Factors Impacting Bacterial Biofilms Formation

#### **Chapter 2**

## Bacterial Biofilm and the Medical Impact

*Norzawani Jaffar*

#### **Abstract**

Most pathogenic bacteria species form biofilm as their protective mode of growth, which helps them survive from the bactericidal effect of the antimicrobials or the killing activity of the host immune cells. The bacteria cells' survivability via biofilm formation creates challenges in the medical field in terms of the device and also disease-related to biofilm. The impact of the bacterial biofilm issue is worsening over time, and the association to the high tolerance to the antimicrobial agents leads to increased morbidity and mortality worldwide. This review will highlight the main characteristics of the biofilm, the issue of biofilm in clinical practice, which also covered the pertinence of the biofilm in clinical practice, device-related biofilm disease, oral disease, and the significant bacterial species involved in the biofilm-related infections. Knowledge about the vital role of bacterial biofilm in related disorders will give new insight into the best approaches and alternative treatments for biofilm-related disease.

**Keywords:** antibiotic resistance, medical device, chronic infections, oral disease

#### **1. Introduction**

Microbial biofilm is a microscopic entity that significantly affects human health. It is composed of bacterial colonies within a matrix of extracellular polymeric substances, which protect them from environmental stress, shear stress, detergents, antimicrobial agents, and the host's immune cells. According to the National Institute of Health (NIH), 65% of microbial diseases and 80% of chronic infection is related to biofilm formation [1]. Antibiotics cannot treat several conditions related to biofilm formation due to the high level of biofilm resistance activity. An antibiotic concentration killing effect toward a biofilm might require 1000 times greater than those required to kill the planktonic bacteria cells [2]. In addition, bacterial biofilm causes several diseases in response to both device-related and non-device-related infections. This situation creates challenges for the medical team to provide the best solution or treatment.

Broad heterogeneity of phenotypes developed within a biofilm contributes to the recalcitrance of the sessile bacteria. This condition evolves the bacteria cells inside the biofilm to coordinate and differentiate through the communication system and the releasing of quorum sensing small signaling molecules called autoinducers. Interbacterial communication allows the decision of their density and regulation of the virulence gene expression. This is also the indicator of antibiotic susceptibility profiles of a biofilm. Due to biofilm-cell physiological states, biofilm usually shows high resistance toward most antibiotics. Antibiotics might be effective against the active cells located at the top of the biofilm, in contrast to nutrient-depleted zones at the middle and bottom of the biofilm in which the cell is in the state of dormancy and lack of metabolic activity [3].

The emergence of antibiotic resistance toward biofilm leads to various chronic diseases and is very difficult to treat with efficacy. Most of the recently available antibiotics are not able to resolve the infection. In addition, higher values of minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) used to treat biofilm may result in in-vivo toxicity and other complications. Thus, biofilm formation issues significantly impact human health and the health care industry.

#### **2. Biofilm in clinical practice**

A meta-analysis study by Malone et al. (20017) reported the prevalence of biofilms in chronic wounds was 78.2% [4]. This finding supports the clinical assumptions that biofilms appear and are significant in human chronic non-healing wounds. Besides, one of the most prominent clinical-level species is *Staphylococcus aureus* affecting both hospitalacquired and community-acquired infection. The biofilm production of *S. aureus* cells isolated from clinical samples shows the association of the biofilm with methicillin and inducible clindamycin resistance [4]. In addition, MRSA strains showed a higher biofilm production than MSSA strains. Suggesting strong biofilm formation increases the possibility of antibiotic resistance and leads to treatment failures in MRSA infections [4].

Other than that, *Escherichia coli* is reported to lead the urinary tract infection (UTI), contributing to 80 to 90% of all community-acquired and 30 to 50% of all hospital-acquired cases of UTIs [5]. The study of the uropathogenic *E. coli* revealed a high prevalence of biofilm-forming strains of this group of bacteria that are also highly associated with the multi-drug resistant (MDR) phenotype. Out of 200 *E. coli* clinical isolates, 62.5% can produce biofilm, with 93% of the isolates showing varied resistance with amoxicillin and co-trimoxazole, followed by gentamycin (87%), cefuroxime (84%), Nalidixic acid (79%), Amoxicillin clavulanic acid (62.5%), Ciprofloxacin (62%), ceftriaxone (55%), Ceftazidime (54%), chloramphenicol (28%), Nitrofurantoin (25.5%) and Imipenem (0.5%) [5].

This finding represents the burden of the biofilm formation issues, which are highly associated with increased antibiotic resistance. In addition, another metaanalysis study concludes that biofilm formation production by microbial species impacts the blood system infection leads to resistance, persistence, and mortality. Staphylococci biofilm producer shows significantly higher prevalence in the resistant strain, whereas *Candida species* biofilm production highly impacted mortality [6]. High cell density within the biofilm facilitates high rates of horizontal gene transfer between microorganisms through the conjugation process, more frequent within the community inside biofilm than the planktonic bacteria [7].

#### **3. The main characteristic of bacterial biofilm and their resistance to antimicrobial agents**

In general, bacterial biofilm shows resistance against antibiotics and human immune systems. The process of biofilm formation initiates with the attachment of the planktonic bacterial cells on the living or non-living surfaces. The attachment will lead to the

#### *Bacterial Biofilm and the Medical Impact DOI: http://dx.doi.org/10.5772/intechopen.103171*

construction of the micro-colony of the bacteria cells and rise to a three-dimensional structure, followed by biofilm maturation and detachment. The process of biofilm formation until a detachment of the cells is regulated by the cell-to-cell communication known as the quorum-sensing system. Extracellular polymeric substance (EPS) is one of the main components in a biofilm, strengthening the interaction of the microorganism in the biofilm [8]. Typically 65% of the biofilm volume is constituted by the extracellular matrix, partially or mainly composed of polysaccharides, proteins, and nucleic acid [9]. The EPS protects bacteria from environmental stress such as salinity, UV exposure, dehydration, antimicrobial, and phagocytes [10]. Besides, some channels separate the microcolonies inside the biofilm structure to be attached to new niches [1].

There are studies on the resistant mechanism of the bacterial biofilm toward antibiotics. Most of the studies suggest that the production of glycocalyx or EPS matrix and other functions play a prominent role that prevents the penetration of the antimicrobial agents inside the biofilm. Common disinfectant such as chlorine is only 20% or less of the total concentration in the bulk liquid measured inside the biofilm of *P. aeruginosa* and *Klebsiella pneumoniae*. Interestingly, a complete equilibrium with the bulk liquid did not reach even after 1 to 2 hour incubation time [11]. Another study also showed the same finding when the biofilm production of *P. aeruginosa* on a dialysis membrane showed retarded piperacillin diffusion [12].

In contrast, evaluation on *Staphylococcus epidermidis* biofilm that were grown in the same manner show diffusion of rifampicin and vancomycin across the membrane [13]. Thus, this finding might suggest that inhibition of antibiotic absorption cannot be explained by antimicrobial resistance. Other pathways and mechanisms might be occurring inside the biofilm.

In addition, the difference between thin and thick biofilm formation toward antibiotic resistance has been explored. Penetration of the hydrogen peroxide in the thin biofilm of *P. aeruginosa* was observed compared to a viscous biofilm, which shows no penetration of that chemical compound inside the biofilm [14, 15]. Interestingly, the penetration of the hydrogen peroxide in the thick biofilm was observed in the mutant strains of *P. aeruginosa* without *katA* gene, which is the calatase gene that functions to neutralize the hydrogen peroxide [14].

Furthermore, depletion of the nutrient level inside the biofilm will influence the interaction of the bacteria cells against antimicrobials. Generally, during bacterial growth, the transition from exponential to stationary or no growth leads the bacteria to resistance to antibiotics [3]. Due to low nutrient level and high cell density, the planktonic cell of the bacteria starts to aggregate and initiate attachment and biofilm formation. In the biofilm community, bacteria begin to change their mode to slowgrowing. These physiological changes might play a role in the insensitivity of the bacterial cells inside the biofilm toward antibiotics.

Biofilm disease includes device-related infection, chronic infection with the absence of a foreign body, and malfunction of medical devices. Biofilm-related disease or infection is complicated to treat and detect at early stages by microbiological analyses. Thus, characterization of the chemical composition of the EPS might expedite the development of new therapies against biofilm related-infection.

#### **4. Biofilm and device-related infection (DRI)**

The emergence of device-related infections is highly associated with biofilmproducing bacteria among critical patients in the intensive-care units. DRI is defined as an infection that occurs in a patient with any device (for example, endotracheal tube, intravascular catheter, or indwelling urinary catheter) for at least 48 hours in use before the onset of infection [16]. Most of the DRI reported in the developed country is led by catheter-related bloodstream infections (CRBSI), followed by catheter-associated urinary tract infections (CAUTI) and ventilator-associated pneumonia (VAP) [17]. In addition, another study of the biofilm formation on or in the medical devices that were examined upon removal from the patients or were tested in animal or laboratory systems. Several medical devices may involve biofilm formation, such as central venous catheters, central venous catheter needleless connectors, contact lenses, mechanical heart valves, pacemakers, peritoneal dialysis catheters, prosthetic joints, tympanostomy tubes, and voice prostheses (**Table 1**) [18].

Biofilm formation on medical devices is related to the substratum and cell surface properties. For instance, the characters of glass and various metals that are highly charged hydrophilic materials, water pipes, and environmental surfaces are pretty rough or textured. Some materials might be coated with antimicrobial, such as antibiotic-impregnated catheters [24]. The characteristic of the substratum might have a significant effect on the rate of bacterial adhesion and biofilm formation. The rougher and more hydrophobic materials will develop rapid biofilm formation.

Hydrophobicity of both bacteria and material surfaces may influence the adherence capacity of bacterial cells. Hydrophilic material surfaces are usually more resistant to bacterial attachment than hydrophobic materials [25]. Fletcher and Loeb's (1978) study reported that many marine *Pseudomonas sp*. are attached to hydrophobic plastics with little or surface charge-free like Teflon, polyethylene, polystyrene, poly (ethylene terephthalate). At the same time, very few are attached to hydrophilic and negatively charged substrata like glass, mica, and oxidized plastics [26]. However,


**Table 1.**

*Common devices related diseases and the microbial etiology.*

*Bacterial Biofilm and the Medical Impact DOI: http://dx.doi.org/10.5772/intechopen.103171*

dental plaque formation in the human oral cavity is reported as far less on hydrophobic compared to hydrophilic surfaces, even after nine days without oral hygiene [27]. In addition, another study by Everaert et al. (1997) showed less biofilm formation on hydrophobic silicone rubber voice prosthesis of laryngectomized patients compared to the hydrophilic surfaces after six weeks in the human body [28]. Thus, the role of hydrophobic material surfaces toward rapid biofilm formation is still unclear.

#### **5. Biofilm in chronic infections**

Chronic infections are a significant burden to patients and the healthcare systems. Besides, the economy is also impacted and varies depending on chronic infection due to several treatments failure. It is expected that there will be an increase in chronic infection cases in the future due to an aging population concurrent with the rise in lifestyle diseases such as diabetes which is a significant cause of chronic wounds [29]. Bacterial biofilm has been recognized as responsible for most chronic infections, including otitis, diabetic foot ulcer, rhinosinusitis, chronic pneumonia in cystic fibrosis patients, osteomyelitis, and infective endocarditis [30]. These infections affect millions of people each year, with high mortality and morbidity rate as a consequence. The worse issue of biofilm involvement in infection is due to undetectable species responsible as swabs and scrapes of biofilm samples often show culture-negative. This might be due to the strong association of bacteria within the biofilm or their uncultivability. The same problems occur for implant and catheter-related infections; identifying the bacteria has been almost impossible. Up to this date, bacteria species from a biofilm were considered unculturable. In addition, some pathogenic bacteria that cannot grow the culture media are believed to be activated when present in the host system or environment, and later they can initiate infection [31]. The biofilm infection often finalizes as untreatable, leading to the chronic state of bacterial infections. However, chronic infection will lead to an adaptive inflammatory response, characterized by a high level of mononuclear leucocytes and IgG antibodies [32]. In some cases, such as the cystic fibrosis patient suffering chronic lung infection, the inflammatory response shows the chronic response with continued recruitment of


#### **Table 2.**

*Examples of biofilm-related chronic infections and suggestive pathogenesis.*

polymorphonuclear neutrophils (PMNs) [32]. PMN are the leukocytes critical to the innate immune response against invading pathogens (**Table 2**).

#### **6. Oral diseases**

An oral disease associated with bacterial biofilm is periodontal disease. Periodontal disease has been reported by Global Burden of Disease (GBD) 2010 as a global prevalence of 35% for all ages combined and the sixth-most prevalent condition in the world [38]. Initiating biofilm formation at the periodontal area by various pathogenic species of oral bacteria may lead to severe inflammatory disorders that reduce the gum line, bleeding of the gum, and tooth loss. The issue of periodontal disease is not limited to the antibiotic resistance properties of the biofilm but also the aggressive pro-inflammatory response toward the virulence activities of the pathogenic species that reside in the biofilm. In addition, there are associations between periodontal disease and other systemic diseases such as respiratory tract infection, cardiovascular disease, Alzheimer's disease, gastrointestinal and colonrectal cancer, diabetes and insulin resistance, and adverse pregnancy outcomes [39]. The association of periodontal disease with systemic disease is possible when the progressive inflammatory activity releases toxins or leakage of microbial products enter the bloodstream thru the blood vessel in the pulp chamber of an infected tooth. This agrees with a meta-analysis of 5 prospective cohort studies (86,092 patients) that indicates that individuals with periodontal disease had 1.14 times higher risk of developing coronary heart disease [40]. Whereas for the case of respiratory tract infection and pneumonia, the lung infection might occur due to the accumulation of the pathogens from saliva or oral cavity at the lower airways. Genetically identical respiratory pathogens isolated from dental plaque and bronchoalveolar lavage fluid from the same patient in the ICU indicate that respiratory pathogens' significant reservoir might be associated with dental plaque [41].

#### **7. Significant bacterial species related to a biofilm infection**

Biofilm-producing bacteria play a significant role in biofilm-related diseases. The biofilm's high resistance against antimicrobial agents and the host immune system contribute to considerable treatment challenges. Generally, the ability of a microorganism to form biofilms on the human tissue or related medical devices will lead to the association of chronic infection. The most common bacterial species related to biofilm formation in hospital settings are *Enterococcus faecalis, S. aureus, S. epidermidis, Streptococcus viridans, E. coli, K. pneumoniae, Proteus mirabilis, Acinetobacter baumannii,* and *Pseudomonas aeruginosa* [42]. These species may originate from the skin of healthcare workers or patients or might be from the surrounding as simple as tap water to which entry ports are exposed or other sources in the environment. For instance, Staphylococcus species mainly colonize humans' skin and mucous membrane. *S. aureus* and *S. epidermidis* are the prominent aetiologic agents for nosocomial infection, surgical site, and bloodstream infection [43, 44]. The persistence of *S. aureus* biofilm formation is related to antibiotic pressure. This species own the ability to stay in the viable state but is not culturable [45]. Recently, daptomycin has been used as the last resort for treating Gram-positive bacterial infections, including MRSA and Vancomycin-resistant Enterococcus. This is due to its bactericidal activity against

these bacteria [46, 47]. Enterococci cause a wide variety of infections in humans, including infection of the endocardium, urinary tract, bloodstream, biliary tract, abdomen, burn wounds, and medical devices [48]. However, the most prevalent is *E. faecalis* due to its biofilm formation ability and several virulence factors related to the persistence of biofilm formation and heterogeneity in antimicrobial resistance acquiring activity [49].

On the other hand, a study of attributable mortality dan morbidity caused by carbapenem-resistant *K. pneumonia* showed that 50% of the 391 patients ended with mortality, with 12.2% of the case being bloodstream infections [50]. In addition, *K. pneumonia* is responsible for many cases of nosocomial infection related to a pyogenic liver abscess or endophthalmitis [51]. Besides that, *P. aeruginosa* and *E. coli* are most prevalent for medical device-associated pathogens. *P. aeruginosa* contributes to 10 to 20% of all nosocomial infections, whereas *E.coli* contributes to 50% of the infections associated with urinary catheters [52, 53]. At the same time, *A. baumannii* emerges with significant pathogenicity due to its multi-drug resistant capacity and the ability to form biofilm on several biotic and abiotic surfaces [54]. This species is rapidly spread in the health care facilities and can stay months on the dry surface on insensate objects [55].

#### **8. Conclusion**

Biofilm formation is a natural process employed by several bacteria species. This is part of the adaptation process and survival mechanism in response to their environment. Unfortunately, bacterial biofilm formation develops to impact human health and industries. Evolution to adapt toward the surroundings triggered by an antimicrobial substance during a treatment intervention leads the bacteria cell to manage their survival by acquiring the resistant genes thru several pathways and mechanisms. Applying antibiotics to treat bacteria's biofilm-related infection will lead to another level of resistance activity in the biofilm community as well as toxic effects to the host system. A comprehensive understanding of the biofilm structure organization and the prominent chemical involved might help the researcher elucidate a potent compound or chemical that can degrade or interact with the bacterial biofilm. Alternative methods or therapies other than antibiotics application must be explored to reduce the impact of the bacterial biofilm on human health and the health care industry.

#### **Author details**

Norzawani Jaffar Faculty of Health Sciences, University Sultan Zainal Abidin, Terengganu, Malaysia

\*Address all correspondence to: zawanijaffar@unisza.edu.my

© 2022 The Author(s). Licensee IntechOpen. 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.

