Demystifying the biofilm in canine otitis

Introduction

Otitis is a common condition encountered in first-opinion veterinary practice, with allergy being the most common trigger in dogs. The inflammation of the ear canal can then lead to secondary overgrowth of bacteria or yeasts, and when left unmanaged, otitis can progress to a chronic condition. In such cases, perpetuating factors may come into play, for example the presence of otitis media, calcification of the ear canal, or changes in the ear’s microbial flora, resulting in the emergence of more virulent strains of bacteria 1.

In certain cases, particularly when specific pathogens such as Pseudomonas spp. are involved, biofilm formation can be observed 1. Biofilm is a complex, living biomass with a specific structure that makes its elimination very difficult once established. Microorganisms within the biofilm employ various factors to make them more resistant to both the immune system and antibiotic treatment 2. This article aims to clarify the concept of biofilms, providing readers with the knowledge necessary to recognize their presence, thereby facilitating the implementation of appropriate treatment strategies.

What lies beneath: unraveling the mystery of biofilms

Biofilms are complex structures formed by the accumulation of microorganisms, creating a bacterial aggregate with a unique composition. A biofilm consists predominantly of water (90%), with the remaining 10% comprising the microbial biomass 2. Other than water, the main components of biofilm matrix are extracellular polysaccharides (EPS), DNA and proteins. This combination of components imparts remarkable properties to a biofilm, which has significant implications for the survival and persistence of its bacteria 3.

The development of the three-dimensional structure of a biofilm occurs in several stages. Initiation begins with the attachment or adhesion of free-living bacteria, known as planktonic bacteria, to a surface. This attachment becomes irreversible, and the bacteria then undergo a transition to a sessile state. Subsequently, they aggregate to form a microcolony and initiate the production of the extracellular matrix by activating specific genes. Once the biofilm matures, fragments containing planktonic bacteria detach and disperse into the surrounding environment, facilitating biofilm dissemination 2. A notable advantage of biofilm formation is its ability to establish a gradient from the outermost to the innermost layers, in terms of nutrients, oxygen levels, growth rates, and genetics 4 (Figure 1).

One of the key factors of bacterial biofilms is their ability to create a protective barrier that is resistant to antibiotics. Bacteria embedded in a biofilm are better protected against the effects of antimicrobial treatments, making biofilm-associated infections more challenging to eliminate 5. Furthermore, biofilms exhibit increased resistance to environmental stresses, including attacks by the host’s immune system – for example, they can be resistant to phagocytosis by leukocytes 6. Another critical aspect of biofilms is their capacity for horizontal gene transfer. Due to the close proximity of bacterial cells within the biofilm, genetic exchanges can occur more efficiently, facilitating the dissemination of beneficial traits or antibiotic resistance within the bacterial population 7. 

Quorum sensing (the ability to detect and respond to cell population density by gene regulation) plays a major role in the complex process that is biofilm formation. Essentially, quorum sensing allows bacteria to coordinate their behavior based on their cell density, enabling bacteria in a biofilm to synchronize the production of key components of the extracellular matrix, such as EPS 2.

Exploring biofilm formation: which organisms are involved?

Both Gram-positive and Gram-negative bacteria demonstrate the ability to produce biofilms. Among the bacteria commonly reported or observed to do so in canine ear infections, Pseudomonas spp. stands out as a frequent culprit, with a high incidence of biofilm production 8. Biofilms can be composed of a single organism, or may include multiple organisms, a phenomenon known as polymicrobial biofilm formation. This diversity in biofilm composition highlights the complexity of these structures and their role in various infections 9.

