**3. Biofilm**

Biofilms have been around for billions of years. They have been identified in 3.2 – 3.4 billion year old South African Kornberg formation, and in deep-sea hydrothermal rocks [55]. Similar biofilms can be found in modern hot springs and deep-sea vents [124, 160]. The presence of biofilms in both ancient fossils and in similar modern environments indicates that biofilm formation is an ancient and integral characteristic of prokaryotes. It is likely that biofilms provided homeostasis during the harsh and fluctuating conditions of the primitive earth such as extreme temperatures, pH and exposure to UV light, thus enabling complex interactions between individual cells. It is, however, generally accepted that planktonic cells existed before the development of biofilm communities. The concomitant development of both planktonic and sessile bacteria in biofilm communities could be attributed to the conditions offered by life on surfaces [151]. The ability of bacteria to adhere to surfaces and form biofilms in different environments is due to the selective advantage that surface association offers the bacteria.

### **3.1. Definition**

*Escherichia coli* is the predominant uropathogen responsible for almost 80% of all cases, followed by *Staphylococcus*, *Klebsiella*, *Enterobacter*, *Proteus* and *Enterococci* species [128]. The financial implications of UTIs are enormous due to high incidence. UTIs account for a total

In addition to being the most common bacterial infection, UTIs are also the most common type of hospital acquired infections (HAI). HAIs can be defined as a localized or systemic condition resulting from an adverse reaction to the presence of an infectious agent or toxin, which occurs in a patient in a health care setting and was not present or incubating at the time of admission [64, 66]. UTIs account for 30% of all HAI [77]. Of these 30% infections, 80% of them are estimated to be catheter-associated [89]. According to the CDC, CAUTIs are defined as an UTI in a patient who had an indwelling urinary catheter in place at the time of or within 48 hours prior to infection onset. CAUTI can lead to complications such as cystitis, pyelonephritis, gramnegative bacteremia, prostatitis, epididymitis, endocarditis, vertebral osteomyelitis, septic arthritis, endophthalmitis and meningitis [20]. Additionally CAUTIs also result in prolonged hospital stay, increased cost and mortality [77]. An estimated 15-25% of hospitalized patients will have a urinary catheter at some point during their hospital stay [175]. Obstruction of indwelling catheters can lead to sepsis, even resulting in mortality [174]. Each year around 13,000 deaths are attributed to UTIs in the United States [77]. The cost associated with CAUTI episodes is about \$750-\$1000 per infection, and the estimated total cost in the United States

Millions of transurethral, suprapubic and nephrostomy catheters or urethral stents are used in patients every year. These devices overcome several host defenses and enable bacterial entry at a rate of 3 to 10% (cumulative rate) per day, which leads to bacteriuria in patients after a month [8]. In intubated patients, bacteria frequently ascend from the urethral meatus into the bladder between the mucosal and catheter surfaces. In certain cases, bacteria may ascend through the drainage system due to contamination of the drainage bag or disruption of the tubing junction. The presence of a device enables the persistence of the etiologic organism in the urinary tract. Several studies have demonstrated that bacteria exist as biofilms on these devices [53]. Formation of a biofilm and incrustation with calcium and magnesium struvites has a significant role in the pathogenesis and treatment of catheter-associated infections.

Biofilms have been around for billions of years. They have been identified in 3.2 – 3.4 billion year old South African Kornberg formation, and in deep-sea hydrothermal rocks [55]. Similar biofilms can be found in modern hot springs and deep-sea vents [124, 160]. The presence of biofilms in both ancient fossils and in similar modern environments indicates that biofilm

annual cost of more than \$ 3.5 billion in the United States [87].

**2. Catheter associated urinary tract infection**

56 Recent Advances in the Field of Urinary Tract Infections

ranges from \$340-\$450 million annually [132].

