Different infections and involved microorganisms .
Biofilms are the aggregation of microbial cells, which are associated with the surface in almost an irreversible manner. It exists in variety of forms like dental plaque, pond scum, or the slimy build up in sink. Biofilm formation involves sequence of steps like conditioning, attachment, metabolism, and detachment. Biofilm consists of water channels, EPS (Exopolysaccharide), and eDNA (Environmental DNA), which plays an important role in nutrient circulation, its development, and structure stabilization. Resistance of planktonic bacteria against antimicrobial agents gets increased on the formation of biofilm, which may be the presence of diffusive barrier EPS or neutralizing enzyme, cells undergoing starvation, or due to spore formation. There are numerous factors, which affects biofilm formation such as substratum effects, conditioning film on substratum, hydrodynamics, characteristics of the aqueous medium, cell characteristics, and environmental factors. Biofilm can cause industrial, medical, and household damage and is a reason for loss of billions of dollars every year. Development of biofilm on catheters, medical implants, and devices is a major cause of infections and diseases in humans. Examples include Plaque, Native Valve Endocarditis, Otitis media, Prostatitis, Cystic fibrosis, Periodontitis, Osteomyelitis, and many more.
- medical implants
Biofilms are the aggregation of microbial cells, which are associated with the surface in almost an irreversible manner, i.e. cannot be removed by gently rising . They are attached with a biotic or abiotic surface integrated into the matrix that they have produced . An accustomed biofilm provides favorable conditions for genetic material mobility between the cells and has a defined architecture. It is also reported that these surface-associated microorganisms possess definite phenotype with reference to growth rate and gene transcription .
The credit of discovery of microbial biofilm can be given to Van Leeuwenhoek who, with his simple microscope first observed the microorganisms on tooth surface .
2. Biofilm formation
A biofilm may be composed of one microbial species or many microbial species found on a variety of living or nonliving surfaces. However, mixed species biofilms form the majority in most of the environments and single species biofilms host the surface of medical implants and hence being the reason of infections.
The initiation of biofilm formation have some requirements as the bacteria must be capable of attaching itself to and moving on the surface, detecting their cell density and ultimately to form a 3-D mesh of cells enclosed by exo-polysaccharide . There is also an important role of cell membrane proteins, extracellular polysaccharides and signaling molecules  ( Figure 1 ).
2.1 Biofilm formation steps
3. The composition of biofilm
Biofilm is primarily composed of bacterial micro-colonies which are nonrandomly distributed in a shaped matrix or glycocalyx . Mostly, these micro-colonies are rod-like or mushroom-shaped or they can have one or more types of bacteria. Based on bacteria type, the composition of micro-colonies contains 10–25% (by volume) of microbial cells and 79–90% (by volume) of the matrix [2, 6]. Extensive bacterial growth assists in the rapid formation of visible layers of microbes accompanied by excretion of EPS in an abundant amount . At bottom of most of the biofilms, a dense layer of microorganism is bound together in polysaccharide matrix with other organic and inorganic components. The successive layer is highly irregular and loose and may extend into surrounding medium .
3.1 Water channels
These are present in between the micro-colonies which act as the simple circulatory system for distributing nutrients and receiving harmful metabolites .
Exopolysaccharide which is produced by the bacteria, are the major component of a biofilm. It constitutes about 50–90% of the total organic matter in a biofilm . It is mainly composed of polysaccharides, some of which may neutral or polyanionic in case of Gram-negative bacteria or cationic as in case of Gram-positive bacteria. The anionic property of polysaccharide is confirmed by the presence of uronic acids (such as D-glucuronic, D-galacturonic, and mannuronic acids) or ketal-linked pyruvate. This anionic property plays an important role in the association of divalent cations like calcium and magnesium that have been shown to provide greater binding force in developed biofilm by cross-linking with polymer strands . Along with the polysaccharide (which constitutes 1–2% of EPS), EPS also contains proteins [<1–2% (including enzymes)], DNA (<1%), RNA (<1%) as well as some lipids and humic substances .
The microbial genetics and the environment in which bacteria grows are the determining factors for the composition of a biofilm.
One of the common mechanism by which eDNA is released is Autolysis. Released eDNA plays an important role in the development of the biofilm, biofilm structure stabilization as well as in gene transfer mechanisms. This genetic transfer is responsible for spreading of virulence and antibiotic resistance genes in circulating strains exposed to the selective pressure of medical treatment.