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#### **Chapter 3**

## Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical Interactions

*Theerthankar Das and Brandon C. Young*

#### **Abstract**

Pathogenic bacteria cause infectious diseases, mainly when the host (humans, animals, and plants) are colonised by bacteria, especially in its biofilm stage, where it is known to cause chronic infections. Biofilms are associated with resistance to antimicrobial agents, including antibiotics, antiseptics, detergents, and other therapeutic approaches. *Antimicrobial resistance (AMR)* is one of the biggest public health challenges of our time and is termed a 'silent pandemic' by the United Nations. Biofilm formation, pathogenicity and the associated AMR are regulated through a bacterial cell-to-cell communication system termed "Quorum Sensing (QS)'. As the bacterial cells sense the fluctuations in their population, they biosynthesise and secrete the signalling molecules called autoinducers (AI). In gram-negative, the signalling molecules are primarily homoserine lactones (AHL) whereas in grampositive the signalling molecules are autoinducing peptides. The AI binds to receptor and regulator proteins in the bacterial cells to activate the complete QS system, which controls the regulations of various genes that are essential for the biosynthesis of virulence factors, extracellular biopolymers (EPS) production, biofilm formation and bacterial fitness.

**Keywords:** bacterial biofilms, antibiotic resistance, quorum sensing, *Pseudomonas aeruginosa*, *Staphylococcus aureus*, pyocyanin

#### **1. Introduction**

Infectious diseases of humans, animals and plants are caused by the spread of microorganisms, including bacteria, fungi, viruses, protozoa and parasites. Microorganisms that cause disease are called pathogens. Our body (gastrointestinal tract, skin, mucosa of mouth, nose and vagina) is inhabited by numerous bacterial species that form part of the host commensal microflora [1]. However, under certain circumstances, when the host immune system is compromised due to diseases such as HIV, cancer, COVID-19, cystic fibrosis or when the individual has burn injuries, blunt trauma or penetrating trauma (such as through surgery), bacteria can breach the

host barriers and colonise to cause infection. Such bacteria are called opportunistic pathogens. Pathogenic bacteria cause infectious diseases, often when they colonise and form biofilms. Biofilms significantly impact human health; it is estimated that 65% of all microbial infections and more than 80% of chronic infections involve biofilm-associated microorganisms [2]. In this chapter, we have discussed a few of the clinically important biofilm-associated infections.

Urinary tract infections (UTIs) are infections involving any part of the urinary tract. They are one of the most common infections, resulting in an estimated 7 million office visits, 1 million emergency department visits and over 100,000 hospitalisations annually in the United States [3]. UTIs are caused by both gramnegative and gram-positive bacteria, with the most common causative agent for both complicated and uncomplicated UTIs being uropathogenic *Escherichia coli* (UPEC), causing approximately 75% and 65% of these cases, respectively, with other notable contributors including *Staphylococcus saprophyticus*, *Enterococcus faecalis*, Group B *Streptococcus* (GBS), *Proteus mirabilis* and *P. aeruginosa* [4]. UPEC, as well as many of the other common uropathogens, establish biofilms on the bladder wall and surfaces of indwelling urinary catheters as a strategy to protect the encased bacteria from the host immune response and intervention with antimicrobial therapy [5, 6].

Microbial keratitis is an infection of the cornea; when mismanaged, this infection can result in scarring of the cornea, permanent loss of vision and even total loss of the eye [7]. In the United States alone, there are nearly 1 million clinical visits for keratitis annually at an estimated cost of US\$175 million in direct health care expenditures [8]. Biofilms play an essential role in bacterial keratitis as their presence on contact lenses as well as their storage cases can allow bacteria to survive and eventually spread to corneal epithelium [9]. Biofilm populations have increased resistance to antibiotics and host immune response [10]. Bacterial keratitis is significantly more prevalent than fungal keratitis in the United States and other developed countries and is commonly caused by *S. aureus* and *P. aeruginosa*.

Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease characterised by poorly reversible airway obstruction and is currently the third leading cause of death worldwide [11]. The lower respiratory tract of COPD patients is often colonised by bacteria, such as *P. aeruginosa*, *Haemophilus influenzae* and *Streptococcus pneumoniae* [12, 13]. Chronic bacterial colonisation is a major factor driving chronic inflammation in COPD patients [14]. Exacerbations are one of the most important manifestations of COPD and are defined as an increase in the inflammation present above the stable state of COPD, and COPD patients are estimated to suffer 1−4 exacerbations annually [15]. Exacerbations are thought to worsen the decline in lung function with increasing exacerbation frequency, are responsible for much of the morbidity and mortality of COPD [16], account for 50%−75% of the total economic burden due to COPD [17] and estimated to be US\$32 billion annually in the United States alone [18]. Respiratory infections are the most common cause of severe exacerbations in COPD, with *P. aeruginosa* being one of the most frequently isolated causative microorganisms in severe COPD patients [19, 20].

Seasonal respiratory viruses such as influenza virus and respiratory syncytial virus (RSV) as well as respiratory viruses that have spread in major outbreaks such as SARS-CoV, H1N1 Influenza, MERS-CoV and SARS-CoV-2 are a significant cause of morbidity and mortality worldwide. Following the primary viral infection, disruption of the airway epithelium barrier and dysregulation of immune responses promote the colonisation of various bacteria to establish secondary bacterial infections, also known as superinfections, which can have significantly worse clinical outcomes

*DOI: http://dx.doi.org/10.5772/intechopen.106686 Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical…*

when compared to the initial primary infection [21, 22]. Among COVID-19 patients, secondary bacterial infections can arise due to subsequent colonisation by *Klebsiella pneumoniae*, *Acinetobacter baumannii*, *P. aeruginosa* and other bacteria [23], and it has been observed that patients with these superinfections are seen to have mortality rates twice as high as those without secondary bacterial infections [24].

#### **2. Multiple stages in biofilm formation**

Biofilm formation is the most complex stage in the bacterial lifestyle [25]. Compared to the planktonic stage or free-living bacterial cells, bacterial cells encased within biofilms are highly resistant to antimicrobial agents, detergents, host immune responses and environmental and physical stress [26, 27]. Researchers in many publications have widely described the mechanism of biofilm formation [28]. **Figure 1**, in brief, represents schematically bacterial biofilm formation in a hierarchical process.


#### **Figure 1.**

*Schematic showing the five major steps involved in the biofilm formation cycle. The cycle begins with mobility and initial adhesion to the substratum and eventually results in a mature biofilm in which bacteria can disperse as planktonic cells to colonise new sites and repeat the cycle.*


In the final stage, biofilm ageing and dispersion of mature biofilm as planktonic bacterial cells occur, allowing for bacterial attachment and biofilm formation at new sites through a repeat of the biofilm cycle. The dispersion stage is essential for expanding bacterial colonisation and survival and is triggered through active and passive mechanisms. In the active mechanism, bacteria produce various enzymes/ proteins (e.g., DNase I, Alginate lyase, Dispersin B, Exopolysaccharide lyase, protease, surface-protein-releasing enzyme, etc.). These enzymes cleave the biofilm matrix and trigger the release of bacterial cells. The passive dispersal mechanism is mainly the external environment, including nutrient deficiency, QS signals, phagocytosis and antimicrobial agents [32].

#### **3. Physical: Chemical forces influence bacterial adhesion and program biofilm formation**

Many studies have acknowledged that the fundamental physical-chemical interaction forces observed throughout the biofilm formation cycle are essential for mature biofilm formation. The physical-chemical interaction forces mediated by bacterial cells or substratum surfaces are purely dependent on the presence of chemical functional groups and the charge of molecules on surfaces. For instance, Das et al. 2012 showed that removing eDNA from *Streptococcus mutans* cell surface via DNase I treatment significantly decreases short-range acid-base interaction forces between bacteria and surface and consequently impaired *S. mutans* adhesion to the glass substratum surface [33, 34]. In another study, Swartjes et al. 2015 showed similar inhibition of *P. aeruginosa* and *S. aureus* adhesion and biofilm formation on DNase I immobilised surfaces [35].

Thermodynamics and extended Derjaguin−Landau−Verwey−Overbeek (DLVO) analyses theoretically revealed that long-distance van der Waals interaction forces are always favourable or attractive due to the induced dipole interactions. These forces are weak and can range up to hundreds of nanometres and are essential to initially bringing bacteria closer to the substratum [29].

Electrostatic interactions are purely dependent upon the surface charge of bacteria and substratum. Bacterial cell surfaces are generally negatively charged due to the presence of negatively charged biopolymers and cell appendages. Electrostatic interactions would predict repulsion between bacteria and surfaces if the substratum

#### *DOI: http://dx.doi.org/10.5772/intechopen.106686 Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical…*

surface also exhibits a negative charge [29, 34], whereas bacteria should rapidly attach to positively charged substratum surface. It is to be noted that many antibiotics (e.g., Gentamicin, tobramycin, etc.) or antimicrobial peptides (bacitracin, colistin/ polymyxin E and B) are naturally or engineered to be cationic charged to enhance their interactions with bacterial cells [36]. Also, antimicrobial surfaces are made by immobilising cationic antimicrobial polymers to attract bacterial adhesion and kill without inducing biofilm formation [37]. Electrostatic forces are also influenced by the presence of nutrients such as divalent cations (Ca2+ and Mg2+), which promote bacterial interactions, aggregation and biofilm matrix stability by interacting between negatively charged biopolymers within the matrix [38, 39].

Short, ranged acid-base interactions come into action when bacteria are at very close range to the substratum (below 5 nanometres). These forces are influenced by the presence of polar moieties in the molecules; polar groups promote electronaccepting or electron-donating parameters that are essential for bond-strengthening and transition from reversible bacterial adhesion to irreversible adhesion stage. An atomic force microscopic study performed by Das et al. 2011 revealed that bacterial cell surfaces containing eDNA had more vital adhesion forces, multiple minor peaks (due to bond breakage) and a more significant separation distance than DNase I treated bacterial cells [34]. This means eDNA favours bond-strengthening mediated by close-range acid-base interactions (triggers by electron donation and accepting moieties in the eDNA) [29, 34].

Hydrophobic forces are also one significant factor determining bacterial adhesion to the surface and biofilm formation. Studies have shown that hydrophobicity of surfaces (bacteria or substratum) promotes bacterial adhesion and biofilm formation [34, 40, 41]. Hydrophobic forces are strong interactive solid forces compared to van der Waals and hydrogen forces. Garcia-Fernandez et al. 2021 showed that EPS-producing strains of *Streptococcus thermophilus* and *Lactococcus lactis* spp. have a higher water contact angle (hydrophobicity) than EPS-negative mutants [42]. EPS production by these strains is directly related to its robust biofilm formation ability [42]. Contact angle analysis has also revealed a significant change in bacterial cell surface hydrophobicity when subjected to DNase I treatment: *P. aeruginosa* PAO1 strain water contact angle is 65O when exposed to exogenous DNA whereas, when not exposed to exogenous DNA the water contact angle is 44O [34]. Hydrophobic and van der Waals interactions are essential for maintaining biofilm stability by interacting with different biopolymers within the matrix, e.g., carbohydrates and proteins [43]. A study revealed that in Burkholderia *multivorans,* EPS component polysaccharide (EpolC1576) holds many non-polar rhamnoses (6-deoxy sugar) units in its primary structure; these non-polar units influence rhamnose binding with many hydrophobic molecules and are essential for the architecture of three-dimensional biofilm matrix [44].

Mirani et al. 2016 have shown that bacteria can change their cell surface phenotype i.e., hydrophilic to hydrophobic and vice versa when exposed to antibiotics [45]. Their study showed that when *S. aureus* is exposed to a sub-inhibitory concentration of oxacillin, *S. aureus* changes to biofilm mode and its cell surface hydrophobicity increases in contrast to its planktonic phase characterised by more hydrophilic character [45]. Another interesting finding is that in *S. aureus* and *P. aeruginosa* biofilms, the small colony variants (SCVs), which are metabolically inactive (but viable and non-culturable bacterial cells), exhibited hydrophobic properties [46]. These SCVs play a critical role in the persistence of infection and pathogenicity [47, 48].

#### **4. QS mechanism in bacteria**

Through the decades of research, it has been well acknowledged that the QS system is an essential phenomenon for the bacterial biofilm lifestyle. The principal purpose of bacterial QS is to control the regulation of gene expression related to bacterial biosynthesis of numerous endo and exogenous molecules critical for necessary bacterial fitness, survival, virulence production, biofilm formation, infection of the host, evading host immune response and antimicrobial agents. QS is a step-bystep mechanism that begins with bacterial population density fluctuations triggering the release of signalling chemical molecules called "autoinducers'. Studies suggest that autoinducers influence bacterial communication (i.e., 'calling distance') at ranges between 5 and 200 μm [49, 50]. Autoinducers could be of different types and classes [51]. For example, most gram-negative bacteria (e.g., *P. aeruginosa, E. coli, A. baumannii, Vibrio Cholera*, etc.) produces homoserine lactone molecules of different molecular weight and carbon length. At the same time, gram-positive bacteria (e.g., Staphylococcus sp. and Streptococcus sp.) produce autoinducing peptides and competence stimulating peptides as their signalling molecules. Once secreted, autoinducers get recognised by bacterial cell membrane-associated or intracellular receptor proteins. In addition to population-based naturally secreting autoinducers/signalling molecules, many other environmental factors, including oxidative stress, antibiotics or antimicrobial chemicals or nutrients, trigger QS in bacteria.

The typical gram-negative and gram-positive QS mechanisms have been illustrated in **Figure 2**.

#### **4.1** *P. aeruginosa* **is a classic example of a hierarchical QS system**

In most gram-negative bacterial species, *luxI-luxR* genes or homologous genes regulate the QS system. In *P. aeruginosa,* there are four principal QS systems.

#### **Figure 2.**

*Schematic showing the quorum sensing (QS) mechanism in gram-negative and gram-positive bacteria. In gram-negative bacteria, the signalling molecule is primarily AHLs, whereas in Gram-positive bacterial species, signalling molecule is primarily by AIPs, followed by bindings of signalling molecules to the receptors in a bacterial cell and triggering activation of QS-controlled genes. Regulation of Qs genes influences virulence factor production and biofilm formation.*

*DOI: http://dx.doi.org/10.5772/intechopen.106686 Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical…*

First, *lasI/lasR* genes are homologous to the *lux* system and are responsible for the biosynthesis of the chief lactone-based signalling molecule/autoinducer *N*-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HL). The gene *lasI* encodes the autoinducer enzyme LasI, which acts to catalyse the synthesis of the lactone autoinducer (also called AI-1) from substrates 3-oxo-C12-acyl-carrier protein (acyl-ACP) and S-adenosyl-L-methionine [52, 53]. The homoserine lactone molecules are generally lipophilic and freely diffuse through the lipopolysaccharides in the *P. aeruginosa* cell membrane out to the immediate external microenvironment. The AI-1 then binds with the intracellular transcriptional LasR protein (in this case, LasR functions as both AI binding protein and regulatory protein) to activate various virulence factors genes, including *exoprotease (lasA), elastase (lasB), alkaline protease (aprA) and endotoxin A (toxA),* Phospholipase C, heat-labile hemolysin *(plC)*, and *lasI* (for positive autoregulation) [54, 55].

Next in the QS hierarchy is the RhIl-RhIR system. The RhlI (encoded by rhlI) autoinducer synthase enzyme synthesises N-butyryl homoserine lactone (C4-HSL) binds with transcriptional regulatory protein RhlR. RhlR- C4-HSL interactions lead to the activation of several other virulence genes, including *rhlAB* (rhamnolipids) and *lasB* (elastase B) in Pseudomonas species [53–55].

The PQS-PqsR QS system is a late QS system responsible for producing a phenazine-based cytotoxic metabolite 1-hydroxy-N-methylphenazine (pyocyanin) [54]. Operons *pqsABCDEHR* and *phnAB* and genes outside these operons are responsible for synthesising the pseudomonas quinolone signal (PQS) autoinducer in a complex multistep process [56]. The receptor for PQS is the PqsR protein (*pqsR*), which is regulated through the AHL-LasR QS system [54, 57–59]. The binding of the PQS autoinducer to the PqsR receptor/regulator protein activates the expression of virulence factors, including *phz* (pyocyanin), which are critical for causing infection. PQS signalling molecules also act as siderophores in chelating ferric ion (Fe3+) and activate the production of siderophore genes *pvd* (pyoverdine) and *pch* (pyochelin) [57–62].

A newly identified class of autoinducer, termed IQS (2-(2-hydroxyphenyl) thiazole-4-carbaldehyde), has been recognised in *P. aeruginosa* and categorised into a fourth QS system known as the AmbBCDE/IqsR system [63, 64]. This system can integrate environmental stress cues such as phosphate depletion into QS signalling to activate PQS-PqsR signalling in the absence of LasI-LasR activity [65].

QS-mediated toxin biosynthesis induces a severely detrimental effect on the host body. For instance, endotoxin A constrains protein synthesis in the host by impeding protein elongation factor 2 [66]. Exoenzyme S quests on low molecular weight proteins in the host, consequently hindering DNA synthesis and cell morphology [67]. Elastase from *P. aeruginosa* cleaves human leukocyte elastase, human neutrophil elastase and collagens, destroying host tissue elastic properties and impairing wound healing [68, 69]. Production of hemolytic phospholipase C (PlcHR) by *P. aeruginosa* directly interferes with the host protein kinase C signalling pathway (PKC), thus restraining neutrophil burst activity and superoxide (O2 .−) production [70]. Neutrophil assembly and production of superoxides at the infection site are essential to fight against *P. aeruginosa* pathogenicity. Thus, PlcHR promotes *P. aeruginosa* survival in host tissue by evading host inflammatory response by restraining neutrophil burst activity [70].