**3. Biofilm**

The definition of biofilm has evolved over the years. Marshal in 1976 [94] observed the presence of fine extracellular polymer fibrils that anchored bacteria to the surface. Costerton and coworkers [1978; 28] defined biofilms as communities of attached bacteria that were found to be encased in a glycocalyx matrix of polysaccharide that mediates adhesion [28]. They also stated that biofilms consist of single cells and microcolonies which are embedded in the matrix [26]. This definition was later modified to include the ability of biofilms to adhere to surfaces and to each other forming microbial aggregates and floccules [29]. The adhesion to a surface also triggers the expression of genes controlling production of bacterial components required for biofilm formation, thus including the role of gene modulation in the definition [29]. Consequently, a definition of biofilm must include the ability of cells to attach to a surface, extrapolymeric encasing, presence of noncellular and abiotic components in the matrix, physiological attributes of these organisms and the differential gene expression in biofilm cells versus planktonic cells. Taking all this into account, biofilms can be defined as a microbially derived sessile community consisting of cells that are attached to an interface or to each other, are embedded in an extracellular polymeric matrix that they have produced and demonstrate altered phenotype associated with differential gene expression [38]. This definition also applies to biofilm cells that have broken off from a biofilm on a colonized medical device and circulate in the body fluids with the ability to establish itself in another niche.

### **3.2. Biofilm formation and structure**

Biofilms can form on abiotic surfaces such as minerals, air-water interfaces, and biotic surfaces such as plants, other microbes and animals. In the human body, bacteria reside as biofilms on skin, oropharynx and nose, intestine and indwelling medical devices. To form a biofilm, bacteria are attracted to the surface by environmental signals. On reaching the surface, the bacteria attach to it as single cells or as clusters. When single cells attach to a surface they form a monolayer biofilm. A monolayer biofilm can be defined as one in which the bacteria attach only to the surface [75]. When bacteria attach to a surface as a cluster, they form a multilayer biofilm. Multilayer biofilms can be defined as a microbial community, where the bacteria are attached both to the surface and the neighboring bacterial cells [75]. The type of biofilm formed depends on the environmental conditions and surfaces that favor their development, the genes that are activated, the architecture of the biofilm and the matrix composition [75].

Monolayer biofilms are composed of a single layer of cells attached to a surface. These biofilms are favored when cell-surface interactions predominate. Since monolayer biofilms offer bacteria more proximity to surfaces, they commonly occur during the interaction of the bacterial pathogen with the host. In flagellate motile bacteria, monolayer formation occurs in two steps, where bacteria first become attached to a surface when they come in close proximity to it. After attachment, the bacteria break the forces tethering them to the surface, resulting in transient attachment. However, a few bacteria that have transitioned from transient to permanent attachment remain attached to the surface. Multilayer biofilms form when bacteria adhere to the surface as well as to each other. Several adhesion factors are known to mediate this transition, including preformed adhesins, conditionally synthesized adhesins and specific adhesins.

*3.3.1. Matrix components*

biofilm.

Exopolysaccharides are a major component of the biofilm matrix. The absence of polysacchar‐ ide synthesis and export leads to an inability to form multilayer biofilms in most bacteria. Bacteria capable of forming biofilms possess distinct genetic loci that encode for the synthesis of polysaccharides. One of the most common exopolysaccharides in the biofilm matrix is a polymer of β-1, 6-N-acteyl-D-glucosamine called PGA or PNAG. Several bacterial species, including *E. coli*, *S. aureus*, *Actinobacillus* spp., and *Bordetella* spp. make use of PGA to construct their matrix [30, 70, 71, 114, 173]. The synthesis and export of PGA is carried out by the *icaADBC* locus in Staphylococcal species and the *pgaABCS* locus in *E. coli*. PGA is required for bacterial attachment and biofilm formation in *E. coli*. Mutations in this locus prevent attachment even after prolonged incubation [173]. In *S. aureus*, the *icaADBC* locus is important for attachment and biofilm formation on indwelling medical devices [42]. In *S. epidermidis*, this locus is also shown to be required for virulence and immune evasion, thus emphasizing the role of biofilms in disease [172]. Another commonly found polysaccharide in the biofilm matrix is cellulose which has been identified as a major component of the matrix in *E. coli*, *Salmonella*, *Citrobact‐ er*, *Enterobacter* and *Pseudomonas* [140, 142, 181, 182]. In *E. coli* and *Salmonella* Typhimurium, cellulose synthesis is made possible by the *bcsABZC-bcsEFG* locus [140, 182]. In addition to PGA and cellulose, some *E. coli* strains also make colanic acid, which is a branched chain polymer synthesized by the *wca* locus [146]. Mutants that are defective in colonic acid forma‐

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tion can attach to surfaces, but are incapable of forming multilayer biofilms [32].

