**4. Urinary catheter biofilms**

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

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

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

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

potential approach for the treatment of biofilm associated infections.

in another niche, thereby resulting in secondary infections.

**3.4. Medical device associated biofilms**

vesicles containing DNA [6].

60 Recent Advances in the Field of Urinary Tract Infections

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 urinary tract via indwelling devices.

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

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].

### *4.1.1. Crystalline biofilms*

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*, *Morganella morganii* and *K. pneumoniae* produce urease and form a unique type of crystalline biofilms on catheters. Urease production by these organisms enables them to break down the urea in urine [86] and releases ammonia, which raises the urine pH resulting in calcium and magnesium phosphate crystal formation within the biofilm matrix [149]. Studies have also demonstrated that biofilm formation is a prerequisite for crystal formation since the matrix may act as a nucleation site for crystal development [106]. Stickler and others have shown that *P. mirabilis* biofilm formation on catheter surface starts near the eye-hole in the form of microcolonies [150]. Following this, due to production of urease by these colonies, calcium and magnesium phosphate crystals begin to form and the biofilm extends down the luminal surface. The crystal formation is medically significant because of the blockage of catheters due to crystallization and encrustation, which can lead to bladder distention, urine leakage and pyelonephritis when urine from the distended bladder refluxes into the kidney. Additionally, crystalline biofilms that form on the outside of the catheter can lead to irritation and trauma of the urethral mucosa [58].

and recombinant FimH have shown that uroplakin UP1a is the unique bacterial receptor for FimH adhesion [180]. It has been shown that commensal and pathogenic *E. coli* contain type I pili and bind to trimannose receptors via FimH adhesion [139]. However, type I pili in UPEC strains also have a high affinity for binding monomannose units [180], which potentially provides a selective advantage during pathogenesis by increasing specific binding on the

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In addition to their role in adherence, type I pili are also essential for the invasion of bladder epithelial cells by UPEC. TEM and SEM imaging have revealed that bladder cells internalize UPEC through interactions between FimH and UP1a [99]. Other studies have also demon‐ strated that type I pili carrying bacteria interact with plasma membrane micro domains knows as lipid rafts [39]. More specifically, caveolae, a subtype of the lipid rafts with a cave-like appearance have been shown to associate with intracellular bacteria during UPEC invasion. Besides the bladder cells, UPEC can also bind and invade macrophages [10] and mast cells [136], thereby serving as a source of chronic UTIs. The ability of UPEC to invade macrophages allows the bacteria to survive within them and evade phagocytosis. Besides tiding over phagocytosis, ability to survive inside bladder cells also helps to avoid host defenses, including urine flow, secretion of adhesion-binding competitors such as Tamm-Horsfall protein, IgA, chemokines, and exfoliation of superficial bladder cells [113, 155]. UPEC sequestered within the bladder cells are also protected from antibiotic treatments that sterilize the urine, and are provided a rich environment in which the bacteria replicate [100]. UPEC has the ability to form biofilms on abiotic surfaces such as polypropylene, polyvinylchloride, polycarbonate and borosilicate glass when grown statically [120]. Using transposon mutagenesis, Pratt and Kolter demonstrated that Fim mutants were defective in initial attachment and biofilm formation was severely impacted. This indicates that type I pili are essential for the initial attachment of UPEC to abiotic surfaces. Besides type I pili, motility also plays an important in biofilm formation. Non motile strains were severely defective in the initial attachment and consequently in biofilm

UPEC are capable of attaching and invading uroepithelial cells, persisting and forming intracellular reservoirs that help them escape host defenses [100]. Anderson and coworkers [2003; 7] hypothesized that UPEC reservoirs are established by the formation of biofilm-like pods or intracellular bacterial communities (IBC) within the bladder cells. Replication of UPEC in the superficial bladder cells leads to the formation of tightly packed biofilm-like pods that protrude into the lumen. Bacteria inside these pods undergo continuous development leading to the maturation of the IBCs. The development of IBC can be divided into four phases. The first phase begins 1-3 h after infection. The type I pili bind and invade the superficial bladder epithelial cells [74]. At this stage the bacteria are non-motile and divide rapidly and by 8 h post infection, they form loosely organized colonies that resemble microcolonies of abiotic biofilms, known as early IBC. The next phase leads to the formation of middle IBCs, which is charac‐ terized by a reduction in cell proliferation and cell size. Each pod corresponds to a single epithelial cell tightly packed with bacteria forming an intracellular biofilm. Within the pods,

uroepithelium.

formation [120].

