Urinary tract infections (UTIs) are one of the most common bacterial infections in humans, and account for 40% of all nosocomial infections ^[1][2][3][4][1,2,3,4]. The incidence of UTIs among the population has increased by 60% between the years 1990 and 2019, confirming that UTIs represent a common public health problem that is far from being eradicated [5]. Typical symptoms include bacteriuria accompanied by urinary frequency, urgency, dysuria and suprapubic discomfort [6]. UTIs can be classified into complicated and uncomplicated urinary tract infections. Uncomplicated UTIs, commonly called cystitis, are a lower urinary tract infection, which only involves the bladder. Such infections are simple and easily resolved with antibiotic treatments. On the other hand, a complicated urinary tract infection is a simple infection that has progressed to the upper urinary tract and is usually associated with other risk factors. Complicated UTIs usually require longer antibiotic courses and are associated with high rates of treatment failure, recurrent infection, sepsis, and significant morbidity and mortality ^[6][7][8][6,7,8]. Uncomplicated UTIs are common in females, affecting 40–60% of females at least once in their lifetime, while all urinary tract infections in males are considered complicated ^[6][8][6,8]. Recurrent UTIs are defined as at least two acute episodes of UTIs occurring within a 6-month period or three episodes in a 12-month period, with much higher incidence in females than males ^[9][10][9,10]. The infection can be community-acquired, hospital-acquired or self-infected ^[10][11][12][10,11,12]. Most community-acquired infections result from poor personal hygiene, low sanitary precautions, or multiple sexual partners. Self-infection usually arises in immunocompromised individuals or high-risk cases, usually caused by commensal inhabitants from the periurethral, vaginal or rectal flora ^[13][14][13,14].

Biofilm formation represents the cornerstone in the pathogenesis of UTIs, since bacterial biofilms play a pivotal role in catheter-associated UTIs (CAUTIs), which account for 40% of all nosocomial infections ^[15][16][17][15,16,17]. Furthermore, biofilm formation is considered one of the most important mechanisms behind the high rates of recurrence and antimicrobial resistance that are often associated with UTIs ^{[15][17][18][19]}[15,17,18,19]. Bacterial pathogens, both Gram-negative and Gram-positive, are the most common suspects behind UTIs ^[9][19][20][9,19,20]. Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus aureus and Enterococcus faecalis are among the most frequent bacterial pathogens that are common in uncomplicated UTIs and complicated UTIs, as well as in CAUTIs ^{[5][6][14][21][22][23]}[5,6,14,21,22,23]. This is not to mention the increasing role of Pseudomonas aeruginosa and Acinetobacter baumannii in complicated nosocomial UTIs ^[24][25][24,25]. However, some fungi, such as Candida spp., are also associated with high incidence of UTIs. Although using different pathogenic pathways, Candida spp. share bacterial pathogen with their ability to form persistent biofilms that contribute to chronic recurrent UTIs ^{[11][26][27][28]}[11,26,27,28].

2. Urinary Tract Infections (UTIs)

2.1. Risk Factors

UTIs are very common infections, and one of the most common infections encountered in health care facilities. However, some risk factors are associated with high infection rates. The female gender represents an important risk factor; it is estimated that 40–60% of females will get a urinary tract infection at least once in their life, and half of them will experience recurrence within one year ^[8][9][10][8,9,10]. On the other hand, in males, the incidence of contracting UTI drops to 10–15%, and drops even further for circumcised males [6]. The risk of infection is higher in females due to many factors. First of all, females have a shorter urethra than males, which means a shorter distance that bacteria have to travel to reach the urinary bladder and initiate the infection [29]. Additionally, the incidence of self-infection from the perineal flora is higher in females, due to the anatomical differences between the two genders [8]. Hormonal fluctuations during menopause represent another female-related factor due to the drop in estrogen level, which makes urogenital skin thinner and reduces colonization by Lactobacilli. Colonization of Lactobacilli provides a protective barrier against pathogenic infection mediated by lactic acid production, which reduces vaginal pH, hence rendering the media less suitable for bacterial growth [30]. Pregnancy is another risk factor, due to reduced immunity [31]. Likewise, immunocompromised individuals and geriatrics are at particularly high risk ^[32][33][32,33]. Diabetes mellitus is one of the major risk factors contributing to high rates of complicated urinary tract infections, especially in catheterized cases ^[5][21][5,21]. This can be explained due to nephropathy, glucosuria and reduced immunity, which are common complications found in most diabetic patients [17]. Other risk factors include structural abnormalities of the urinary tract, renal stones, physical interventions like the use of catheters, spermicides, contraceptive diaphragms, intrauterine devices, and frequent pelvic examinations ^[6][14][6,14].

