Antimicrobial Resistance and Anti Biofilm Strategies

#### **Chapter 8**

## Bacterial Biofilm: Contribution to AMR and Approaches to Tackle

*Meenakshi Sharma, Pragati Yadav and Deepika Tripathi*

#### **Abstract**

The brisk emergence of resistant microbes is occurring worldwide, endangering the efficacy of various antimicrobial agents. The overprescription of antimicrobial drugs results in the emergence of mutant strains of drug-resistant pathogens challenging the existing antimicrobial regime. Moreover, the outbreak of the pandemic has emphasized the necessity to consider the coinfections and antimicrobial resistance crisis as a vital motive of morbidity and mortality. Therefore, the prevention of such infections is much better than the eradication of the same. Thus, herein, we aim at providing a comprehensive list that can be used as an alternative class of antibacterial agents by exploiting the activity of various phytochemicals. The antibiofilm activity of various classes of phytochemicals would be projected for both the eradication and the prevention of biofilm formation in the presence of selected compounds. This chapter visualizes antimicrobial resistance as a matter of grave concern and one of the greatest threats to global health, food security, and development today.

**Keywords:** biofilm, antimicrobial resistance, phytochemicals, antibacterial resistance

#### **1. Introduction**

Antimicrobials can be synthetic or natural molecules that have the efficacy to kill microorganisms effectively. The tolerance toward antimicrobials has emerged as a major challenge for scientists and doctors across healthcare sectors, and it is becoming a serious threat worldwide. Since the late 1960s, the situation is intensified by decline in the search of novel drugs, as testing new drugs and finally its acceptance requires long time periods by the authorities for commercialization [1]. Antimicrobial resistance (AMR) in pathogenic microbes is the threatening global health problem with the biggest threat to human health, and the world is suffering without any significant and effective antibiotics [2]. It occurs when bacteria, viruses, fungi, and parasites change over time and, now, no longer respond to antibiotics.

In other words, microbes become resistant to antibiotics and cause reinfection. Sometimes, it is impossible to treat such infection, and it ultimately increases the risk of disease spread, severe illness, and even becomes fatal day by day. According to recent studies and World Health Organization (WHO)'s reference, the antimicrobial resistant microbes are also referred to as "superbugs" sometimes. According to 2014 World Health Organization (WHO) report, "Antimicrobial Resistance: Global Report on Surveillance," the problem is "so serious that it threatens the achievements of

#### **Figure 1.**

*The number of deaths per year (in millions) as per data provided by the report on the AMR review by Hala Audi in 2014.*

modern medicine. A post-antibiotic era—in which common infections and minor injuries can kill—is a very real possibility for the 21st century" [3]. **Figure 1**, based on the report presented by Hala Audi in 2014 [3], shows the number of deaths (in million) versus various causes of death in the present age, and the number of deaths due to AMR is estimated to be increased from 700,000 at present to 10 million deaths per year in 2050 [4].

One of the major reasons contributing to the emergence of AMR is the overuse of antibiotics. At present, most of the antimicrobial compounds target the necessary microbial physiological processes, thereby exerting strong selection pressure on microbes that promote the emergence and spread of drug-resistant strains. Recently, researchers have targeted their research toward finding novel solutions to overcome AMR by targeting the cause of resistance. Phytochemicals, such as alkaloids, flavonoids, quinones, tannins, coumarins, terpenes, lectins, and saponins, have exerted potential antibacterial activities against sensitive as well as resistant pathogens [5, 6]. In this chapter, we have focused on AMR in bacteria, their mechanism of action specifically biofilm formation, and the probable ways to tackle them with emphasis on phytochemicals.

#### **2. Antibacterial resistance**

With the discovery of new antibiotics, resistance closely follows and develops continuously. The first antibiotic, penicillin (discovered in 1928), was soon followed by the identification of penicillinase, which led to the discovery of new β-lactams. Similarly, the discovery of sulfonamides, in 1937, led to the resistance in late 1930s. Therefore, each and every new discovery of antibiotics led to the emergence of resistance, leading to decreased treatment options and ultimately rise in morbidity

and mortality [7]. The antibacterial resistance is an ever-evolving genetic phenomenon that may be due to genetic mutations or horizontal gene transfer.

The multidrug resistance (MDR) in bacteria is increasing rapidly (**Table 1**), and in 2017, the WHO has categorized and prioritized the drug-resistant bacteria as "critical, high, and medium" for research of new antibiotics. The list includes carbapenem



#### **Table 1.**

*List of bacteria showing antibacterial resistance and the illness caused by them.*

resistant (*Acinetobacter baumannii*, *Pseudomonas aeruginosa*, *Enterobacteriaceae*, ESBLproducing) as critical priority; *Enterococcus faecium* (vancomycin-resistant), *Staphylococcus aureus* (methicillin-resistant and vancomycin-intermediate and resistant), *Helicobacter pylori* (clarithromycin-resistant), *Campylobacter* spp. (fluoroquinoloneresistant), *Salmonellae* (fluoroquinolone-resistant), and *Neisseria gonorrhoeae* (cephalosporin-resistant, fluoroquinolone-resistant) as high priority; and *Streptococcus pneumoniae* (penicillin nonsusceptible), *Haemophilus influenzae* (ampicillin-resistant), *Shigella* spp. (fluoroquinolone-resistant) as medium priority drug-resistant bacteria.

The antibiotics have a specific site of action in the bacterial cells, as shown in **Figure 2**. The antibiotic can cause defect in cell wall synthesis, inhibition of DNA

**Figure 2.** *Different classes of antibiotics and their site of action in the bacterial cell.*

gyrase, topoisomerase IV, and translocation inhibition (via 30S ribosome subunit) leading to formation of nonfunctional proteins or protein synthesis inhibition (via 50S ribosome subunit) [44].

#### **3. Mechanism of antibacterial resistance**

Antibacterial resistance means that the bacterial cell is capable of escaping the effects of drugs by various mechanisms. These resistant mechanisms can be general like modification in structure, which results in the hindrance of drug attachment to bacterial cells, attainment of aminoglycoside modifying enzyme, neutralizing or pumping the antibodies outside by efflux pumps, mutation of DNA gyrase, decrease in the affinity to antibiotics, methylation and/or mutation of 23S rRNA, alteration of target sites like penicillin-binding proteins (PBPs), and inactivation of antibiotics. The specific mechanisms, such as the production of lactamases for the enzymatic degradation of lactam antibodies and affecting the susceptibility and affinity of the target sites as in grampositive bacteria [45, 46], are also present. The mechanism can be either intrinsic or extrinsic resistance, which helps bacteria to acquire new resistant genes. Apart from these well-known genetic mechanisms, biofilm-formation- and quorum sensing (QS) related responses are other important features that help bacteria to gain resistance. In this chapter, we will discuss about the role of biofilm and its formation in detail.

#### **3.1 Biofilm**

Biofilms are a complex three-dimensional densely packed architectural network of microbes residing inside the polymeric matter secreted by them on several biotic and abiotic surfaces. The biofilm concept was given in 1971 by Marshall et al. [47], and later, Fletcher, Characklis, and Costerton described it as follows: "Biofilm is the unique pattern of growth in the life cycle of microbes that provides specific properties, advantages, and a higher level of organization to the free-living bacterial cells during colonization" [48]. According to the National Institutes of Health (NIH), 65% of microbial and 80% of chronic infections are linked to biofilm forming bacteria as compared to planktonic cells. The biofilm formation gives bacteria protection from antibiotics, disinfectants, and host defense system, thus showing resistance to them. For biofilm production, some bacteria adjust their gene expression and some use quorum-sensing systems. In both the gram-negative and gram-positive bacteria, quorum-sensing (QS) mechanisms exist, but the signal molecules used by them to transmit information are different. The QS signals of bacteria participate in various physiological processes such as motility, plasmid conjugation, biofilm formation, and antibiotic resistance to help them cope in the adverse environmental situations. The QS system comprises autoinducing peptides (AIPs), autoinducer-2 (AI-2), and acyl-homoserine lactones (AHLs) [49]. The presence of glycocalyx, outer membrane structure, efflux pumps, heterogeneity in growth rate, genetic adaptation, metabolic state, and metabolism of cells within a biofilm are the leading causes of biofilm that acquire resistance against antimicrobials [50]. As biofilms have extracellular polymeric substances (EPSs) that surround the cells, they provide protection to the microbial cells against harsh growth conditions [51]. EPSs are constituted of lipids, proteins, extracellular DNA, and polysaccharides. The biofilm formation is a multistep process, starting with attachment to the biotic or abiotic surface, forming a microcolony and then finally forming a threedimensional structure, which, after maturation, starts the detachment of bacterial cells for another cycle of biofilm formation via attachment (**Figure 3**).