**4.3. Biofilm formation in urinary tissues**

### **4.2. Uropathogen specific factors that contribute to biofilm formation**

Uropathogenic *E. coli* (UPEC) are the most common etiology of UTIs [65]. Consequentially, UPEC biofilms are responsible for many CAUTIs [108]. Therefore this section will focus on the specific factors associated with UPEC that aid its biofilm formation. UPEC has several virulence factors such as α-hemolysin, cytotoxic necrotizing factor I, lipopolysaccharide capsule, siderphore aerobactin and enterobactin, proteases and adhesive organelles [109]. The presence of a different repertoire of virulence factors with each UPEC strain could be the reason for the high number of cases associated with UPEC [93]. The single most important virulence factor of UPEC significant to biofilm formation and the associated illness could be type I pili. Type I pili have been shown to play an important role in bacterial adhesion to biotic and abiotic surfaces, and invasion and persistence in the bladder.

Type I pili are pertrichously present on the cell surface of many members of the Enterobac‐ teriaceae, which includes both pathogenic and commensal strains of *E. coli* [179]. Type I pili in *E. coli* is encoded by nine genes of the fim gene cluster which have structural and regulatory roles. The *fimAFGH* genes are structural genes that encode the protein components of the pilus rod and tip [58], whereas FimB and fimE encode the regulatory proteins that control phase variation of type I pili [46]. Phase variation helps *E. coli* to reversibly switch on/off the expres‐ sion of type I pili, and a stringent regulation of phase variation is critical for successful UPEC infection [138]. The FimH adhesion confers mannose-specific binding property to the type I pili. FimH can recognize the terminal mannose residues on various cell types and secreted glycoproteins such as superficial bladder umbrella cells [39] and CD48 on macrophages and mast cells [136]. Langermann and others reported that FimH is essential for colonization of the murine bladder and immunization with FimH protected the animals from UPEC colonization and infection [80, 81]. Scanning electron microscopy (SEM) revealed that type I pili are in close contact with uroplakin-coated superficial bladder membrane [99]. Uroplakins are proteins that cover the apical surface of superficial umbrella cells and give strength to the bladder epithe‐ lium to create a permeability barrier [152]. *In vitro* studies using mouse uroepithelial plaques and recombinant FimH have shown that uroplakin UP1a is the unique bacterial receptor for FimH adhesion [180]. It has been shown that commensal and pathogenic *E. coli* contain type I pili and bind to trimannose receptors via FimH adhesion [139]. However, type I pili in UPEC strains also have a high affinity for binding monomannose units [180], which potentially provides a selective advantage during pathogenesis by increasing specific binding on the uroepithelium.

In addition to their role in adherence, type I pili are also essential for the invasion of bladder epithelial cells by UPEC. TEM and SEM imaging have revealed that bladder cells internalize UPEC through interactions between FimH and UP1a [99]. Other studies have also demon‐ strated that type I pili carrying bacteria interact with plasma membrane micro domains knows as lipid rafts [39]. More specifically, caveolae, a subtype of the lipid rafts with a cave-like appearance have been shown to associate with intracellular bacteria during UPEC invasion. Besides the bladder cells, UPEC can also bind and invade macrophages [10] and mast cells [136], thereby serving as a source of chronic UTIs. The ability of UPEC to invade macrophages allows the bacteria to survive within them and evade phagocytosis. Besides tiding over phagocytosis, ability to survive inside bladder cells also helps to avoid host defenses, including urine flow, secretion of adhesion-binding competitors such as Tamm-Horsfall protein, IgA, chemokines, and exfoliation of superficial bladder cells [113, 155]. UPEC sequestered within the bladder cells are also protected from antibiotic treatments that sterilize the urine, and are provided a rich environment in which the bacteria replicate [100]. UPEC has the ability to form biofilms on abiotic surfaces such as polypropylene, polyvinylchloride, polycarbonate and borosilicate glass when grown statically [120]. Using transposon mutagenesis, Pratt and Kolter demonstrated that Fim mutants were defective in initial attachment and biofilm formation was severely impacted. This indicates that type I pili are essential for the initial attachment of UPEC to abiotic surfaces. Besides type I pili, motility also plays an important in biofilm formation. Non motile strains were severely defective in the initial attachment and consequently in biofilm formation [120].