Among other species known for biofilm production, researchers in human medicine have identified Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus viridans, Staphylococcus aureus, and Enterococcus faecalis 2. In veterinary medicine, biofilm-forming bacteria at sites other than from ear infections have been encountered, including, among others, Staphylococcus spp. and E. coli 10. And the concept of biofilm is not limited to bacteria alone; fungi such as Malassezia spp. also have the capability to form biofilms 11. This emphasizes the broader significance of biofilm formation across microbial species and underscores the importance of understanding and managing biofilms in various clinical and veterinary contexts. It also emphasizes that when dealing with biofilms, it is essential to not focus exclusively on Pseudomonas spp. but to investigate the possible presence of other microorganisms; pathogen identification is essential to optimize effective management of these infections. Biofilms can be complex communities comprising various species, making it crucial to adopt a thorough and multifaceted approach to address the diverse challenges they may pose by pinpointing the responsible pathogens.

The extent of bacterial biofilm production is often categorized on a scale of weak to strong, and depends on the bacterial or fungal strains involved 12; for instance, a study has highlighted that Pseudomonas aeruginosa is a strong biofilm producer compared to other species 13. This classification of biofilm production levels could potentially serve as a tool for assessing the potential virulence and treatment resistance of various bacterial strains, helping in clinical decision-making and therapeutic approaches, but it remains to be studied if it really could have a clinical and therapeutic impact 12.

Revealing biofilms: methods for detection and visualization

Detecting a biofilm can be challenging, as these structures are often not easily visible to the naked eye and require specific detection methods 14. Clinically, the macroscopic aspect of biofilms can vary depending on their maturity, the type of microorganisms involved, and what site in the body is involved (Figures 2 and 3). Different characteristics can be observed 14:

• Color: can range from white/translucent to gray/green
• Texture: may appear slimy, sticky or mucoid
• Shape: can exhibit a flat or a three-dimensional aspect

A clinical observation in the case of Pseudomonas spp. infection is the appearance of a classic grayish/greenish, sticky, or slimy biofilm. This may be attributed to the presence of alginate, as well as the production of pyocyanin, which imparts these characteristics 15.

The cytological appearance of biofilms depends on the microorganisms involved and the sample preparation technique. Special stains such as Periodic Acid Schiff that highlight the polysaccharide matrix can be used, but these are generally not available in routine practice, making identification challenging 16. However, it is important to note that biofilms may appear as clusters of cells (bacteria, spores, fungal hyphae) surrounded by an extracellular matrix (which is not always visible) and may or not be accompanied by polymorphonuclear neutrophils or mononuclear cells 17 (Figure 4).

Bacterial or fungal culture of microorganisms able to form biofilm is one of the diagnostic criteria for biofilm identification. However, it should not be considered a gold standard, as false negative cultures can be observed and discrepancies have been noted between bacterial culture and 16S amplicon profiling 18. Traditional culture methods primarily support the growth of planktonic bacteria, failing to accurately mimic biofilm conditions. Consequently, therapy decisions based on antibiograms derived from planktonic bacteria may not reflect the true antimicrobial susceptibility profile. Moreover, replicating biofilm conditions from planktonic cultures can be challenging due to potential differences in biofilm maturity between the sampled and cultured bacteria. Bacteria within biofilms exhibit considerably higher antibiotic resistance compared to their planktonic counterparts, rendering antibiotic susceptibility assessments less predictive of treatment efficacy 15. 

This limitation underscores the need for alternative methods, such as the Crystal Violet biofilm assay using broth media (e.g., Luria-Bertani Broth, Mueller-Hinton Broth or Tryptic Soy Broth), to assess biofilm production based on optical density readings. These methods offer a more comprehensive understanding of bacterial biofilm presence and its potential impact on antibiotic treatment outcomes 8, but they are not typically employed for routine bacterial culture. Other methods, again not commonly used for routine diagnosis, include 19:

• Scanning electron microscopy
• Transmission electron microscopy 
• Confocal laser scanning microscopy
• Fluorescent in situ hybridization
• Molecular biology, e.g., polymerase chain reaction (PCR), which detects specific genes associated with the formation of biofilm

Given the challenges in directly visualizing biofilms, it is often necessary to use multiple methods in combination to achieve accurate and comprehensive identification. 

