Biofilm Formation For Antibiofilm Agents

Biofilm Formation Introduction And Characteristics Pdf Biofilms are a collective of one or more types of microorganisms that can grow on many different surfaces. Microorganisms that form biofilms include bacteria, fungi, and algae. The biofilm matrix is an important part of the biofilm containing the microbial cells, exopolysaccharides, and water. Usually, the microbial cells in a biofilm are embedded in the extracellular polymeric substances (EPS) Produced by themselves which is also called Slime.

EPS contains extracellular DNA, proteins, and polysaccharides which form slime. Therefore biofilms are also called the collection of microorganisms surrounded by the slime they secrete. Microbial cells in the biofilm are different from the planktonic cells that are single cells and can float on a liquid medium.

When a single cell changes into biofilm, there’s a shift in the phenotypic behavior due to the change of expression in its genetics. The biofilms allow the microorganism to adhere to any surface, living or nonliving. The adaptive and genetic changes of the microorganisms within the biofilm make them resistant to all known antimicrobial agents. Most environmental biofilms contain multiple spp. It has been involved in various microbial infections, some even extending up to lethal infections, ranging from UTI to the infections of the implant such as catheters, prostheses, etc.

A biofilm may also be considered a hydrogel, which is a complex polymer that contains many times its dry weight in water. Biofilms are not just bacterial slime layers but biological systems; the bacteria organize themselves into a coordinated functional community. Biofilms can attach to a surface such as a tooth, rock, or surface, and may include a single species or a diverse group of microorganisms. The biofilm bacteria can share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and a host body’s immune system. A biofilm usually begins to form when a free-swimming bacterium attaches to a surface,

Biofilms CHARACTERISTICS

Biofilms are dynamic and responsive to their environment; that is, they can adapt to changes in their environment. A phenomenon known as detachment seems to be common among all biofilms. Bacterial cells can detach from their biofilm colony individually or in clumps. When individual microorganisms detach from a biofilm, these isolated microorganisms are relatively easy to kill with chemicals designed for this purpose.

When microorganisms detach from their biofilm colony in clumps, the clumps are pieces of the biofilm that are at the moment not attached to a surface; in this case, they maintain the protective properties of the original biofilm and are thus much more difficult to kill. In the right conditions, biofilms can migrate across surfaces over some time in a variety of ways.

Biofilms trap nutrients for microbial growth and help prevent the detachment of cells on surfaces present in the flowing system. It contains several porous layers and the cells in each layer can be examined by scanning laser confocal microscopy. Usually grown on a solid substrate that is submerged in or exposed to a liquid medium. Under certain conditions, they form a monospecies culture. Capable of exchanging antibiotic-resistant genes with one another, thus producing antibiotic degrading enzymes that can entirely nullify the effect of the incoming antibiotics.

FORMATION OF BIOFILMS

Biofilm formation begins with the attachment. Mainly there are 5 steps for the formation of biofilm as follows:

Step 1: Initial Attachment

Free-floating microorganism attaches to a solid substrate, exposed in a moist environment through a weak Vander Waals force which is reversible. The attachment also takes place due to the hydrophobic effect. Increased hydrophobicity reduces the repulsion between the bacterium and the extracellular matrix.

Step 2: Irreversible attachment

After the attachment using weak Vander Waals force. if the bacteria is not removed, it will completely anchor itself using cell adhesion structures such as pilla. This would be an irreversible attachment.

Step 3: Expansion

After irreversible attachment, the bacterial/microbial colony expands either by cell division or combining cell division with the recruitment of the other microbial cells. Motile cells can easily recognize and attach themselves to the matrix whereas the non-motile cannot perform this function easily. Such cells that are not able to attach themselves to the substrate successfully, attach themselves to initially colonized cells.

Step 4: Maturation

Once the microbial cells have aggregated and colonized themselves to form a biofilm, they communicate with each other using a Quorum sensing product (N-acyl homoserine lactone ). This facilitates them to perform different biochemical functions and release Extracellular polymeric substances (EPS). It acts as a slime for the protection of biofilm and facilitates communication.