### **4.3. Biofilm formation in urinary tissues**

*Morganella morganii* and *K. pneumoniae* produce urease and form a unique type of crystalline biofilms on catheters. Urease production by these organisms enables them to break down the urea in urine [86] and releases ammonia, which raises the urine pH resulting in calcium and magnesium phosphate crystal formation within the biofilm matrix [149]. Studies have also demonstrated that biofilm formation is a prerequisite for crystal formation since the matrix may act as a nucleation site for crystal development [106]. Stickler and others have shown that *P. mirabilis* biofilm formation on catheter surface starts near the eye-hole in the form of microcolonies [150]. Following this, due to production of urease by these colonies, calcium and magnesium phosphate crystals begin to form and the biofilm extends down the luminal surface. The crystal formation is medically significant because of the blockage of catheters due to crystallization and encrustation, which can lead to bladder distention, urine leakage and pyelonephritis when urine from the distended bladder refluxes into the kidney. Additionally, crystalline biofilms that form on the outside of the catheter can lead to irritation and trauma

Uropathogenic *E. coli* (UPEC) are the most common etiology of UTIs [65]. Consequentially, UPEC biofilms are responsible for many CAUTIs [108]. Therefore this section will focus on the specific factors associated with UPEC that aid its biofilm formation. UPEC has several virulence factors such as α-hemolysin, cytotoxic necrotizing factor I, lipopolysaccharide capsule, siderphore aerobactin and enterobactin, proteases and adhesive organelles [109]. The presence of a different repertoire of virulence factors with each UPEC strain could be the reason for the high number of cases associated with UPEC [93]. The single most important virulence factor of UPEC significant to biofilm formation and the associated illness could be type I pili. Type I pili have been shown to play an important role in bacterial adhesion to biotic and abiotic

Type I pili are pertrichously present on the cell surface of many members of the Enterobac‐ teriaceae, which includes both pathogenic and commensal strains of *E. coli* [179]. Type I pili in *E. coli* is encoded by nine genes of the fim gene cluster which have structural and regulatory roles. The *fimAFGH* genes are structural genes that encode the protein components of the pilus rod and tip [58], whereas FimB and fimE encode the regulatory proteins that control phase variation of type I pili [46]. Phase variation helps *E. coli* to reversibly switch on/off the expres‐ sion of type I pili, and a stringent regulation of phase variation is critical for successful UPEC infection [138]. The FimH adhesion confers mannose-specific binding property to the type I pili. FimH can recognize the terminal mannose residues on various cell types and secreted glycoproteins such as superficial bladder umbrella cells [39] and CD48 on macrophages and mast cells [136]. Langermann and others reported that FimH is essential for colonization of the murine bladder and immunization with FimH protected the animals from UPEC colonization and infection [80, 81]. Scanning electron microscopy (SEM) revealed that type I pili are in close contact with uroplakin-coated superficial bladder membrane [99]. Uroplakins are proteins that cover the apical surface of superficial umbrella cells and give strength to the bladder epithe‐ lium to create a permeability barrier [152]. *In vitro* studies using mouse uroepithelial plaques

**4.2. Uropathogen specific factors that contribute to biofilm formation**

surfaces, and invasion and persistence in the bladder.

of the urethral mucosa [58].

62 Recent Advances in the Field of Urinary Tract Infections

UPEC are capable of attaching and invading uroepithelial cells, persisting and forming intracellular reservoirs that help them escape host defenses [100]. Anderson and coworkers [2003; 7] hypothesized that UPEC reservoirs are established by the formation of biofilm-like pods or intracellular bacterial communities (IBC) within the bladder cells. Replication of UPEC in the superficial bladder cells leads to the formation of tightly packed biofilm-like pods that protrude into the lumen. Bacteria inside these pods undergo continuous development leading to the maturation of the IBCs. The development of IBC can be divided into four phases. The first phase begins 1-3 h after infection. The type I pili bind and invade the superficial bladder epithelial cells [74]. At this stage the bacteria are non-motile and divide rapidly and by 8 h post infection, they form loosely organized colonies that resemble microcolonies of abiotic biofilms, known as early IBC. The next phase leads to the formation of middle IBCs, which is charac‐ terized by a reduction in cell proliferation and cell size. Each pod corresponds to a single epithelial cell tightly packed with bacteria forming an intracellular biofilm. Within the pods, a polysaccharide matrix surrounds the bacteria [7, 74]. At around 12 h post infection, late IBCs are formed, when UPEC regain their rod shape and motility and flux out of the bladder cells. Fluxing aids UPEC in infecting neighboring cells [74]. The last phase of IBC formation results in UPEC filamentation which occurs 24 to 48 h post infection, where filamentation helps UPEC evade host immune responses. The filamentous bacteria can also separate to form rod-shaped daughter cells. The appearance of filamentous cells also coincides with the appearance of small groups of UPEC on newly infected healthy cells [74].

leakage and pyelonephritis when urine from the distended bladder refluxes in to the kidney

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**Infected urinary caliculi**: In case of urease positive bacteria, biofilm formation is accompanied by the deposition of calcium and magnesium crystals. This crystallization occurs only after the

