**1. Introduction**

[141] Falagas ME, Giannopoulou KP, Kokolakis GN, et al. Fosfomycin: use beyond urinary

[142] Rahal JJ. Novel antibiotic combinations against infections with almost completely re‐ sistant Pseudomonas aeruginosa and Acinetobacter species. Clin Infect Dis 2006; 43.

[143] Zuravleff JJ, Yu VL, Yee RB. Ticarcillin-tobramycin-rifampin: in vitro synergy of the triple combination against Pseudomonas aeruginosa. J Lab Clin Med 1983;101. 896–

[144] Fish DN, Choi MK, Jung R. Synergic activity of cephalosporins plus fluoroquino‐ lones against Pseudomonas aeruginosa with resistance to one or both drugs. J Anti‐

[145] Gunderson BW, Ibrahim KH, Hovde LB, et al. Synergistic activity of colistin and cef‐ tazidime against multiantibiotic-resistant Pseudomonas aeruginosa in an in vitro

[146] Saiman L, Chen Y, San Gabriel P, et al. Synergistic activities of macrolides antibiotics against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas malto‐ philia, and Alcaligines xylosoxidans isolated from patients with cystic fibrosis. Anti‐

[147] Perez Urena MT, Barasoain I, Espinosa M, et al. Evaluation of different antibiotic ac‐

[148] Chini NX, Scully B, DellaLatta P. Synergy of polymyxin B with imipenem and other antimicrobial agents against Acinetobacter, Klebsiella, and Pseudomonas species. Program and abstracts of the 38th Interscience Conference on Antimicrobial Agents and Chemotherapy (San Diego). Washington, DC: American Society for Microbiolo‐

[149] Pankuch GA, Lin G, Seifect H, et al. Activity of meropenem with and without cipro‐ floxacin and colistin against Pseudomonas aeruginosa and Acinetobacter baumannii.

tions combined with rifampicin. Chemotherapy 1975;27. 82–89.

pharmacodynamic model. Antimicrob Agents Chemother 2003;47. 905–909.

tract and gastrointestinal infections. Clin Infect Dis 2008;46. 1069-1077

Suppl 2:S95-99.

microb Chemother 2002;50. 1045–1049.

microb Agents Chemother 2002;46. 1105–1107.

Antimicrob Agents Chemother 2008;52. 333-336.

gy 1998; Abstract E-56.

902.

56 Infection Control

Microbes have been characterized as planktonic, free-floating single cells. The morphological and physiological properties of microbes have been described as they grow in nutritionally rich culture media. Earlier very little thought have been given how microbes survive in the environment. But, the fact is, in natural environment, microbes are commonly found to be attached to surfaces as biofilms. Hence, the formation of surface attached microbial cells known as biofilms open a new horizon to study the micro-organisms.

Automatically, the question arises, "What is biofilm?" According to the recent definition, Biofilms can be defined as sessile communities of microbial cells irreversibly attached to a surface or interface or to each other which are embedded in a self produced matrix of extrac‐ ellular polymeric biomolecules and are physiologically different from planktonic cells with respect to growth rate and gene transcription [1]. While studying Pseudomonas aeruginosa Davis and Geesay have shown that gene *alg*C controlling phosphomannomutase involved in alginate (exopolysacharide) synthesis is upregulated within 15 minutes of adhesion to a solid surface [2].

Biofilms are ubiquitous. They can be present on any surface – biotic or abiotic. Biofilms can be found on ship hulls, dairy and petroleum pipeline and rocks or pebbles at the bottom of streams or rivers. They can grow in hot acidic pools in Yellowstone National Park (USA) and on glaciers in Antarctica. Biofilms can form anywhere with easy access to water e.g. on tiles of floor, kitchen platform or clogged sink etc. They are also found on plants and can remain symbiot‐ ically or cause crop diseases like citrus canker, Pierce's disease of grapes etc [3]. Fossilised bioilms with 3.5 billion years are among the oldest records of life on earth [4]. Biofilms are also

© 2013 Basak et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

associated with biocorrosion of metals(microbiologically influenced corrosion.i.e.MIC) which affect kinetics of cathodic and or anodic reactions [5]. Biofilms can also grow in contact lenses, biomedical implants and transcutaneous devices.