Step 5: Dispersion

The final stage of biofilm formation. Here in the biofilm only change in size and shape. Dispersal helps to colonize new surfaces inhibition of biofilm. EPS degrading enzymes such as dispersion-B and deoxyribonuclease degrades the EPS hence increasing the “NO”.
Nitric oxide (NO) triggers the dispersal of biofilms formed by several bacterias. It has the potential for the treatment of patients with chronic infection due to biofilms, chances of biofilm destruction. EPS degrading enzymes can be used as anti-biofilm agents & inhibit the growing colonies.

BIOFILMS CAUSING INFECTIOUS DISEASES

As research has progressed over the years, biofilms — bacterial and fungal — have been implicated in a variety of health conditions. In a 2002 call for grant applications, the National Institutes of Health (NIH) noted that biofilms accounted “for over 80 percent of microbial infections in the body.”

Biofilms can grow on implanted medical devices such as prosthetic heart valves, joint prosthetics, catheters, and pacemakers. This, in turn, leads to infections. The phenomenon was first noted in the 1980s when bacterial biofilms were found on intravenous catheters and pacemakers. Bacterial biofilms have also been known to cause infective endocarditis and pneumonia in those with cystic fibrosis, according to the 2004 article in Nature Reviews Microbiology, among other infections.

Fungal biofilms can also cause infections by growing on implanted devices. Yeast species such as the members of the genus Candida grow on breast implants, pacemakers, and prosthetic cardiac valves. Candida species also grow on human body tissues, leading to diseases such as vaginitis (inflammation of the vagina) and oropharyngeal candidiasis (a yeast infection that develops in the mouth or throat).

1) Plaque

The formation of dental plaque bacteria found in the mouth during 1st year of life is aerotolerant anaerobes such as Streptococci and lactobacilli. Bacterial colonization of tooth surfaces begins with the attachment of single bacterial cells. Even on a freshly cleaned tooth surface, an acidic glycoprotein from the saliva forms a thin organic film several micrometers thick.

This film provides an attachment site for bacterial microcolonies. Extensive growth of these organisms results in a thick bacterial layer called dental plaque. If plaque continues to form filamentous anaerobes such as Fusobacterium spp begin to grow. Filamentous bacteria embedded in the matrix formed by the streptococcus extend perpendicular to the tooth surface, making a thicker bacterial layer. Associated with the filament bacteria are spirochetes such as Borrelia species gram-positive rods and gram-negative cocci. In plaque filamentous obligately anaerobic organisms Actinomyces may predominate.

Thus dental plaque consisting of a relatively thick layer of bacterial several different genera consider as mixed culture biofilm. Dental plaque is a biofilm or mass of bacteria that grows on surfaces within the mouth. It is a sticky colorless deposit at first, but when it forms tartar, it is often brown or pale yellow. It is commonly found between the teeth, on the front of teeth, behind teeth, on chewing surfaces, along the gum line, or below the gum line cervical margins. Dental plaque is also known as microbial plaque, oral biofilm, dental biofilm, dental plaque biofilm, or bacterial plaque biofilm. Bacterial plaque is one of the major causes of dental decay and gum disease.

Progression and build-up of dental plaque can give rise to tooth decay – the localized destruction of the tissues of the tooth by acid produced from the bacterial degradation of fermentable sugar – and periodontal problems such as gingivitis and periodontitis; hence it is important to disrupt the mass of bacteria and remove it.

Plaque control and removal can be achieved with correct daily or twice-daily tooth brushing and the use of interdental aids such as dental floss and interdental brushes. Oral hygiene is important as dental biofilms may become acidic causing demineralization of the teeth (also known as dental caries) or harden into dental calculus (also known as tartar). Calculus cannot be removed through tooth brushing or with interdental aids, but only through professional cleaning.

2) Infectious bacterial endocarditis

Endocarditis occurs when bacteria enter the bloodstream and attach to a damaged portion of the inner lining of the heart or abnormal heart valves. Not all bacteria entering the bloodstream are capable of causing endocarditis. Only those bacteria that can stick to the surface lining the heart and abnormal valves tend to cause endocarditis.

Most often occurs in people with preexisting heart disease. The pathogen causing endocarditis: Streptococcus sanguis, Streptococcus mutans, Staphylococcus aureus.

The heart valves are not supplied directly with blood. Therefore, the body’s immune response system, such as the infection-fighting white blood cells, cannot directly reach the valves through the bloodstream. If bacteria begin to grow on the valves (this occurs most often in people with already diseased heart valves), it is difficult to fight the infection, whether through the body’s immune system or through medications that rely on the blood system for delivery.