#### *3.1.1 Attachment to the surfaces*

The first initial step is the attachment step, which is further divided into a twostage process: initial reversible attachment and irreversible attachment [52]. Biofilm

#### **Figure 3.**

*Stages of biofilm formation: the formation begins with a reversible attachment of the planktonic cells (dark brown ovals) followed by the adhesion to the surface (light brown). The bacteria then form a monolayer and irreversibly attach by producing an extracellular matrix. Next, a microcolony is formed where multilayers appear. During later stages, the biofilm matures, and finally, some cells start to detach and the biofilm (shown in yellow) disperses, releasing planktonic cells for re-attachment.*

formation begins by the preliminary reversible attachment of the planktonic microbial cells to the biotic or abiotic surface followed by adhesion. Bacteria will then start to form a monolayer and will produce an extracellular matrix (also known as slime) for protection. In this stage, the formation of microcolonies takes place, which shows significant growth and cell-cell communication for example quorum sensing. Now, the biofilm grows and the attachment is irreversible.

#### *3.1.2 Maturation*

This step initiates the cell growth that results in small colonies of microorganisms forming a characteristic "toadstool"-like structure. Bacteria within biofilm communities perform specialized functions after communicating via QS to each other. As the biofilm matures, more DNA, proteins, polysaccharides, etc., also known as biofilm scaffolds, are secreted by the bacteria residing within the biofilm. As the stage progresses, a heterogeneous physicochemical environment—mediated by van der Waals forces and hydrophobic and electrostatic interactions—is developed via the cell-to-cell interaction, which provides the embedded-cell-specialized physiological features. This environment inside the biofilm leads to specialized characters to the residing microbes for differentiation into the mature bacterial community for the final dispersion of the planktonic form [53].

#### *3.1.3 Dispersion*

After the biofilm maturation, some cells of mature biofilm start detaching and disperse into the environment as planktonic cells; this planktonic stage is considered as more sensitive to antimicrobials and immune responses. Therefore, dispersion is a very promising path for biofilm control. This mechanism is cyclic as the released microbial planktonic cells have the potential to again start a new biofilm formation cycle.

#### **4. Approaches to tackle**

The resistance of pathogenic microbes against the known drug is becoming a global problem. These pathogens also acquire resistance toward various drugs and, thus, termed as multidrug resistance (MDR). These MDR bacteria pose a major threat to community and health care as hospital-acquired secondary infections lead to longer stay in hospitals and complications. The common examples are *S. pneumoniae*, *E. faecium*, and *S. aureus*. Thus, active research for novel antibiotics or novel targets such as dodecyl deoxy glycosides, teixobactin, 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl) amino)-2 (hydroxymethyl)propane1,3-diol (DCAP), and malacidins to combat such bacterial infections is the need of an hour. Moreover, natural compounds of either plant origin or microbial by-products as antimicrobials, such as cannabinoids, antimicrobial peptides, and odilorhabdins, are promising aspects of this research. The combinatorial strategy giving synergistic effect is also being used to tackle AMR such as probiotics and bacteriophages. Of these various strategies, this chapter will focus on plant products or phytochemicals that are being researched for their use to combat AMR by targeting various resistance mechanisms such as biofilm, quorum sensing, etc. (**Figure 4**).

Many present studies focus on the strategy for screening various phytochemicals, the method in the identification of their bioactive components, their further investigations, and various approaches that could be adopted to prevent the lethal

**Figure 4.**

*Different types of phytochemicals and their site of action in the bacterial cell.*

consequences of multidrug resistance. Phytochemicals have an immense potential to combat bacterial infections by disrupting the bacterial membrane, inhibition of cell wall or protein synthesis, interference with intermediary metabolism, damage to the synthesis and function of DNA/RNA, and normal cell communication interruption and induction of coagulated cytoplasmic constituents without any pronounced side effect. Major phytochemical classes studied are alkaloids, flavonoids, quinones, tannins, coumarins, terpenes, lectins, and saponins [5, 6]. **Table 2** depicts in detail the structure and common name of phytochemicals with their known mechanism of action.

#### **4.1 Phenolics and polyphenols**

These is a diverse group of aromatic secondary metabolites consisting of flavonoids, quinones, tannins, and coumarins involved in plant defense mechanisms. They exhibit antibacterial properties against various bacteria. Among all flavonols, phenolic acids show maximum activities because they can interact with the cytoplasmic membrane, inhibit bacterial virulence factors including enzymes and toxins, suppress biofilm formation, reduce the pH values, reduce the extracellular polysaccharide activity, exert synergistic effects with conventional antibiotics, and finally can act as EP inhibitors [77].

#### *4.1.1 Flavonoids*

Flavonoids are the main constituent of common edible part of plant, such as fruits, vegetables, nuts, and seeds. These are known to possess various biological activities, such as anti-inflammatory, antioxidant, and antitumor activity, which is now a new

#### *Focus on Bacterial Biofilms*


**Table 2.** *Listofphytochemicals, theirsource,modeofaction,*

 *and the sensitive microbe.*

therapeutic interest. Flavonoids are the pigments that are responsible for colors in fruits, leaves, and flowers and belong to the polyphenol family. Flavonoids show interesting properties in controlling plant growth and development by interacting in a complex manner with the various plant growth hormones [78].

Flavonoid can be classified on the basis of biosynthesis such as chalcones, flavanones, flavan-3-ols, and flavan-3,4-diols, which are both intermediates in biosynthesis and end products that can accumulate in plant tissues. Other classes are only known as end products of biosynthesis such as anthocyanidins, proanthocyanins, flavones, and flavanols. Two additional classes of flavonoids are those in which the 2-phenyl side chain of flavanone isomerizes to the third position, giving rise to isoflavones and related isoflavonoids. Flavonoids have many medicinal activities; therefore, they have been reported to have many useful properties including anti-inflammatory activity, enzyme inhibition, and antimicrobial activity [79, 80].

#### *4.1.2 Quinones*

Quinones are aromatic ring compounds with two ketone substitutions. The major targets of quinones in the microbial cells are cell wall polypeptides, surfaceexposed adhesin proteins, and membrane-bound enzymes. Naphthoquinones is one of the largest groups of plant secondary metabolites that exhibit many biological activities.

#### *4.1.3 Tannins*

Tannins are found in almost all plant parts, and they possess different antibacterial and antifungal activities. The possible mechanism of antimicrobial efficiency is due to the inactivation of cell envelope transport proteins and microbial adhesins [5].

#### **4.2 Alkaloids**

Alkaloids contain variable chemical structures and generally are heterocyclic nitrogen compounds. They tend to exhibit different biological activities, including analgesic effects and antibacterial properties. Therefore, they play a significant role in treating many infectious diseases. The most critical alkaloid groups are aporphines, isoquinolines, quinolones, and phenanthrenes exhibiting suitable antibacterial activities [81]. Their mode of action might be due to the inhibition of repair mechanisms and DNA synthesis, the enzymatic alterations affecting physiological processes, the inhibition of the bacterial nucleic acid and protein synthesis, the modification of the bacterial cell membrane permeability, the damage of the cell membrane and cell wall, the inhibition of bacterial metabolism, and the inhibition of efflux pumps [82–84]. The alkaloids, such as harmane and berberine, results in impaired cell division and ultimately cell death as they possess the ability to intercalate with DNA [85].

#### **4.3 Coumarins**

Coumarins are produced naturally by many plants as well as microorganisms, and chemically, they are aromatic benzopyrones, benzene fused with alpha pyrone rings. Some recent studies also have suggested that coumarins are capable of suppressing

quorum-sensing meshwork of bacterial pathogens and affect their ability to form biofilm and virulence factor formations.

#### **4.4 Terpenes**

Terpenes are naturally occurring hydrocarbons of either cyclic or open-chain structure, such as sesquiterpenes and monoterpenes. Their oils and compounds have several pharmacological activities, such as antitumor, antiviral, antibacterial, antifungal, anti-inflammatory, antiparasitic, and antioxidant properties [86]. Essential oils (EOs) from medicinal plants have shown anti-QS effects, and EOs produced by aromatic plants have been observed to be effective against biofilms. Preferentially, monoterpenes could impact the membrane structures via increasing the permeability and fluidity, thereby changing the topology of proteins leading to the disturbances in the respiratory chain [87].