Microbial cells in biofilms undergo cell density-dependent gene regulation i.e. quorum sensing and thus coordinate through signalling molecules called autoinducers. Autoinducers increase in concentration as a function of cell density [13]. Usually Gram positive bacteria use processed oligopeptides to communicate, where as Gram negative bacteria use N- acyl homoserine lactones (AHLs) as autoinducers [14]. The widespread AI-2 quorum-sensing system is found in several, Gram positive and Gram negative bacteria also [15]. For acyl-HSL quorum-sensing, an enzyme belong to Lux I family is required for synthesis of signal from cellular metabolites [16]. For AI-2 quorum-sensing system which has been implicated in interspecies communica‐ tion, the synthesis of signalling molecule is directed by the Lux S gene product [17]. The ahyR/ I acyl- HSL quorum sensing system of Aeromonas hydrophila has been shown to be required for biofilm maturation [12]. Similarly the Lux S type quorum sensing system in *Streptococcus mutants* is also involved in biofilm development. Lux S system of *Salmonella* enterica serovar

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59

Duenne described biofilm architecture as underwater coral reef with pyramid or mushroom shaped projections from the surface and channels and caverns running through out [19]. Using CLSM, Lawrence et al has shown that Pseudomonas biofilms were more tightly packed at the surface and less dense near the periphery whereas *Vibrio parahaemolyticus* biofilms show

The adherent cells in a biofilm are embedded with a self produced matrix of extracellular polymeric biomolecules. 97% of a biofilm matrix is water. A complex of secreted polymers, absorbed nutrients and metabolites, cell lysis product and even particulate materials from the surrounding environment can form matrix. Actually the matrix surrounds, anchors and protects surface-bound microbes. The matrix actually prevent the access of antimicrobials and disinfectants and confer protection against environmental stresses such as UV radiation, pH

Besides microbial cells all major classes of macromolecules i.e. proteins, polysaccharides, nucleic acids can be observed within a biofilm. Even transformation, transduction and

Biofilms are formed by many bacterial species of medical importance e.g. *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Enterococci*, *Streptococcus mutans*, *Pseudomonas aeruginosa*, *E.coli* O157:H7, *Neisseria gonorrhoeae*, *Vibrio cholerae*, *Nontuberculous mycobacteria* (NTM) etc [6]. Amongst fungi - Candida albicans can usually form biofilm [22]. The two most intensely studied biofilms are produced by: *Staphylococcus epidermidis* and *Pseudomonas aeruginosa*.

The microbial biofilm has received much attention recently because biofilm mode of growth may be the key factor in persistent or chronic infections. The biofilms can act as nidus of acute infections and the microbial cells from biofilm are released at any one time during chronic infection [23]. Clinicians are very much concerned about the fact that it is really difficult to eradicate biofilm bacteria with antibiotics. Even in immunocompetent host the biofilm growth are rarely resolved by host's immune system as antigen may be hidden and key ligands may

Typhimurium is required for biofilm formation on human gallstones [18].

greatest cell density near the periphery [20].

shifts, osmotic shock and dessication [21].

**1.1. Biofilms and human disease**

conjugation result in gene transfer amongst the cells in biofilm.

Nearly every species of microorganisms e.g. bacteria, fungi, algae and protozoa have mecha‐ nisms to adhere to surfaces and to each other. It has been found that over 90% of all bacteria live in biofilms. Biofilms can be formed by single species of microorganism or by multiple species of bacteria, fungi, protozoa etc. Mixed species biofilms predominate in environment. Single species biofilm usually exist in a variety of infections and on medical implants and are the focus of current research [6].

Study of biofilm began when it was discovered that in natural aquatic system bacteria predominantly remain attached to surfaces [7]. The first recorded observation of biofilm was presented by Henrici in 1933 as 'it is quite evident that for the most part water bacteria are not free floating organisms, but grow upon submerged surfaces' [8]. The fouling of ship hulls by microbes in marine environment was already known to mankind. Hence, the study of biofilm has been started with marine bacteria, followed by fresh water microbial ecosystem and formation of biofilm on surface of eukaryotic tissue.

In early part of 20th Century it was difficult to observe biofilm as electron microscopy required complete dehydration of highly hydrated bioilm matrices and light microscopy was badly distorted by out-of-focus effects [1]. Though Confocal Laser Scanning Microscope (CLSM) was invented in 1950s it was never used to study bacteria. CLSM produces optical slices of complex structures, so out of focus effects are removed and it requires no sample preparations, so living microorganisms can be observed if fluorescent dye is introduced to observe the cells [1]. Hence, the modern biofilm era began with the use of Confocal Laser Scanning Microscope (CLSM) which showed the image of biofilm as sessile microbial cells embedded in matrix interspersed between open water channels [9].