Pyocyanin, a hallmark metabolite of *P. aeruginosa*, gives a unique greenish-blue colour when grown in the lab and is also visible at the infection site. For instance, Green Nail Syndrome (GNS) is a nail infection caused by *P. aeruginosa,* and the

presence of pyocyanin (also siderophore pyoverdine) causes the greenish colourisation of nails (chloronychia) [71]. Pyocyanin diffuses into host cells and reduces intracellular thiol antioxidant (glutathione) levels in mammalian cells [72]. *In vitro* study showed pyocyanin induces oxidative stress in cells, hinders human nasal ciliary beat frequency, declines intracellular cyclic AMP and damages epithelium [73]. Pyocyanin has been found in burn wound exudates; from burn wound patients and is known to impair wound healing by triggering cell-cycle arrest and premature senescence (ageing of cells) [74, 75]. Pyocyanin is essential for biofilm matrix stability via intercalation with eDNA [76]. Pyocyanin-DNA binding is necessary to prevent the loss of pyocyanin to the external environment and supports *P. aeruginosa* cells in inner biofilm layers that lack oxygen [77].

#### **4.2 Highlighting QS regulation in gram-positive bacteria**

In gram-positive bacteria, the peptide-based QS system is critical in virulence factor production and biofilm formation. For instance, in *Streptococcus* species (*Streptococcus pneumoniae* and *S. mutans*), competence stimulating peptide (CSP) is the primary autoinducer whose synthesis is regulated by *comE* [78]. The CSP gets released extracellularly via the transporter protein ComAB. In the extracellular microenvironment, CSP autoinducers bind with bacterial membrane-bound receptor ComD (transmembrane histidine kinase), causing the phosphorylation (i.e., transfer of phosphate group) of the regulatory protein ComE [78]. ComE undergoes structural modulation and binds with the promoter region of DNA to promote QS regulation genes and virulence factors [79]. CSP-Com mediated QS induces bacterial cell lysis proteins, including murein hydrolases autolysin A and C (LytA and LytC) and Choline-Binding Protein D (CbpD) [80]. These proteins trigger fratricide in the pneumococcal population and trigger virulence factors pneumolysin and *Streptococcus* cell wall constituent lipoteichoic acid (LTA) into the host cell to trigger an immune response [80]. CSP is essential for *Streptococcus-mediated* DNA binding, uptake and transformation from the microenvironment [81] and eDNA-mediated biofilm formation [81]. Other receptors and transcriptional regulatory proteins have also been identified that bind signalling peptides or activate through external environmental factors (oxygen, acid, oxidative stress) and coordinate QS systems in the *Streptococcus* species, including BlpABCSRH, *CiaRH,* HK11/RR11, VicK/VicR and LytST [82, 83]. This QS system is essential for other virulence factor synthesis such as capsular polysaccharides to evade the host immune response (phagocytosis) in *S. pneumoniae*, antibiotic resistance, acid and oxidative stress tolerance and biofilm integrity [84–87].

In *S. aureus*, multiple QS systems have been reported. The primary QS system is coordinated by the global regulatory QS system called accessory gene regulator (agr). Through agr QS system this bacterium deploys a wide collection of virulence factors to establish biofilms and infections [88]. One of the crucial roles of the agr QS system is to encode a signalling circuit that biosynthesis and sense the autoinducers (AI and AIP) and the intracellular effector RNAIII [89]. The autoinducing peptides and agrABCD proteins coordinate the QS system and are essential for expressing exotoxin hemolysin (*hla* and *hlb*), toxic shock syndrome toxins (*tsst*) and controlling biofilm formation and dispersion [90–93]. Other autoinducer binding proteins in *S. aureus* include KdpD/E, KdpD being a receptor protein that binds with autoinducer-2, whereas KdpE is a regulatory protein triggered via phosphorylation [94].

*DOI: http://dx.doi.org/10.5772/intechopen.106686 Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical…*

Autoinducer-KdpD/E system regulates capsular polysaccharide biosynthesis in *S. aureus*. VraSR is another two-component signalling system that gets activated via environmental factors, i.e., by sensing the presence of bacterial cell wall inhibitor compounds such as antibiotics [95]. This system's primary role is to regulate cell wall biosynthesis, impair antibiotic effects and develop resistance [95, 96].

#### **5. Anti-QS strategy to encounter bacterial biofilms and their pathogenicity**

The introduction of antibiotics (e.g., discovery of penicillin in 1928) into clinical medicine has drastically improved human health, allowing for effective treatment of life-threatening infectious diseases and the ability to perform medical procedures previously avoided due to the high risk of postoperative infections [97, 98]. However, with the immense rise of AMR, existing antibiotics show less effectiveness in treating microbial infections. Developing novel antimicrobial agents and new strategies are critical to overcome biofilms and associated AMR in the medical arena. Antibiotic resistance is rapidly spreading and a major concern, with estimates that by the mid-21st century, antimicrobial resistance could contribute to 10 million deaths each year and cost the global economy US\$100 trillion [98].

Widespread antibiotic resistance is driving an intense search for novel therapeutic approaches. Interfering with QS, termed quorum quenching (QQ ), has been an area of interest in this space with the aim of inhibiting bacterial virulence and biofilm formation [99]. QS inhibitors can reduce bacterial virulence and alleviate symptoms


#### **Table 1.**

*Highlighting the anti-QS molecules and their mechanism of action against various bacterial pathogens.*

of microbial infections in a non-bactericidal or bacteriostatic manner, hence relaxing selection pressure for resistance to these molecules while also not affecting beneficial bacteria [100, 101]. **Table 1** summarises a few examples of QS inhibiting molecules and their mechanism of action against different pathogenic bacteria.

One historic discovery in QS inhibition was halogenated furanones derived from red alga *Delisea pulchra* [116] and early work demonstrating their impact on QS behaviours such as inducing irregular non-coordinated swarming in *P. mirabilis* [102]. Many furanones are now known to act as competitive inhibitors of LuxR-type receptors in gram-negative bacteria by competing with AHL for binding to reduce QS signalling [103]. Following the discovery of halogenated furanones impact on QS, much research was carried out to test synthetic furanones as a potential treatment for microbial infections and it has shown success within mouse models to reduce *P. aeruginosa* pathogenicity and enhance bacterial clearance within lungs [104].

Ascorbic acid (vitamin C) is a natural furanone relevant to human health. Ascorbic acid has long been known as an important molecule for normal physiological functions, playing important roles as an antioxidant to protect the body from free radicals and improving immune system function by increasing lymphocyte proliferation, natural killer activity and aiding in chemotaxis [117]. Ascorbic acid is now known to be a potent inhibitor of QS within *P. aeruginosa*. It has been shown to inhibit pyocyanin production and attenuate biofilm formation [105].

Flavonoids are a class of polyphenolic secondary metabolites found in plants. Quercetin is a flavonol ubiquitous in vegetables, fruits and plant-derived drinks such as tea and wine [118]. Flavonoids such as quercetin have been extensively studied for their cardioprotective, anticarcinogenic, antioxidant and anti-inflammatory effects [119–121]. Additionally, quercetin is an effective QS inhibitor in *P. aeruginosa,* with research showing it can inhibit biofilm formation and initial bacterial adherence and reduce virulence factor expression [106]. Evidence suggests that quercetin acts as a competitive inhibitor of the LasR receptor, competing with AHL for binding to reduce QS signalling in *P. aeruginosa* [107].

Curcumin is another polyphenol and is the distinctive yellow pigment and a major constituent of turmeric derived from the *Curcuma longa* plant. Curcumin has a rich history in traditional medicine for its use in anti-inflammatory and antimicrobial roles. Recent research has proven curcumin anti-QS in numerous pathogens. In *Chromobacterium violaceum,* curcumin inhibits violacein pigment production controlled by QS [108]. In *Salmonella* serovar Montevideo, curcumin is seen to inhibit biofilm formation, and in *Serratia marcescens*, it can completely inhibit swarming motility [109]. In *P. aeruginosa,* curcumin attenuates biofilm formation and down-regulates virulence factors such as pyocyanin and elastase [110]. Silico analysis suggests that curcumin also acts as a competitive antagonist of LuxR-type receptors [111].

Gram-positive bacteria such as *S. pneumoniae* participate in QS through secreting oligopeptides as autoinducers. The competence regulon is a QS circuit present within *S. pneumoniae* and is centred on the competence stimulating peptide (CSP), the AI oligopeptide [122]. Two main CSP variants exist, CSP1 and CSP2, which bind to their corresponding histidine kinase receptors ComD1 and ComD2 to drive virulence factor production and biofilm formation [123, 124]. Synthetic peptide analogues have been explored to inhibit QS in peptide-based QS systems. Dominant-negative competencestimulating peptides (dnCSPs) are one such example. They can reduce virulence factor expression *in vitro* and attenuate pneumococcus infections in mice by competing with CSP for ComD binding [81, 112].

*DOI: http://dx.doi.org/10.5772/intechopen.106686 Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical…*

QS inhibition can also be achieved by enzymatic degradation of AIs. This mechanism has been a major focus within QS inhibition research for gram-negative bacteria, and many QQ enzymes from prokaryotic and eukaryotic origins have been discovered [125]. QQ enzymes targeting AHL in gram-negative principally involve four types of enzymes, AHL-lactonases and decarboxylases hydrolyse the lactone ring, whilst AHL-acylase and deaminase cleave the acyl side chain, ultimately leading to reduced AHL-Lux receptor binding and decay of the QS signalling [125]. Many research examples of QQ enzymes show success in QS inhibition within many different bacteria; in one example, an engineered lactonase originally isolated from *Sulfolobus solfataricus* was seen to reduce virulence in clinical isolates of *P. aeruginosa* with pyocyanin production, protease secretion and biofilm formation all inhibited [113].

QQ antibodies are a novel approach to QS inhibition. AHLs and autoinducing peptides have low molecular weights; consequently, they are poorly immunogenic and not expected to elicit an antibody-based immune response [125]. However, hapten–carrier strategies can overcome this lack of immunogenicity by attaching AHL molecules to carrier proteins before immunisation. Miyairi et al. synthesised a carrier protein-conjugated 3-oxo-C12-HSL (*P. aeruginosa* HSL) and immunised mice prior to intranasal challenge with *P. aeruginosa* [114]. Immunisation generated high titres of specific antibodies to 3-oxo-C12-HSL, which was strongly associated with a survival benefit in mice [114]. Bacterial numbers in the lungs did not differ between control and immunised groups, and the increased survival of immunised mice was suggested to be through blocking an excessive pro-inflammatory host response through suppression of virulence factors under QS control [114]. In a similar approach, antibodies targeting Staphylococcal autoinducing peptides (AIPs) show potent QQ abilities and increasing protection of mice challenged with *S. aureus* [115].

#### **6. Concluding remarks**

Biofilm formation by opportunistic pathogens and its associated AMR has a catastrophic effect on society. Despite extensive research on bacterial biofilms carried out over the past century and AMR in the past few decades, we are yet to fully understand bacterial biofilms and the bacterial strategy to evade host immune responses and antibiotic therapy. The discovery of the QS mechanism in bacterial lifestyle is ground-breaking research that has revealed various behaviours and processes under its control, including adaption to physical and chemical stress, expression of genes that regulate extracellular polymeric substances, metabolite production, the integrity of biofilm matrix, efflux pumps to reduce intracellular antibiotic concentration and various antibiotic cleaving enzymes such as beta-lactamase and macrolide esterases, etc. The discovery and use of natural QS inhibiting molecules such as plant-based curcumin, vitamin C, polyphenols (flavonoids) from green tea and furanone from red algae, as well as the subsequent development of synthetic molecules have provided an innovative strategy to tackle bacterial infection and AMR and may play a critical role in the future to address to the continual spread of AMR in many clinically important bacteria and their increasing burden on human health.

There is a multitude of factors that influence the rise of bacterial-associated infections, AMR and consequently mortality. In developing countries, the burden is disproportionately high due to various factors, including high population density, inadequate and unaffordable healthcare, poor education leading to inappropriate use of antibiotics (e.g., prescribing antibiotics against common cold and seasonal

#### *Focus on Bacterial Biofilms*

viral infections), political factors including poor governance that does not provide the necessary infrastructure and policies related to healthcare, sanitation, hygiene, etc. Tangible measures are essential for governments and corporate sectors to ensure the availability of basic facilities to circumvent the increase in bacterial-associated infections, AMR and its associated mortality and morbidity. Developing innovative ideas, new drugs or improving existing drugs through increased financial support to research institutes, universities and the pharmaceutical industry is critical to addressing AMR and ultimately improving global health.

#### **Author details**

Theerthankar Das1 \* and Brandon C. Young2

1 Infection, Immunity and Inflammation Theme, Sydney Institute for Infectious Diseases, Charles Perkins Centre, School of Medical Science, The University of Sydney, Sydney, Australia

2 Infection, Immunity, and Inflammation Theme, Charles Perkins Centre, School of Medical Science, The University of Sydney, Sydney, Australia

\*Address all correspondence to: das.ashishkumar@sydney.edu.au

© 2022 The Author(s). Licensee IntechOpen. 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.

*Biofilm Formation by Pathogenic Bacteria: The Role of Quorum Sensing and Physical - Chemical… DOI: http://dx.doi.org/10.5772/intechopen.106686*

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**Chapter 4**

## Biofilm and Quorum Sensing in *Helicobacter pylori*

*Tarik Aanniz, Wissal Bakri, Safae El Mazouri, Hajar Wakrim, Ilham Kandoussi, Lahcen Belyamani, Mouna Ouadghiri and Azeddine Ibrahimi*

#### **Abstract**

*Helicobacter pylori* (*H. pylori*) is a gram-negative bacterium living in the human gastrointestinal tract considered as the most common cause of gastritis. *H. pylori* was listed as the main risk factor for gastric cancer. Triple therapy consisting of a proton pump inhibitor and combinations of antibiotics is the main treatment used. However, this line of therapy has proven less effective mainly due to biofilm formation. Bacteria can regulate and synchronize the expression of multiple genes involved in virulence, toxin production, motility, chemotaxis, and biofilm formation by quorum sensing (QS), thus contributing to antimicrobial resistance. Henceforth, the inhibition of QS called quorum quenching (QQ ) is a promising target and alternative to fight *H. pylori* resistance to antimicrobials. Many phytochemicals as well as synthetic compounds acting as quorum quenchers in *H. pylori* were described *in vitro* and *in vivo.* Otherwise, many other compounds known as quorum quenchers in other species and inhibitors of biofilm formation in *H. pylori* could act as quorum quenchers in *H. pylori.* Here, we summarize and discuss the latest findings on *H. pylori*'s biofilm formation, QS sensing, and QQ mechanisms.

**Keywords:** biofilm, *Helicobacter pylori*, quorum sensing, bacterial resistance, chemoreceptor, quorum quenching

#### **1. Introduction**

*Helicobacter pylori* (*H. pylori*) is a microaerophilic, spiral-shaped, gram-negative bacterium that belongs to Epsilonproteobacteria [1]. *H. pylori* establishes about 50% life-long infections. While it is asymptomatic in 85% of cases, individuals with chronic gastritis linked to *H. pylori* have a 10–20% chance to develop peptic ulcers and 1% chance to develop gastric carcinoma [2]. Barry Marshall and Robin Warren were the first to successfully isolate and culture *H. pylori* from the human stomach in 1983 [3]. The pair later conducted self-ingestion experiments that confirmed *H. pylori*'s colonization of the human stomach, thereby inducing inflammation of the gastric mucosa. Marshall first reported the development of persistent gastritis after ingestion, which was treated with doxycycline and bismuth subsalicylate [4].

These findings promoted more research, which ended up showing that high amount of H. pylori in the stomach promotes multiple gastrointestinal troubles, including chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer [3].

In the early 1980s, Robin Warren and Barry Marshall showed for the first time that a bacterium named H. pylori could be associated with cancer development. In 2005, the Nobel Prize in Physiology or Medicine was awarded to R. Warren and B. Marshall for the "*discovery of the bacterium H. pylori and its role in gastritis and peptic ulcer disease*."

Furthermore, the International Agency for Research on Cancer classified *H. pylori* in group 1 of carcinogens [5]. It has been shown that *H. pylori* infection may as well be correlated with insulin resistance, the increase of total and low-density lipoprotein cholesterol, and the decrease of high-density lipoprotein [6]. Due to differences in socioeconomic and hygienic conditions, *H. pylori* prevalence varies between and within countries. In general, it is estimated to range from 85–95% in developing countries and between 30% and 50% in developed countries [7]. The prevalence of the infection cannot be summarized in a single figure due to unreliable diagnostic methods in some regions, poor representation of some countries, and differences in data quality [8].

Currently, the first line therapy used to treat *H. pylori* infection is a combination of proton pump inhibitors (PPIs) with amoxicillin or metronidazole and clarithromycin. This triple therapy fails in about 20–30% of cases, requiring the use of a quadruple therapy consisting of a PPI, bismuth, tetracycline, and metronidazole [9, 10]. Nevertheless, an alarming increase in multidrug-resistant strains of *H. pylori* to ampicillin, penicillin, co-amoxiclav, amoxicillin, clarithromycin, metronidazole, tetracycline, doxycycline, erythromycin, and doxycycline has been reported [11–13]. This is ascribed to antibiotic abuse, therapeutic failures, and phenotypical mechanisms promoting resistance and/or tolerance to antimicrobials, notably, biofilm formation [14, 15]. Biofilm formation is a process in which organisms firmly adhere to abiotic, and/or biotic surfaces then grow together to form a complex community that often forms a special structure through four stages: (i) reversible bacterial adhesion; (ii) irreversible adhesion; (iii) formation and maturation of matrix; and (iv) dispersal of cells [16]. Biofilms mainly consist of extracellular polymeric substances composed of polysaccharides, proteins, nucleic acids, and lipids forming a protective barrier against adverse conditions and decreasing the penetration of antibiotics [17]. In *H. pylori,* flagella play a major role in biofilm formation in the gastrointestinal tract [18].