Taming the biofilm: effective management strategies

As previously mentioned, biofilms confer bacteria with significantly increased resistance to antibiotics, often exhibiting 100-1,000 times greater resistance than planktonic bacteria 5. Consequently, it becomes imperative to develop strategic approaches to disrupt biofilms, thereby enabling antibiotic and antifungal treatments to effectively combat bacterial and fungal infections alike. The pursuit of innovative solutions to reduce biofilms not only enhances the efficacy of antibiotics, but also holds significant promise in addressing the challenges posed by antibiotic-resistant bacteria, and underscores the critical importance of ongoing research and development efforts in veterinary medicine. A review of some treatments that are commonly employed as adjuvant and have demonstrated effectiveness against biofilms is, therefore, worthwhile.

NAC (N-acetylcysteine) is a mucolytic agent that also possesses antimicrobial properties. While the exact mechanisms of its action on biofilms is only partially understood, it has been demonstrated to function as a biofilm-dissolving molecule. Specifically, it inhibits bacterial adhesion, decreases the production of extracellular polysaccharide matrix, and promotes the disruption of mature biofilms by breaking disulfide bonds within the extracellular matrix. This renders the biofilm more permeable and susceptible to antimicrobial treatments 20. Furthermore, its ability to destroy biofilms has been demonstrated in vitro with bacterial strains from the ear canal, namely Staphylococcus pseudintermedius and Pseudomonas aeruginosa. Its effectiveness varies depending on the concentration of NAC used, with approximately 1-2% recommended 13. Importantly, NAC has been proven safe during intratympanic injection in experimental conditions, making it a potentially non-ototoxic and viable option for treating chronically discharging ears 21.

Tris-EDTA (ethylenediaminetetraacetic acid-tromethamine) exerts its antimicrobial effects through a well-defined mechanism of action. The EDTA component functions as a chelating agent, sequestering divalent cations, which in turn disrupts the outer cell membrane of Gram-negative bacteria. This disruption leads to the release of lipopolysaccharides and renders the bacterial cells more permeable to other antimicrobial agents. Simultaneously, the Tris component acts as a buffer, enhancing the chelation capabilities of EDTA 22. While Tris-EDTA has demonstrated antibiofilm activity against P. aeruginosa, its effects on Staphylococcus spp. biofilms differ, often inhibiting their growth rather than eradicating them 13. It is worth noting that the combination of Tris-EDTA with certain antimicrobial agents might lead to a reduction in antibacterial efficacy, and further research is needed to fully understand these interactions. However, Tris-EDTA shines as an adjuvant in combination with some antimicrobial agents, where it exhibits synergistic antibiofilm activity. Studies have demonstrated that Tris-EDTA can reduce minimum bactericidal concentrations (MBCs) and minimum inhibitory concentrations (MICs), thereby enhancing the efficacy of antibiotics like marbofloxacin and gentamicin, especially in cases of multidrug-resistant P. aeruginosa in vitro 22.

In addition to Tris-EDTA, several other compounds have shown activity against biofilms, diversifying the arsenal of tools available in the battle against bacterial infections. For example, silver nanoparticles, povidone iodine and honey all provide alternatives for biofilm management 23,24. Moreover, ongoing research in the biofilm field continues to yield exciting discoveries. Innovations such as cold atmospheric microwave plasma, quorum sensing inhibitors and bacteriophages are emerging as potential agents with antibiofilm effects 23,25. These approaches hold the promise of enhancing our ability to combat biofilm-related challenges, offering hope for more effective treatment strategies in the future.

Conclusion

Biofilm formation represents an important virulence factor for various bacterial infections, including Pseudomonas spp. and Staphylococcus spp., especially in the context of chronic otitis. However, it is crucial not to neglect the role of yeasts such as Malassezia spp. in the role of biofilm production. When facing treatment failures characterized by specific macroscopic and microscopic features, considering the potential involvement of biofilm formation is paramount. Detecting and suspecting biofilm presence can lead to more adequate therapeutic strategies and increased hope of eradicating these stubborn infections.