3) Catheter-associated UTI

The predominant form of life for the majority of microorganisms in any hydrated biologic system is a cooperative community termed a “biofilm.” A biofilm on an indwelling urinary catheter consists of adherent microorganisms, their extracellular products, and host components deposited on the catheter. The biofilm mode of life conveys a survival advantage to the microorganisms associated with it and, thus, biofilm on urinary catheters results in persistent infections that are resistant to antimicrobial therapy.

Because chronic catheterization leads almost inevitably to bacteriuria, routine treatment of asymptomatic bacteriuria in persons who are catheterized is not recommended. When symptoms of a urinary tract infection developed in a person who is catheterized, changing the catheter before collecting urine improves the accuracy of urine culture results. Changing the catheter may also improve the response to antibiotic therapy by removing the biofilm that probably contains the infecting organisms and that can serve as a nidus for reinfection.

BIOFILM FORMATION ON IMPLANTS

Dental implant

Dental implants are used to replace the teeth in the case of edentulous and partially edentulous patients. Dental implants support single or several tooth restorations. Materials that are currently used for dental implants include metals, ceramic, polymer, and vitreous carbon. A dental bacterial plaque is the colonization of bacterial cells around the tooth surface, crown, or implant.

It is estimated that one million dental implants are placed annually, with a success rate reported as high as 90–95%, despite bacterial infection. However, the prevalence of implant mucositis is reported to be greater than 60%. The various parts of the dental implant include an implant post, abutment, and crown.

The implants are classified as

(i) endosteal (root form or plate form) implant, which is inserted directly into the jaw bone,

(ii) subperiosteal implant, placed on and around the bone,

(iii) transosteal implant, inserted through the chin and supported by a plate, and iv) temporary implant, used for a temporary purpose.

Orthopedic implants

Orthopedic implants can be broadly classified as shoulder, elbow, hip, knee, and spine implants. Some of the materials that are widely used include stainless steel, titanium alloys, tantalum, titanium, cobalt-chromium alloys, and polyethylene.

Most bacteria isolated from orthopedic implants are not susceptible to common antibiotics, even in the sessile form. Orthopedic implant-related infections are of great importance because of their significant morbidity rate. Free-floating species bind to the native or prosthetic joint and transforms into small colony variants. This results in an additional modification that results in a mature, antimicrobial-resistant biofilm.

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus are the predominant bacteria associated with most of the orthopedic implant-related infections, whereas in prosthetic joints, low-grade infection, which commences from few months to a year after implantation, is caused by coagulase-negative Staphylococci and Propionibacterium acnes.

The risk of infection is higher in the case of knee joints than in the case of hip or shoulder prosthesis followed by fracture plates. The adherence of Mycobacterium tuberculosis to stainless steel and titanium is poor, which makes them a better substitute in the case of spinal tuberculosis.

Cardiac implants

Prosthetic valves, ventricular-assisted devices, and coronary stents are some of the predominant cardiac implants. The environment of most of these implants is blood. Coagulase-negative Staphylococcus aureus is a primary causative agent in the case of heart valves and ventricular-assisted devices. Staphylococcus aureus, Streptococcus sp., Gramnegative Bacilli, Enterococci, Pseudomonas-aeruginosa are other colonizers found in these implants. CoNS, a normal skin flora, also adheres to the medical devices and the adjacent tissues,

predominantly to fibronectin. Staphylococcus aureus, which is a nasopharyngeal and nosocomial pathogen, produces multiple toxins that degrade the tissues and stimulate the host immune response.

Non-implant-associated biofilms

Not all infection is due to the presence of an implant. The formation of a native biofilm is due to the presence of viable bacteria in the blood and its duration of growth (physiological bacteremia period). Although implant-associated biofilms have been well documented, the non-implant-associated biofilms leading to infection are not well studied.

Patients with cystic fibrosis, in which lung sections have been reported with a dense colony of Pseudomonas aeruginosa with a well-defined EPS alginate, is an example of a nonimplant-associated biofilm. The exact role of alginate in the biofilm architecture and antimicrobial resistance is not well established. Otitis media is another infection that is likely to be caused by biofilms. Haemophilus influenza, Streptococcus pneumoniae, and Moraxella catarrhalis are responsible for the infection of the middle ear.