The biofilm matrix is also composed of proteins exported to the matrix by cells within the biofilm. Proteinaceous appendages such as fimbriae and pili confer adhesive properties in bacteria. In *E. coli* and *Salmonella*, curli fimbriae produced by the *csgBAC* and *csgDEFG* operons are part of the biofilm matrix [57]. Transcriptional profiling studies have demonstrated that fimbria and pili gene expression is upregulated in biofilms compared to planktonic cells [12]. Another group of proteins associated with the matrix are the *Bap* or Biofilm-associated proteins. These proteins hold bacterial cells together in the biofilm by interacting with similar proteins on the surface of neighboring cells. Bap proteins have been shown to be critical for biofilm production in *S. aureus* [82]. Besides proteins that bind other proteins on neighboring cells, the biofilm matrix also contains lectins and sugar binding proteins. These proteins recognize sugar moieties on the surface of eukaryotic cells and bind to them, thereby facili‐ tating cell-cell interactions [163]. Besides the above mentioned proteins, autotransporter proteins have been identified to be part of the biofilm matrix. The proteins can transport themselves to the cell surface without the need for other transport systems [48]. In *E. coli* autotransporters proteins such as *ag43*, AIDA and TibA have been shown to promote biofilm formation [135]. These proteins serve to maintain close-range interactions between cells in the

Another major component of the biofilm matrix is eDNA (extracellular DNA). In *P. aerugino‐ sa*, the biofilm matrix has significant amounts of DNA that is essential for biofilm integrity [95]. Addition of DNase to the culture media resulted in an inhibition of biofilm formation and dissolution of preformed biofilms [177]. It is hypothesized that DNA could serve as a grid that enables bacteria to move using type IV pili. The ability of type IV pili to bind DNA has been

Preformed adhesins include flagellum and pili. Motility is believed to increase the initial interaction between bacteria and the surface. Several studies have also demonstrated that flagellar motility promoted surface adhesion in bacteria [76, 85, 167]. However, under certain conditions, flagellar mutants that are defective in the synthesis of flagellar components have shown an increased synthesis of adhesive matrix that promotes bacterial attachment and multilayer biofilm formation [83, 176]. These observations indicate that flagellar impedence may be important in priming the bacteria for the formation of a multilayer biofilm. Neverthe‐ less, mutants lacking the flagellum or the flagellar motor are completely defective in monolayer and multilayer biofilm formation [83], implying that flagellar motor plays a vital role in biofilm formation independent of flagellar motility. Retractable pili are critical for gram-negative bacteria to attach to surfaces [75]. It is hypothesized that these structures pull bacteria along surfaces by attaching to the surface and retracting, thus helping the bacteria approach the surface more closely [75].

Bacteria can also conditionally synthesize adhesins to promote surface attachment. In *Pseudomonas fluorescens*, the transition from transient to permanent attachment is mediated by LapA (Large adhesion ProteinA) that associates with the bacterial surface [62]. In *E. coli*, a similar function has been attributed to the exopolysaccharide adhesin, PGA (poly-β-1,6-*N*acetyl-d-glucosamine) which mediates the transition from temporary to permanent attach‐ ment [2]. Following the transient attachment which is accomplished through the array of adhesins such as flagella and pili, bacteria form stable and specific binding through interactions with eukaryotic cell receptors [59]. These interactions are mediated by specific adhesins which aid in internalization.

### **3.3. Biofilm matrix**

Bacterial cells in the biofilm are surrounded by a variety of molecules that make up the matrix of the biofilm. The matrix is highly hydrated and can contain up to 97% water [154]. In addition, the matrix is composed of polysaccharides, proteins, DNA, surfactants, lipids, glycolipids, membrane vesicles and ions like calcium. This composition varies with different conditions or stages during biofilm maturation. The biofilm matrix is dynamic and interactive, and is essential to the integrity and function of the biofilm.