Most bacteria use quorum sensing (QS) as a communication system, relying on the secretion and perception of small molecules called auto-inducers (AIs) [19, 20]. The QS system can activate and/or regulate gene expression of many phenotypes that can be problematic for humans, i.e., biofilm formation, so that bacteria as a group can jointly cope with changes in the surrounding environment, resulting in adverse consequences such as drug resistance and virulence [21, 22]. A new tactic for outsmarting bacteria called quorum quenching (QQ ) is currently explored to reduce their virulence without interfering with their growth, causing less Darwinian selection pressure for bacterial resistance [23]. This paradigm shift has become a promising antibacterial strategy, which not only prevents the development of antimicrobial resistance but also the disturbance of human gastrointestinal microflora, as well as the prevention of adverse side effects commonly associated with the available treatment [24]. Since the main steps of QS are the production and detection of signal molecules, QQ can interfere with this system in different

*Biofilm and Quorum Sensing in* Helicobacter pylori *DOI: http://dx.doi.org/10.5772/intechopen.104568*

ways, either intracellularly or extracellularly by application of inhibitors of AI biosynthesis and perception [25], application of AI antagonists (mimicking AIs), chemical inactivation of AI, sequestering antibodies [26] or macromolecules such as cyclodextrins [27], and degrading enzymes [28]. This strategy showed promising effect *in vitro* and *in vivo*, as well as synergistic effects with antibiotics by increasing bacterial susceptibility to antibiotics [29].

Here, we summarize the biofilm formation regulated by the QS system involved in the antimicrobial resistance in *H. pylori*. Meanwhile, we also provide the latest development of QS inhibitors (QSIs) or QQ enzymes (QQEs) as a potential strategy for the design of new antimicrobial agents to manage *H. pylori* infections.

#### **2. Biofilm formation in** *H. pylori*

Biofilms have been recognized as a microbial sessile community, irreversibly attached to either animate and inanimate objects [30]. Biofilms are contained in a self-produced extracellular polysaccharide (EPS) layer. This matrix is commonly rich in proteins including enzymes, polysaccharides (1–2%), nucleic acids (<1%), and water (up to 97%) [31]. Temperature, pH, osmolarity, UV radiation, desiccation, oxygen tension, and nutrient availability are all environmental stressors that directly affect the phenotype of biofilms [16, 32]. *In vitro* analyses have further confirmed that *H. pylori* biofilms reduce drug permeability and decrease the susceptibility to antibiotics. In fact, cells in the bacterial biofilm are 10–100 times more resistant toward antimicrobial agents than cells in a planktonic state [33, 34]. *H. pylori* colonizing the stomach has developed three patterns of drug resistance, including single drug resistance (SDR), heteroresistance (HR), and multidrug resistance (MDR), which probably overlap and are linked in their molecular mechanisms and their clinical implications [35–42].


#### **Table 1.**

*Factors involved in the formation of biofilms in Helicobacter pylori.*

In the human stomach, *H. pylori* biofilms are found on the surface of gastric mucosa*.* Once introduced into the stomach, *H. pylori* appears in a spiral form, which is very mobile and associated with the colonization of new niches [43–46]. Subsequently, it comes into contact with the mucin layer that covers the epithelial cells, resulting in tension-dependent adhesion between the mucin and *H. pylori* [47]. After an efficient adhesion and multiplication, a morphological transformation occurs, which is accompanied by the creation of multiple shapes (spiral, rod, curved, coccoid, and filamentous forms) to establish a biofilm [48]**.** However, in the case of prolonged colonization, all biofilm cells eventually transform into a coccoid form involved in survival and greater tolerance to adverse environmental factors [49, 50]. Biofilm formation in *H. pylori* involves many factors shown in **Table 1***.*

#### **3. Biofilm formation and QS in** *H. pylori*

The discovery of QS in *Vibrio fischeri* and *Vibrio harveyi*, two species that achieve bioluminescence using QS signaling molecules, sparked research into this complex signaling system [58]. The regulation of gene expression under QS control was investigated in multiple gram-negative bacteria species, including *H. pylori* [52, 59, 60]. For *H. pylori*, QS is involved in motility, biofilm development, and antibiotic resistance [32, 47, 59, 61]. Once biofilm formation is elicited from planktonic cells, the aggregated cells surrounded with extracellular polymeric substances (EPS) modify their phenotype, exchange genetic material, produce AI, and provide physical protection [33]. Owing to the formation of biofilms, *H. pylori* infections became typically persistent and rarely resolved by traditional antimicrobial therapies [34].

Overall, the QS system includes the following steps: (i) AI production; (ii) excretion of AI to the surrounding environment; (iii) sensing and binding of the AI to receptors at high cell density; (iv) retrieval of the receptor-signal complex from the cell and its binding to the promoter region; and (v) activation of genes expression [62, 63]. There are four different signals involved in QS. The most common are N-acyl homoserine lactones (AHLs), also known as autoinducer-1 (AI-1), which are fatty acid derivatives produced and used by gram-negative bacteria [64], while gram-positive bacteria use peptides or modified peptides. Furanosyl borate diesters or autoinducer-2 (AI-2) are derived from the recycling of S-adenosyl-homocysteine and used by both gram-positive and gram-negative bacteria [64]. There is also the autoinducer-3 (AI-3), which allows the cross-talking with mammalian epinephrine host cell signaling systems [65].

*H. pylori*, when located in the gastric mucosa, responds to several specific chemical signals. The chemotactic response is mediated by chemoreceptors called chemotaxis proteins [59]. *H. pylori* genome encodes four chemoreceptors: TlpA (effector; arginine, bicarbonate), TlpB (effector; AI2, urea, hydroxyurea, formamide acetamide.), TlpC (effector; unknown), and TlpD (effector; hydrogen peroxide) [66]. The *H. pylori* QS network involves the chemoreceptor TlpB responding to the AI-2 signaling molecule, a class of furanosyl borate diesters synthesized by the LuxS protein [59, 66] (**Figure 1**). The 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of AI-2 in H. pylori, is produced by LuxS protein [67]. First, LuxS produces the homocysteine through the cleavage of S-ribosylhomocysteine (SRH), which is a part of the S-adenosylmethionine (SAM) pathway. The process involves two main enzymes, i.e., 5′-methylthioadenosine/adenosylhomocysteine nucleosidase (MTAN) and metalloenzyme [68]. The DPD generated is rearranged into an assortment of

*Biofilm and Quorum Sensing in* Helicobacter pylori *DOI: http://dx.doi.org/10.5772/intechopen.104568*

#### **Figure 1.**

*QS in Helicobacter pylori: LuxS produces AI-2 from the methyl cycle. At high cell density, high concentration of AI-2 in the environment bind to TlpB to active chemotaxis. The binding to the periplasmic proteins AibA and AibB active chemorepulsion. Moreover, AI-2 signals upstream of FlhA manages the branching pathways of gene expression under control of FlgS, FlgM, and σ28 proteins.*

chemically related molecules known as AI-2 through a process of dehydration and cyclization [69]. Usually, there are two types of chemoreceptor binding to their AIs, either through direct binding with AI or through interactions with AI binding proteins that transduce signals to the chemoreceptor [70]. In *H. pylori*, TlpB does not bind to AI-2 *in vitro* with high affinity and requires two periplasmic binding proteins, AibA and AibB, which bind to AI-2 independently. AibA and AibB are conserved at greater than 95% identity at the amino acid sequence level in all species of *H. pylori* [32]. The structures of AibA and AibB are not yet elucidated. However, protein sequence homology identifies AibA as homologous to dipeptide binding proteins (39% identity to *E. coli* dipeptide binding protein (PDB ID: 1DPP) and AibB as homologous to proteins of *E. coli* molybdate binding (36% identity to the periplasmic molybdate binding protein of *Azotobacter vinelandii* (PDB ID: 1ATG) [32].

The QS system regulates several mechanisms to assure *H. pylori* colonization in the harsh conditions of the stomach. These include flagellar motility, chemotaxis, and the cag pathogenicity island (Cag PAI) expression, which are all involved in biofilm formation [18, 32, 60]. This indicates that the QS system regulates the various stages of biofilm development from the initial adhesion to the final detachment of the cells [46, 69]. The deletion of luxS gene altered the expression of flagellar genes, i.e., flaA, flaE, flhA, and fliI [69]. Otherwise, the addition of AI-2 or DPD restored the altered phenotype and transcription of these genes. This evidenced that AI-2 is involved in flagellar morphology in H. pylori as it influences the first steps of the flagellar gene expression (**Figure 1**) [69]. The presence of flagella provides motility that enhances the recruitment of planktonic cells to the biofilm, a crucial step in biofilm formation [18].

CagA protein, encoded by cag PAI, has been identified to be induced in *H. pylori* biofilms [54]. A significant decrease in biofilm biomass was observed following mutations in cagA and cag PAI, confirming its important role in biofilm formation [52]. The QS system regulates the cag PAI through its repression by AI-2, which, in turn, attenuates inflammatory response [60]. The type IV secretion system (T4SS), also encoded by cag PAI, is essential in direct cell–cell contact [71]. It is believed that this direct cell–cell contact can also control the biofilm behavior in *H. pylori* [33]. While cag PAI is involved in bacteria-host interaction, it could also be involved in H. pylori bacteria-bacteria interaction, as well as biofilm formation. Besides, bacterial outer membrane proteins (OMPs) are crucial for ion transport, osmotic stability, bacterial virulence, and adherence. Adhesion to gastric cell mediated by Omp18, a peptidoglycan-associated lipoprotein precursor, was reported in H. pylori [72]. After adhesion, the cell envelope gene (lpxD) is upregulated [73]. *H. pylori* urease enzyme (ureA) is important for pH regulation; it prevents the acidification of the biofilm, increasing its stability [74, 75]. Thus, omp18, lpxD, and ureA genes could be directly involved in *H. pylori* biofilm formation [76].

#### **4. QQ in** *H. pylori*

In *H. pylori*, AI-2 has been involved in the regulation of motility, type IV secretion, and, most importantly, biofilm formation [32]. The QS plays a critical role in multidrug resistance of *H. pylori* by upregulating both biofilm-associated matrix and efflux pump genes to improve bacterial resistance [77]. Cells in the bacterial biofilm are 100–1000 times more resistant toward antimicrobials than cells in a planktonic state [34]. The inhibition of QS results in a decrease in biofilm formation, making bacteria more susceptible [78].

Since the main component of QS is the production and detection of signal molecules, QQ can interfere with this system in different ways, either intracellularly or extracellularly. It includes: (i) the inhibition of signal synthesis; (ii) the inhibition of signal transmission; (iii) the enzymatic degradation of AI; and (iv) the inhibition of signal detection [25, 28] (**Figure 2**). These strategies showed promising effect *in vitro* and *in vivo*, as well as synergistic effects with traditional antibacterial treatments by increasing bacterial susceptibility to antibiotics [79].

To date, few *H. pylori* QSIs were described, whether synthetic or produced by living organisms, such as plants, animals, and bacteria [80–82]. Flavonoids, i.e., naringenin, quercetin, myricetin, baicalein, catechin, flavone, and turmeric, exhibited promising antibiofilm and antiadhesive properties against *H. pylori* [83–88] (**Table 2**). Notably, a study conducted to assess the effect of *Acorus calamus* on *H. pylori* cultures demonstrated strong antibiofilm and antiadhesive properties [89]. Molecular interaction studies were later performed by the same group of researchers through molecular docking of β-sitosterol, a phytobioactive component of *A. calamus*, toward QS proteins ToxB, DnaA, PhnB, and Sip. Exceptionally high binding affinity and molecular interaction were exhibited, linking the antibiofilm properties of *A. calamus* to the inhibition of QS proteins by β-sitosterol [89]. The most direct and effective way to inhibit the QS system is the enzymatic degradation of the QS molecules, which stops signal transduction [93]. In gram-negative bacteria, two types of hydrolases were described, namely, AHL-lactonase and AHL-acylase. Today, few studies investigated the enzymatic lysis of QS signals in H. pylori. By degrading AHL produced by H. pylori, N-acylhomoserine lactonase produced by Bacillus licheniformis inhibited the biofilm formation and attenuate virulence [90].

*Biofilm and Quorum Sensing in* Helicobacter pylori *DOI: http://dx.doi.org/10.5772/intechopen.104568*

#### **Figure 2.**

*Different ways to inhibit QS in Helicobacter pylori.*


#### **Table 2.**

*QSIs and QQEs in Helicobacter pylori.*

Another effective way to inhibit QS is the blockage of signaling cascade through the inactivation of downstream response regulators. The precursor SRH of AI-2 results from the action of MTAN on SAH. The inhibition of MTAN induces an accumulation of 5-methylthioadenosine (MTA) and SAH, which, in turn, inhibits AI-2 production [91, 94]. *In silico* testing of DADMe-ImmA derivatives further confirmed this as a viable QQ technique, since it displayed MTAN inhibition by tight binding to the receptor [95]. More *in silico* studies investigated the possibility of designing furanosyl borate diester derivatives from its pharmacophore modeling by substituting the –OH groups of AI-2 and DPD by -SH making it a potent competitive inhibitor to AI-2 [92].

Based on previous studies, various phytochemicals from medicinal plants with known antibiofilm activity could act *via* inhibition of QS in *H. pylori* (**Table 3**). Baicalin


#### **Table 3.**

*Inhibitors of biofilm formation potentially via inhibition of QS in Helicobacter pylori.*

from medicinal plants exhibited, *in vivo*, bactericidal and antiadhesive activities as well as limited urease production and reduced vacA gene expression, leading to virulence reduction. Baicalin limited the bacterial adhesion and colonization and enhanced bacterial sensitivity *via* suppression of urease and blockage of the sulfhydryl group. This makes Baicalin a potential quorum quencher in H. pylori [83, 88]. Quercetin from *Vitis rotundifolia* inhibited the growth of *H. pylori* [84], while in *P. aeruginosa,* quercetin inhibited AHL production suggesting its action through QQ against *H. pylori.* In parallel, catechin was described as a quorum quencher in *P. fluorescens* suggesting its potential inhibition of QS in *H. pylori.* Catechin from *Chamomilla recutita* inhibited the growth of *H. pylori* and urease production in *H. pylori* (which increases bacterial sensitivity) as well as caused membrane disruption [86]. Naringenin produced by *Hibiscus rosa sinensis* showed a potent bactericidal effect to MDR bacteria and also the inhibition of growth and biofilm formation in *H. pylori* [96]. Moreover, naringenin exhibited a potent competition with AHL for binding in *P. aeruginosa*. Taken together, it seems that naringenin

*Biofilm and Quorum Sensing in* Helicobacter pylori *DOI: http://dx.doi.org/10.5772/intechopen.104568*

inhibits biofilm formation in *H. pylori* by acting as quorum quencher. Turmeric (*Curcuma longa*) exhibited a good antibiofilm effect toward *H. pylori* [97, 101]. Besides, turmeric decreased AHL production in Aeromonas sobria and limited interaction with LuxI-type synthases and downregulated LuxI-type and LuxR-type genes in various bacterial species. This makes turmeric a potential quencher toward H. pylori. Vaccinium oxycoccus produces proanthocyanidins with antibiofilm and bacteriostatic activities against H. pylori [98]. Proanthocyanidins also limited the siallylactose-specific (S-fimbriae) adhesion of H. pylori to human mucus, erythrocytes, and gastric epithelial cells. In P. aeruginosa, proanthocyanidins was shown to inhibit AI production and to limit the activation of QS transcriptional regulators*.* Taken together, proanthocyanidins could be considered as a potent quorum quencher in H. pylori*.*

#### **5. Conclusion**

Despite the advancements in the medical field, the treatment of *H. pylori* infections has lost its efficacy. *H. pylori* QS-mediated behavior is the main contributor to bacterial survival and pathogenicity. The significance of bacterial communication in the expression of pathogenic factors makes QS a great target to treat *H. pylori* infection or increase antibiotic efficacy by synergy. In the past two decades, researchers have discovered plenty of QSI agents that can prevent biofilm formation and decrease virulence. The development of new QSI/QQE that can be combined with antibiotics has been a hot topic in the antibacterial research field. More studies are required to demonstrate their mechanisms of action and the optimal doses of the QS inhibitory compounds that are safe and effective.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Focus on Bacterial Biofilms*

#### **Author details**

Tarik Aanniz1 \*, Wissal Bakri1 , Safae El Mazouri1 , Hajar Wakrim1 , Ilham Kandoussi1 , Lahcen Belyamani2 , Mouna Ouadghiri1 and Azeddine Ibrahimi1

1 Medical Biotechnology Laboratory (MedBiotech), Bioinova Research Center, Rabat Medical and Pharmacy School, Mohammed Vth University, Rabat, Morocco

2 Emergency Department, Military Hospital Mohammed Vth, Rabat Medical and Pharmacy School, Mohammed Vth University, Rabat, Morocco

\*Address all correspondence to: tarik.aanniz@gmail.com

© 2022 The Author(s). Licensee IntechOpen. 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.

*Biofilm and Quorum Sensing in* Helicobacter pylori *DOI: http://dx.doi.org/10.5772/intechopen.104568*

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#### **Chapter 5**

## Mechanism Involved in Biofilm Formation of *Enterococcus faecalis*

*Ajay Kumar Oli, Palaksha K. Javaregowda, Apoorva Jain and Chandrakanth R. Kelmani*

#### **Abstract**

Enterococci are commensal bacteria in the gastrointestinal flora of animals and humans. These are an important global cause of nosocomial infections. A Biofilm formation constitutes an alternative lifestyle in which microorganisms adopt a multi-cellular behavior that facilitates and prolongs survival in diverse environmental niches. The species of enterococcus forms the biofilm on biotic and abiotic surfaces both in the environment and in the healthcare settings. The ability to form biofilms is among the prominent virulence properties of enterococcus. The present chapter highlights the mechanisms underlying in the biofilm formation by enterococcus species, which influences in causing development of the diseases.