#### **5. Conclusion**

AMR is becoming a primary cause of morbidity and mortality worldwide, and the resistant microbes are mounting and phenomenal according to the geographic area and the extent of resistance [88]. The infectious agents and diseases that were thought to be controlled by drugs are again emerging with more force against these treatments. The recurrence of resistant microbes, importantly in developing countries, is due to the accessibility of drugs without valid prescription. The golden example is the re-emergence of tuberculosis (in 1980s), which has emerged as multidrug resistant and escalated by HIV infection [89, 90]. The trouble and seriousness in treating MDR strains requires the utilization of a few, some of the time six to seven distinct, drugs. Few mechanisms leading to resistance are the modification of drug targets, the limiting uptake of drug, the active efflux of drug, or the inactivation of drug. Another major well-known resistance mechanism is the biofilm formation.

The protective layers build in the biofilm are a major setback in the treatment of biofilm-related infections, which leads to the ineffectiveness of the existing antibiotics. These layers limit the antibiotic penetration, and thus, the community of sedentary cells survives even in the presence of antibiotics effective against their motile counterparts [53]. Many pieces of evidence suggest that the medicinal plants hold great promise in search of novel antimicrobial agents, and the phytochemicals obtained are very effective in the treatment of infections. Moreover, the plants are cheap, readily available, and almost have minimum side effects. These properties of medicinal plants have gained attention in recent years, for the herbal-based medicines as therapeutics. However, studies are still needed to ensure the safety of antimicrobial phytochemicals and its mechanism of action. Till date, the mechanism of action and the activity related to the structure of phytochemicals have been largely elusive and need further attention [91].

To overcome AMR effectively, all combating new strategies should be practically delivered at all levels, such as community, national, and global levels. Active research to investigate the AMR, its mechanism, strategies to overcome resistance, and leading the novel antimicrobial candidates to clinical practice should be continued. It is important to understand that the distribution, driving force, and the solutions for AMR are different in different countries. Therefore, different approaches are required in high-income countries as compared with low-income countries.

### **Acknowledgements**

Meenakshi Sharma acknowledges the support of grant received from IOE, University of Delhi, Delhi [No./IoE/2021/12/FRP].

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Meenakshi Sharma\*, Pragati Yadav and Deepika Tripathi Genetics of Aging, Dr. B.R. Ambedkar Center for Biomedical Research (ACBR), University of Delhi, Delhi, India

\*Address all correspondence to: meenakshisharma@acbr.du.ac.in

© 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 9**

## The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative Antibiofilm Agents

*Zeuko'O Menkem Elisabeth* 

#### **Abstract**

Biofilms are a community of microorganisms with accretions of their extracellular matrix that attach both to biological or non-biological surfaces, conferring a significant and incompletely understood mode of growth for bacteria. Biofilm formation represents a protected mode of growth of bacteria that allows cells to survive in hostile environments, facilitating the colonization of new areas. This biofilm formation appears to be produced by microorganisms to resist drug action, causing them to become resistant. Therefore, the search for alternative agents is necessary to counteract and reduce this production, creating suitable drugs against these biofilms. Natural products from medicinal plants possess an array of secondary metabolites and bioactive compounds that could have bioactive potentials that inhibit and eradicate biofilms.

**Keywords:** biofilms, inhibition, eradication

#### **1. Introduction**

Biofilms are complex communities of microbes found attached to a surface or may form aggregates without adhering to a surface. Biofilms also display unique properties, such as multidrug tolerance and resistance to both opsonization and phagocytosis, enabling them to survive in hostile environmental conditions by resisting selective pressures [1]. Sometimes, the host immune system is immunocompromised, making it ineffective in clearing biofilms with evidence that immune cells are paralyzed with disrupted phagocytosis capacities or decreased burst responses, lowering the production of reactive oxygen species [2, 3]. Moreso, these communities of microorganisms are unique since they involve several species in a cooperative. The biofilm thus constitutes a microbial society, with its own set of social rules and patterns of behavior, including altruism and cooperation, both of which favor the success of the group with task-sharing behavior. All of these characteristic patterns are orchestrated by chemical or genetic communication. The biofilm thus constitutes a unique way to stabilize interactions between species, inducing marked changes in the symbiotic

relationships [3, 4]. Moreover, biofilms protect invading bacteria against the host's immune system via impaired activation of phagocytes and the complement system [5]. The use of antibiotics such as imipenem and colistin mostly reduces biofilms but does not eliminate the entire biofilm in most cases [6]. Due to their toxicity and side effects, it is not possible to reach the minimal concentration of antibiotics *in vivo*. This chapter describes the mechanisms of bacterial biofilm inhibition and eradication with the search for alternative antibiofilm agents.

### **2. Stages of biofilm formation**

Bacteria form complex multicellular structures called biofilms. Biofilm formation is commonly considered to occur in four main stages [7]: (1) adhesion of planktonic cells, (2) microcolony formation, (3) biofilm maturation and (4) detachment (also termed dispersal) of bacteria, which may then colonize new areas (**Figure 1**). Sessile bacterial cells exist in the stationary or dormant growth phase, exhibiting phenotypes distinct from planktonic bacteria [8]. In biofilms, bacteria display exceptional resistance to environmental stresses, especially antibiotics. This makes biofilms a major public health problem, as they account for 60–80% of human microbial infections [9]. The different stages in biofilm formation involve different environments, as shown in **Figure 1**.

#### **2.1 Attachment of planktonic cells**

Biofilm formation starts with the attachment of microbial cells to abiotic or biotic surfaces. These biotic surfaces are living tissues such as endothelial lesions, mucosae, and nervous tissues, while abiotic surfaces are non-living cells including indwelling devices, prostheses, clinical environment surfaces, vascular and urinary catheters [10]. This initial attachment depends on the motility and adhesins expression

**Figure 1.** *Stages of biofilm development.*

*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

(microbial factors). The extension is influenced by the planktonic strains migrating to specific sites to either adhere to existing lesion or surface or directly cause tissue infection [11]. The physiology of the cell's changes affecting the surface membrane proteins making the removal of the attached cells laborious, necessitating the action of specific enzymes, sanitisers and detergent. The physicochemical properties of the surfaces (biotic and abiotic) controls microbial adherence making biofilms independent of surface extension [12].

#### **2.2 The extracellular polymeric substance (EPS) matrix**

The genes responsible for attachment and matrix assembly are activated when stimulated by factors such as population density and nutrient limitation [7]. The EPS matrix is composed of a mixture of biopolymers. The matrix produced is different and is surface- or medium-specific and differs between *in vivo* and *in vitro* conditions [11]. EPS is produced by planktonic cells, resulting in enhanced extension [13, 14].

#### **2.3 Accumulation of multi-layered clusters of microbial cells**

The microbial assembly development process results in simultaneous bacterial aggregation and growth. This disposition is entrenched as a distinct model with the aid of a confocal laser microscopy. The distinct model indicates that active metabolism is exhibited by the cells in the outer biofilm layers while those deeper inside the biofilm downregulate their metabolism, making them inactive in a persistent state [12, 15, 16]. This accumulation mostly involves intercellular adhesion. Specific genes and polysaccharide intercellular adhesin (PIA) are responsible for their accumulation on a polymer surface. However, the purification and structural analysis of these clustered microbial cells indicate the presence of two forms of that PIA, major polysaccharide I (>80%) and a minor polysaccharide II [17].

#### **2.4 Biofilm maturation**

In the biofilm maturation phase, the canals are created in the biofilm structure, allowing gradient-based passage of nutrients and signaling molecules based on their metabolic state, favoring the organized agglomeration and differentiation of cells [7, 12, 18]. These gradient passages are necessary for nutrients to enter the cells inside the biofilm layers. Biofilm structuring is a disruptive process causing the detachment of cell clusters controlling the biofilm invasion during *in vivo* biofilm infection leading to systemic dissemination [19].

#### **2.5 The disentanglement and scattering of planktonic bacteria**

The biofilms grow more thicker and compact on the interior, while external layers begin separating. The disentanglement and scattering occurs as a results of nutritional imbalance with insufficient carbon accessibility, increasing the synthesis of extracellular polymeric substances [20]. The scattered cells or clusters travel as septic emboli colonizing new sites, causing infection with possibly novel biofilms [2]. The dispersed cells form biofilms as a result of growth and may return quickly to their normal planktonic phenotype.