The development of biofilm is a 5 stage process – 1) reversible attachment 2) irreversible attachment 3) early development 4) maturation 5) detachment or dispersal of cells. When the microbial cell reaches very closer to a surface (<1nm), the initial attachment depends upon the total attractive or repulsive forces between two surfaces. These forces include electrostatic and hydrophobic interactions, steric hindrance, van der Waals forces etc. Probably hydrophobic interactions play important role in primary adhesion [10]. The second stage of irreversible attachment employs molecular binding between specific adhesins and the surfaces [11].

The factors controlling biofilm formation are: i) recognition of attachment sites on a surface ii) nutritional cues iii) change of pH and temperature iv) exposure to antibiotics, chemical biocides, and host defense mechanisms e.g. complement system etc.

The gene expression in biofilm cells differ from planktonic cells and by 2D gel electrophoresis it had been found that in mature biofilm of *Pseudomonas aeruginosa* >300 proteins were detectable that were undetectable in planktonic cells [12].

During colonisation, microbial cells communicate via quorum sensing. In mature biofilm quorum sensing regulates formation of channels and pillar like structure for nutrient delivery. Microbial cells in biofilms undergo cell density-dependent gene regulation i.e. quorum sensing and thus coordinate through signalling molecules called autoinducers. Autoinducers increase in concentration as a function of cell density [13]. Usually Gram positive bacteria use processed oligopeptides to communicate, where as Gram negative bacteria use N- acyl homoserine lactones (AHLs) as autoinducers [14]. The widespread AI-2 quorum-sensing system is found in several, Gram positive and Gram negative bacteria also [15]. For acyl-HSL quorum-sensing, an enzyme belong to Lux I family is required for synthesis of signal from cellular metabolites [16]. For AI-2 quorum-sensing system which has been implicated in interspecies communica‐ tion, the synthesis of signalling molecule is directed by the Lux S gene product [17]. The ahyR/ I acyl- HSL quorum sensing system of Aeromonas hydrophila has been shown to be required for biofilm maturation [12]. Similarly the Lux S type quorum sensing system in *Streptococcus mutants* is also involved in biofilm development. Lux S system of *Salmonella* enterica serovar Typhimurium is required for biofilm formation on human gallstones [18].

Duenne described biofilm architecture as underwater coral reef with pyramid or mushroom shaped projections from the surface and channels and caverns running through out [19]. Using CLSM, Lawrence et al has shown that Pseudomonas biofilms were more tightly packed at the surface and less dense near the periphery whereas *Vibrio parahaemolyticus* biofilms show greatest cell density near the periphery [20].

The adherent cells in a biofilm are embedded with a self produced matrix of extracellular polymeric biomolecules. 97% of a biofilm matrix is water. A complex of secreted polymers, absorbed nutrients and metabolites, cell lysis product and even particulate materials from the surrounding environment can form matrix. Actually the matrix surrounds, anchors and protects surface-bound microbes. The matrix actually prevent the access of antimicrobials and disinfectants and confer protection against environmental stresses such as UV radiation, pH shifts, osmotic shock and dessication [21].

Besides microbial cells all major classes of macromolecules i.e. proteins, polysaccharides, nucleic acids can be observed within a biofilm. Even transformation, transduction and conjugation result in gene transfer amongst the cells in biofilm.

Biofilms are formed by many bacterial species of medical importance e.g. *Staphylococcus epidermidis*, *Staphylococcus aureus*, *Enterococci*, *Streptococcus mutans*, *Pseudomonas aeruginosa*, *E.coli* O157:H7, *Neisseria gonorrhoeae*, *Vibrio cholerae*, *Nontuberculous mycobacteria* (NTM) etc [6]. Amongst fungi - Candida albicans can usually form biofilm [22]. The two most intensely studied biofilms are produced by: *Staphylococcus epidermidis* and *Pseudomonas aeruginosa*.