**Keywords:** biofilm, *Enterococcus faecalis*, pathogenesis, microcolony, quorum sensing

#### **1. Introduction**

Gram Positive bacterium has been renowned as a pathogen of hospitals acquired infectious. One among these bacteria is *Enterococcu*s species. *Enterococcus* species are ubiquitous, commensally inhabitants of the gastrointestinal tract of humans and animals. These can be frequently isolated from the environmental sources such as soil, surface water, raw plant and animal products. Even these can screen from female genital tract, oropharynx and skin. *Enterococcus sps* belongs to the gram positive, facultative anaerobic cocci with an optimum growth temperature of 35°C [1]. There are around 36 species of enterococci have been reported; conversely 26 species are associated with human infection. The most predominant human pathogen is *Enterococcus faecalis*, even *Enterococcus faecium* is one of the important pathogen which is prevalent increasing as hospital acquired infections. The other remaining enterococci species only accounts 5% of infections [2–4]. Some few examples of enterococcus species which are associated with human infections, *E. avium, E. cecorum, E. cassseliflavus, E. durans, E. gallinarum, E. raffinosus* [5, 6].

*E. faecalis* has now become the most common nosocomial pathogen and its virulence is increasing in clinical isolates. The presence and function of different suggested characteristics related virulence have been reported [7, 8]. The factor which influences the virulence is mediated through gelatinase production, enterococcus surface protein (ESP), aggregation substance (AS), and biofilm formation [9].

It cause the following infections such as pelvic and abdominal infections, infections in the mouth especially after root canal surgery, infections in open wounds, a lesser known form of meningitis called enterococcal meningitis, infections in the blood called bacteremia and urinary tract infections.

Biofilms are surface attached, organized microbial communities made up of sessile cells (bacteria and /or fungi) embedded in an extracellular matrix composed of polysaccharides, DNA and other components.

#### **2. Chronological background on biofilm**

Generally bacterial cell grow in two modes; biofilm formation through aggregate and planktonic cell. It associated with microorganism in which cells stick to each other on a surface encased within matrix of extracellular polymeric substance produced by bacteria itself [10]. Antoni van Leeuwenhoek, the Dutch research, who discovered the simple microscope and observed 'animalcule' on surfaces of tooth and this event is known as discovery of biofilm. Characklis, in the year 1973 phrase that biofilms are not only tenacious but even resist to disinfectants (e.g. chlorine). In 1978, Costerton, defined the term biofilm and explained the importance of biofilm. Biofilms can be found in nature in all places like waste water, labs, and hospital settings. It forms as floating mat on the surface of liquid on both living and non-living surfaces [11].

#### **3. Components of biofilm**

Biofilm are produced from different group of organisms, the microbes cells produces the extracellular polymeric substances (EPS) such as DNA <1%, Polysaccharides 1–2%, proteins(includes enzymes) with <1–2%, RNA <1% and water with 97% are the major part of biofilm which is responsible for the flow of nutrients inside biofilm matrix [12]. The main two components of the biofilm that is water channel for nutrients transport and a region of densely packed cells having no prominent pores in it [12]. Another way microbial cells in which biofilms are arranged with significant different physiology and physical properties. They will access of antibiotics and human immune system. The organism that produces biofilm has capability to bear and neutralize antimicrobial agents and result in prolonged treatment. The bacteria which produces the biofilm, switch on the genes that can activate the expression of stress genes which in turn switch to resistant phenotypes due to certain changes examples are as follows cell density, nutritional, temperature, pH and osmolarity. When the biofilm water channels are compared with system of circulations showed that biofilms are considered primitive multi-cellular organism [13, 14]. The compositions of biofilms like DNA, proteins, polysaccharides and water will signify the biofilm integrity and making it resistant against different environmental factors [15].

#### **4. Epidemiology of biofilm formation by** *Enterococcus faecalis*

In the worldwide, the prevalence of production of biofilm varies to different part. The study reported in Rome, Italy, 80% of *E. faecalis* isolates have ability to form biofilms in the infected patients [16]. In India, a study has showed that 52% of *E. faecalis* isolated screened from clinical samples has showed the biofilm formation [17]. In China, Shenzhen Nanshan Hospital, the prevalence of *E. faecalis* biofilm formation

#### *Mechanism Involved in Biofilm Formation of* Enterococcus faecalis *DOI: http://dx.doi.org/10.5772/intechopen.103949*

has showed 50.4% (57/113) in urinary tract infection isolates [18]. The biofilm formation in case of food isolates were less with 60% non-biofilm producers. The major ability in formation of biofilm was endodontic isolates with 73.7% was observed in the Department of Operative Dentistry and Periodontology, University of Freiburg Medical Center, Germany [19].

A study carried out Ahvaz teaching hospital, Iran demonstrated that high frequency 63% of biofilm formation in clinical isolates [20]. The *E. faecalis* bacterial isolated from patient with complicated UTI from department of Urology, Okayama University, Japan has showed the biofilm formation 64 (18.2%) and 156 (44.3%) exhibited strong and medium respectively [21]. A study reported at Malaysia, the *E. faecalis* isolates has showed the biofilm formation of 49% [22]. In the United Kingdom, 100% *E. faecalis* isolates produced biofilms, these isolates were from intravascular catheter-related bloodstream infections (CRBI) found to produce more biofilm than enterococcal isolates that cause non-CRBI [23]. A 93% of *E. faecalis* strains isolated from clinical samples especially fecal isolates have showed more biofilm formation in the United States [24]. In Spain, 57% of *E. faecalis* clinical isolates represent the biofilm production [25]. Tertiary care hospital in India showed 26% isolates of *E. faecalis* having capability in forming biofilm [26].

#### **5. Pathogenesis of biofilm in causing disease**

Generally infectious is connected with biofilm primarily confine to particular location and though time detachment may occur. Further, the detached biofilms may result in bloodstream or urinary tract infections or in the production of blockage of blood flow [26]. In another side cells in biofilms are mostly resistant to antimicrobial agents and the host immune system. *E. faecalis* isolates which produces biofilms is 1000 times more resistant to antibodies, antimicrobial agents and phagocytosis process than non-biofilm producers. Consequently, infections caused from *E. faecalis* associated with biofilm aggravated in this case [27, 28].

In endocarditis infection a complex biofilm formed by *E. faecalis* and host components will be formed on cardiac valve. These biofilms causes disease is through three basic mechanisms. Firstly, the biofilms physically disrupts valve function and may cause leakage. Second, detachment of biofilm can be carried to a terminal point in the circulation and formation of emboli (blockage of the blood vessel). Finally, the biofilm provides continuous infection of the bloodstream even during antibiotic treatment. These can cause recurrent fever, chronic systemic inflammation and lead to other infection also [27, 29].

#### **6. Mechanism steps involved in** *E. faecalis* **biofilm formation**

It comprises of four stages; initial attachment, microcolony formation, biofilm maturation (which is in part governed by quorum sensing) and dispersal.

#### **7. Initial attachment**

A surface adhesion is the first step in establishing a biofilm, and a number of surface adhesions, proteases, and lipids are involved. The endocarditis and biofilm-associated pilus (Ebp), which is composed of subunits A, B, and C, mediates the adherence of biofilms on surface *in-vitro* and *in-vivo* [30–35]. The deletion of ebpABC attenuates binding to platelets, fibrinogen and collagen, reduces initial attachment, and thus impairs biofilm formation *in-vitro* [30, 32, 33].

In addition, Ebp contributed to early biofilm formation in *in-vivo* models of urinary tract infection (UTI), catheter associated UTI (CAUTI), and infectious endocarditis, in which bacteria with deletions of pilus components were substantially attenuated [30, 32, 33, 36]. Additionally, the absence of surface adhesions, such as aggregation substance (Agg), enterococcol surface protein (ESP), and adhesion to collagen from *E. faecalis* (Ace), reduced adhesion to cultured human cells and prevented biofilm formation *in-vivo* [37–41]. Bacteria deficient for Esp showed reduced initial attachment and decreased bladder colonization in a UTI ascending model, which is not unexpected since Esp binds fibrinogen and collagen, and these ligands are present in the bladder because Esp binds fibrinogen and collagen, and these ligands are present in the bladder [41, 42].

Ace is also involved in interacting with collagen, laminin, and dentin and deletion of Ace resulted in reduced colonization in rat endocarditis and UTI models [43–47]. As a result, Ace deletion in the peritonitis model did not reduce bacterial burden suggesting Ace-mediated biofilm formation is not relevant to peritoneal infection. By disparity, deletion of Agg reduced adherence to renal epithelial cells [38, 39], binding to lipoteichoic acid (LTA) of other *E. faecalis* cells (and therefore inter-bacterial clumping) and bacterial titers recovered from endocarditis vegetation on aortic heart valves. Agg cannot colonize the urinary tract, suggesting that Agg-mediated biofilms aren't necessary for ascending UTI's [48, 49].

*In-vitro*, biofilm associated glycolipid synthesis A (BgsA) contributes to initial adhesion and biofilm development, but its role *in-vivo* is unknown [50]. The extracellular secreted protein encoded by salB (Saga-Like Protein B) increased fibronectin and collagen binding but decreased biofilm formation paradoxically, which has hypothesized to be owing to the salB mutant cells decreased hydrophobicity. These investigations suggest that a variety of variables play a role in the initial attachment of bacteria, and that their contribution is likely to vary depending on the surface to which the bacteria adhere. As a result, focusing on a single component as anti-adherence or anti-biofilm strategy is unlikely to totally prevent enterococcal biofilm formation [37].

#### **8. Microcolony formation**

Bacteria proliferate and produce modest amounts of biofilm matrix to form aggregates known as microcolonies after first adhesion [51]. However, the enterococcal mechanisms that drive the establishment of microcolonies are unknown, and no transcriptome data from early-stage biofilms or microcolonies is available. The importance of microcolonies for gut colonization has been demonstrated. *E. faecalis* colonization of the stomach of germ free mice resulted in discrete microcolonies covered in a fibrous sweater-like matrix within a week, rather than the largely 2D biofilm sheets (2–3 cells high) that are normally observed in biofilm models *in-vitro* [52].

Despite the fact that microcolonies are commonly assumed to be a temporary stage of early biofilm production, these data imply that microcolonies may represent a mature biofilm stage in this niche that is particularly crucial for gut colonization. In addition, *in-vitro* enterococcal microcolonies emerge in response to antibiotic

therapy [53, 54]. Biofilms treated with sub-inhibitory levels of daptomycin began to restructure extensively into microcolonies as early as 8 hours after drug exposure, in contrast to typical biofilm sheets. Even in the absence of antibiotics, deletion mutants of eapOX, which encodes a glycosyl-transferase involved in the formation of cell wall associated rhamnopolysaccharide (Epa), developed microcolonies *in-vitro.* In contrast to the monolayer biofilms, these epaOX microcolonies had lower structural integrity, as shown by their facile separation following washing.

#### **9. Biofilm growth and maturation**

Active growth and synthesis of extracellular matrix components such as extracellular DNA (eDNA), polysaccharides, LTA, and extracellular proteases are required for biofilm development. eDNA is the best studied matrix component of enterococcal biofilms:eDNA can be found at the bacterial septum, as part of intercellular filamentous structures, and as part of the larger biofilm matrix, and its release from cells is controlled by autolysin Atla [55–57].

eDNA-associated cells showed no significant cell lysis and had a membrane potential [55], implying that eDNA is liberated from metabolically active cells. As a result, DNase treatment decreased biofilm stability and increased detachment [58, 59], whereas atlA deletion decreased eDNA release and biofilm formation [56]. Despite the lack of evidence that eDNA influences the spatial organization of enterococcal biofilms (as has been postulated for other bacterial species), eDNA remains a potential therapeutic target.

Biofilm production is also aided by non-proteinaceous cell surface components such as glycoproteins, polysaccharides, and modified lipids. The dltABCD operons are involved in the production of D-alanine esters of LTA, which are an important component of Gram-positive bacteria's cell wall, and deletion of this operons decreased biofilm formation *in-vitro*, decreased adherence to epithelial cells, and increased susceptibility to antimicrobial peptides [60]. Biofilm on plastic D (BopD), a potential sugar-binding transcriptional regulator, also promotes to biofilm development *in-vitro* [61].

The deletion of bopABC, which is located upstream of bopD, boosted biofilm growth in glucose but decreased biofilm growth and colonization levels in the murine gut, implying that the ability to utilize maltose is required for biofilm growth in the gut. MprF2, a paralogue of multiple peptide resistance factor (MprF), was likewise found to promote eDNA release and biofilm formation [61–63]. MprF2 reduces the net positive charge of the membrane via aminoacylating phosphatidylglyceroal to mediate electrostatic repulsion of cationic antimicrobial peptides.

While deletion of MprF2 had no effect on biofilm persistence in a mouse bacteremia model, deletion of both MprF1 and MprF2 reduced biofilm persistence in a wound infection model, suggesting that cell membrane charge may play a role in biofilm formation and pathogenicity *in-vivo* [63, 64]. These findings back up the theory that cell surface glycoproteins, membrane phosphatidylglycerol, and polysaccharides all play a role in biofilm development.

The quorum sensing response regulator FsrA regulates matrix remodeling by upregulating the expression of gelE, SprE, and altA [57, 58, 65–67]. The proteases gelE and sprE were found to diminish biofilm formation *in-vitro* and bacterial load in numerous *in-vivo* models [68–71]. However, in a rabbit endocarditis model, loss of gelE alone increased fibrinous matrix formation in aotic vegetation, leading to endocarditis as shown in the **Table 1** [70].


#### **Table 1.**

*Different quorum sensing genes signaling molecules involved in Enterococcus quorum sensing system and virulence factors production.*

*In-vitro*, sprE deletion increased autolysis and eDNA release and accelerated biofilm development, but gelE deletion inhibited eDNA releaseand elevated ace expression, which may increase surface attachment but make the biofilm detachable [71, 72].

#### **10. Quorum sensing**

Population density-dependent signaling influences biofilm formation [73, 74]. Despite the fact that quorum sensing and peptide pheromone signaling are known to coordinate gene expression and direct enterococcus biofilm growth, there have been few research on these tiny signaling molecules and secondary messengers in

#### *Mechanism Involved in Biofilm Formation of* Enterococcus faecalis *DOI: http://dx.doi.org/10.5772/intechopen.103949*

enterococci. The cCF10 peptide pheromone, which facilitates the transfer of the conjugative plasmid pCF10, is an exception. This plasmid has the ability to transfer antibiotic resistance genes as well as virulence determinants like Agg across cells [75–79]. The buildup of cCF10, which stimulates conjugation proteins, is required for pCF10 transfer. The mechanism underpinning peptide pheromone-mediated gene regulation and plasmid transfer has been well documented, and it was recently demonstrated in mice to promote pCF10 transmission between *E. faecalis* cells in the gut [79, 80]. The immature peptide pheromones cAD1 and cCF10 are processed by the membrane protease Eep. Eep also facilities the proteolytic processing of RsiV, the anti-sigma factor for sigV, resulting in improved stress resistance. A sigV mutant showed similar symptoms, indicating that Eep is involved in the regulation of sigV production [81–83].

*In-vitro*, Eep, together with AhrC and the ArgR family transcriptional regulators, leads to biofilm formation, and deletion of the genes encoding either protein lowered bacterial burden in UTI and endocarditis models [84–86]. Furthermore, eep deletion mutants develop tiny aggregates unlike wild-type biofilms. FsrABC is another quorum-sensing system. FsrC is a membrane sensor kinase that detects density-dependent accumulation of the FsrB peptide and triggers a signal to the FsrA response regulator [87]. Because this system controls multiple biofilm-related genes and operons (such as bopABCD, ebpABC, GelE, and SprE), knocking down fsrABC entirely eliminates biofilm formation [88]. FsrD, a precursor for the cyclic peptide gelatinase biosynthesis activating pheromone (GBAP), is also controlled by the Fsr quorum sensing system as shown in the **Table 1** [89]. Finally, autoinducer 2 (Al-2) is involved in *E. faecalis* biofilm formation and is produced by S-ribosylhomocysteinelyase (LuxS). *In-vitro* biofilm development of *E. faecalis* is increased by Al-2 supplementation, while luxS deletion causes aberrant biofilm production with aggregation a dense structure, in contrast to the confluent monolayers of wild type *in-vitro* biofilms [90, 91].

#### **11. Factors influencing for the formation of biofilms in** *E. faecalis*

#### **11.1 Dlt gene**

A Lipoteichoic Acid, component of *E. faecalis*, the most common organism in root canals, develops colonies on the dentin surface (LTA). LTA is a biofilm-forming component of *E. faecalis* that functions as a receptor molecule on receptor cells during the aggregation process. *E. faecalis* antigen recognizes immune cells via pattern recognition receptors (PRRs) and induces the release of proinflammatory cytokines like TNF alpha (TNFα), interleukin 1 beta (IL-1β), IL-6, and IL-8 [92]. LTA causes cells to produce cytokines, which is followed by the activation of Nuclear Factors kβ (NF-kβ), which promotes cytokines release as shown in the **Table 2** [93].

The release of these cytokines causes the dlt gene in LTA to fabricate D-alanine instantly, causing other bacteria to assist in the formation of biofilms [94, 95]. The D-Ala-LTA gene is triggered by the surface protein of Gram-Positive bacteria. Cationic homeostasis and autolytic activity are controlled by this gene. Additionally, it is involved in the assimilation of metal cations as well as the electromechanical repair of bacterial cell walls [94]. These capabilities will enhance bacterial cell system transfer while even increasing autolytic activity. The host's defense system will be weakened by the modified tick.


#### **Table 2.**

*Factors influencing for the formation of biofilms in* E. faecalis.

#### **11.2 Cytolisin lytic enzymes**

A lytic enzyme operated on by cytolysin is the one of *E. faecalis* bacteria's virulence factors. Apart from lysing erythrocytes, collagen fragmentation caused by this enzyme can cause tissue injury at the site of inflammation. The cylLL and cylLs genes on cytolysin promote this role, allowing *E. faecalis* to survive longer. *E. f*aecalis is the most common microbe found in root canals [92, 96]. Other bacteria will be inhibited by *E. faecalis* cytolysin. The cylLL and cylLS genes in *E. faecalis* cytolysin encode structural cytolysin subunits. They create cytolysin in anaerobic circumstances and respond to oxygen depletion in root canals by producing cytolysin as shown in the **Table 2**.