2.2. Microbial Etiology of UTIs

Most urinary tract infections would result from self-infection; others are transferred from sexual partners, bad hygiene habits or hospital-acquired. UTIs can be caused by a wide range of Gram-negative and Gram-positive bacteria as well as fungi (Figure 1) ^{[6][8][11][23][25]}[6,8,11,23,25]. The source of infection mostly arises from the normal commensal inhabitants of the urogenital, rectal and vaginal flora, with intestinal microbiota acting as the main reservoir of infection ^[8][23][8,23]. E. coli is by far the most commonly isolated uropathogen, accounting for 75% of uncomplicated UTIs and 65% of complicated UTIs [34]. K. pneumonia is the second-most common uropathogen, causing 6–8% of all UTIs, while E. faecalis and C. albicans are major causative agents of complicated UTIs, accounting for 11% and 7% of infections, respectively [14]. Proteus mirabilis, Staphylococcus saprophyticus, Staphylococcus epidermidis, Staphylococcus aureus, group B Streptococcus, Pseudomonas aeruginosa and Acinetobacter baumannii are less frequently isolated from UTIs [35].

2.3. Pathogenesis of UTIs

The infection starts by the ability of some bacteria to cross the urethral sphincter muscle, which acts as a natural barrier against pathogens, followed by adherence to the urethral epithelium [19]. Adhesion is mediated by bacterial fimbriae and adhesins, which play a pivotal role in the initiation of urinary tract infections. Later, most pathogens would climb up the urethra by swimming to the urinary bladder ^[14][22][14,22]. Colonization in the urinary bladder is attained by simultaneous expression of multiple virulence factors – mainly motility, adhesion and secretion of toxins like proteases, hemolysins, urease, colony-necrotizing factors, siderophores, and polysaccharide coatings. These toxins would reflect tissue necrosis and facilitate invasion, giving rise to a typical cystitis [23]. Common symptoms are bacteriuria accompanied by fever, dysuria, pyuria, itching, urinary frequency, urgency lower abdominal pain (LAP), suprapubic pain, burning sensation during urination, blisters and ulcers in the urogenital area ^[7][36][7,36].

2.4. Catheter-Associated Urinary Tract Infections (CAUTIs)

Urinary catheters are tubular devices made of latex or silicone that are widely used in hospitalized patients. Catheter-associated urinary tract infections (CAUTIs) are the most common nosocomial infections, accounting for approximately 40% of all hospital-acquired infections ^[5][21][5,21]. Catheters are foreign bodies that induce local mechanical stress which in turn activates a wide range of inflammatory responses at the adjacent tissues such as exfoliation, oedema, and mucosal lesions, in addition to localized deposition of serum fibrinogen on the catheter surface [37]. Fibrinogen acts as a lubricating layer with the aim of reducing abrasion, but also provides an ideal niche for the attachment of pathogens and as a nutrient source [22]. Urinary catheters can be contaminated during insertion due to inadequate application of aseptic techniques; additionally, long-term catheterization (more than seven days) represents a significant risk factor for development of urinary tract infections, since the surface of urinary catheters acts as an attractive niche for bacterial adhesion and proliferation [17].