4. Drug resistance and biofilm
In a biofilm, rendering biofilm becomes ten to thousand times less prone to several antimicrobial agents than the same planktonic culture grown bacterium. As an example, it has been seen that there is an increase of 600-fold concentration in sodium hypochlorite (an oxidizing biocide that is counted in most effective antibacterial drugs) for killing biofilm cells of
Slow or partial diffusion of antibiotics into inner layers of biofilm. This is due to EPS matrix which has biofilm entrenched bacteria, act as a diffusive barrier .
In the biofilm microenvironment, some microbial cells fall into a state of slow growth or starvation due to nutrient limitation or accumulation of harmful metabolites. These are not vulnerable to many antimicrobial agents [2, 11].
The differentiation of a bacterial subpopulation resembles the process of spore formation. It has a distinctive and highly resistance phenotype (a biologically programmed response to bacterial sessile life form) that protects them from antibacterial effects .
Presence of neutralizing enzymes also contributes to the antibiotic resistance in the biofilm. These proteinaceous enzymes degrade or inactivate antibiotics by mechanisms like hydrolysis and modification of antimicrobials by different biochemical reactions .
Although, intensive and insistent treatment of antibiotic is effective in reducing the biofilm and controlling the exacerbations of chronic biofilm infections but are not able to eliminate biofilm infections it is possibly because the minimal concentration of antibiotic (required to eliminate a mature biofilm) is challenging to reach
Experimental studies suggested that in most of the cases antibiotic treatment alone is not sufficient to eliminate infections of biofilm . In a study, a nanoparticle called ciprofloxacin-loaded poly (lactic-co-glycolic acid), that were functionalized with DNase I, were prepared to observe their antibiofilm activity against
5. Factors affecting biofilm formation
A number of factors such as substratum effects, hydrodynamics and various properties of cell surface play an important role in microbial attachment .
5.1 Substratum effects
As the surface roughness increases microbial colonization increases because as the roughness increases, surface area increases and shear forces get diminished. And considering extent and rate of attachment, it has been seen that microorganisms get attached to more rapidly to hydrophobic and nonpolar surfaces as Teflon and other plastics rather than to glass and other materials having hydrophilic properties.
5.2 Conditioning films forming on the substratum
When a material surface gets exposed to any aqueous medium, it gets immediately coated with polymers from that surface or become conditioned. The coating or film is found to be organic in nature formed within minutes of exposure. The nature of these films is found to be quite different for surfaces exposed in the human host. As an example, “acquired pellicle,” a proteinaceous conditioning film, develops on tooth enamel surface. A pellicle is composed of glycoprotein, lysozymes, phosphoproteins, albumin, lipids and gingival crevice fluid. Oral cavity bacteria get adhered within hours of exposure to this pellicle conditioned surface.
The hydrodynamic flow layer is the zone of negligible flow which is found at the immediately adjacent to the substratum/liquid interface. The flow velocity of this zone is negligible and its thickness is inversely proportional to the linear velocity. Substantial mixing or turbulence is the main characteristics shown by the region outside the boundary layer. The hydrodynamic boundary layer can considerably affect the interaction between cells and substratum. The velocity characteristic of the liquid governs the association of cells with the submerged surfaces. At, very low linear velocities, the cells must navigate through the hydrodynamic boundary layer, and cell size and cell motility govern its association with the surface. The boundary layer decreases, as the velocity increases and cells will be exposed to progressively larger turbulence and mixing. Therefore, higher linear velocities would be supposed to form a more rapid association with the surface, at least until velocities become high enough to apply abundant shear forces on the attaching cells, that results in detachment of these cells .