#### **11.3 Hyaluronidase**

Hyaluronidase is a protein to be found in *E. faecalis* that helps the bacteria and toxins progress to the host tissue. Other bacteria will continue to migrate from the root canal to the periapical lesions as a result of hyaluronidase. Furthermore, hyaluronidase stimulates the production of toxins by other bacteria, which increases damage and inflammation. This stipulation is very beneficial for the development of *E. faecalis* [97, 98].

#### **11.4 Dentine matrix structurization**

*E. faecalis* will increase resistance to antimicrobial treatments by increasing the biofilm structural characteristics at the primary site of *E. faecalis* invasion, notably dentin. As a result, *E. faecalis* is known to delay antimicrobial agent penetration through the biofilm matrix by altering the growth rate of other microbes in biofilm development and encouraging changes in the physiological shape of biofilm growth in dentin.

When *E. faecalis* is cultivated in nutrient-poor media, it forms thicker biofilms than when cultured in nutrient-rich media [99]. Under stress inducing mechanism in other bacteria that can cause a more resilient *E. faecalis* biofilm. Besides *E. faecalis* biofilms profitably renew themselves. Furthermore, *E. faecalis* will receive vital carbon by hydrolyzing the substrate required for survival [23].

*E. faecalis* will continue to grow and develop in environments with or without oxygen with extreme alkaline pH by penetrating cell membrane ions and increasing the cytoplasmic's buffer capacity [100]. The pH balance of the biofilm is always maintained by bacteria by assimilation of protons into the cell, resulting in a lower internal cell pH. As a result, the dentin buffer capacity is unable to keep the pH in the dentinal tubule constant, and *E. faecalis* survives [101].

Other investigations found in *E. faecalis* that the ability to promote apatite re-deposition in the forming biofilm is responsible for its persistence after root canal therapy. Besides this, the dentin matrix is composed of chlorapatite Ca5 (PO4)3 [102]. Different varieties of apatite have different dissolving tolerances. Till date, chlorapatite has been considered as a weaker apatite than hydroxyapatite and fluorapatite in terms of nanostructure [102, 103]. Although it is known that calcium hydroxide can stimulate the formation of hard tissue by raising the Ca2+ ion to increase defense through dentin mineralization, the type of apatite that makes up the host dentin will influence the results [104, 105].

However, no further research into the drug resistance of this inorganic dentin material's nanostructures has been done. Furthermore, dentin deterioration is not solely dependent on inorganic elements. Collagen makes up 20% of the organic dentin, which accounts for 85% of the total [103]. Gelatinase, an *E. faecalis* virulence component, is required for hydrolyzing host collagen, High gelatinase levels have been linked to dentin organic matrix degradation [106, 107].

#### **11.5 Tolerance for antimicrobial therapy**

Antimicrobial therapy is known to be limited to eliminating free microbes but not to remove cells bound to the biofilm so that re-infection can occur [100]. As a root canal medication, calcium hydroxide is currently the most popular option among dentists. *E. faecalis* is known to be resistant to calcium hydroxide. This is a serious clinical problem. Every root canal treatment failure, which is documented widely, has linked to *E. faecalis* [101]. Calcium hydroxide is known to prevent the acid reaction that happens as a result of the inflammatory response. This lactic acid generated by osteoclasts to absorb hard tissue will be neutralized by the alkaline pH [102, 103].

#### **12. Conclusion**

*Enterococcus faecalis* is one of the most predominant organism in nosocomial infection and also developed the drug resistance. The intrinsic virulence factors *E. faecalis* are associated in biofilm formation and other environmental factor and signals are alarming the biofilm formation. A genome wide study is required to know the role of genetic and environmental factors in development of biofilm and mounting the superior strategies for biofilm control in *E. faecalis* isolates.

*Focus on Bacterial Biofilms*

#### **Author details**

Ajay Kumar Oli1 \*, Palaksha K. Javaregowda1 , Apoorva Jain1 and Chandrakanth R. Kelmani2

1 Department of Biomedical Science, SDM Research Institute for Biomedical Sciences, Shri Dharmasthala Manjunatheshwara University, Dharwad, Karnataka, India

2 Department of Biotechnology, Gulbarga University, Jnana Ganga campus, Kalaburagi, Karnataka, India

\*Address all correspondence to: ajay.moli@hotmail.com

© 2022 The Author(s). Licensee IntechOpen. 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.

*Mechanism Involved in Biofilm Formation of* Enterococcus faecalis *DOI: http://dx.doi.org/10.5772/intechopen.103949*

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#### **Chapter 6**

## Biofilm Development in Gram-Positive and Gram-Negative Bacteria

*Deepak Dwivedi and Trishla Sehgal*

#### **Abstract**

Biofilms are the communities of microorganisms, especially bacteria attached to a biotic or abiotic surface. These biofilms live in a self-sustained matrix and produce different substances called extracellular polymeric substances (EPS) which are responsible for the pathogenicity of a number of bacteria such as *Pseudomonas aeruginosa, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Escherichia coli,* etc. These EPS substance makes it difficult to eradicate the biofilm present on the surface. Biofilm formation is a five-step process. Biofilms can be monospecies or multispecies. In biofilms, cells communicate via Quorum Sensing (QS). QS is the regulation of gene expression in bacteria with respect to changes in cell population density. In QS, bacteria produce various signaling molecules called Auto-inducers (AI). AI concentration increases as the bacterial population increases. Bacteria respond to these AIs results in an alteration of gene expression, which results in the release of various virulence factors. QS involves a two-component signaling process which is different for both Gram-positive and Gram-negative bacteria. QS and EPS make the bacteria resistant to various antibiotics, which make the eradication difficult and hence requires more effective treatment. This article discusses the biofilm structure, phenomenon of biofilm formation, signaling, and pathogenicity to highlight the understanding of processes involved in biofilm formation.

**Keywords:** biofilm, exopolysaccharides, quorum sensing, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, pathogenicity

#### **1. Introduction**

Microorganisms exist in nature primarily attached to biotic and abiotic surfaces. This is possible due to the development of biofilm. Biofilms are the group of microorganisms living within a self-produced matrix of polymeric substances which get attached to several surfaces [1]. Biofilms are different from the planktonic form of bacteria. Planktonic forms are the free-living forms of bacteria. Bacteria try to switch this planktonic form to biofilm due to a number of advantages which includes protection against environmental stresses such as extreme pH, oxygen, osmotic shock, heat, freezing, UV radiation, predators, etc [2]. Biofilm contains a group of microorganisms irreversibly attached to and grow on a surface. The substances produced

by these microbes are known as extracellular polymeric substances (EPS) result in the alteration in the phenotype of the organism with respect to growth rate and gene transcription [3].

Biofilms are found to be present on liquid surfaces as floating mat and in a submerged state as well [4]. Biofilms appear either beneficial or detrimental. Biofilms are considered beneficial as these degrade hazardous substances which are present in the soil, but are detrimental to food and slaughterhouse equipment and are also found responsible for the pathogenesis of a number of diseases [5]. Biofilm has been used for the remediation of heavy metals for a long time. EPS as being poly-anionic in nature, forms complexes with positively charged metals (cations) result in metal immobilization within the exopolymeric network. Extracellular enzymatic activities in EPS assist the detoxification of heavy metals by transforming and subsequently participating in exopolymeric mass [6]. Microorganisms in biofilm help in the production and degradation of organic matter, remediation of environmental pollutants, nitrogen cycle, sulfur, and many metals. Some of the literature revealed that microbial biofilms are involved in sewage purification also [7].

Biofilms can grow on surfaces of many medical implants such as sutures, catheters, dental implants, etc [8]. Biofilm formation is an important virulence mechanism in the pathogenesis of many medically important organisms such as *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Escherichia coli*, etc [9–11] infections including biofilm formation such as vaginitis, colitis, gingivitis, otitis, urethritis, etc [12–14]

Biofilms are communities of bacteria embedded in the EPS matrix. EPS is composed mainly of a complex mixture of proteins, lipids, nucleic acids i.e. extracellular DNA (e-DNA) and polysaccharides [15]. EPS helps the biofilm to withstand mechanical stress. Biofilms are viscoelastic in nature and EPS provides physical support against mechanical and chemical stresses [16].

Depending on the interaction between surface and constituent cells, biofilms can be categorized as monolayer or multilayer [17]. Flagellum and pilus present on the surface of cells increase the attachment of bacteria to the surface which accelerates the formation of biofilm monolayer. In another type, the microbial adhesion is synthesized with the simultaneous transition to the permanent attachment [17]. When microorganisms are able to adhere to a surface and also to each other, they often develop multilayer biofilm. It has been noted in many cases that the bacterial surface characteristics lead to repulsion [17].

#### **2. Biofilm structure**

The structure of biofilm consists of matrix of EPS which comprises e-DNA, polysaccharides, and proteins [18]. Channels in this biofilm allow water, air, and nutrients transport to all parts of the biofilm [19].

Exopolysaccharides: These are the high molecular-weight sugar polymers that are secreted outside the matrix act as a scaffold for proteins, nucleic acids, carbohydrates, and lipids to adhere to the surface [20]. Mannose, galactose, and glucose are the most abundant carbohydrates in EPS. Most of the exopolysaccharides are not biofilm specific but their production increases as an environmental stress response.

Extracellular Proteins: This is another major class of EPS. These are found attached to the surface and polysaccharides to help with biofilm formation and stabilization. E.g. Amyloids play a supportive role in biofilm formation. Fap amyloids in *P. aeruginosa* lead to cell aggregation and increased biofilm formation [21]. The dispersal and

*Biofilm Development in Gram-Positive and Gram-Negative Bacteria DOI: http://dx.doi.org/10.5772/intechopen.104407*

detachment of biofilm also require some enzymes which release biofilm cells and initiate a new biofilm lifecycle. For E.g. Dsp B protein is responsible for the detachment of *Actinobacillus pleuropneumoniae* biofilms [22].

e-DNA: It comes from both lyzed cells and also actively secreted [23]. It plays an important role in biofilm formation critical for attachment. It interacts with receptors present on the substratum surface to facilitate adhesion [24]. It also coordinates with the cell movement in twitching motility mediated *P. aeruginosa* biofilm expansion [25]. It also inhibits the transportation of antibiotics within biofilm thus protects the bacteria within the biofilm. E.g. In *Staphylococcus epidermis*, e-DNA inhibits the transportation of vancomycin and thus protect the biofilm [26]. Vancomycin is a glycopeptide antibiotic that penetrates the biofilm and kills the growing biofilm including gram-positive bacteria. **Figure 1** shows components of the EPS matrix.

#### **3. Steps of biofilm formation**

Biofilms are three-dimensional communities of microorganisms that adhere to a surface and form a matrix of EPS. Both gram-positive and gram-negative bacteria develop biofilm but the most common species are *E. faecalis*, *S. aureus*, *S. epidermidis*, *S. viridans*, *E. coli*, *K. pneumoniae*, *P. mirabilis,* and *P. aeruginosa* [27]. Biofilm formation takes place over five main stages including: 1. Initial reversible attachment; 2. Irreversible attachment; 3. Maturation Stage I; 4. Maturation Stage II and 5. Dispersion [28, 29].

1.*Initial reversible attachment*: Bacteria generally adhere to a surface that is rich in organic molecules (e.g. nutrients, salivary proteins, large macromolecules). These molecules promote the adherence of bacteria to the surface. Initial attachment is mediated through weak van der Waals force which later turns to stronger dipole-dipole interaction, hydrogen, ionic or hydrophobic interactions. There is

a stronger adhesin-receptor mediated attachment. It is an attachment between adhesins, adhesive structures present on the surface of microorganisms and receptors, complementary adhesive structures present on the surface of host cells [6]. These interactions are mediated through the surface structures present on the bacterial cell such as fimbriae, flagella, lipopolysaccharides (LPS), outer membrane proteins (OMPs), and exopolysaccharides [30].

2.*Irreversible attachment*: Initial reversible attachment further changes to the irreversible attachment. In this stage, the forces of attraction are greater than the forces of repulsion. Initially immobilized bacterial cells attach to the surface irreversibly [31]. The structures present on the surface overcome the physical repulsive forces of the electrical double layer of the cell and consolidate the interaction between bacteria and the surface [32]. The hydrophobic interactions between the surface and bacteria also reduce the repulsive forces between them [4].

In the first and second stages, bacteria reversibly adhere to the surface which is further replaced by irreversible interaction.

	-
	-
	-
	-
	-

**Figure 2.** *Stages of biofilm formation.* increase in substrate exchange between bacteria, distribution of metabolic products, and removal of toxic end-products produced by the bacteria [34]. Syntrophic association develops between distinct bacteria in which these utilize certain substrates as energy sources [34]. In this stage, biofilm adapts with the external conditions by manipulating its structure, physiology, and metabolism.

5.*Dispersion*: In this stage, dispersion of bacteria takes place and bacteria return to motile form [35]. In this stage, the microbial community produces different saccharolytic enzymes which break the biofilm stabilizing polysaccharides that releases the bacteria present on the top of the biofilm and colonize to the new surface. The microorganism upregulates the expression of flagella proteins and bacteria return to motile form to translocate to the new site. **Figure 2** shows the process of biofilm formation.

#### **4. Quorum sensing**

QS in bacteria is the regulation of gene expression with respect to the fluctuations in the cell-population density. In QS, bacteria produce chemical signal molecules called AI which increase in concentration as a function of cell density [36]. Bacterial populations coordinate their gene expression by producing and responding to a variety of intra and inter-cellular signals called AIs [37]. Microorganisms communicate by producing and responding to small diffusible molecules AIs that acts as signals. When a single bacterium releases AIs into the environment, the concentration is too low to be detected but when mass bacteria releases AIs, the concentration reaches a threshold level which allows the bacteria to sense a critical cell mass, and in response to this it activates or represses target genes. Many classes of AIs have been described to date and N-acyl homoserine lactones (AHLs) are most studied AIs of gram-negative bacteria. A class of AIs termed AI-2 with unknown structure in most cases and the peptides of gram-positive bacteria are most studied [38].

#### **5. Quorum sensing in gram-negative bacteria**

In gram-negative bacteria, the QS circuit involves at least two regulatory proteins called LuxR and LuxI. These proteins bind with the protein receptor bound to the bacterial cell membrane/wall. The signaling molecules bind with the receptor proteins then enter the cell. The LuxI protein is responsible for the biosynthesis of AHL, which is utilized as signaling molecules. The AHL concentration increases with the increase in cell population density. The LuxR protein is responsible for binding to cognate AHL AIs that have achieved a threshold concentration; these complexes also activate target gene transcription. The following **Figure 3** shows protein involved in QS and signaling pathway in gram-negative bacteria.

#### **6. Quorum sensing in** *Pseudomonas aeruginosa*

*P. aeruginosa* can be best understood in terms of the virulence factors regulated and the role of QS plays in pathogenicity. *P. aeruginosa* is found to be an opportunistic pathogen as it primarily infects individuals who are immune-compromised, such as

#### **Figure 3.**

*Proteins & two-component signaling pathway in gram-negative bacteria.*

patients with cancer or AIDS or those having breaches in normal barriers caused by burns, indwelling medical devices, or prolonged use of broad-spectrum antibiotics [39]. *P. aeruginosa* is an impressive armament of both cell-associated and extracellular virulence factors. *P. aeruginosa* involves two intertwined QS systems in virulence, biofilm development, and many other processes. Iglewski and colleagues discovered the first system (Las) consists of LasI encoded acyl-HSL synthase and the LasR encoded transcriptional activator. LasI is homologous to LuxI. A number of investigators found the second system (Rhl) consists of an rhlI-encoded acyl-HSL synthase and an rhlR-encoded transcriptional activator. In the respective QS systems, each produces and responds to a specific acyl-HSL; LasI directs the synthesis of 3-oxo-dodecamoyl-HSL (3-oxo-C12-HSL) and RhlI directs the synthesis of butyryl-HSL (C4-HSL) [40].

Using *P. aeruginosa,* lasI, and rhlI double mutant recently, Whiteley et al identified nearly 40 QSc genes that showed a fivefold or greater response to exogenously added acyl-HSL signals. On the basis of the pattern of the responses to cells grown in presence of Las signal, 3-oxo-C12-HSL and/or the Rhl signal, CH-HSL, the QSc genes were classified. A number of early QSc genes were found that responded immediately to exogenously added signals suggesting that these genes behave like the Lux genes of *V. fischeri* and the carbapenem biosynthesis genes of Ervinia. By seminal observations, a number of proteins have been found that support this hypothesis including the stationary phase sigma factors RpoS, RsmA, a third LuxR homolog (QScR), and stringent response proteins RelA, all of them are involved in modulating the expression of genes. QScR gene was found to be the negative regulator of both rhlI and

*Biofilm Development in Gram-Positive and Gram-Negative Bacteria DOI: http://dx.doi.org/10.5772/intechopen.104407*

#### **Figure 4.**

*Quorum sensing in* P. aeruginosa.

lasI genes. In *P. aeruginosa,* early activation of QSc genes and premature synthesis of signals like C4-HSL and 3-oxo-C12-HSL were found in QScR mutant varieties. Overexpression of rsmA gene product resulted in decreased production of QSc virulence factors and acyl-HSLs whereas rsmA deletion led to early activation of LasI and thus the early synthesis of 3-oxo-C12-HSL [41].

