Biofilm: microorganisms working cooperatively

Dr. Chiara Noli, DVM, Dip. ECVD, European Specialist in Veterinary Dermatology

In the world of microorganisms, survival is often a result of cooperation and interaction. A perfect example of this type of teamwork is found in biofilms, complex structures formed by colonies of microorganisms. A biofilm is a community of microorganisms, mainly bacteria but also yeasts, that grow on a surface and surround themselves with a self-produced mucinous material, consisting mainly of polysaccharides, proteins and DNA. This layered structure allows microorganisms to adhere to different surfaces - both inert surfaces such as glass or steel, and organic surfaces such as living mucous cells or skin. The biofilm provides a kind of protective shield for the microorganisms it harbours, offering an effective defence against adverse environmental conditions and antimicrobial agents, including systemic and topical disinfectants and antibiotics.

However, in many environments, biofilms play crucial ecological roles. For example, they are responsible for the biodegradation of many pollutants in aquatic ecosystems, such as in wastewater treatment.

How does a biofilm form?

Biofilms form when microorganisms in the planktonic phase (single floating cells) perceive that they have reached a high density (quorum sensing). In these situations, intercellular chemical communication mechanisms are activated that change their behaviour. The first stage of biofilm formation is the adhesion of planktonic bacteria onto a substrate, initially by means of weak, reversible bonds and then by more powerful adhesive structures such as pili. These first cells provide adhesion sites for other microorganisms, even of different species, and initiate the production of a water-rich mucinous matrix, which may account for 50 to 90% of the mass. This protects the members of the colony from aggression by external agents (desiccation, bacteriophage viruses, natural or synthetic antimicrobial agents, antibodies and phagocytosis by the cells of the immune system) and promotes their multiplication and intercellular communication. Occasionally, some microorganisms may break away from the biofilm to disperse into the environment and find other niches to colonise.

The role of biofilms in medicine

In the medical context, biofilms are present in various pathological situations and can present a significant challenge, as their unique structure offers significant resistance to antibiotics. They are often responsible for chronic and persistent infections such as sinusitis, tonsillitis, endocarditis, pneumonia, osteomyelitis, dental plaque, urinary tract and wound infections and otitis.

Biofilm in otitis

In veterinary medicine, one of the most common problems in dogs is otitis, a multifactorial ear inflammation due to bacterial or fungal infections, allergies, parasites or anatomical problems. Recently, the important role of biofilms in dog ear infections has been noted, and it has been shown that many common bacteria and yeasts responsible for otitis, such as Pseudomonas aeruginosa, E. coli, Staphylococcus pseudintermedius, S. aureus, but also Malassezia pachydermatis, are capable of forming biofilms. In a study of Pseudomonas aeruginosa strains isolated from cases of otitis in dogs, as many as 40% were capable of producing biofilms (10).

These complex aggregates of microorganisms can complicate the management of otitis, representing a significant obstacle to treatment, as they make infections more resistant to therapy and promote relapses. Antibiotics have difficulty penetrating the biofilm matrix and killing the bacteria within it, such that bacteria in biofilm condition require 2 to 4000 times higher antibiotic concentrations than bacteria in planktonic form (not in biofilm) to be inhibited (5-7). Many antibiotics are mainly active on the replicative phase of the micro-organisms, but most of the bacteria present in the biofilm are in a quiescent stage, and the antimicrobial substances that manage to penetrate do so in a limited concentration, favouring the development of bacterial resistance and predisposing to a relapse of the infection with resistant strains.

How to identify a biofilm during otitis

The presence of biofilm should be suspected during otitis refractory to common therapy and/or relapsing, especially if bacterial cultures and repeated antibiograms always report the same micro-organism with the same sensitivity profile. A dark mucinous exudate is also an indicator of the presence of biofilm.

Cytological examination of this exudate is often confirmatory. Samples are obtained with a Q-tip from the depth of the vertical auricular duct. The Q-tip should then be rolled gently over the surface of the slide to deposit the material, avoiding swiping it, as this could produce cell-breakage artefacts (nuclear striae). After staining, the biofilm appears as mucin streaks in the background, with bacteria and/or yeasts mostly in aggregates. It should be noted that Malassezia is also often capable of producing a biofilm, and therefore the finding of this yeast in the cytological sample does not exclude its presence.

Management and treatment of auricular biofilm

However, it has recently been reported that auricular biofilm is often underestimated and identified less frequently than its true presence (8), and should instead be suspected in all cases of otitis with the presence of micro-organisms.

To manage otitis with the presence of biofilm, a multimodal approach must be adopted. First of all, it is essential to mechanically remove the mucinous patina through the use of so-called 'biofilm disrupters', before intervening with disinfectant agents and/or antibiotics. Biofilm disruptors break down the biofilm structure and make microorganisms more susceptible to antibiotics. The best known of these are N-acetylcysteine (NAC) and tris-EDTA, both of which are safe for intra-auricular therapy. Interestingly, both substances have also been described to have their own intrinsic antimicrobial activity, both against bacteria and Malassezia (1).

NAC is commonly used to dissolve catarrh during bronchitis, and several studies have also proven its efficacy in dissolving biofilms and promoting their penetration by antibiotics (2). NAC has also shown synergism of action with certain betalactam and quinolone anitbiotics (3,11).

Tris-EDTA is able to promote the penetration of antibiotics into the wall of Gram-negative bacteria, such as Pseudomonas spp. (4,12), even those organised in biofilms (9), and enhance their effect.