#### **1.1. Biofilms and human disease**

associated with biocorrosion of metals(microbiologically influenced corrosion.i.e.MIC) which affect kinetics of cathodic and or anodic reactions [5]. Biofilms can also grow in contact lenses,

Nearly every species of microorganisms e.g. bacteria, fungi, algae and protozoa have mecha‐ nisms to adhere to surfaces and to each other. It has been found that over 90% of all bacteria live in biofilms. Biofilms can be formed by single species of microorganism or by multiple species of bacteria, fungi, protozoa etc. Mixed species biofilms predominate in environment. Single species biofilm usually exist in a variety of infections and on medical implants and are

Study of biofilm began when it was discovered that in natural aquatic system bacteria predominantly remain attached to surfaces [7]. The first recorded observation of biofilm was presented by Henrici in 1933 as 'it is quite evident that for the most part water bacteria are not free floating organisms, but grow upon submerged surfaces' [8]. The fouling of ship hulls by microbes in marine environment was already known to mankind. Hence, the study of biofilm has been started with marine bacteria, followed by fresh water microbial ecosystem and

In early part of 20th Century it was difficult to observe biofilm as electron microscopy required complete dehydration of highly hydrated bioilm matrices and light microscopy was badly distorted by out-of-focus effects [1]. Though Confocal Laser Scanning Microscope (CLSM) was invented in 1950s it was never used to study bacteria. CLSM produces optical slices of complex structures, so out of focus effects are removed and it requires no sample preparations, so living microorganisms can be observed if fluorescent dye is introduced to observe the cells [1]. Hence, the modern biofilm era began with the use of Confocal Laser Scanning Microscope (CLSM) which showed the image of biofilm as sessile microbial cells embedded in matrix interspersed

The development of biofilm is a 5 stage process – 1) reversible attachment 2) irreversible attachment 3) early development 4) maturation 5) detachment or dispersal of cells. When the microbial cell reaches very closer to a surface (<1nm), the initial attachment depends upon the total attractive or repulsive forces between two surfaces. These forces include electrostatic and hydrophobic interactions, steric hindrance, van der Waals forces etc. Probably hydrophobic interactions play important role in primary adhesion [10]. The second stage of irreversible attachment employs molecular binding between specific adhesins and the surfaces [11].

The factors controlling biofilm formation are: i) recognition of attachment sites on a surface ii) nutritional cues iii) change of pH and temperature iv) exposure to antibiotics, chemical

The gene expression in biofilm cells differ from planktonic cells and by 2D gel electrophoresis it had been found that in mature biofilm of *Pseudomonas aeruginosa* >300 proteins were

During colonisation, microbial cells communicate via quorum sensing. In mature biofilm quorum sensing regulates formation of channels and pillar like structure for nutrient delivery.

biocides, and host defense mechanisms e.g. complement system etc.

detectable that were undetectable in planktonic cells [12].

biomedical implants and transcutaneous devices.

formation of biofilm on surface of eukaryotic tissue.

the focus of current research [6].

58 Infection Control

between open water channels [9].

The microbial biofilm has received much attention recently because biofilm mode of growth may be the key factor in persistent or chronic infections. The biofilms can act as nidus of acute infections and the microbial cells from biofilm are released at any one time during chronic infection [23]. Clinicians are very much concerned about the fact that it is really difficult to eradicate biofilm bacteria with antibiotics. Even in immunocompetent host the biofilm growth are rarely resolved by host's immune system as antigen may be hidden and key ligands may be repressed [24]. Biofilms are associated with kidney stones of infective origin, formation of dental plaques, infections in cystic fibrosis, infections of permanent indwelling devices such as joint prosthesis & heart valves, intrauterine devices (IUDs) and urinary catheters etc [25].

interesting finding is emergence of *P. aeruginosa* with mucoid phenotype in late stage CF patients [28]. This mucoid material is a polysaccharide i.e. alginate which probably prevent antibody coating and opsonic phagocytosis. In fact, biofilm protects *P. aeruginosa* from antimicrobials and host defenses. Genetic fingerprinting studies show same strain of *P. aeruginosa* can persist in CF patients for decades leading to chronic inflammation and decline

Biofilms: A Challenge to Medical Fraternity in Infection Control

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61

Biofilms also play a major role in causing dental caries, gingivitis, periodontitis, apical periodontitis etc [30].The anatomical complexities in root canal system provide favourable condition for biofilm formation, which is actually initiated by invasion of pulp chamber by oral flora after tissue breakdown. Facultative or strict anaerobes are more frequently associated than aerobic microorganisms. *Porphyromonas gingivalis* is the primary agent responsible for periodontitis [31]. Endodontic biofilm can be—i) intracanal, ii) extraradicular, iii) periapical and iv) foreign body centered. Foreign body centered biofilm is a major complication associ‐