### *3.3.1. Matrix components*

Monolayer biofilms are composed of a single layer of cells attached to a surface. These biofilms are favored when cell-surface interactions predominate. Since monolayer biofilms offer bacteria more proximity to surfaces, they commonly occur during the interaction of the bacterial pathogen with the host. In flagellate motile bacteria, monolayer formation occurs in two steps, where bacteria first become attached to a surface when they come in close proximity to it. After attachment, the bacteria break the forces tethering them to the surface, resulting in transient attachment. However, a few bacteria that have transitioned from transient to permanent attachment remain attached to the surface. Multilayer biofilms form when bacteria adhere to the surface as well as to each other. Several adhesion factors are known to mediate this transition, including preformed adhesins, conditionally synthesized adhesins and specific

Preformed adhesins include flagellum and pili. Motility is believed to increase the initial interaction between bacteria and the surface. Several studies have also demonstrated that flagellar motility promoted surface adhesion in bacteria [76, 85, 167]. However, under certain conditions, flagellar mutants that are defective in the synthesis of flagellar components have shown an increased synthesis of adhesive matrix that promotes bacterial attachment and multilayer biofilm formation [83, 176]. These observations indicate that flagellar impedence may be important in priming the bacteria for the formation of a multilayer biofilm. Neverthe‐ less, mutants lacking the flagellum or the flagellar motor are completely defective in monolayer and multilayer biofilm formation [83], implying that flagellar motor plays a vital role in biofilm formation independent of flagellar motility. Retractable pili are critical for gram-negative bacteria to attach to surfaces [75]. It is hypothesized that these structures pull bacteria along surfaces by attaching to the surface and retracting, thus helping the bacteria approach the

Bacteria can also conditionally synthesize adhesins to promote surface attachment. In *Pseudomonas fluorescens*, the transition from transient to permanent attachment is mediated by LapA (Large adhesion ProteinA) that associates with the bacterial surface [62]. In *E. coli*, a similar function has been attributed to the exopolysaccharide adhesin, PGA (poly-β-1,6-*N*acetyl-d-glucosamine) which mediates the transition from temporary to permanent attach‐ ment [2]. Following the transient attachment which is accomplished through the array of adhesins such as flagella and pili, bacteria form stable and specific binding through interactions with eukaryotic cell receptors [59]. These interactions are mediated by specific adhesins which

Bacterial cells in the biofilm are surrounded by a variety of molecules that make up the matrix of the biofilm. The matrix is highly hydrated and can contain up to 97% water [154]. In addition, the matrix is composed of polysaccharides, proteins, DNA, surfactants, lipids, glycolipids, membrane vesicles and ions like calcium. This composition varies with different conditions or stages during biofilm maturation. The biofilm matrix is dynamic and interactive, and is

adhesins.

58 Recent Advances in the Field of Urinary Tract Infections

surface more closely [75].

aid in internalization.

**3.3. Biofilm matrix**

essential to the integrity and function of the biofilm.

Exopolysaccharides are a major component of the biofilm matrix. The absence of polysacchar‐ ide synthesis and export leads to an inability to form multilayer biofilms in most bacteria. Bacteria capable of forming biofilms possess distinct genetic loci that encode for the synthesis of polysaccharides. One of the most common exopolysaccharides in the biofilm matrix is a polymer of β-1, 6-N-acteyl-D-glucosamine called PGA or PNAG. Several bacterial species, including *E. coli*, *S. aureus*, *Actinobacillus* spp., and *Bordetella* spp. make use of PGA to construct their matrix [30, 70, 71, 114, 173]. The synthesis and export of PGA is carried out by the *icaADBC* locus in Staphylococcal species and the *pgaABCS* locus in *E. coli*. PGA is required for bacterial attachment and biofilm formation in *E. coli*. Mutations in this locus prevent attachment even after prolonged incubation [173]. In *S. aureus*, the *icaADBC* locus is important for attachment and biofilm formation on indwelling medical devices [42]. In *S. epidermidis*, this locus is also shown to be required for virulence and immune evasion, thus emphasizing the role of biofilms in disease [172]. Another commonly found polysaccharide in the biofilm matrix is cellulose which has been identified as a major component of the matrix in *E. coli*, *Salmonella*, *Citrobact‐ er*, *Enterobacter* and *Pseudomonas* [140, 142, 181, 182]. In *E. coli* and *Salmonella* Typhimurium, cellulose synthesis is made possible by the *bcsABZC-bcsEFG* locus [140, 182]. In addition to PGA and cellulose, some *E. coli* strains also make colanic acid, which is a branched chain polymer synthesized by the *wca* locus [146]. Mutants that are defective in colonic acid forma‐ tion can attach to surfaces, but are incapable of forming multilayer biofilms [32].