In severe cases Tris-EDTA and NAC should be left in situ for at least 5 minutes, with repeated washing and suction cycles, preferably under general anaesthesia and endotracheal intubation, until the mass of biofilm and exudate is completely removed from the ear canal.

Afterwards, washing with disinfectants and desiccants, usually chlorhexidine-based, can be performed before instilling antibiotic and/or antifungal drops, always accompanied by a topical steroid. This procedure should then be repeated at home by the owner daily until no more biofilm formation is observed.

In less severe situations, Tris-EDTA and NAC can be applied in the ear canal in the awake subject, after proper cleansing, massaging the base of the ear, with a contact time of 5 minutes, then removing excess fluid with sterile gauze and waiting at least 30 minutes before applying antibiotic and/or antifungal drops, always accompanied by a topical steroid. In vitro work has shown that the presence of Tris-EDTA and NAC compromises neither the efficacy nor the stability of topical steroids (13).

The skin biofilm

It is perhaps less well known that biofilms can also form in other body areas and not only in the ears, e.g. in the skin folds. These include the interdigital spaces, the muzzle folds in brachycephalic dogs, the vulval plica, the labial plica, in the ventral neck of dogs with abundant skin folds and salivation (cocker, dogue de Bourdeaux), the umbilical, subcaudal or pericaudal folds of corkscrew-tailed dogs.

Colonisation by bacteria and/or yeasts in these areas is pathological, and only occurs if there are predisposing factors: e.g. an allergic state of the skin in the interdigital spaces with consequent lapping and increased dampness; in the plica of the snout excessive lacrimation; in the vulvar plica urinary incontinence, etc. Certainly the anatomical "plica" conformation of these body districts does not allow proper aeration of the skin and favours an increase in humidity, which may be worsened by licking or stagnation of urine, saliva or faeces.

The clinical aspect in these cases is represented by the presence of creamy material in the plica (Figures 5 a,b), which on cytological examination shows an abundant and , in the absence of inflammatory cells (it is therefore not pus).

These may be bacteria, but it should be remembered that Malassezia is also capable of forming a biofilm (Figure 6), which is often difficult to eradicate with common therapies.

Often topical therapy with antibiotic creams or disinfectant sprays or gels may not yield the desired results, due to the presence of the biofilm. As with the ears, therapy in these cases is based on breaking up the biofilm with Tris-EDTA and NAC solutions, subsequent disinfection of the part and application of an antimicrobial cream or gel to inhibit the formation of new biofilm. However, it is also essential to identify and resolve predisposing factors (anatomical conformations with particularly pronounced folds, frequent bathing, etc.) or primary factors (underlying allergic condition, urinary incontinence, diarrhoea, etc.) for a definitive resolution of the problem.

Conclusion

Otitis and skin fold dermatitis are common conditions in dogs and the presence of biofilms can make them a complicated problem to manage. Recognising the importance of biofilms in ear infections and intertrigo in dogs is a key step in developing more effective treatment strategies, such as the use of Tris-EDTA and NAC-based disrupting agents.

Bibliographic references

1. Chan WY, Khazandi M, Hickey EE, et al. In vitro antimicrobial activity of seven adjuvants against common pathogens associated with canine otitis externa. Vet Dermatol 2018, DOI: 10.1111/vde.12712.

2. Dinicola S, De Grazia S, Carlomagno G, Pintucci JP. N-acetylcysteine as powerful molecule to destroy bacterial biofilms. A systematic review. Eur Rev Med Pharmacol Sci. 2014; 18:2942-2948.

3. El-Feky MA, El-Rehewy MS, Hassan MA. Effect of ciprofloxacin and N-acetylcysteine on bacterial adherence and biofilm formation on ureteral stent surfaces. Pol J Microbiol 2009: 58:261-267.

4. Farca AM, Piromalli G, Maffei F, Re G. Potentiating effect of EDTA-Tris on the activity of antibiotics against resistant bacteria associated with otitis, dermatitis and cystitis. J Small Anim Pract. 1997; 38:243-245.

5. Hoffman LR, D'Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 2005; 436:1171-1175.

6. H?iby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010; 35: 322-332.

7. Karatan E, Watnick P. Signals, regulatory networks, and materials that build and break bacterial biofilms. Microbiol Molecular Biol Rev 2009; 73:310-347.

8. Luciani L, Stefanetti V, Rampacci E et al. Comparison between clinical evaluations and laboratory findings and the impact of biofilm on antimicrobial susceptibility in vitro in canine otitis externa. Vet Dermatol 2023; DOI: 10.1111/vde.13197, e-pub ahead of print.

9. Pye CC, Singh A, Weese JS. Evaluation of the impact of tromethamine edetate disodium dihydrate on antimicrobial susceptibility of Pseudomonas aeruginosa in biofilms in vitro. Vet Dermatol 2014; 25:120-123.

10. Pye CC, Yu AA, Weese JS. Evaluation of biofilm production by Pseudomonas aeruginosa from canine ears and the impact of biofilm on antimicrobial susceptibility in vitro. Vet Dermatol 2013; 24:446-449.

11. Roberts D, Cole P. N-acetylcysteine potentiates the anti-pseudomonas activity of carbenicillin in vitro. J Infect 1981: 3: 353-359.

12. Wooley RE, Jones MS. Action of EDTA-Tris and antimicrobial agent combinations on selected pathogenic bacteria. Vet Microbiol. 1983; 8:271-280.

13. Milanesi N, Ghibaudo G, della Mira T. The comparative cerumenolytic activity of otic preparations, an in vitro study (abstract). Vet Dermatol 2021; 32: 422