Similarly during acute phase of osteomyelitis, microscopical examination have shown biofilm formation on infected bone surfaces [33]. In chronic prostatitis, adherent bacterial colonies on the surface of prostatic duct have been observed on microscopical studies, even in culture

Biofilms can develop on indwelling medical devices like prosthetic heart valve, pacemakers, central venous catheter, urinary catheter, contact lenses, intrauterine devices etc. and can cause persistent infections which are usually lethal. Scanning electron microscopy clearly shows biofilm formation at the tip of urinary catheter kept for 7 days. On medical devices, biofilms are most commonly formed by coagulase negative Staphylococci (CoNS) especially *S. epider‐*

Biofilms can develop on both types of contact lenses i.e. soft and hard and also on contact lens storage cases. *Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, E.coli,* Candida species can adhere to contact lenses [35]. Evidence of biofilm on contact lenses and it's storage cases have been reported from patients with microbial kerattis [36]. The rate of prosthetic valve endocarditis (PVE) range from 0.5% to 4% [37]. Coagulase negative Staphy‐ lococci are the commonest early colonizers after surgical implantation of prosthetic valve whereas *Streptococcus viridans* most commonly colonize during late PVE (i.e. 12 months following valve replacement) [38]. Though *S. aureus*, Gram negative coccobacilli or fungi may

Infection with central venous catheter is a quite common device related infection. Biofilms

have been shown by CLSM to be present outside the catheter or inner lumen [34].

in lung function and ultimately respiratory failure [29].

ated with prosthesis and implant supported prosthesis [32].

*midis* followed by *S. aureus, Enterococci, Pseudomonas aeruginosa* etc.

*1.1.4. Endodontics*

*1.1.5. other conditions*

negative cases [34].

*1.1.6. Indwelling medical devices*

also be responsible for PVE.

However in many chronic infections both the biofilm and planktonic growth may coexist. Parsek and Singh in 2003 have proposed few criteria to define the role of biofilms in human diseases [12]: a) the causative bacteria are surface associated b) examination of infected tissue shows bacteria living in microcolonies and embedded in extracellular matrix c) infection is usually confined to a particular site and dissemination occurs as a secondary phenomenon d) the infection is difficult to eradicate with antibiotics though the causative bacteria are suscep‐ tible to that antibiotics in planktonic state.

#### *1.1.1. Infection-related kidney stones*

15-20% of kidney stones occur in the setting of urinary tract infections. Infact, infection stones are produced by interplay between infecting bacteria and mineral substrates derived from urine resulting in formation of a complex biofilm. Microscopic analysis of stone has revealed that bacteria are organized in microcolonies and surrounded by an anionic matrix composed of both polysaccharides and crystallized minerals [26]. It requires an alkaline environment to decrease solubility of phosphate, increased concentration of NH4 <sup>+</sup> for struvite and CO3 - for carbon apatite formation as these are major constituents of this type of stone. The normal urine is not saturated with struvite and carbon apatite. The alkaline pH of urine occurs in infection with urease producing organisms like *Proteus, Providencia, Klebsiella* and *Pseudomonas* species. It is hypothesized that biofilms provide localized and concentrated urease activity to form stones [26].

#### *1.1.2. Bacterial endocarditis*

The primary lesion in endocarditis is due to vegetation (valve biofilm), which is composed mainly of bacteria and their products, platelets and fibrin derived from circulation with the damaged endothelial surface as substratum. Durack in1975, developed nonbacterial throm‐ botic endocarditis by leaving a polyethylene catheter in contact with aortic valve of a rabbit and showed how bacterial microcolonies were formed within 24 hours [27].