Expression of a number of virulence factors is regulated by QS in *P. aeruginosa* and QS plays an important role in the pathogenicity of this organism. This presumption has been confirmed by using a number of different animal models. A lasR deficient strain of *P. aeruginosa* was found to have decreased virulence compared to that of the parent in a neonatal mouse model of pneumonia. Analysis of the *P. aeruginosa* mutant varieties such as lasI mutant, rhlI mutant, and a lasI, rhlI double mutant in the same model revealed markedly decreased virulence and the most remarkable reduction was found in the double I mutant variety [42]. **Figure 4** shows the QS in *P. aeruginosa.*

#### **7. Quorum sensing in gram-positive bacteria**

QS systems are found to be involved in the pathogenicity and biofilm formation of a number of gram-positive bacteria and these systems use different signal molecules from those of gram-negative bacteria which produce AHLs as AIs. In gram-positive bacteria, no AHL production has been observed in biofilm. Small post-translationally processed peptide signal molecules are used by the gram-positive bacteria QS system. These peptide signals interact with the sensor element of a histidine kinase two-component signal transduction system. Development of bacterial competence in *B. subtilis* and *S. pneumoniae*, conjugation in *E. faecalis,* and virulence in *S. aureus* is regulated by using QS system. A wide variety of disease states caused by *S. aureus* ranges from mild skin infections to life-threatening endocarditis. The virulence of this organism is dependent on the temporal expression of a diverse array of virulence factors which include cell-associated products, such as collagen and fibronectinbinding protein A, and secreted products including lipases, proteases, alpha-toxin, toxin-1, beta-hemolysin, and enterotoxin [43]. **Figure 5** shows the signaling pathway in gram-positive bacteria.

**Figure 5.** *Signaling pathway in gram-positive bacteria.*

### **8. Quorum sensing in** *Staphylococcus aureus*

Surface proteins involved in attachment during the early stages of *S. aureus* infection (collagen and fibronectin-binding protein) and defense protein (protein A) predominate. Expression of *S. aureus* surface proteins is decreased and secreted

**Figure 6.** *Quorum sensing in* S. aureus.

*Biofilm Development in Gram-Positive and Gram-Negative Bacteria DOI: http://dx.doi.org/10.5772/intechopen.104407*

proteins are preferentially expressed when once a high cell density is achieved at the infection site. Two pleiotropic regulatory gene loci called agr (accessory gene regulator) and sar (staphylococcus accessory gene regulator) determine the genetic basis for this temporal gene expression [44].

The agr locus of *S. aureus* consists of two promoters P2 and P3 with two divergent operons, RNAII and RNAIII. The RNAII operon contains the agr BDCA genes which encode the response regulator (AgrA) and signal transducer (AgrC), and AgrB and AgrD which are involved in generating the QS signal molecule. *δ*-hemolysin is encoded by the RNAIII and is itself a regulatory RNA that plays a key role in agr response. In response to the octapeptide signal molecule, the AgrC signal transducer is autophosphorylated during *S. aureus* QS, which in turn leads to the phosphorylation of the AgrA response regulator. The transcription of RNAIII is stimulated by phosphorylated AgrA and in turn RNAIII upregulates the expression of numerous *S. aureus* exoproteins as well as the agr BDCA locus. The latter leads to a rapid increase in the synthesis and the export of the octapeptide signal molecules. The AgrA gene product (AgrA) functions as a regulatory DNA-binding protein to induce the expression of both RNAII and RNAIII operons of the agr locus at the second regulatory locus [45, 46]. **Figure 6**: Showing the QS in *S. aureus.*

#### **9. Role of biofilm in pathogenesis**

Biofilms play a major role in the pathogenesis of many diseases [47]. A large number of nosocomial infections result due to the colonization of bacteria on the surface. Almost 95% of urinary tract infections are associated with urinary catheters which include *S. aureus* infections. *S. aureus* and *P. aeruginosa* are responsible for frequent biofilm infections.

#### **10.** *Pseudomonas aeruginosa* **pathogenicity**

*P. aeruginosa* is a gram-negative bacterium that is found to be responsible for a number of infections. It is an opportunistic human pathogen capable of causing both acute and chronic infections [48]. The lungs are one of the common niches for its colonization. It is found to be associated with respiratory infections like cystic fibrosis, lung infections [49]. Its greater adaptability and opportunistic sense enable its association with other infections also like wounds, burns, etc. [50]. Multidrugresistant *P. aeruginosa* is emerging nowadays which makes the treatment more difficult. *P. aeruginosa* shows resistance to a number of antibiotics like β-lactams, aminoglycosides, quinolones, etc due to mechanisms such as low outer membrane permeability, efflux system, inactivating enzymes like β-lactamases [51]. It can also acquire resistance genes from other micro-organisms by horizontal gene transfer such as in the case of biofilm [52].

*P. aeruginosa* shows adaptation which is related to complex mechanisms. A number of factors are found to be responsible for the pathogenic potential of bacteria which play a key role in biofilm formation and dispersion. These include flagella, pili, enzymes like proteases, siderophores like pyoverdine, surfactants like rhamnolipids and toxins like exotoxin A and pyocyanin, etc. [53].

#### **11.** *Staphylococcus aureus* **pathogenicity**

Both gram-positive and gram-negative bacteria are found to be pathogenic in nature. *S. aureus* is a gram-positive bacteria frequently found on the mucosal surface of the nose and respiratory tract and skin [51]. It is easily transmitted by direct contact. It is also found to be methicillin-resistant which makes it difficult to treat. Methicillin is a narrow-spectrum β-lactam antibiotic of the penicillin family. *S. aureus* is very often found to be associated with nosocomial infections. Multidrug-resistant *S. aureus* (MRSA) has the ability to evolve and adapt easily which is being considered as a threat according to W.H.O [54]. In addition to this, MRSA is also developing resistance to other antibiotics via mutations and horizontal gene transfer [55]. It has been reported that the presence of *S. aureus* in heterogeneous biofilms increases the rate of plasmid horizontal transfer which increases the resistance of antibiotics in biofilm [56]. *S. aureus* shows the ability to survive host-defense mechanisms through different factors such as cell wall-anchored proteins like clumping factors, fibronectin-binding protein A, collagen adhesion which enables tissue attachment, evasion, and biofilm formation [57]. Extracellular toxins (including hemolysin, leukotoxin, entero-toxin) and enzymes (including coagulase, proteases, staphylokinase) help in tissue penetration and host invasion [58]. Surfaceassociated factors are down-regulated and surfactants are also expressed in the later stages which lead to biofilm dispersion and the spread of infection [59].

#### **12. Conclusion**

Biofilms are made up of bacteria that consist of monospecies or multispecies. Bacterial biofilms are found to be present on a number of surfaces and for this purpose, bacteria secrete and produce EPS matrix which makes adherence easier. Biofilm formation has become a ubiquitous phenomenon found on both living and non-living surfaces. In this biofilm, bacteria interact by producing various toxins, virulence factors that are pathogenic in nature. Both gram-positive and gram-negative bacteria show different QS systems. QS leads the bacteria to evade the immune response and increase cell density. QS is found to be responsible for the virulence shown by the bacteria. Many bacteria show virulence characteristics such as *S. aureus*, *P. aeruginosa*, *E. faecalis, V. cholerae*, *S. pneumoniae*, etc. *S. aureus* produces alpha-hemolysin, toxins, various proteases whereas *P. aeruginosa* is found to produce exoenzymes, cell-cell spacing and sis also resistant to chloramphenicol. *S. aureus* and *P. aeruginosa* are two of the most common bacteria which show biofilm formation. These bacterial biofilms are difficult to eradicate from the surface due to strong adhesive forces and resistance against a number of antibiotics. Current therapeutic approaches are not effective to prevent biofilm formation and thus there is a requirement for new strategies and drugs for the treatment of biofilm infection.

#### **Abbreviations**


*Biofilm Development in Gram-Positive and Gram-Negative Bacteria DOI: http://dx.doi.org/10.5772/intechopen.104407*

#### **Author details**

Deepak Dwivedi1 \* and Trishla Sehgal2

1 Minor Forest Produce Processing and Research Center, Bhopal, Madhya Pradesh, India

2 Vedanta Testing and Research Laboratory, Bhopal, Madhya Pradesh, India

\*Address all correspondence to: microbio.deep@gmail.com

© 2022 The Author(s). Licensee IntechOpen. 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.

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#### *Focus on Bacterial Biofilms*

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[59] Hartmann A, Rothballer M, Hense BA, Schröder P. Bacterial quorum sensing compounds are important modulators of microbe-plant interactions. Frontiers in Plant Science. 2014;**5**:131. DOI: 10.3389/fpls.2014.00131

### **Chapter 7**

## Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery

*Roger Bayston*

#### **Abstract**

Biofilms are responsible for chronic persistent infections and are a major problem in implant surgery. The microbial pathogenesis, treatment and prevention of biofilm infections is reviewed.

**Keywords:** biofilm infections, biofilm phenotype, small colony variants, prevention of biofilm infections

#### **1. Introduction**

Though the "discovery" of biofilms is ascribed to Anton van Leeuwenhoek in 1676 using a novel magnifying device, and possibly to Robert Hooke two decades earlier, and biofilms were recognised in a marine setting about a century ago, they were of no medical interest until two studies described them in a medical device and in sputum in 1972 and 1974 respectively. The latter was a description of aggregates of *Pseudomonas aeruginosa* in secretions from the lungs of people with cystic fibrosis [1], and led to a burgeoning of research into *Ps aeruginosa* infection in that field.

**Figure 1.** *Examples of implantable devices.*

**Figure 2.** *Anatomical sites of common implantable devices.*

Through a meeting with Costerton, Højby studied these aggregates and the term "Biofilm" was made popular by Costerton in 1987 [2], though the term was originally used by Mack et al. [3] to describe "biofilm" on a water filter. However, many biofilm infections occur in association with implanted materials and devices, and their use has become much more common since the middle 1900's. The first biofilm reported in a medical device was found in a shunt to treat hydrocephalus in 1972 [4]. This discovery explained the difficulty in successfully treating these infections non-surgically with antibiotics alone, and the report demonstrated the extracellular matrix of the biofilm in vitro and in vivo and carried out investigations to suggest that it was a glycosaminoglycan. This was later confirmed by important studies in 1996 [5]. Implantable biomaterials and devices are now widely used in modern surgery, and the list is extensive (**Figures 1** and **2**).

#### **2. Biofilm definitions**

Many definitions of "biofilm" are found in the literature, and they can be based on either structure or function. Many of the definitions and their accompanying images are derived from in vitro models, and the appearance of mushroom-like structures and water-channels are not seen in biofilms occurring in vivo [6]. A definition based on functional aspects of biofilms is more useful in a medical context. This could be reduced to a population of bacteria or other micro-organisms, often associated with a surface, and enveloped in an extracellular matrix, showing insusceptibility to antimicrobials and to the host immune system, and ability to persist for long periods.

#### **2.1 Biofilm phenotypes**

The basis of this functional definition is the paucity of nutrients, including iron, and oxygen in the depths of the biofilm leading to a bacterial stress response caused by a crisis in energy generation and transport [7]. The bacterial stress response is mediated by the intracellular signal sigma-B. The bacterial response to this is to downregulate all synthetic functions not needed in biofilm mode, such as cell wall material, toxin and other non-essential protein synthesis, and DNA replication. These are the targets for common antibiotics, and beta-lactams, glycopeptides, aminoglycosides, macrolides and fluoroquinolones all become significantly less effective against biofilm bacteria. Other factors contribute to the lack of effect of antibiotics, including a slowing of their penetration into the biofilm, though this is rarely a major factor. The bacterial stress response results in significantly reduced cell metabolic activity and loss of some synthetic activities leading to auxotrophy for heme and menadione, and sometimes other substances such as thymidine. This biofilm phenotype is crucial to the clinical impact of biofilm infections; the colonies of biofilm bacteria when grown from clinical samples in the laboratory are typically less than ten times the size of their planktonic counterparts, and are known as small colony variants or SCV. The molecular control and regulation of biofilm phenotype has been described in detail by Proctor et al. [8]. SCV are important in biofilm infections not only because their metabolism leads to antibiotic insusceptibility, but because, though they can be internalised by professional and non-professional phagocytes, they are not killed and survive inside the phagocytic cells. Auxotrophic SCV of *Staphylococcus aureus* for heme and menadione, that do not produce alpha-toxin, are more able to survive intracellularly, and supplementation of intracellular populations of *S aureus* in vitro with menadione resulted in restoration of alpha-toxin production and reduced intracellular survival [9, 10]. SCV are not always auxotrophic and considerable variation occurs, but intracellular survival is a common feature. Many also show reduced susceptibility to aminoglycosides, and exposure to gentamicin can induce SCV formation [11]. Some SCV are the result of mutations in the genes concerned with electron transport, and these do not revert to parent forms whereas other forms of SCV appear to be phenotypic variants that revert to parent forms when the stress factor is withdrawn [8, 12]. SCV of gram negative bacteria have been known for decades, having been produced in the laboratory from exposure to antibacterial chemicals [13, 14]. However, more recently capnophilic (carbon dioxide—dependent) SCV of *Escherichia coli* have been isolated from a patient with a urinary tract infection, though no information on biofilm involvement was given [15]. A report of septic shock in a patient from whose urine capnophilic *Proteus mirabilis* SCV were isolated again did not state that biofilms were involved [16] but the patient had chronic renal stones, known to be associated with biofilms [17]. *P mirabilis* is an important uropathogen as it is highly motile and is capable of enzymatically hydrolysing urea into ammonia, thus being highly inflammatory as well as alkalinising the urine. The rising pH causes crystallisation of calcium and magnesium phosphates [18], and the *P mirabilis* biofilm typically consists of a mesh of bacteria, their extracellular matrix and phosphate crystals. These biofilms are obviously different in composition from those consisting mainly of bacteria and their products, and another example of such complex biofilms is the vegetations found in native valve endocarditis. Here the lesion consists largely of a matrix of platelets and fibrin, with bacteria, usually viridans streptococci, embedded in it. The lesion usually begins as a response to damage to the endocardium, which is then colonised by bacteria from the bloodstream, becoming progressively built up

of fibrin and platelets with rafts of bacteria interspersed [19, 20]. A similar situation arises with prosthetic heart valves. In both cases, SCVs have been reported [21, 22] as well as other auxotrophic variants [23].

The biofilm phenotype, and SCV in particular, are important in treatment of biofilm infections. Surviving intracellular bacteria are protected from further immune assault and from most therapeutic antibiotics, which do not accumulate inside host cells sufficiently to kill SCV [24]. These factors mean that the amount of antibiotic required to kill bacteria in biofilm mode is typically 500–1000 times the minimum inhibitory concentration as measured in the clinical laboratory. Such concentrations are not achievable by intravenous or oral therapy, and eradication of biofilm infection usually requires extensive surgery to debride the site and to remove all surgical hardware.

#### **2.2 Biofilm development**

Development of biofilms in surgery depends on a sequence of events. Initially, the causative bacteria must be able to gain access to the site of biofilm formation, usually an implantable device. In modern surgery most device pathogens originate on the patient's skin or mucous membranes, consisting mainly of coagulase-negative staphylococci (CoNS), typically *Staphylococcus epidermidis*, and *Cutibacterium acnes*. Conventional pre-operative skin preparation reduces but does not eradicate these bacteria, and the importance of relatively small numbers of bacteria in the operation field has been shown by an experiment in human volunteers, where various "doses" of *S aureus* were inoculated into incisions to determine how many bacteria were necessary to produce an abscess [25]. In one group, "foreign" material in the form of sutures were also introduced into the incision, and the number of bacteria required to form an abscess in those cases was 10,000 times fewer. This study, which is unlikely to be repeated in a modern setting, is extremely important in illustrating the role played by implantable materials and devices in infection in modern surgery.

The sequence of events involved in development of a biofilm infection involving a surgically implanted device are (**Figure 3**):

Access to the device from the source. Though heavy contamination of the air in the operating environment has historically been associated with surgical infection, modern operating room design and ventilation has meant that this source has declined in importance, and most surgical infections are caused by bacteria originating on the patient's skin or mucous membranes. Bacteria reach the incision from the cut edges of

#### **Figure 3.**

*Sequence of events in development of biofilm infection. Here implant has an antimicrobial coating, but within minutes this is covered by a glycoprotein conditioning film produced by the patient. This usually prevents the activity of the coating and bacteria now adhere to the conditioning film. Within a few hours the attached bacteria begin to produce an extracellular matrix and to multiply. Powerful antibacterial activity is essential now, as after this point, it is almost inevitable that a biofilm will develop, within a few weeks.*

#### *Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery DOI: http://dx.doi.org/10.5772/intechopen.104526*

the skin, or from contamination from surrounding skin surfaces, during surgery. The causative bacteria are therefore often present when the device is implanted.

Attachment to the device. Many bacteria possess adhesins on their surfaces that allow them to attach to biomaterials (vitronectin—binding protein etc) but more often they employ specific adhesins for the glycoproteins, platelets and other host-derived materials that rapidly coat all implanted materials [26, 27]. *S aureus* possesses specific adhesins for fibrinogen, fibronectin, laminin, thrombospondin, bone sialoprotein and other host-derived components of the conditioning film. These bacterial surface adhesins are known as MSCRAMMs (Microbial Surface Component Recognising Adhesive Matrix Molecules) [28] and they can be found in other organisms such as *S epidermidis* and enterococci [29]. Gram negative bacteria often attach by means of swarming or twitching motility over the new surface [30], some using twitching motility by Type IV pili [31, 32], and this might be particularly important in biofilm formation on urinary catheters. In addition, *Ps aeruginosa* uses a von Willebrand Factor-like surface factor in twitching motility over biomaterial surfaces [33].

Once bacteria have attached to the surface or conditioning film, they begin to proliferate and to develop intercellular adhesins such as polysaccharide intercellular adhesin (PIA) in staphylococci. This substance is integral to further development of biofilm, and is encoded by the *ABDC* operon, and regulated by *icaR*. At this stage, bacterial stress responses are operating in response to limitation of nutrients and oxygen and the biofilm phenotype is appearing [34]. It is important to note that the bacterial stress response, mediated by Sigma B, downregulates *icaR* and increases PIA production, and the stress response can be provoked by external factors such as antibiotics as well as nutrient starvation. Once the biofilm phenotype has developed, the biofilm is stable and is not susceptible to host immune activity or to antimicrobials. There is often a lag phase of about 14–28 days before the biofilm reaches functional maturity, during which it might be more susceptible to antimicrobials [35].