Periodontitis is another nonimplant-associated infection, which arises due to the presence of an oral biofilm, particularly containing Gram-negative bacteria. Porphyromonas gingivalis, a Gram-negative pathogen, together with primary tooth colonizers co-aggregate and produce protease, which interferes with the host cytokine signaling pathway. Salmonella enterica, Shigella sp., and Yersinia sp. are the infectious agents of arthritis.

RESISTANT TO ANTIMICROBIAL AGENTS

The biofilm mode of growth confers on the associated organisms a measurable decrease in antimicrobial susceptibility. For example, Ceri et al. found that biofilm-associated Escherichia coli required >500 times the MIC of ampicillin to provide a 3-log reduction. Williams et al. found that Staphylococcus aureus biofilms required >10 times the MBC of vancomycin to provide a 3-log reduction.

The effect on susceptibility may be intrinsic (i.e., inherent in the biofilm mode of growth) or acquired (i.e., caused by the acquisition of resistance plasmids). There are at least 3 reasons for the intrinsic antimicrobial resistance of biofilms as follows: First, antimicrobial agents must diffuse through the EPS matrix to contact and inactivate the organisms within the biofilm.

EPSs retard diffusion either by chemically reacting with the antimicrobial molecules or by limiting their rate of transport. Hoyle et al. showed that the EPSs of Pseudomonas aeruginosa were capable of binding tobramycin; dispersed cells were 15 times more susceptible to this agent than were cells in intact biofilms. Second, biofilm-associated organisms have reduced growth rates, minimizing the rate that antimicrobial agents are taken into the cell and therefore affecting inactivation kinetics.

It was found that an increase in growth rate increased the susceptibility of Staphylococcus epidermidis biofilms. Third, the environment immediately surrounding the cells within a biofilm may provide conditions that further protect the organism.

Tresse et al. found that agar-entrapped E. coli demonstrated a decreased susceptibility to aminoglycoside antibiotics as a result of decreased uptake of the antibiotic by the oxygen-deprived cells. Concerning acquired resistance, research has shown that plasmids can be exchanged in biofilms under several conditions. Plasmids are extrachromosomal circles of DNA that may encode resistance to several antimicrobial agents, including β-lactams, erythromycin, aminoglycosides, tetracycline, glycopeptides, trimethoprim, and sulfonamides

CONTROL OR TREATMENT OF BIOFILM

1) Quorum sensing

It is the ability to detect and respond to cell population density by gene regulation. As one example, quorum sensing (QS) enables bacteria to restrict the expression of specific genes to the high cell densities at which the resulting phenotypes will be most beneficial. Many species of bacteria use quorum sensing to coordinate gene expression according to the density of their local population.

Similarly, some social insects use quorum sensing to determine where to nest. In addition to its function in biological systems, quorum sensing has several useful applications for computing and robotics. In general, quorum sensing can function as a decision-making process in any decentralized system in which the components have:

(a) a means of assessing the number of other components they interact with and

(b) a standard response once a threshold number of components is detected.

2) Use of heavy metal in treatment of biofilm

Sometimes, biofilms are useful. “Bioremediation, in general, is the use of living organisms, or their products — for example, enzymes — to treat or degrade harmful compounds.

Biofilms are used in treating wastewater, heavy metal contaminants such as chromate, explosives such as TNT, and radioactive substances such as uranium. “Microbes can either degrade them or change their mobility or their toxic state and therefore make them less harmful to the environment and humans”. Nitrification using biofilms is one form of wastewater treatment.

During nitrification, ammonia is converted to nitrites and nitrates through oxidation. This can be done by autotrophic bacteria, which grow as biofilms on plastic surfaces. Microorganisms degrade TNT by reduction. Most microorganisms reduce the three nitro groups, while some attack the aromatic ring.

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REFERENCE :

Biofilm Formation Introduction And Characteristics

• https://www.livescience.com/57295-biofilms.html

• https://www.cs.montana.edu/webworks/projects/biofilmbook/contents/chapters/chapter001/section004/green/page001.html

• https://www.nature.com/articles/pj2012130

• https://www.researchgate.net/figure/Biofilm-formation-steps-i-Reversible-attachment-of-planktonic-bacteria-to-surfaces_fig1_273611983

• https://en.wikipedia.org/wiki/Dental_plaque#Consequences_of_plaque_build-up

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