The biofilm matrix is also composed of proteins exported to the matrix by cells within the biofilm. Proteinaceous appendages such as fimbriae and pili confer adhesive properties in bacteria. In *E. coli* and *Salmonella*, curli fimbriae produced by the *csgBAC* and *csgDEFG* operons are part of the biofilm matrix [57]. Transcriptional profiling studies have demonstrated that fimbria and pili gene expression is upregulated in biofilms compared to planktonic cells [12]. Another group of proteins associated with the matrix are the *Bap* or Biofilm-associated proteins. These proteins hold bacterial cells together in the biofilm by interacting with similar proteins on the surface of neighboring cells. Bap proteins have been shown to be critical for biofilm production in *S. aureus* [82]. Besides proteins that bind other proteins on neighboring cells, the biofilm matrix also contains lectins and sugar binding proteins. These proteins recognize sugar moieties on the surface of eukaryotic cells and bind to them, thereby facili‐ tating cell-cell interactions [163]. Besides the above mentioned proteins, autotransporter proteins have been identified to be part of the biofilm matrix. The proteins can transport themselves to the cell surface without the need for other transport systems [48]. In *E. coli* autotransporters proteins such as *ag43*, AIDA and TibA have been shown to promote biofilm formation [135]. These proteins serve to maintain close-range interactions between cells in the biofilm.

Another major component of the biofilm matrix is eDNA (extracellular DNA). In *P. aerugino‐ sa*, the biofilm matrix has significant amounts of DNA that is essential for biofilm integrity [95]. Addition of DNase to the culture media resulted in an inhibition of biofilm formation and dissolution of preformed biofilms [177]. It is hypothesized that DNA could serve as a grid that enables bacteria to move using type IV pili. The ability of type IV pili to bind DNA has been demonstrated in *P. aeruginosa* [171]. The eDNA is similar in composition to the genomic DNA, and is hypothesized to be released from whole cell lysis or secretion from outer membrane vesicles containing DNA [6].

multispecies biofilms [148]. There are several factors that influence the rate and extant of biofilm formation on devices. First the bacteria must attach to the surface of the device long enough to result in permanent attachment. This initial rate of attachment depends on the number and type of bacterial cells in the fluid in which the device is exposed to, the flow rate through the device and the physicochemical characteristics of the exposed surface [37]. On indwelling devices, the components in the fluid milieu to which the device is exposed to can change the surface properties and influence bacterial attachment. Following permanent attachment to the surface, the bacteria produce exopolysaccharides to form the biofilm. The rate of growth and establishment of a biofilm depends on flow rate, nutrient availability,

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CAUTIs account for around 80% of all nosocomial UTIs [89]. The risk of developing an UTI significantly increases with the use of indwelling devices. It has been reported that the risk of developing CAUTI increases 5% with each day of catheterization, and virtually all patients are colonized by day 30 [91]. Several studies also support the role of biofilm in the establishment of CAUTIs [161, 167]. The predominant pathogens associated with UTIs include *E. coli* (25%), *Enterococci* (16%), *P. aeruginosa* (11%), *Klebsiella pneumonia* (8%), *Candida albicans* (8%), *Entero‐ bacter* (5%), *P. mirabilis* (5%) and coagulase-negative *Staphylococci* (4%) [40]. These pathogens are normally found in the lower intestinal tract of humans, and can be introduced into the

Prior to the initial attachment of bacteria to the device surface, it is critical that the surfaces are conditioned, where the attachment of proteins and polysaccharides from the fluid environ‐ ment form a film on the exposed surface of the device [161, 167]. This conditioning film facilitates the initial bacterial attachment, which normally adhere poorly on uncoated surfaces [58]. Indwelling devices used in the urological settings include open and closed catheters, urethral stents and sphincters and penile prostheses. Biofilm formation has been documented from infection sites associated with all of these device types [24, 161]. Among all these devices, urinary catheters serve as the common substrate for the development of UTIs [166]. Numerous studies have demonstrated the presence of adherent biofilms on catheters removed from patients [104]. Additionally, scanning electron microscopy studies have documented extensive biofilm formation on urinary catheters [111]. Such catheters recovered from patients that failed antibiotic therapy were shown to contain *P. aeruginosa*, *E. fecalis*, *E. coli* and *P. mirabilis* [103].