### **3. Bacterial biofilm structure, characteristics and chemical composition**

#### **3.1 Bacterial biofilm structure and characteristics**

The basic structural units of a biofilm are microcolonies and separate communities of bacterial cells embedded into the EPS matrix. These microcolonies are in most cases mushroom-shaped or rod-like and can consist of one or more types of bacteria. The microcolonies consist of 10–25% cells and 79–90% EPS matrix depending on the bacterial type. This EPS matrix protects biofilm cells from various environmental conditions, such as UV radiation, changes in pH values, draining and temperature. There are channels through which water flows between microcolonies. These water channels function in distributing nutrients to microcolonies and receiving harmful metabolites as a simple circulatory system. Biofilms under different hydrodynamic conditions, such as laminar and turbulent flow, show changes in biofilm structure depending on the flow type. In laminar flow, bacterial microcolonies become round, and in turbulent flow, they extend in the downstream direction [21].

#### **3.2 Chemical composition**

The matrix of extracellular polymeric substances (EPSs) are self-secreted substances that keep bacterial cells in a compact structure attaching them to surfaces which makes the physical aspect of a biofilm [16]. The major constituent of the biomass of the biofilm is the hydrated EPS ranging between 2–15% of the total biofilm mass [4]. The EPS contains mostly extracellular DNA (eDNA), polysaccharides, proteins and lipids (**Table 1**) [22]. The EPS matrix exhibit three important characteristic features which are enhancing antimicrobial resistance, nutrient capture and social cooperation [14]. The tissues of higher organisms are similar to biofilms structures which are architecturally different and extremely heterogeneous in gene expression, all participating to the resistance mechanisms of biofilms [5, 23].


*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*


#### **Table 1.**

*Chemical composition of biofilms.*

Thus, the enzymatic activity within the biofilm provides nutrients to bacterial cells and promotes biofilm reorganization and dispersal [29]. In addition to polysaccharides and proteins, eDNA also contributes to the structural integrity of the matrix. The contribution of this component to the three-dimensional structure of the biofilm differs greatly among species [29]. The EPS matrix has an important role in biofilm formation, progression and durability as a result to its multiplex constitution and organization. It is also a protective barrier against external factors, a source of nutrients, enzymes and an intercellular connector. These unique features of the matrix participate in the high antimicrobial forbearance and/or recalcitrance of biofilms [15, 29].

#### **4. Factors influencing bacterial biofilm formation and development**

The formation of biofilms is a dynamic and complex process that includes the initial attachment of bacterial cells to the substratum, physiological changes within the microbe, multiplication of adhered cells to form microcolonies and finally biofilm maturation [30]. Biofilm-associated bacteria demonstrate distinct features from their free-living planktonic counterparts, such as different physiologies and high resistance to immune systems and antibiotics that render biofilms a source of chronic and persistent infections [2, 31]. It is known that the change in phenotype from planktonic to the sessile form occurs in response to changes in environmental conditions [3].

**Figure 2.** *Factors affecting biofilm formation.*

**Environmental factors**, such as nutrient level, temperature, pH, and ionic strength, can influence biofilm formation, as shown in **Figure 2** [30]. These factors influence bacterial adhesion; cell surface properties, such as hydrophobicity, flagellation, and motility; surface properties, such as hydrophobicity and roughness; and environmental factors, such as temperature, pH, availability of nutrients and hydrodynamic conditions [21, 30, 32]. The cell surface properties, specifically the presence of extracellular appendages, such as fimbriae and flagella, the interactions involved in cell-to-cell communication and EPS production, such as surface-associated polysaccharides or proteins, possibly provide a competitive advantage for one organism in a mixed microbial community [3, 12]. Bacteria with hydrophobic properties are more likely to attach to surfaces than hydrophilic bacteria; however, the attachment of biofilms will occur readily on surfaces that are rough, hydrophobic, and coated by surface conditioning films.

**The physicochemical properties** of the substratum, such as texture (rough or smooth), hydrophobicity and charge, can also be modified by environmental conditions, such as pH, temperature, and nutrient levels [4, 10, 30]. In aquatic environments, the rate of microbial attachment can be increased by increasing the velocity of the flow, water temperature or nutrient concentration, providing that these factors do not exceed critical levels [6, 15].

**Quorum Sensing**: This is a bacterial cell–cell communication process that involves the production, detection, and response to extracellular signaling molecules called autoinducers (AIs) [33]. In Gram-positive bacteria, oligopeptides are used as signaling molecules to form biofilms, and quorum sensing is used for intraspecies communication. Quorum sensing controls processes such as bioluminescence, sporulation, competence, antibiotic production, biofilm formation, and virulence factor secretion [34]. Three main types of quorum sensing systems exist:


*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

• Autoinducer-2 (AI-2) system in both gram-negative and positive bacteria [34].

The acyl homoserine lactone-dependent QS system is a prominent cellular signaling molecules of homoserine lactones involved in quorum sensing regulation used primarily by Gram-negative bacteria. The AHL molecules have the homoserine lactone ring in common varying in length and substituents, synthesized by a specific AHL synthetase. The concentration of AHL contributes to bacterial growth. Autoinducing peptide (AIPs) are signal molecules secreted by membrane transporters and synthesized by Gram-positive bacteria. The AIPs bind to the histidine kinase sensor phosphorylating, consequently altering gene expression as the environmental concentration of AIPs augments [32, 35, 36]. These genes control the formation of innumerable toxins and decomposable exoenzymes [21, 36, 37]. The microorganisms can sense and translate the signals from distinct strains in AI-2 or autoinducer-2 interspecific signals, catalyzed by LuxS synthase as part of their cooperation and communication strategies [6, 25, 38]. Moreover, LuxS is involved in the activation of the methylation cycle and has been demonstrated to control the expression of hundreds of genes associated with the microbial processes of surface adhesion, detachment, and toxin production [24, 39, 40]. The QS system is a paramount target for the treatment of biofilm-associated infections [12].

#### **5. Biofilm-producing bacteria and infections**

Biofilm formation is present in approximately 65% of all bacterial infections and approximately 80% of all chronic infections according to the statistics of the National Institute of Health (NIH) (**Table 2**) [12]. Indwelling devices by bacteria settlement was associated with infections in 4% of the cases when pacemakers and inhaler were utilized and 2% in breast implant cases [35]. The device-related infections were estimated to be about 40% in ventricular-assisted devices, 2% in joint prostheses, 4% in mechanical heart valves and 6% in ventricular shunts [12, 25]. The heart infection (infective valve endocarditis) occurs as a result of the adherence of bacteria cells to the endothelium. The most frequent microbes being staphylococci and streptococci, members of the HACEK group, gram-negative bacteria and fungal strains [42]. The implanting of the endothelium generally occurs from colonization or the infection of different tracts (the genitourinary and gastrointestinal tract) or through the direct crossing of the skin barrier, either due to wounds or through injecting drugs [41]. Some biofilm-driven infections are chronic wounds, diabetic foot infections, and pulmonary infections in patients with cystic fibrosis and specific bacterial species (**Table 2**) [21, 37, 43].

#### **6. Mechanisms of biofilm inhibition and eradication**

i.Antibiofilm molecules and their mechanism of action:

The material matrix of implanted medical devices and biomaterials provides an ideal site for bacterial adhesion promoting mature biofilm formation [3]. Methods that prevent bacterial attachment to these materials represent a preventative strategy. The most common method for preventing bacterial extension is a surface modification (**Table 3**). The exterior surface of the implanted medical device or biomaterial


**Table 2.**

*Examples of bacterial species involved in biofilm formation with their biological effects.*

is altered, either directly or with the aid of a cover-producing barrier that is hostile to bacteria [45, 46]. This strategy has shown significant promise for preventing biofilmrelated infections resulting from orthopedic implants. Thus, the area of surface modification to prevent biofilm formation is a large field [46–48]. The use of small molecule biofilm inhibitors is another approach used to prevent biofilm formation (**Figure 3**). The antibiofilm properties of a biofilm inhibitor are often employed to passivate the surface of an implanted medical device or biomaterial [41, 49, 50]. The use of biofilm inhibitors is one of the largest areas in biofilm remediation research, with a plethora of unique biofilm inhibitors currently described (phenols, imidazoles, furanone, indole, bromopyrrole) [51].

Anti-biofilm molecules are diverse compounds that inhibit biofilm formation. The identified anti-biofilm compounds are mainly isolated from natural sources, and some synthetic compounds, chelating agents, and antibiotics possess antibiofilm activity. The different antibiofilm molecules along with their target microorganisms are listed in **Table 2**. These antibiofilm molecules follow different mechanisms to inhibit biofilm formation in different bacteria, as listed in **Table 3**.

