Biofilms: A microbial home

INTRODUCTION

Oral cavity is an open growth system with an uninterrupted introduction and removal of microbes and their nutrients. It offers diverse habitats where-in different species of micro-organisms can prosper. The primary requisite for any group of microbes to flourish in a niche is their ability to adhere to the tooth surfaces and multiply in shielded environments like periodontal pockets and tooth crevices. Such an aggregation of microbes on tooth surfaces has been traditionally referred to as ‘plaque’ because of its yellowish color, reminiscent of mucosal plaques caused by syphilis.

Dental plaque has been defined as “a specific but highly variable structural entity consisting of micro-organisms and their products embedded in a highly organized intercellular matrix.” It represents a true biofilm consisting of a variety of micro-organisms involved in a wide range of physical, metabolic and molecular interactions. The cooperative nature of a microbial community provides advantages to the participating organisms such as a broader habitat range for growth, enhanced resistance to antimicrobial agents and host defenses and enhanced pathogenicity.[1]

Biofilms have been implicated as the chief culprit in the etiopathogenesis of dental caries and periodontal disease. Though uncalcified biofilms can be removed by routine oral hygiene aids or professional dental instruments, they have the potential to calcify into dental calculus making their removal difficult. Hence, these biofilms pose a great challenge to the dental clinician in the control and eradication of biofilm-associated diseases.

HISTORICAL PERSPECTIVE

Biofilms are nothing new. The first description dates back to the 17^thcentury, when Anton Von Leeuwenhoek - the inventor of the Microscope, saw microbial aggregates (now known to be Biofilms) on scrapings of plaque from his teeth.

The term ‘Biofilm’ was coined by Bill Costerton in 1978.

In 2002, Donlan and Costerton offered the most salient description of a biofilm. They stated that biofilm is “a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene transcription.”[2]

WHAT IS A BIOFILM?

The term Biofilm (Wilderer and Charaklis 1989) describes the relatively indefinable microbial community associated with a tooth surface or any other hard non-shedding material, randomly distributed in a shaped matrix or glycocalyx.[2]

In the lower layers of a biofilm, microbes are bound together in a polysaccharide matrix with other organic and inorganic materials. Above it, is a loose amorphous layer extending into the surrounding medium. The fluid layer bordering the biofilm has stationary and dynamic sub layers.

CLASSIFICATION OF BIOFILMS

FORMATION OF A BIOFILM

Formation of a biofilm is a complex process that follows several distinct phases, beginning with adsorption on to the tooth surface of a conditioning film derived from bacterial and host molecules, which forms immediately following tooth eruption or tooth cleaning. This adsorption is followed by passive transport of bacteria mediated by weak long-range forces of attraction. Covalent and hydrogen bonds create strong, short-range forces that result in irreversible attachment.

The primary colonizers form a biofilm by autoaggregation (attraction between same species) and coaggregation (attraction between different species). Coaggregation[4] results in a functional organization of plaque bacteria and formation of different morphologic structures such as Corncobs and Rosettes. The microenvironment now changes from aerobic/capnophilic to facultative anaerobic. The attached bacteria multiply and secrete an extracellular matrix, which results in a mature mixed-population biofilm.

After one day, the term Biofilm is fully deserved because organization takes place within it. Transmission occurs from other sites, leading to incorporation of new members into the biofilm and the formation of a climax community. The thickness of the plaque increases slowly with time, increasing to 20 to 30 μm after three days.

Four stages of dental plaque biofilm growth (as shown in Figure 1)

Stage I - Attachment (lag - not inert, but metabolically reduced)

Stage II - Growth (log - exponential growth)

Stage III - Maturity (stationary)

Stage IV - Dispersal (death)

MICROBIOLOGY OF BIOFILMS

Plaque biofilm consists of different complexes of periodontal microorganisms which are based on the frequency with which microorganisms are recovered together. These complexes have been depicted in Figure 2.

PROPERTIES OF BIOFILMS

Biofilms are ubiquitous; they form on virtually all surfaces immersed in natural aqueous environments. A biofilm confers certain properties to bacteria that are not seen in the planktonic state, a fact that justifies recognition of dental plaque as a biofilm.

A major advantage is the protection that biofilm provides to the colonizing species from competing micro-organisms, environmental factors such as host defense mechanisms and potentially toxic substances like lethal chemicals or antibiotics. Biofilms also facilitate processing and uptake of nutrients, cross feeding and removal of potentially harmful metabolic products through the voids or water channels between the micro-colonies, acting as a primitive circulatory system.[1] They also create an appropriate physicochemical environment such as a properly reduced oxidation reduction potential.