Foley catheters are commonly used to manage urinary incontinence in elderly patients and those with bladder dysfunction. These devices besides helping the patient also put them under high risk for the development of UTIs. Uropathogens such as *P. mirabilis*, *Providencia stuartii*,

antimicrobial concentration and temperature.

**4. Urinary catheter biofilms**

urinary tract via indwelling devices.

*4.1.1. Crystalline biofilms*

**4.1. Biofilm formation on indwelling urinary tract devices**

An important characteristic of bacterial cells within the biofilm is the chemical mediated cellcell crosstalk known as quorum sensing. Quorum sensing allows bacteria to coordinate their gene expression in a density-dependent manner [75]. These circuits involve chemical media‐ tors or autoinducers that are secreted by the bacteria and accrue in the extracellular environ‐ ment. When the autoinducer concentration exceeds a certain threshold, quorum sensing is activated. In most gram negative bacteria, the prototype quorum sensing system is the LuxI/ LuxR system [61]. LuxI proteins synthesize the autoinducer such as acylated homoserine lactone (AHL), which modulates the activity of LuxR to activate gene expression upon binding. In case of gram positive bacteria, oligopeptides serve as autoinducers which then activate gene expression in a two component system [61]. Activation of quorum sensing has been shown to stimulate biofilm formation in *P. aeruginosa*. Quorum sensing mutants of *Pseudomonas* make biofilms that are sensitive to detergents such as sodium dodecyl sulfate indicating that the matrix synthesis is defective [34]. In light of the role that quorum sensing plays in the formation and regulation of biofilms, it is proposed that use of quorum-sensing inhibitors may be a potential approach for the treatment of biofilm associated infections.

Existence as a biofilm is advantageous to the bacterium since it enables its survival under a variety of conditions. However when the environmental conditions change or their microen‐ vironment becomes unfavorable, bacteria can return to their planktonic state. This is referred to as dispersion of biofilms. Dispersion of biofilms can be brought about by degradation of the biofilm matrix, which will lead to disruption in cell to cell adhesion and escape from the biofilm. Several bacteria have been shown to produce enzymes that can degrade matrix components and result in biofilm dispersion [15, 69]. Another mechanism of dispersion is through the induction of motility. Onset of dispersal has been shown to coincide with a return in motility of the biofilm associated cells [72]. Certain bacterial biofilms also produce surfac‐ tants such as rhamnolipids. Biofilms formed by strains of *P. aeruginosa* with increased rham‐ nolipid production dispersed after 2 days, whereas wild type biofilms under the same conditions did not disperse until day 10 [14]. Biofilm dispersal is of medical significance as the bacterial cells released from the biofilm can enter the body fluids and can establish themselves in another niche, thereby resulting in secondary infections.

### **3.4. Medical device associated biofilms**

The biofilms on medical devices can be composed of gram-positive and gram-negative bacteria, or yeast. Commonly isolated bacteria include gram-positive organisms such as *E. fecalis*, *S. aureus*, *S. epidermidis*, *Streptococcus viridians* and gram- negative organisms like *E. coli*, *Klebsiella pneumonia*, *P. mirabilis* and *P. aeruginosa*. These organisms can reside on the skin of healthy patients or health-care workers, in the water to which entry ports are exposed or in the environment, from where they eventually contaminate the medical device. Indwelling devices can be colonized by single or multispecies biofilms. In the case of urinary catheters, initially the biofilms are composed of a single species and continued further exposures lead to multispecies biofilms [148]. There are several factors that influence the rate and extant of biofilm formation on devices. First the bacteria must attach to the surface of the device long enough to result in permanent attachment. This initial rate of attachment depends on the number and type of bacterial cells in the fluid in which the device is exposed to, the flow rate through the device and the physicochemical characteristics of the exposed surface [37]. On indwelling devices, the components in the fluid milieu to which the device is exposed to can change the surface properties and influence bacterial attachment. Following permanent attachment to the surface, the bacteria produce exopolysaccharides to form the biofilm. The rate of growth and establishment of a biofilm depends on flow rate, nutrient availability, antimicrobial concentration and temperature.