*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

**Figure 3.**

*The different steps in biofilm formation.*



#### **Table 3.**

*Mechanism of biofilm-mediated antimicrobial resistance.*

#### ii.Using Natural Products:

The formation and development of biofilms is a complicated procedure involving different stages that can be the target of natural antibiofilm agents for the prevention of biofilm development. Natural anti-biofilm agents either act solely or synergistically by diverse mechanisms.

There are five broad classes of natural compounds that have high antibiofilm properties, including phenolics, essential oils, terpenoids, lectins, alkaloids, polypeptides, and polyacetylenes [52]. Phenolics are a group of compounds. It has seven subclasses, which include phenolic acids, quinones, flavonoids, flavones, flavonols, tannins, and coumarins, out of which tannins, specifically condensed tannins, have anti-biofilm activity. These compounds act on biofilms by six main mechanisms, such as substrate deprivation, membrane disruption, binding to the adhesin complex and cell wall, binding to proteins, interacting with eukaryotic DNA, and blocking viral fusion [52, 53].

*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

Many bioactive compounds from medicinal plants for the discovery of novel natural antibiofilm compounds are ongoing. The antibiofilm properties of Indian medicinal plants were studied with Cinnamomum *glaucescens (Nees) Hand.-Mazz, Syzygium praecox Roxb. Rathakr. & N. C. Nair, Bischofia javanica Blume, Elaeocarpus serratus L., Smilax zeylanica L., Acacia pennata (L.) Willd., Trema orientalis (L.) Blume, Acacia pennata (L.) Willd., Holigarna caustica (Dennst.) Oken, Murraya paniculata (L.) Jack, and Pterygota alata (Roxb.)* R. Br. extracts have promising antibiofilm activity against *S. aureus* [36, 53, 54]. Phytochemicals inhibit the quorum sensing mechanism mainly by blocking quorum sensing inducers such as AHL, autoinducers, and autoinducer type 2. Garlic extracts play a vital role in the inhibition of quorum sensing signaling molecules of *Pseudomonas* and *Vibrio* spp. Biofilms [5, 36, 52, 55]. Phytochemicals also play a significant role in inhibiting bacterial adhesion and suppressing genes related to biofilm formation. Biofilm development at the initial stages can be outlined by interfering with the forces (van der Waals force of attraction, electrostatic attraction, sedimentation and Brownian movements) that are responsible for the support of bacterial attachment to various surfaces [56]. Some phytocompounds have the potential to interfere with the extension along with the capability to stop the accessibility to nutrients essential for adhesion and bacterial growth. An alkaloid (norbgugaine) had a significant effect on *P. aeruginosa* biofilms by preventing adhesion due to loss of cell motility [9, 24, 55, 57]. A very recent study on *Adiantum philippense* L. crude extract showed a promising role in decreasing the content of biofilm exopolysaccharides [44, 58, 59]. It was reported that *A. philippense* L. crude extract restrained biofilms at the initial stages by targeting adhesin proteins, destroying the preformed biofilms inhibiting EPS assembly. Diverse group of phytocompounds especially polyphenols such as 7-epiclusianone, tannic acid, and casbane, have been identified and proved to protect cell surface. Members of Enterobacteriaceae express curli, an amyloid fiber on the cell surface that helps in attachment to characteristics and cell aggregation and enhances biofilm formation as well as a cellular invasion [41, 49, 60]. The phytocompounds of curlicide and pilicide nature can be exploited in therapeutic strategies of Enterobacteriaceae biofilm prevention [57, 61, 62]. These phytocompounds with fewer side effects are better therapeutic agents for biofilm-related infections, but recent reports suggest a combined approach that is always better than the individualistic approach [24, 44, 50, 51]. A few plant-based antimicrobials with the potential of anti-biofilm activity are summarized in **Table 4** [53].

#### **7. Conclusion**

Biofilm infections are highly resistant to antibiotics and physical treatments. Many strategies support biofilm antibiotic resistance and tolerance, such as persistent cells, adaptive responses, and limited antibiotic penetration. Thus, the underlying mechanisms of antibiotic forbearance and recalcitrance in biofilms are controlled by genes. In human infections, most organized bacterial cells gradually induce immune responses to form biofilms causing chronic infections leading to tissue destruction with permanent pathology. Therefore, biofilms arrangement is a vital perturbation in medical care environment. The exploration of alternative treatment procedures for biofilm-associated infections is of utmost importance. There are little novel and efficient antibiotic strategies which are scattering of biofilms, merging of antimicrobials with quorum sensing inhibitors, and a mixture of these procedures. Although the mentioned anti-biofilm strategies are key research areas, they are still in their infancy


#### *Focus on Bacterial Biofilms*


**Table 4.**

*Anti-biofilm activity of phytocompounds and their mechanism of action [53].*

*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

and has to be improved to upgrade and implement the strategies. The administration of a single antibiotic is often not enough to eradicate bacterial invasions, and a high concentration of the antibiotic can be extremely toxic. Also, some natural compounds as well as quorum sensing inhibitors, may be toxic and less effective. A possible solution might be the coadministration of antibiotics with antibiofilm peptides that allow the use of low antibiotic concentrations. New anti-biofilm molecules from natural substances with low or no harmful effects and synergistic effects with commonly used antibiotics are necessary. Moreso, natural products from medicinal plants and quorum sensing inhibiting compounds with little or no toxic effects will be of great importance in the fight against biofilms.

### **Author details**

Zeuko'O Menkem Elisabeth1,2

1 Faculty of Health Sciences, Department of Biomedical Sciences, University of Buea, Buea, Cameroon

2 Faculty of Science, Antimicrobial and Biocontrol Agents Unit, Laboratory for Phytobiochemistry and Medicinal Plant Studies, Department of Biochemistry, University of Yaoundé I, Yaoundé, Cameroon

\*Address all correspondence to: mllemenkem@gmail.com; zeukoo@yahoo.com; zeuko'o.elisabeth@ubuea.cm

© 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.

*The Mechanisms of Bacterial Biofilm Inhibition and Eradication: The Search for Alternative… DOI: http://dx.doi.org/10.5772/intechopen.104772*

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#### **Chapter 10**

## Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms

*Sankar Veintramuthu and Selliamman Ravi Mahipriya* 

#### **Abstract**

Biofilm may be a consortium of microbial species where the cells of microbes attach to both life form and inanimate surfaces inside a self-made matrix of extracellular polymeric substance (EPS). Biofilm matrix surrounding the polymicrobial environment makes them highly resistant to harsh conditions and antibacterial treatments. The two significant factors that differentiate planktonic from biofilm resident microbes are EPS containing a variety of macromolecules and a diffusible molecule for transferring signals known as quorum sensing (QS). Against this backdrop of microbial resistance and cell signaling, different approaches have been developed to interfere with the specific mechanisms of intracellular and extracellular targets that include herbal active compounds and synthetic nanoparticles. This chapter outlines the features of biofilm development and the approaches with the evidence that can be incorporated into clinical usage.

**Keywords:** biofilm, antimicrobial resistance, quorum sensing, herbal compounds, nanoparticles

#### **1. Introduction**

In seventeenth century, Antonie von Leeuwenhoek saw microbial aggregates on the scrapings of the plaque from his teeth that was termed as "biofilm" by Bill Costerton in 1978 [1]. The biofilms were not characterized for their physical and chemical properties until the end of 1960 [2]. The evolution of scanning electron microscopy and transmission electron microscopy allowed for identifying the biofilm from wastewater treatment plant [3] after when Heukelekian and Heller identified the *"Bottle effect"* on marine microbes where there is a significant difference in the microbial population between *in situ* and *in vitro* due to environmental or man-made changes*.* Biofilm is an aggregation of microbially derived sessile communities having various bacterial colonies or individual cells in the group, which adheres to the surface. This group of cells attaches on an extracellular polymeric substance (EPS), a matrix that is mostly comprised of environmental DNA (eDNA), proteins, and polysaccharides, which provides significantly excessive resistance to antibiotics [4]. Bacterial biofilm can be formed in response to various factors such as high salt

#### **Figure 1.**

*Biofilm life process. (1) Planktonic bacteria attaches to the exterior face. (2) Adhesion, irreversible attachment occurs at this phase. (3) EPS is secreted and results in a matrix that forms the basis for biofilm's structure and initiates the onset of biofilm maturation. (4) The biofilm becomes completely matured, with the tower-like structures dispersed with water channels for the movement of oxygen, nutrients, and for discharging waste products.*

concentration, restricted nutrients, high pH and pressure, and UV radiation. Biofilm life process is depicted in **Figure 1**.