CAUTI is the most common hospital acquired infection and accounts for up to 40% of all health care associated infections in the United States [102, 156]. About 15-25% of hospitalized patients have an urethral catheter in place during some point of their stay. It is estimated that around 30 million bladder catheters are placed annually in the United States, resulting in several hundred thousand cases of CAUTI [156]. A systemic review of the proportion of health care associated infections that can be prevented revealed that CAUTI was the most preventable nosocomial infection [170]. An estimate of the number of avoidable cases ranged from 95,483 to 387,550 per year and associated lives saved ranged from 2225 to 9031 annually. This prevention could also avoid the annual cost of these illnesses which is estimated at \$1.8 million to \$115 million [170]. This underscores the need for control strategies to prevent CAUTI. Prevention of CAUTI is primarily based on reviewing the criteria for appropriate placement and early removal of catheters. The advances in our understanding of the pathogenesis and key factors that influence the onset of infection are also critical in the development of adequate and effective control strategies [137]. Several protective strategies have been suggested for CAUTI, some of which are already in place for patient care, whereas others are still in

It is estimated that about 21-50% of catheters are placed without justified need and catheters are inappropriately retained for 33-50% of total device days [73, 101]. The most effective ways for the preventing CAUTI are by reducing the duration of catheterization and its early removal [51]. Use of interventions such as nurse prompted removal suggestions and computer based reminders to the patients have resulted in a decline in catheter retention and a concomitant reduction in bacteriuria [164]. Thus, it is important to refrain from using an indwelling catheter without an appropriate indication. A study conducted in an emergency department indicated that use of pre-insertion checklists have led to an improved adherence to indications for placement resulting in the increase in the number of appropriately placed catheters from 37%

Since the catheter provides a connection between the highly colonized perineum and the sterile bladder, sterility during catheter handling and placement is of greatest importance. In this regard, hand hygiene plays a vital role in the prevention of CAUTI [16]. Insertion of a catheter

biofilm is formed, since the biofilm serves as a nucleation site [106].

**5. Control strategies to prevent CAUTI**

development. The control strategies include:

**5.1. Need for and duration of catheterization**

**5.2. Catheter placement and management**

to 51% [50].

[162].

### *4.3.1. Pathogenesis of catheter-associated biofilm*

The pathogenesis of CAUTI depends on the physicochemical properties of the catheter material and its susceptibility to bacterial colonization. Bacterial binding to the bladder mucosa triggers an inflammatory response that leads to neutrophil influx and sloughing of the infected epithelial cells [78]. This helps to clear the bacteria from the mucosal surface. In the case of a catheter, besides the absence of inherent defense mechanisms, they also provide a survival advantage to the bacteria which become difficult to eradicate. The advantages include resistance from being swept away by the urine flow, resistance to phagocytosis and antimi‐ crobials [167]. In addition to the catheter providing an environment for biofilm formation, the presence of a catheter helps to weaken many normal defenses of the bladder. The catheter helps to connect the heavily colonized perineum with the sterile bladder, thus providing a route for bacterial entry into the bladder. Urine pools in the bladder or in the catheter and the resulting urinary stasis promote bacterial growth. Additionally, the catheter also damages the bladder mucosa by triggering inflammatory response and mechanical erosion [175]. Once bacteria gain entry into the urinary tract, low level bacteriuria progresses within 24 to 48 h in the absence of an antimicrobial therapy [145].

### **4.4. Biofilm related UTIs**

**Chronic bacterial prostatitis**: The prostatic ducts and acini provide a safe environment for bacteria to multiply and induce host response. If the bacteria are not eradicated by the immune response, it leads to their persistence and formation of bacterial microcolonies. The presence of microcolonies induces persistent immunological stimulation and chronic inflammation [105].

**Recurrent cystitis**: UPEC binds to superficial bladder epithelial cells resulting in neutrophil recruitment and influx into the bladder lumen. Neutrophil recruitment occurs due to the recognition of bacterial LPS by the toll-like receptors. Additionally, interaction between type I pili and the uroepithelium results in exfoliation of the superficial epithelial cells causing pathogen shedding into the urine [129]. When IBCs form in the epithelial cells, they persist as a chronic reservoir, which leads to recurrent cystitis.

**Pyelonephritis**: Once the bacteria reach the kidney, they adhere to the uroepithelium and form thin biofilms before invading the renal tissue [106]. Additionally encrustation and obstruction to the catheter flow due to formation of crystalline biofilms leads to bladder distention, urine leakage and pyelonephritis when urine from the distended bladder refluxes in to the kidney [162].

**Infected urinary caliculi**: In case of urease positive bacteria, biofilm formation is accompanied by the deposition of calcium and magnesium crystals. This crystallization occurs only after the biofilm is formed, since the biofilm serves as a nucleation site [106].