An important characteristic seen in Biofilm-associated bacteria is Quorum sensing, or cell density mediated gene expression.[5] This involves the regulation of expression of specific genes through the accumulation of signaling compounds that mediate intercellular communication. Quorum sensing may give biofilms their distinct properties. Eg.- Expression of genes for antibiotic resistance at high cell densities may provide protection. It also has the potential to influence community structure by encouraging the growth of species beneficial to the biofilm and discouraging the growth of competitors.

Another important characteristic of biofilm associated bacteria is the gene transfer[6] through which bacteria communicates with each other. In S. mutans, quorum sensing is mediated by competence stimulating peptide, wherein genes are responsible for multiple functions - biofilm formation, competence and acid tolerance.

Biofilm related Regulation of gene expression has been shown in certain bacteria. eg. Exposure of S. gordonii to saliva results in induction of genes that mediate host surface binding and coaggregation with P. gingivalis and Actinomyces. Similarly, genes encoding glucan and fructan synthesis are differentially regulated in Biofilm-associated S. mutans.

MECHANISM OF INCREASED ANTIBIOTIC RESISTANCE IN BIOFILMS

Organisms in a Biofilm are 1000-1500 times more resistant to antibiotics than in their planktonic state. The mechanisms[2] of this increased resistance differ from species to species, antibiotic to antibiotic and for biofilms growing in different habitats. This antibiotic resistance in bacteria is thought to be affected by their nutritional status, growth rate, temperature, pH and prior exposure to sub-effective concentrations of antimicrobial agents. Another important mechanism appears to be the slower rate of growth of bacterial species in a biofilm, which makes them less susceptible to bactericidal antibiotics. Biofilm matrix can resist diffusion of antibiotics. Eg. strongly charged or chemically highly reactive agents can fail to reach the deeper zones of the biofilm as it acts as an ion exchanger in removing such molecules from solution.

‘Super-resistant’ bacteria have been identified within a biofilm, which have multidrug-resistance pumps that can extrude antimicrobial agents from the cell. Since these pumps place the antibiotics outside the outer membrane, the process offers protection against antibiotics that target cell wall synthesis. Above mentioned observations are critical to the use of antimicrobials in the treatment of Biofilm-associated infections.[7]

BIOFILMS AND INFECTIOUS DISEASES

Biofilms have been found to be involved in a wide variety of microbial infections (by one estimate 80% of all infections). These include dental caries, periodontal disease, otitis media, musculoskeletal infections, necrotizing fascitis, biliary tract infection, osteomyelitis, bacterial prostatitis, native valve endocarditis, meloidosis, cystic fibrosis pneumonia and peri-implantitis. Salient features of these infections are persistence and chronicity.[2]

MANAGEMENT PROTOCOL

Detection and assessment of biofilms: Biofilms can only be revealed by staining with dyes – either basic fuschin or erythrosine. These are called disclosing agents.[7] Two tone dyes contain FDC Red no.3 and FDC Green no.3, which stain immature and mature plaque respectively. Laser confocal microscopy is the latest method for plaque detection.

Criteria utilized in the assessment of Dental Biofilm: (Ribeiro et al. 1999):

• 0

  No visible biofilm

• 1

  Thin biofilm only on anterior teeth

• 2

  Easily removed thin biofilm distributed on anterior and posterior teeth

• 3

  Firmly adhered thick biofilm only on anterior or posterior teeth

• 4

  Firmly adhered thick biofilm on anterior teeth and thin biofilm on posterior teeth, or firmly adhered thick biofilm on posterior teeth and thin biofilm on anterior teeth

• 5

  Firmly adhered thick biofilm on anterior and posterior teeth

THE FUTURE

Biofilms have become a hot topic in environmental and infectious disease microbiology as well in the popular press. In recent years, dental researchers have begun to view plaque as a biofilm. This shifting view on plaque has important implications for future efforts in its prevention and treatment.

CONCLUSION

It is universally acknowledged that oral diseases are multifactorial, with each factor exerting its own influence thereby propelling the host towards diseased state. Even then, dental plaque is recognized as the single most important etiological factor. Dental health professionals have been waging a constant battle against biofilms.

Biofilms are difficult to locate, remove and penetrate and have an uncanny adaptive ability or to create their own environment. The need of the hour is to supplement the conventional treatment strategies like Scaling, Root planing and Surgery with Chemical Plaque Control Agents, Antimicrobials (e.g. chemically Modified Tetracyclines and Low Dose Tetracyclines), irrigational devices and Local Drug Delivery (LDD) systems to fight the menace of biofilms. Above all, the role of the dental office and the patient himself in maintaining adequate plaque control cannot be overemphasized. Conventional mechanical plaque control methods like efficient tooth brushing and interdental cleaning aids are still the easiest, cheapest and the most convenient weapons against oral biofilms. The role of a dentist extends much beyond the removal of biofilms but encompasses educating and motivating the patient to do so. This would go a long way in creating and maintaining a disease-free oral environment.