The biofilm formation can be described in three steps:


#### **2. Biofilm: a threat to antibiotics and infections caused by biofilm**

Around 80% of chronic and periodic microbial infections in the human bodies are caused by bacterial biofilm. Bacteria's present inside the biofilm aids to the chronic phase of infection, when released from the biofilm can cause an acute phase of infection [6]. The infections caused by bacterial biofilm can be placed in two broad categories such as device and non-device-associated infections. They can develop on or inside medical devices that are built in body such as central venous catheters, mechanical heart valves, pacemakers, urinary catheters, which cover both Gram-positive and Gram-negative bacteria or yeasts. These organisms on the medical devices may cause blood stream and urinary tract infections in the patient [7]. **Table 1** shows the microbial species that colonizes the devices based on the type of medical device and time taken for their action.

Microbial biofilm show 10–1000 times more antibiotic resistance than the planktonic species [12]. Bacterial biofilm offers huge evolutionary advantage for the *Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.104470*


**Table 1.**

*Medical devices and associated biofilm organisms.*

bacteria including changes in environmental pH, resistance to antimicrobial agents, and phagocytic attack [13].

#### **3. Quorum sensing (QS) and interaction**

The bacterial cells have intercellular communication that is delivered through the extracellular signaling molecules known as autoinducers. The collection of signaling molecules enables individual bacterial cells to analyze the total number of bacteria, that is, cell density known as quorum sensing. In low-density planktonic populations, bacteria releases low-molecular-weight, highly diffusible, signal molecules (autoinducers, such as oligopeptides in Gram-positive bacteria and N-acyl-L-homoserine lactones in Gram-negative bacteria) at very low levels to produce changes in gene expression. When critical mass of bacterial population becomes high, the concentration of autoinducer molecules increases in the EPS followed by allowing individual bacteria to sense the presence of other bacterial species [14].

#### **4. Conventional treatments and antimicrobial resistance**

Biofilms are considered to be important owing to their potency in showing resistance toward antibiotics and antifungals. Once routed within the wound infection, biofilm shows enhanced tolerance to conventional treatments. Antibiotics work by deranging the cell wall of bacteria and affecting the DNA replication, repair, and protein synthesis. Apparently biofilm has various mechanisms through which they resist the effectiveness of antibiotics [15]. The primary defense mechanism involves EPS, which is capable of restricting the permeability of antibiotics into the cell thereby trapping them in the pores, followed by acidic internal environment and lack of oxygen. Ultimately, the lysis of genetic component can be carried between the cells to extend antimicrobial resistance.

The persister cells within the biofilm have the potency to restrict the effects of antibiotics targeting the cell division [16]. **Figure 2** illustrates the mechanism through which biofilm develops resistance to conventional antibiotics [17].

The resistance can be developed through persistent cells, phenotype of the biofilm, inhibition in antibiotics penetration, production of enzymes that resist the action of antimicrobial agents.

**Figure 2.** *Antibiotic resistance associated with biofilm.*

### **5. Nanoparticles (NPs) as antibiofilm agents**

Nanotechnology is fascinating, which likely benefited the field of biomedical and became widely conceded for the treatment of various diseases. Numerous resistance mechanisms set biofilm as one of the major disputes in infection treatment, which can be addressed by the strategy of using nanoparticles. NPs have two or three dimensions in the size range of 1 to 100 nm. They are of various types based on their size, shape, and composition [18]. Their higher surface area built them as suitable drug career, which has the capability to immobilize the compounds on their surface to increase their solubility and targeted delivery [19]. They can be of two types, polymer NPs and metallic NPs. Polymer NPs also possess the advantage of retaining the drug inside

*Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.104470*

the cavity and delivers the drug at the target area in either entangled or immobilized form. Reports suggest that NPs disrupt the integrity of biofilm by interacting with EPS, eDNA, proteins, lipids, and biofilm release reactive oxygen species (ROS) on interaction with NPs that can damage the cell envelope, cell membranes, cell structures, and biomolecules of the microbes. **Figure 3** represents the general mechanism involved in combating biofilm through NPs [20].

The nanoparticles can restrict biofilm by disruption electron transport between cell membrane, damaging the peptidoglycan layer, breaking through the cell membrane, denaturation of proteins, and DNA damage.

#### **6. Synthesis of nanoparticles**

Nanoparticles can be synthesized in laboratory broadly using two different approaches, that is, bottom-up and top-down techniques. The top-down approach implies breaking the bulk material into nanosized structures, which is based on miniaturizing the bulk substance through fabrication process and produces the NPs of appropriate properties. Bottom-up technique is an alternative approach because it creates less waste and involves building up of a material from the bottom [21].

#### **7. Types of nanoparticles**

Polymeric NPs can be engineered to release antibiotics, antibacterial agents, and bacteriostatic peptides or by modifying their chemical surface. The antibacterial activity of these organic NPs is due to polycationic groups accountable for cell damage through ion exchange interaction between bacteria and polymer surface with charges [22]. Metals are used in the synthesis of nanoparticles because of their antibacterial property broadly used in managing infections. Metallic nanoparticles can exert physical disruption to bacterial biofilms. **Table 2** enlists the types of metallic nanoparticles and their potential antimicrobial property [23].

The pH of micro-environment, magnetic field, or light can be used to turn on the nanomaterials or transform it to more active species enhancing their antibiofilm activity. These are often metallic nanoparticles due to their broad-spectrum


#### **Table 2.**

*Metallic nanoparticles and their antimicrobial property.*

antimicrobial activity and rich surface chemistry [24]. For negatively charged bacteria, the adhesion property rises because of the positively charged surface of NPs and the binding takes place through electrostatic interactions and Van der Waals interaction especially to cell membrane proteins [25].

#### **8. Metal NPs against biofilm**

CuO NPs inhibit formation of biofilm that was studied by *Agarwal et al.* that concluded eradication of biofilm formed by MRSA and *E. coli* with the exposure period of 4 days to CuO NPs at the concentration of 50 μg/ml [26]. ZnO NPs can exhibit antibacterial action between the concentration of 20–500 μg/ml for *E. coli* and *S. aureus* that can be enhanced by additional physical exposure and amplified by ultrasound [27]. MgO NPs can act against Gram-positive and Gram-negative bacteria, bacterial spores, and viruses at higher concentration of 100–1200 μg/ml. TiO2 NPs can destroy biofilms of both Gram-positive and Gram-negative bacteria but the latter being more sensitive due to the sturdy layer of peptidoglycan that increases the absorption of reactive radicals [28]. However, their toxicity to humans and environment outweighs their advantages. Gold and silver NPs offer huge advantages such as higher surface area to volume ratio, small size, amenability, cheaper method of synthesis. Extensive research studies have been accomplished over the recent areas involving AgNPs and AuNPs. Three important steps involved in their antimicrobial action are a) interaction with biofilm when it comes into contact with the surface, b) subsequent penetration of NPs into the cell based on this interaction, and c) NPs as a whole or ions (Au+ and Ag+ ) reacts with cellular and biofilm components. The factor that plays significant role in penetration includes particle size, surface chemistry, surface charge, and concentration.

#### **8.1 Silver nanoparticles: a biofilm buster**

Silver has been used since remote time because of their therapeutic properties and their antibacterial activity and also explored through extensive research in medical field. Topical ointments and creams contain silver for treating burn wound infection. Several approaches are involved in synthesis of AgNPs, which include the use of microorganisms and plants but one of the easiest and convenient methods is through chemical synthesis [29]. **Table 3** enlists some of the sources that can be used for the synthesis of AgNP.

The synthesis of AgNPs can also be done by utilizing other physical methods such as evaporation-condensation and laser ablation, UV-initiated photo reduction, electrochemical synthetic method, irradiation methods [35]. Studies concluded that geometric mean diameter, shape, pH, and source for synthesis of AgNPs influence their efficiency. The synthesized AgNP can be characterized using UV-visible spectroscopy, X-ray diffraction, scanning electron microscopy, transmission electron microscopy for their structural properties. Although AgNPs were remarkably noted for their potential in pathogenic control, their effect on EPS has not been given sufficient attention [36].