3. Bacterial Biofilms

Bacterial biofilms are closed bacterial communities in which the bacteria are embedded in an extracellular polymer matrix secreted by bacteria itself. This viscous medium anchors the bacterial colony at the infection site and provides a protective shield against host defenses, as well as antimicrobial treatments [13]. Bacterial biofilm formation represents an important virulence factor in most uropathogens, playing an important role in infection persistence and recurrence [19]. Additionally, bacterial biofilms represent the main cause behind CAUTI, which is the most common complication in long-term hospitalized patients [38]. The bacterial population within the biofilm shows an altered behavior to that of planktonic bacteria; bacteria rearrange their priorities in order to fortify their attack plan, where motility and metabolic activities are reduced in order to conserve energy and nutrients ^[19][39][19,39]. At the same time, extracellular toxins are upregulated in order to reflect maximum possible tissue damage; this ensures generous release of nutrients at the infection site and further cements the biofilm in site ^[40][41][40,41]. Meanwhile, the biofilm community would continue to shed daughter planktonic cells that aim to seed adjacent tissues, causing the spread of infection and development of new biofilms [13]. Eventually, a bacterial biofilm would develop into a mature, densely-packed structure that is very difficult to eradicate, hence contributing to chronic and recurrent infections [19].

3.1. Biofilm Formation Mechanism

The stages involved in biofilm formation have been extensively studied in order to identify possible mechanisms for biofilm inhibition (Figure 2) ^{[13][28][40][41][42][43][44]}[13,28,40,41,42,43,44]. The first stage in biofilm formation is the reversible attachment of freely-swimming planktonic bacteria to a suitable surface, which can be found in a wide variety of sites or niches. Inanimate hydrophobic surfaces like polystyrene catheters, prosthetic devices, stents or rough surfaces like renal stones represent an ideal surface for biofilm attachment. This step is driven by different forces like hydrophobic interactions, Van der Waals forces, or electrostatic attraction. The attachment can be easily affected by media pH, temperature and solutes ^[42][45][46][42,45,46]. At some point, attachment becomes irreversible due to the stronger forces of adhesion mediated by bacterial adhesins. Noteworthy is the fact that inhibition of this step (by downregulation of pili formation or by adhesins antibodies) can drastically diminish biofilm formation [47]. The next stage is early development, in which the attached bacteria start replicating at site, causing an increase in population number, which triggers the initiation of QS communication systems; this cell-to-cell communication will result in increased secretion of extracellular polysaccharides. After this is the biofilm maturation stage, in which biofilm biomass grows into a three-dimensional macro-colony in which the dominant bacterial phenotype is the persister cells. At this stage, biofilm eradication is very difficult [48]. Finally, at the dispersion stage, the biofilm starts shedding daughter planktonic cells that seed adjacent sites for the formation of new biofilms ^[49][50][49,50].

3.2. Bacterial Communication within Biofilms

Quorum sensing (QS) is a bacterial communication system which allows the bacteria to coordinate their behavior using chemical molecules as a communication signal. It is important to understand that QS communication system aims at orchestrating a grouped bacterial behavior which results in maximum benefits for the population within the biofilm including optimum use of nutrients, increased pathogenicity, and extension of survival rates ^[18][51][18,51]. That being said, it is logical to expect a certain threshold for activation of the QS system by reaching a minimum population level in order to trigger the onset of the system ^[41][45][41,45]. The mechanisms of QS and its role in controlling group bacterial behavior have been extensively studied, revealing that QS system utilizes an inducer/receptor mechanism to control bacterial gene expression ^{[18][43][51][52][53]}[18,43,51,52,53]. The same system is used in both Gram-negative and Gram-positive bacteria. However, the signaling pathway is different. Gram-negative bacteria recruit a system based on N-acyl-homoserine lactones (AHLs) inducers that are synthesized via AHL synthase genes, such as different luxI genes, and sensed by their cognate receptors LuxR-type QS receptors ^[54][55][54,55]. The AHLs molecules are secreted in minute amounts that are undetected at low bacterial population. However, at high population levels, the amount of secreted AHLs is enough to bind to specific bacterial receptors, forming QS receptor-AHL complex. After hitting a certain threshold concentration, the QS receptor-AHL complexes control the transcription of genes involved in biofilm formation ^{[56][57][58][59][60]}[56,57,58,59,60]. On a parallel basis, QS systems in Gram-positive bacteria employ autoinducing peptides (AIPs) that are detected by two-component membrane-bound signal transduction systems ^{[55][61][62][63]}[55,61,62,63]. The different QS systems in Gram-negative and Gram-positive bacteria are represented in Figure 3. QS plays a crucial role in biofilm formation through controlling the production of extracellular polymer matrix, upregulation of bacterial virulence genes, increasing secretion of exoenzymes, siderophores, antibiotics and bioluminescence ^[50][64][50,64].