic atrophic candidiasis occur since the mucosal barrier gets altered. Dentures are very good abiotic surfaces which harbor C. albicans. The peculiarity of candidal biofilm is that,it is 30-2000 fold more resistant to AMB, fluconazole, itraconazole and ketoconazole.Also it over expresses beta glucan.So we can treat a candidal biofilm only with echinocandins/liposomal AMB/ gentian violet. Gentian violet inhibit germination and form radicals which enhance penetration through EPS. In future,drugs targeting BCR-1,cytokines and QS may also come to the picture for treatment

(5)Acne

There are four ways in which Propionibacterium acne BF is important in pathogenesis of acne.

*  P. acne BF produces EPS which has adhesive properties.So it causes tenacious binding of keratinocytes to infundibular epithelium.Thus P.acne BF is an important reason for follicular plugging in acne.

*  P.acne biofilm produce lipase, hyaluronidase, chemotactic factors etc which increase free fatty acids, available as a nutrient source for bacteria.Thus it further perpetuates inflammation.

*  Toll like receptors 2 and 4 are activated to bind to the BFs which then activate NF-kappa B and thereby proinflammatory response in acne.

*  P.acne is well nested in BFs, hence well protected from antimicrobials.Thus, treatment response will be low.

So then,how do we manage P.acne biofilms?

* Isotretinoin and PDT reduces sebaceous gland size and sebum.So nutrients to P.acne is reduced, so BF formation is hindered.

*  Other drugs-selenium,silver,rifampicin,flavanoids,plant extracts like icariin and resveratrol,0.1% triclosan

*  5% Benzoylperoxide +0.5 % erythromycin /1% clindamycin,may be used .BPO alone is ineffective against BF. It must be combined with clindamycin/erythromycin.

*  Minocyclin is highly effective against P.acnebiofilms and acinetobacter baumanii biofilms.

*  Polymyxin B is also effective.

(6)Onychomycosis

Onychomycosis,especially with candida and nondermatophytes.BF concept explains the rationale of BOAT regime (i.e,Boosted Oral Antifungal Treatment).BFs make onychomycosis and dermatophytosis chronic and treatment resistent. Antifungals with antibiofilm properties are liposomal AMB,echinocandins, miltefosine and propolis resin from bees. Others used to treat are NDYag lasers/ IPL/ near infrared light/PDT/ enzymes like DNAase , Alpha amylase, Chitosin etc.

(7)Dermalfillers

Long acting dermal fillers like polyacrylamide fillers can result in BF formation in the form of erythematous tender nodules,abscesses and sinuses .It may be formed weeks to months after their administration.It is usually formed by P.acne/S.aureus/ Pseudomonas.Earlier we thought,it was an allergic or a foreign body reaction and we gave steroids.But it worsened.Then we understood,it is not an immune granulomatous process,rather it is an infectious process by BFs. So then we started to manage it with broad spectrum antibiotics.Cultures are usually negative but FISH technique detects biofilm.

Now,why is biofilm occurring with dermal fillers?It is due to bacterial contamination during the procedure.Use of increased needle diameter,reduced injection depth and fanning technique are three factors which further increases risk of BFs.

To prevent this,thoroughly cleanse the area with antiseptic before giving injection,may consider giving prophylactic antibiotic especially if it is acne affected skin.

It is treated with broad spectrum antibiotics for 2-3 weeks and I and D if it is a fluctuant lesion.

(8)Miliaria-BFs occlude sweat ducts

(9)Impetigo- Strep.pyogenes and S. aureus form BFs

(10)Pemphigus foliaceous-S. aureus form BFs.

(11)Buruli ulcer-Mycobacterium ulcerans producing mycolactone formsBFs on aquatic plants and in humans.

(12)Rosacea

(13)Bacterial vaginosis- Gardnerella vaginalis and Atopobium BFs

Till now we talked about BF in the worst possible ways.But BFs do have certain virtues as well. It is called Bioremediation. Bioremediation means usage of living organisms or their products like enzymes to treat or degrade harmful compounds.Eg; waste water treatment,heavy metal contamination etc.

So to sum up,

BFs represent densely packed aggregates of microorganisms,encased in a self produced matrix of extracellular polymeric substance,helping in their attachment to biotic and abiotic surfaces conferring them survival advantage in unfavourable condition and implicated in a variety of chronic dermatological and non-dermatological diseases.