#### **8.2 Natural compounds as antibiofilm agents**

Herbal compound aids the determination of novel constituents with interesting structures and biological activity. The antibiofilm properties of natural products rely on the inhibition of polymer matrix formation, resisting cell adhesion and attachment, breaking in ECM generation, and reducing virulence factors generation,

*Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.104470*


#### **Table 3.**

*Sources used for synthesis of AgNP.*

thereby obstructing QS network and biofilm development [37]. The natural compounds that possess antibiofilm properties can be broadly classified into phenolics, essential oils, terpenoids, lectins, alkaloids, polypeptides, and polyacetylenes [38]. They either act merely or synergistically by different mechanisms. Various researches have been carried out with natural products that are discussed below:

#### *8.2.1 Garlic*

*Allium sativum* L has been extensively used in treating numerous diseases such as wound infection, malaria, common cold, sexually transmitted diseases [39]. Garlic possibly has a QS-interfering compound. DNA microarray analysis disclosed that Ajoene, a garlic-derived sulfur-containing compound, restricted QS-regulated gene expression in *P. aeruginosa*. Reasonable designing and biological screening of all compounds from garlic was carried out, resulting in the identification of a potent QS inhibitor N-(heptylsulfanylacetyl)-l-homoserine lactone. This element was found to disrupt the QS signaling by inhibiting transcriptional regulators LuxR and LasR. Recent studies have proved the antiswarming, anti-adherence, and antibiofilm activity of the aquatic extracts of garlic [40]. Ethanolic and methanolic extracts of garlic against six different bacterial species *(Escherichia coli, Staphylococcus aureus, Bacillus cereus, Streptococcus pneumoniae, Pseudomonas aeruginosa,* and *Klebsiella pneumonia*) show antibacterial activity at the concentration of 125–500 mg/mL through disc diffusion, and the *A. sativum* L extracts were potent enough restrict biofilm structures and the concentrations of each extract depend on the inhibitory effect [41].

#### *8.2.2 Onion*

Extracts of onion contains pharmaceutical properties that can be used as one of the promising therapies for the treatment of neoplastic, metabolic, and immunological diseases, which also involves bacterial, viral, and other fungal infections [42]. The anti-adherence, antibacterial, antibiofilm, and antimotility role of aqueous extracts of fresh or powdered onion and onion oil were studied from which the aqueous extracts of fresh and powdered onion showed more powerful inhibitory effects on biofilm than onion oil on the growth of both Gram-positive and Gram-negative bacteria [43]. Systematic assessment of quercetin, total phenolics, flavonoids, antioxidants, antibacterial, and antibiofilm or antibiofouling properties of methanolic extracts of fresh and aging onions of six varieties was studied by Kavitha *et al.,* which concluded that the

onions that had been stored for 3 months showed the best antibiofilm effects. The red variety of *Allium cepa* extract was found to have higher antimicrobial activity when compared with the white and yellow varieties. At the range of about 50 μg mL–1, the extracts were observed to reduce the biofilm growth of *P. aeruginosa* and *S. aureus* [44].

#### *8.2.3 Rhubarb*

Rhubarb is one of the most traditionally available medicinal materials included in Pharmacpoeia due it its bacteriostatic and anti-inflammatory properties. Emodin is the bioactive compound that has the ability to reverse multi-drug resistance. Natural emodin is obtained from Rheum palmatum L., Rheum tanguticum Maxim ex Balf, and Rheum ocinale [44]. Yan *et al.* studied the activity of emodin against *S. aureus* biofilm and confirmed the molecular mechanism that they decrease the release of eDNA and represses the biofilm-forming genes such as *cidA, icaA, dltB, agrA, sortaseA,* and *sarA* [45].

#### *8.2.4 Banana*

Studies concluded the antibacterial properties of banana in traditional medicine across the world. Generally, stem juice, flowers, and fruits of the banana plant are utilized for treating diarrhea and dysentery [46]. Vijayakumar *et al.* studied the antibiofilm properties of *Musa acuminata Colla.* against *P. aeruginosa* and described the mechanism of inhibiting the secretion of biofilm proteins and cell surface hydrophobicity productions [47].

#### *8.2.5 Ginger*

Ginger had been used in food and medicine for thousand years with the evidence demonstrating that it has antibacterial activity against the commercially available


#### **Table 4.**

*Natural compounds with antibiofilm activity.*

*Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.104470*

antibiotics by inhibiting QS signaling pathway [48]. Kim *et al.* initially investigated the inhibition of biofilm with ginger extract in *P. aeruginosa*. The biofilm assay demonstrated that the ginger extract decreased the biofilm development by 39–56% by reducing the formation of extracellular polymeric substances (EPS), which was associated with the suppression in secondary messenger, bis-(3c-5c)-cyclic dimericguagranosine [49]. Studies have shown that ginger essential oil at the biofilm inhibitory concentration (BIC) of 1.56 μL mL−1, *S. aureus* had 94% inhibition of biofilm, and at BIC 0.78 μL mL−1 *Enterococcus faecalis, K. pneumonia, and E. coli* showed 91, 89, and 83% inhibition of biofilm [50].

**Table 4** enlists the natural compounds with antibiofilm activity.

#### **9. Conclusion**

In recent times, the concept of biofilm has influenced almost every treatment step of infection due to high level of protection against antibiotics and antimicrobial agents, being the thrust to human medical management. Hence, there is a crucial demand to develop novel strategies to surpass the antibiotic resistance after understanding the clear mechanisms behind it. The plant compounds, phytochemicals, and nanoparticles can be fused with antimicrobial agents, which have substantial research evidence for their antibiofilm effects through their synergism. In spite of the clinical trials being done on such compounds, further study is required to prove their safety and effectiveness to support the clinical systems.

#### **Acknowledgements**

We would like to thank our principal Dr.M. Ramanathan D.Sc., for providing us with sufficient facilities to complete this book chapter.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Focus on Bacterial Biofilms*

#### **Author details**

Sankar Veintramuthu\* and Selliamman Ravi Mahipriya PSG College of Pharmacy, Coimbatore, Tamil Nadu, India

\*Address all correspondence to: sankarv@psgpharma.ac.in

© 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.

*Approaches to Enhance Therapeutic Activity of Drugs against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.104470*

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#### **Chapter 11**

## Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis on Medicine

*Saulo Henrique Rodrigues, Marcelo Assis, Camila Cristina de Foggi, Andréa Cristina Bogas, Mariana Ottaiano Gonçalves, Lavinia Cipriano, Elson Longo, Evandro Leite de Souza and Cristina Paiva de Sousa*

#### **Abstract**

The growing antimicrobial resistance and persistence of pathogenic microorganisms in infections–particularly in nosocomial infections–have become a major problem for public health worldwide. One of the main causes of these issues is the formation of biofilms, which are microbial communities associated with extracellular polymeric substances (EPS) that form a slimy extracellular matrix, causing the bacteria to become more tolerant to usual drugs in these structures. Thus, the search for new antibiofilm compounds is part of a strategy to deal with this problem. Endophytic microorganisms such as bacteria and fungi, mutualistically associated with plants, are sources of compounds with biological properties, including antimicrobials, and can be important allies in the synthesis of antibiofilm. These secondary metabolites can interfere with cell-to-cell communication and cell adhesion ability, promoting the dispersal of bacterial colonies and affecting biofilm. Since endophytes are cultivable in laboratory conditions, these microorganisms are environmentally friendly, as they do not contribute to pollution, are easy to handle and are produced on a large scale. Furthermore, metabolites from endophytes are of natural origin and may contribute to the reduced use of synthetic drugs. Considering these aspects, this chapter will focus on the characterization of endophytic microorganisms as potential active sources of antibiofilm and antimicrobial compounds with applications in medicine.

**Keywords:** endophytes, biofilms, antimicrobial resistance, antibiofilm activity, anti-quorum sensing activity

#### **1. Introduction**

One of the most worrisome problems in public health nowadays is antimicrobial resistance and multi-resistance (AMR and MDR). This natural process has been

accelerated by the unrestrained and irrational use of antimicrobials, such as antibiotics and antifungals [1]. One of the biggest challenges to overcome this problem is to equate the speed of development of new drugs with the adaptation of pathogens to current drugs, since the development of new compounds does not follow the growing resistance of microorganisms [2]. In addition, there is a large number of resistant pathogens involved in healthcare-related infections (HAI), making the treatment of diseases more difficult and expensive as well as increasing mortality and morbidity rates [3, 4]. Among the most common pathogens in nosocomial infections, bacteria from the ESKAPE group–an acronym used to refer to *Enterococcus faecium*, *Staphylococcus aureus, Klebsiella pneumoniae*, *Acinetobacter baumannii*, *Pseudomonas aeruginosa,* and *Enterobacter* are the most problematic, as they have mechanisms potentially involved in antimicrobial resistance [5]. Nevertheless, it is argued that the main cause of resistance may not be related to the classic mechanisms of microbial adaptation, but to the formation of a structure called biofilm [6].