5.4 Characteristics of the aqueous medium
Characteristics of the aqueous medium such as temperature, pH, nutrient level and ionic strength possibly play an important role in attachment of microbes with the substratum. As an example, it has been found that the attachment of
5.5 Properties of the cells
The rate and extent of adherence of microbes depends on the properties of cells like cell surface hydrophobicity, as hydrophobic interactions tend to increase with an increasing nonpolar nature of one or both involved surfaces and adhesion increases with increase in hydrophobicity, presence of fimbriae and flagella as fimbriae contribute to cell surface hydrophobicity probably by overcoming the initial electrostatic repulsion barrier that exists between the cell and substratum and production of EPS. EPS might be hydrophobic, although mostly they are both hydrophilic and hydrophobic. Numerous bacterial EPS have the backbone of 1,3- or 1,4-β-linked hexose residues and tend to be less deformable, more rigid and inadequately soluble or insoluble in specific cases although other EPS molecules may be water soluble. Researches also showed that different organisms produce different amounts of EPS and the amount of EPS increases with age of the biofilm. Antimicrobial resistance properties in the biofilm are possibly mediated by the EPS by impeding the mass transport of antibiotics through the biofilm, which might be by binding directly to these agents . EPS formation is an essential part of biofilm formation as studies on
5.6 Environmental factors
Different environmental factors affect the biofilm formation; listed below:
5.6.1 Availability of certain nutrients
It has been shown by studies on
5.6.2 Presence of oxygen
Presence of oxygen regulates Biofilm formation in
5.6.3 Environmental pH
Environmental pH effects were observed by studying on
On the other hand, bacteria like
When temperature was kept high,
6. Diseases due to biofilm
Besides infecting the industrial pipelines, waste water channels, oral cavity, ventilators, catheters, and medical implants, they are a major cause of human diseases . Infections and diseases in humans are mostly due to development of biofilm on or within indwelling implants or devices such as contact lenses, bio prosthetic and mechanical heart valves, pacemakers, intra-arterial and intravenous catheters, central venous catheters, peritoneal dialysis catheters, urinary catheters, joint prosthesis, voice prosthesis, penile prosthesis, ureteral stents, biliary stents, endotracheal tubes, nephrostomy tubes, intrauterine contraceptive devices (IUDs) [13, 14]. A biofilm may be composed of gram-positive or gram-negative microorganisms which may arise from the skin of a patient, health worker, tap water or any other environmental source .
Biofilm growth usually was seen in the lungs of cystic fibrosis patients causing chronic bronchopneumonia, in the middle ear in patients with chronic and secretory otitis media, in chronic rhino sinusitis, in chronic osteomyelitis and in chronic wounds . Infections and then diseases occur because of these two reasons: (a) Implantation of any medical device cause tissue damage which attracts platelets and fibrin accumulation at the site of the attachment. The damaged tissue aids in colonizing the microorganisms . (b) Drug resistance and inflammation in host might get stimulated by biofilm formation which results in sustained infections  ( Table 1 ).
|Gram-positive microorganisms||Site of infections and diseases|
|Acidogenic gram-positive cocci (e.g. ||Dental caries|
|Gram-positive cocci (e.g. ||Musculoskeletal infections|
|Group A ||Necrotizing fasciitis|
|Viridans Group ||Native valve endocarditis|
|Sutures, exit sites and arteriovenous shunts|
|Urinary catheter cystitis|
|Central venous catheter|
|Mechanical heart valves|
|Nontypable strains of ||Otitis media|
|Biliary tract infection, bacterial prostatitis|
|Cystic fibrosis pneumonia|
|Melioidosis nosocomial infections|
|Urinary catheter cystitis|
|Central venous catheter|
7. Biofilm on some common medical devices
7.1 Central venous catheter biofilms
Commonly found organisms on catheter biofilm are
Many studies have been done to control or avoid biofilm formation in these devices. Few remarkable results are:
It has been found in a research that microbial colonies of the left arterial catheter can be eliminated by addition of sodium metabisulfite to the dextrose-heparin flush.
Less colonization was seen on catheters coated with minocycline and rifampin than those coated with chlorhexidine and silver sulfadiazine .
7.2 Mechanical heart valve biofilms
To prevent initial attachment of the microbes, anti-microbial agents are provided during valve replacement or any invasive process like dental work. It has also been found out that less inflammation was caused when silver coated sewing cuff of St. Jude mechanical heart valve was implanted than an uncoated one [5, 17].
7.3 Urinary catheter biofilms
Organisms which develop biofilm on these devices are
It has been shown in studies that hydrophobicity of both organism and surface is responsible factors for microbial attachment on the catheter as a wide range of microbial colonies are found to be attached on the catheter’s surface which displays both hydrophobic and hydrophilic regions . Bacterial attachment is also enhanced by an increase in urinary pH and ionic strength by divalent cations (Mg and Ca). Urease is produced by some of the organisms of this biofilm which is responsible for hydrolyzing the urea to ammonium hydroxide. As a result, pH at the biofilm-urine interface gets higher, which causes precipitation of minerals such as struvite and hydroxyapatite. These biofilms having mineral components form encrustations which can completely block the catheter’s inner lumen .