## *1.1.3. Airway infections in cystic fibrosis*

Cystic fibrosis (CF), a common inherited disease of lower respiratory tract is caused by mutation in the gene which encodes Cystic fibrosis transmembrane regulator protein (CFTR). CFTR functions as a chloride ion channel protein [1]. Chloride ion transport is severely impaired when CFTR is defective in CF patients, resulting in hyperviscous mucus. Initially CF patients suffer from intermittent respiratory infections but in late stage permanent infection with *P. aeruginosa* occurs. It has been found that even with higher antibiotics given parenterally *P. aeruginosa* could not be eradicated from sputum of CF patients in the late stage and it may persist for the rest of the patient's life. In permanent infection phase of CF patients, *P. aeruginosa* biofilm may be found in airways. Another interesting finding is emergence of *P. aeruginosa* with mucoid phenotype in late stage CF patients [28]. This mucoid material is a polysaccharide i.e. alginate which probably prevent antibody coating and opsonic phagocytosis. In fact, biofilm protects *P. aeruginosa* from antimicrobials and host defenses. Genetic fingerprinting studies show same strain of *P. aeruginosa* can persist in CF patients for decades leading to chronic inflammation and decline in lung function and ultimately respiratory failure [29].

## *1.1.4. Endodontics*

be repressed [24]. Biofilms are associated with kidney stones of infective origin, formation of dental plaques, infections in cystic fibrosis, infections of permanent indwelling devices such as joint prosthesis & heart valves, intrauterine devices (IUDs) and urinary catheters etc [25].

However in many chronic infections both the biofilm and planktonic growth may coexist. Parsek and Singh in 2003 have proposed few criteria to define the role of biofilms in human diseases [12]: a) the causative bacteria are surface associated b) examination of infected tissue shows bacteria living in microcolonies and embedded in extracellular matrix c) infection is usually confined to a particular site and dissemination occurs as a secondary phenomenon d) the infection is difficult to eradicate with antibiotics though the causative bacteria are suscep‐

15-20% of kidney stones occur in the setting of urinary tract infections. Infact, infection stones are produced by interplay between infecting bacteria and mineral substrates derived from urine resulting in formation of a complex biofilm. Microscopic analysis of stone has revealed that bacteria are organized in microcolonies and surrounded by an anionic matrix composed of both polysaccharides and crystallized minerals [26]. It requires an alkaline environment to

carbon apatite formation as these are major constituents of this type of stone. The normal urine is not saturated with struvite and carbon apatite. The alkaline pH of urine occurs in infection with urease producing organisms like *Proteus, Providencia, Klebsiella* and *Pseudomonas* species. It is hypothesized that biofilms provide localized and concentrated urease activity to form

The primary lesion in endocarditis is due to vegetation (valve biofilm), which is composed mainly of bacteria and their products, platelets and fibrin derived from circulation with the damaged endothelial surface as substratum. Durack in1975, developed nonbacterial throm‐ botic endocarditis by leaving a polyethylene catheter in contact with aortic valve of a rabbit

Cystic fibrosis (CF), a common inherited disease of lower respiratory tract is caused by mutation in the gene which encodes Cystic fibrosis transmembrane regulator protein (CFTR). CFTR functions as a chloride ion channel protein [1]. Chloride ion transport is severely impaired when CFTR is defective in CF patients, resulting in hyperviscous mucus. Initially CF patients suffer from intermittent respiratory infections but in late stage permanent infection with *P. aeruginosa* occurs. It has been found that even with higher antibiotics given parenterally *P. aeruginosa* could not be eradicated from sputum of CF patients in the late stage and it may persist for the rest of the patient's life. In permanent infection phase of CF patients, *P. aeruginosa* biofilm may be found in airways. Another

and showed how bacterial microcolonies were formed within 24 hours [27].

<sup>+</sup> for struvite and CO3


decrease solubility of phosphate, increased concentration of NH4

tible to that antibiotics in planktonic state.

*1.1.1. Infection-related kidney stones*

stones [26].

60 Infection Control

*1.1.2. Bacterial endocarditis*

*1.1.3. Airway infections in cystic fibrosis*

Biofilms also play a major role in causing dental caries, gingivitis, periodontitis, apical periodontitis etc [30].The anatomical complexities in root canal system provide favourable condition for biofilm formation, which is actually initiated by invasion of pulp chamber by oral flora after tissue breakdown. Facultative or strict anaerobes are more frequently associated than aerobic microorganisms. *Porphyromonas gingivalis* is the primary agent responsible for periodontitis [31]. Endodontic biofilm can be—i) intracanal, ii) extraradicular, iii) periapical and iv) foreign body centered. Foreign body centered biofilm is a major complication associ‐ ated with prosthesis and implant supported prosthesis [32].