Clear understanding of the sequence of events and periods of risk is essential for effective planning of preventative measures.

#### **3. Prevention of biofilm infections**

#### **3.1 Surgical considerations**

Since the days of Semmelweis, Lister and others in the mid–to late 1800s, personal hygiene of the surgeon, aseptic technique and antisepsis have become accepted norms. Since the 1950s, when bacteria-laden operating room air was identified as a major factor in surgical infection [36], greatly improved practices and ventilation systems have made this a minor source. Two main forms of ventilation are in use in modern operating rooms: plenum, and laminar flow with high efficiency particulate air (HEPA) filtration. While it is clear that the numbers of airborne bacteria are significantly reduced when laminar flow is used [37] there has never been a clear causative link between either this reduction or the actual bacteria and surgical infection, leading the USA CDC to downgrade their initial recommendation [38]. More recently, reports have appeared of small but significantly increased infection rates when laminar flow is used [39, 40] and this appears to be due to flaws in its design and manner of use [41]. For most types of implant surgery, plenum (conventional) ventilation appears to be satisfactory so long as other precautions are taken (**Figure 4**).

#### **Figure 4.**

*Sequence of surgical preventative events.*

Care bundles have been proposed for infection reduction in various healthcare settings. A bundle is a collection of interventions that are expected to contribute to reduced risk of infection, but which singly might have weak or no evidence base. A measure such as ensuring that only three people are present in the operating room during a procedure is not supported by any clear evidence but it is intuitively likely to be beneficial if only in reinforcing operating room discipline. A bundle must be directed towards behaviour change on the part of relevant staff members, and it works best if they contribute to its content, and formally agree to abide by it. Some bundles insist on contents being evidence-based, but the quality of evidence is usually very weak for individual components. However, when bundles are properly applied, they are often very effective in reducing surgical infection [42, 43] and in any case they and their contents should form part of a well-managed surgical discipline. Usually no single component can be identified to explain their success, but clinical trial evidence has shown that violations of the bundle are associated with re-emergence of infection [43].

As the major source of pathogens is the patient's skin, attention has been directed towards the effectiveness of preoperative skin preparation. Two main antiseptics are in use: chlorhexidine and povidone iodine. Each can be formulated in water or 70% alcohol. A report by the World Health Organisation (WHO) favouring chlorhexidine [44] has been called into question on the basis of quality of evidence [45]. However, sampling is usually by swabbing of the skin surface, and almost none of the many studies on surgical skin preparation explore the effectiveness of any agent on bacteria resident in the dermis, though an early study showed that full thickness skin biopsy was necessary [46]. This has since been confirmed [47, 48]. When skin biopsy is used, neither antiseptic in alcohol is able to eradicate resident skin bacteria, and though reduced, the remaining numbers are often sufficient to cause a biomaterialassociated infection [25]. Two studies on the penetration of both aqueous and alcoholic chlorhexidine into human skin using full thickness biopsy have found it to be minimal [49, 50]. Further measures are therefore necessary. Some researchers have investigated the effect of antiseptic-soaked material to protect the incision from

#### *Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery DOI: http://dx.doi.org/10.5772/intechopen.104526*

the skin edges during surgery, and while this is commonly used, there have been no quantitative studies to show benefit. Intravenous antibiotics are almost universally used in surgery, ideally as a single dose 30–60 min before incision, but extra doses are commonly used postoperatively though they offer no benefit over that of the single pre-operative dose. Antibiotic prophylaxis is undoubtedly highly effective in reducing infection risk in many types of surgery, including colorectal surgery [51] and orthopaedic surgery [52] but probably less so in neurosurgery due to limited penetration of systemic antibiotics intracranially. However, it is probably inevitable that a small number of bacteria will reach the implant during operation, and further measures have been directed to attempts to eradicate these. As knowledge of attached bacteria and biofilms has shown that very high concentrations of antibiotics are necessary, some surgeons have used either antiseptic or antibiotic irrigation [53, 54], or have simply added antibiotic powder to the incision before closure [55–57] with successful reduction in infection rates and complications. This intervention gives extremely high local antibiotic levels not reachable by systemic administration, yet avoids most of the complications associated with the latter method.

#### **3.2 Antimicrobial biomaterials**

Other methods of prevention accept that despite efforts, bacteria will reach the implant, and aim to prevent their attachment or to kill them when attached. Various "anti-fouling" surfaces have been investigated with the aim of allowing host cell and tissue proliferation but preventing bacterial attachment [58, 59] but none of these has yet reached clinical application, largely because of the complex relationship between implant surface, host tissue environment, and bacterial surface adhesins. Biomaterials designed to kill bacteria that do attach to them have generally included coatings of silver, antiseptic or antibiotic and combinations of these, often with a vehicle to bind the antimicrobial to the biomaterial surface. Such coatings have several disadvantages. The normal host reaction to the implant of deposition of plasma proteins [26, 27] also obliterates the antimicrobial coating in many cases, making it ineffective. Silver is susceptible to this due its avidity for proteins [60], and it can also be inactivated by chloride [61] which is abundant in the human body. Silver ions have also been shown to be cytotoxic in certain conditions [62]. Clinical studies on silver-processed devices give very variable results, and there is doubt about their cost-effectiveness in wound dressings [63]. A recent randomised controlled trial of silver-containing catheters intended to reduce ventriculitis in people with hydrocephalus shunts found no difference from plain catheters [64]. Another randomised controlled trial of silverprocessed urinary catheters again found no significant difference from plain catheters [65]. In both of these clinical settings, biofilms play a key role, and the goal is to prevent bacterial proliferation and biofilm development on the catheters. Both have fluid containing proteins and chloride flowing through them.

Another approach has been impregnation of catheter material with antimicrobials. Though the impregnation processes differ, two catheter types can be considered: those containing rifampicin and minocycline, and those containing rifampicin and clindamycin. The first type has been used in central venous catheters [66] and external ventricular drains [67]. The second type has been used in hydrocephalus shunts and external ventricular drains. In all cases they have shown effectiveness in reducing device -related infection. The advantage of impregnation over coatings is that they give a long duration of activity: coatings are usually washed away by fluid after a few days, whereas the surface of an impregnated material is continually replenished by

**Figure 5.**

*Principle of impregnated biomaterial. Antimicrobial molecules are motile within the device matrix and can migrate to the surface to replace those removed by fluid flow.*

migrating antimicrobials until the depot in the material is depleted, usually several weeks later (**Figure 5**). This is important when the implantable device is at risk of contamination for an extended period.

#### **3.3 Importance of source of infection and period of risk**

In order to formulate an effective preventive strategy, knowledge of the source and nature of device pathogens and the period during which the device is at risk is essential (**Table 1**). As many biofilm infections are caused by micro-organisms originating in or on the patient, a knowledge of the distribution of these is useful. The normal bacterial flora of the skin differs according to age and sex, but particularly depending on the anatomical site. The most common bacteria found on the skin are staphylococci, particularly members of the CoNS. These are typified by *S epidermidis* which is broadly distributed over the body surfaces, but other species such as *Staphylococcus* 


#### **Table 1.**

*Periods of risk of infection of common implantable devices.*

#### *Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery DOI: http://dx.doi.org/10.5772/intechopen.104526*

*capitis* have preferred sites such as the head and neck. *C acnes* is an important pathogen in the context of implant infections, but it is a good example of the importance of specific topographical distribution in determining the important pathogens in particular implants. *C acnes* is found on the upper body and head (**Figure 6**) [68], and it is therefore not surprising that devices implanted in these areas show a significantly higher incidence of *C acnes* infection. Examples are neurosurgical shunts and drains [69, 70], spine instrumentation [71], breast implants [72] and shoulder arthroplasty [73, 74]. Implants in other sites such as urinary catheters are at risk from a different microbial profile, as the pathogens originate in the large intestine, and *E coli*, *Klebsiella pneumoniae* and *P mirabilis* are the most common.

The time at which the implant is at risk of microbial contamination also varies. While there is always a risk at the time of implantation, in some implants this is the main time, and the risk of subsequent contamination is proportionally small. Examples of this are hydrocephalus shunts and joint replacements. In other implants the risk at insertion is significantly outweighed by that during use. Examples are external ventricular drains (EVD) for raised intracranial pressure, urinary catheters, venous access catheters and peritoneal dialysis catheters, all of which can be contaminated from environmental sources or from the hands of staff or users during use. Other examples are vascular grafts and prosthetic heart valves, which are at risk from hematogenous seeding from bacteria entering the bloodstream at a distant site.

When planning strategies for prevention of biofilm infections involving antimicrobials, it is therefore important to match the antimicrobial to the most likely pathogen(s). If systemic antimicrobial prophylaxis is contemplated, then the adverse effects of this must be taken into consideration if there is a need for prolonged

administration due to extended period of risk. If antimicrobial materials or devices are to be used, these must address not only the likely pathogen(s) but also the duration of protective activity required.

International guidelines indicate that for most surgical procedures, any systemic antimicrobial prophylaxis should be administered as one dose 30–60 min before start of surgery [75, 76]. Extension of this prophylaxis beyond 24 hours does not reduce surgical infection further, but it does increase the incidence of acute kidney injury and *Clostridioides difficile* infection [77], which is a life-threatening colitis associated with over-use of antibiotics. Where the period of risk extends beyond the insertion procedure, such as in EVD, long courses of systemic antibiotics are often given until the drain is removed. This has been shown in some cases to reduce brain infections, but at a cost. A randomised study comparing the use of plain catheters and prolonged systemic antibiotics with antimicrobial-impregnated catheters and one dose of antibiotic at insertion found no difference in the brain infection rate, which was low in each group, but there were three cases of *C difficile* infection in the prolonged antibiotics group, one patient requiring total colectomy [78].

#### **4. Treatment of biofilm infections**

The difficulty in treating biofilm infections in surgery emphasises the importance of effective prevention. However, this is not always possible. The nature of the biofilm phenotype and its implications for antibiotic treatment mean that further surgery is almost inevitable, and this usually involves removal of the device. This might be relatively simple, as in the case of a venous access catheter or a urinary catheter, but it can be both surgically complicated and hazardous, as in the case of spinal instrumentation or prosthetic heart valves.

Attempts to eradicate established biofilm with antibiotics usually fail. A comparison of treatment regimens for hydrocephalus shunt infections showed that results with shunt removal and antibiotics were significantly superior to those with antibiotics alone [79]. Successful treatment of joint replacement infections relies on device removal and extensive debridement of infected tissue, with prolonged antibiotic therapy. However, understanding of biofilm biology has led to advances in this area. The biofilm phenotype takes a few weeks to "mature" to the point where full insusceptibility to antibiotics is expressed, and this has been exploited in development of a regimen for treatment of prosthetic joint infection when the diagnosis can be made within 3–4 weeks of insertion [80]. In this regimen, known as Debridement, Antibiotics and Implant Retention (DAIR), surgical treatment of the infected joint prosthesis is carried out on a planned basis after careful investigation to establish the causative micro-organism and its antimicrobial susceptibilities, to allow consultation with specialists including Microbiology/Infectious Diseases, and to determine that the implant is stable (**Figure 7**). Infections due to multi-drug-resistant bacteria, fungi or multiple bacteria are not suitable for this approach. During the operation, the prosthetic components are exposed and the acetabular module is removed, leaving the main metal prosthesis in place. All infected tissue is removed and samples are sent for microbiological examination. Copious irrigation with antiseptic is applied, and biodegradable antibiotic—eluting beads can be inserted to provide high local concentrations. The choice of antibiotic in the beads should be made in consultation with a microbiologist. The joint is then closed and a long postoperative course of suitable antibiotics is then started [81]. The success rate of DAIR compared to conventional

*Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery DOI: http://dx.doi.org/10.5772/intechopen.104526*

#### **Figure 7.**

*Possibility of retention of infected implant based on knowledge of biofilm phenotype maturation (based on Zimmerli and Trampuz, 2004) [80].*

full implant removal and replacement is slightly lower. Moreover, despite the very thorough surgical debridement and long courses of antibiotics, often for over a year, relapse can occur [82], illustrating the difficulty in eradication of biofilms. DAIR spares the patient the much more extensive surgical removal of the main implant components, and the second surgery to inert fresh implants a few weeks later.

#### **5. Diagnosis of biofilm infections**

#### **5.1 Clinical features**

Most biofilm infections in surgery are chronic and persistent, sometimes for many years [83]. It is important to distinguish between "late infection," implying an infection contracted long after surgery, such as hematogenously, and "delayed infection," meaning that the infection appears long after surgery even though it was contracted at the operation. Delayed infection in spine instrumentation is usually due to infection with CoNS or *C acnes* [84, 85]. A similar situation is found in shoulder arthroplasty [86]. Generally, more virulent bacteria such as *S aureus* are associated with either early-presenting or with hematogenous infections. The delay of months or years between initial surgical implantation and appearance of symptoms [84] has led to doubt about the surgical origins of some infections but this has now been largely dispelled. However, the need for prolonged follow-up and vigilance must be emphasised.

Acute postoperative biofilm infections usually appear within days or weeks of surgery, with failure of wound healing, drainage of pus or other fluid from the wound, local pain and swelling, fever and general illness. Delayed or chronic infections of joint prostheses present with persistent pain and restricted mobility, local swelling and sometimes a sinus. In the absence of a sinus, diagnosis might be delayed as it is often difficult to distinguish infective from mechanical complications. Aspiration of synovial fluid often gives a diagnosis but sensitivity is low [87, 88]. Delayed infection in spine instrumentation similarly presents with persistent pain, tenderness and possibly a draining sinus. Delayed infections in hydrocephalus shunts are very uncommon now that the preferred route of drainage is to the abdomen (ventriculoperitoneal, VP), but

the ventriculo-atrial (VA) route is still used in some cases. In VP shunts infection usually presents within a few months as it leads to obstruction, but this does not happen in VA shunts and symptoms might not appear, or at least become recognisable, for several years. During this time, bacteria are being discharged from the biofilm in the shunt into the bloodstream, and this might give rise to periods of ill-health or sporadic fevers. It also provokes production of antibodies to the bacteria, and eventually the concentrations of circulating antigen and antibody, and therefore immune complexes, become so high that they precipitate on basement membranes of joints, renal glomeruli, alveoli and microvascular system. The presenting clinical picture can therefore be a confusing array of disorders from hematuria, hemorrhagic skin rashes, arthropathy, and chronic cough [89, 90]. Clinical diagnosis can therefore be very difficult, and a high level of suspicion is needed. Aspiration of cerebrospinal fluid from the shunt often gives the diagnosis, but blood cultures can be negative in the later stages.

#### **5.2 Laboratory methods**

Depending on the site of the infection and presence of an implant, sometimes blood cultures are positive, indicating systemic spread of the infection, and risk of sepsis. Blood inflammatory markers such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels are usually raised. Swab cultures from the wound might yield the infecting pathogen, but they might be misleading due to contamination [91]. Surgical exploration of the incision and deeper layers allows tissue samples to be taken and these are more likely to yield the pathogen(s). Such samples should always be taken during debridement surgery [92], using fresh instruments for each of up to six separate samples [81, 93]. In view of the anaerobic preference of *C acnes* and its slow growth, cultures should be incubated anaerobically for up to 10 days [94]. The way in which tissue samples are processed in the laboratory is important. Simply rubbing them on a culture plate or incubating them in a fluid culture is prone to contamination and gives poor yield, leading to under-diagnosis of infection. Tissue should be homogenised but the method of doing this is also important [95]. When hardware such as joint replacement or spinal instrumentation components are removed, these should be seen as valuable samples. Sonication to remove the biofilm has been shown to significantly increase the culture positivity rate [96, 97]. A further aid to laboratory diagnosis has been PCR [98] especially when applied to tissue homogenates or hardware sonicates. However, if PCR is used in an attempt to certify eradication of infection before re-insertion of a prosthesis, residual DNA from bacteria successfully killed by antibiotic therapy can give false positive results suggesting ongoing active infection. This can be overcome by use of a modified PCR method that detects DNA only from live bacteria [99].

#### **6. Conclusions**

The impact of biofilm infections in surgery on healthcare systems, economies and personal lives of patients is immense. The financial cost can only be estimated and published figures do not usually take into account "unseen" costs such as loss of earnings due to disability, increased dependency, and financial burden on carers.

The physical and mental trauma of surgery such as joint replacement, reconstructive breast implant or hydrocephalus treatment can be made unimaginably worse by postoperative biofilm infection.

#### *Molecular Pathogenesis and Clinical Impact of Biofilms in Surgery DOI: http://dx.doi.org/10.5772/intechopen.104526*

The significant difficulty in successfully treating biofilm infections with antibiotics, due largely to the biofilm phenotype, is now well recognised, and the importance of commensal bacteria previously thought to be harmless, such as *S epidermidis* and *C acnes*, is becoming more widely known. However, surgical device removal remains the mainstay of treatment, and new approaches that allow implant retention are needed. Prevention of biofilm infections is crucial, and biomaterials that either reduce bacterial attachment, such as those coated with novel synthetic polymers [100] or those designed to kill bacteria on contact [66, 67] are now in clinical use. Many other biomaterials approaches are in development, and considerable strides have been made in this direction but further progress is being slowed by unrealistic commercial and regulatory barriers [101].

#### **Author details**

Roger Bayston School of Medicine, University of Nottingham, Nottingham, United Kingdom

\*Address all correspondence to: roger.bayston@nottingham.ac.uk

© 2022 The Author(s). Licensee IntechOpen. 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.

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Section 3