Several approaches have been done to control biofilm formation on urinary catheters like the use of antimicrobial ointments and lubricants, bladder instillation, antimicrobial agents in collection bags, impregnation of catheters by silver oxides like antimicrobial agents or systemic antibiotics. Also, biofilm of many Gram-negative microorganisms can be reduced by exposing to mandelic acid in combination with lactic acid .
7.4 Contact lenses biofilms
Microbes get readily attached to the surface of both type of contact lenses i.e. soft contact lenses and hard contact lenses (differentiated according to the material used, design, wear schedule and frequency of disposal). Nature of substrate, water content, polymer composition, electrolyte concentration and type of bacterial strains governs the degree of adherence of microbes to the lenses. The storage case of a lens has been implicated as the primary source of contamination .
7.5 Intrauterine devices
The tail part of IUDs which is made up of a plastic microfilament surrounded by nylon sheath is possibly the primary source of infection. Microorganisms that contaminate IUDs are
8. Some common biofilm infections
8.1 Dental biofilms
Dental biofilms, commonly known as plaque are the most studied biofilm in human. It involves hundreds of species of bacteria. Some significant microbes include
After a good oral wash or dental cleaning, the tooth enamel acquires a coating called as pellicle which is composed of various proteins and glycoproteins of host origin. Then with the help of adhesion molecules and pilli, first
8.2 Native valve endocarditis (NVE)
This condition arises due to the interaction between bacteria, vascular endothelium and generally of mitral, aortic, tricuspid and pulmonary valves of the heart. The organisms responsible for these conditions are species of
Many antibiotic therapies are suggested depending on the organisms involved as Penicillin is recommended for normal treatment of
8.3 Otitis media
It is a condition of chronic ear infection caused due to inflammation of mucoperiosteal lining . In the middle ear cavity, fluid gets accumulated which ultimately affects speech development and learning capability of the patient. However, its complete etiology is still under research . Various organisms responsible for otitis media include
8.4 Chronic bacterial prostatitis
Prostatitis is the inflammation of the prostate gland which possibly occurs due to the microorganisms that have ascended from the urethra or by the reflux of infected urine into prostatic ducts which vacates into the posterior urethra. Once the microbe gets entered in the prostatic duct, they start multiplying rapidly and can form sporadic micro-colonies and biofilms which gets adhered to the epithelial cells of the system of ducts. Microbes responsible for this infection are
8.5 Cystic fibrosis
Cystic Fibrosis is a chronic bacterial infection of intrapulmonary airways with
Microscopic studies of sputum samples and lung tissue section have shown the presence of biofilm or micro-colonies in the airways. These biofilms are able to grow larger than 100 μm in diameter . Some common cystic fibrosis pathogens include
Early antimicrobial treatment (i.e. during early colonization period of microbes) for preventing chronic infection of
Periodontitis is the infection of supporting tissues of teeth, gums (gingiva) and periodontal tissues (gingiva, alveolar bone, and periodontal ligament). Its chronic form may lead to exfoliation of teeth. The primary site of periodontitis is sub gingival crevice which is the channel between the tooth root and the gum. Organisms responsible for this infection are
Osteomyelitis is an inflammatory bone disorder characterized by infection in bone/bone marrow which leads to necrosis and bone destruction [24, 25]. When complex multi-resistant biofilm has established, treatment of osteomyelitis becomes more challenging. Due to increased bacterial resistance to antibiotics in biofilm mode, they cause persistent infections. It has been found that in more than 50% osteomyelitis cases, causative organisms are
Although, endoprostheses which are found to be an increasingly common source of infection, surgically implanted devices or other implants like orthopedic internal fixation devices also represents a remarkable risk factor for the development of osteomyelitis. Stainless steel, titanium, titanium alloys are most commonly used materials in implants in which stainless steel is found to be associated with greater infection rate as compared to titanium. A possible reason of this is might be that soft tissues get firmly adhered to a titanium-implant surface while a fibrous capsule is formed enclosing a liquid filled space around the steel implants. This un-vascularized space is less accessible to host defense mechanisms where bacteria can multiply and freely spread. Studies showed that