Biofilms are organized, complex and dynamic communities of microorganisms adhered to a biotic or abiotic surface and protected by a polymeric extracellular matrix, which is composed of nucleic acids, polysaccharides, lipids, and proteins, generally called polymeric extracellular substances (EPS) [7]. This characteristic of adhering to different surfaces makes biofilms well disseminated in nature and easily found in different environments, including hospitals [8]. What makes biofilms so problematic for health is the fact that they allow the microorganisms inside them to thrive and persist in their environment. When related to infections, these structures tend to increase the tolerance of pathogens to treatments with conventional antimicrobial drugs, as they often prevent these compounds from reaching target cells [9]. Additionally, biofilms harbor different species of microorganisms that when acting together can lead to the development of chronic diseases [10] as well as to antimicrobial resistance due to horizontal gene transfer [11]. Another important point is the form of communication within biofilms. Through the so-called quorum sensing (QS), an intra and extracellular communication channel of microorganisms, they are able to coordinately regulate their activities in biofilms [12, 13]. Based on these considerations, the search for new compounds with antibiofilm activity becomes essential for combating resistant microorganisms.

A niche that has been gaining space because of its diversified production of biomolecules is endophytic microorganisms. By definition, endophytic microorganisms are bacteria and fungi that live symbiotically associated with healthy plant tissues without causing any apparent damage to their host [14]. Endophytes are a source of several secondary metabolites with, for example, antimicrobial [15], antitumor [16], enzymatic [17], anti-COVID [18], and antibiofilm activities. The main antibiofilm compounds currently sought are those capable of i) preventing or inhibiting microbial adhesion to avoid biofilm formation, ii) dispersing the already formed biofilm, and iii) interfering with intra/extracellular communication for biofilm formation (anti-QS) [19]. It is already known that natural products, such as those produced by endophytes, have advantages over synthetic compounds [20], for instance, rigidity, which provides better protein-protein interactions [21], and the possibility of being structurally shaped by evolution to be used by/in living beings [22]. Endophytic microorganisms can also be used in the synthesis of nanoparticles with antibiofilm activity. Nanoparticles can be defined as particles ranging from 1 to 100 nm and with size-related properties [23], being important allies in public health, as they can be applied in medicine [24]. Thus, the eco-friendliest method for the production of nanoparticles is precisely through the so-called green synthesis, which uses products from biological sources for the biosynthesis of nanoparticles [25].

*Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

This book chapter discusses the use of endophytic microorganisms and their compounds as potential tools for controlling and combating pathogenic biofilms, which are closely linked to antimicrobial resistance.

#### **2. Natural antibiofilm and anti-quorum sensing products synthesized by endophytic microorganisms**

Several studies have reported antibiofilm and anti-QS compounds produced by endophytes, reinforcing and highlighting the potential application of these microorganisms in various areas of health. Some of these studies are presented in **Table 1** and will be fully discussed throughout this chapter.

#### **2.1 Natural antibiofilm agents from endophytic bacteria**

Endophytic bacteria play a significant role in the production of a variety of secondary metabolites with potential applications in medicine [45], opening up new perspectives for the prospection of different bacterial species towards the discovery of novel antibiofilm agents against pathogenic microorganisms.

El-Gendy et al. [46] isolated 51 *Streptomyces* strains from the inner healthy tissue of *Sarcophyton convolutum* and determined the antibiofilm activity of ethyl acetate extracts of these endophytes onto 96-well polystyrene plates against seven methicillin-resistant *S. aureus* (MRSA) strains and nine multidrug-resistant *Pseudomonas* species (MRD). The *Streptomyces* strain MORSY 22 showed destructive activity of the biofilm produced by all *S. aureus* strains (MRSA1 to MRSA7), with values ranging from 87.46 to 95.75%, and all *Pseudomonas* species (MRD 1 to MRD9), with values ranging from 96.58 to 70.38%. These results revealed the potential of the strain MORSY 22 to prevent biofilm formation by bacterial pathogens and to develop antibiotic resistance.

Theodora et al. [47] screened the antibiofilm activity of endophytic bacteria against the pathogenic bacteria *Bacillus cereus* ATCC 14579, *S. aureus* ATCC 29213, *Enterococcus faecalis* ATCC 33186, *P. aeruginosa* ATCC 27853, *Salmonella typhimurium* and *Vibrio cholerae.* Crude extracts of isolates JB 19B and JB 18B showed the highest biofilm inhibition activity (90%) and biofilm destruction (76%), respectively, against *S. aureus*. Through scanning electron microscopy (SEM) analysis it was possible to verify a reduction in the extracellular matrix of the biofilms of *B. cereus* and *S. typhimurium* after treatment with extracts of isolates JB 18B and JB 19 B. The isolate JB 3B also showed inhibition activity against biofilm formation by all pathogenic bacteria. These findings confirmed the potential use of antibiofilm inhibitors from endophytic bacteria as a strategy for the control of bacterial infections.

Sabu et al. [48] isolated 14 endophytic actinomycetes from the rhizomes of *Zingiber officinale*. The crude extract of *Nocardiopsis* sp. ZoA1 at 200 μg/mL caused a reduction of more than 90% biofilm formation by multidrug-resistant coagulase-negative *Staphylococcus capitis* 267 and *Staphylococcus haemolyticus* 41 strains. GC-MS/MS analysis of *Nocardiopsis* sp. also revealed the presence of various compounds with antimicrobial activity, such as phenol, 2,4-bis (1,1-dimethylethyl), and trans-cinnamic acid. These results pointed to the inhibition of the synthesis of exopolysaccharide and proteinaceous factors by tested crude extracts and their potential to prevent biofilm formation by multidrug-resistant biofilm-forming strains.


#### *Focus on Bacterial Biofilms*


**Table 1.** *Antibiofilm and anti-QS activities of natural compounds produced by endophytic fungi and bacteria isolated from different host plants.*

### *Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

Biosurfactants are an important class of natural antibiofilm agents produced by microorganisms. They comprise a structural and heterogeneous group of amphipathic molecules, which include glycolipids, lipopeptides, phospholipids, fatty acids and neutral lipids, polymeric and particulate biosurfactants [49, 50]. These microbial molecules can interfere with cell-to-cell communication mediated by QS and cell adhesion ability, promoting the dispersal of bacterial colonies and affecting biofilm formation through distinct mechanisms, such as cell membrane damage, inhibition of electron transport chain and energy restriction [51, 52]. Additionally, microbial surfactants have been considered an eco-friendly alternative with low toxicity and high biodegradability, selectivity and compatibility when compared to chemically synthesized surfactants [53].

Recently, Ashitha et al. [54] studied the endophyte *Burkholderia* sp. WYAT7 was isolated from the medicinal plant *Artemisia nilagirica* (Clarke) Pamp. in order to evaluate its antibiofilm activity. The biosurfactant present in the culture supernatant was identified and characterized as a glycolipid, and an inhibitory effect on the *S. aureus* (MTCC 1430) biofilm formation was observed. The percentage of biofilm formation suppression by MTCC 1430 was 41.79% and 79.22% when treated with 1 mg/ml and 2 mg/ml, respectively. These results suggested that the surfactant produced by *Burkholderia* sp. WYAT7 could be explored as a therapeutic agent for the control of pathogenic bacteria.

Ceresa et al. [55] reported that lipopeptide biosurfactants produced by the endophytic *B. subtilis* AC7 (AC7BS) isolated from *Robinia pseudoacacia* efficiently reduced *Candida albicans* adhesion to and biofilm formation on medical-grade silicone elastomeric disks (SEDs) by 57–62% and 46–47%, respectively. Chemical analysis of the crude extract revealed the presence of surfactin and fengycin. Since the fungus *C. albicans* is considered responsible for colonizing medical implants and causing a high mortality rate, the authors suggested the potential use of these biosurfactants to coat silicone medical devices in order to limit colonization of the pathogen and prevent infections. Later, Ceresa et al. [56] studied the synergistic effect of lipopeptides of *B. subtilis* AC7 (AC7BS) combined with the QS molecule farnesol to counteract *C. albicans* biofilms on silicone elastomer in simulated physiological conditions. There was a significant reduction of up to 74% in the pathogen adhesion within 1.5 hours and up to 93% and 60% in the biofilm formation within 24 and 48 hours, respectively. These effects were confirmed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). According to the authors, these findings opened up new perspectives for the combination of biosurfactants and farnesol to counteract *C. albicans* adhesion to and biofilm formation on materials for medical use.

Cochis et al. [57] evaluated the preventive anti-adhesion activity of biosurfactants extracted from endophytes from *R. pseudoacacia* (AC5 and AC7) and *Nerium oleander* (OC5) against *C. albicans* biofilm on acrylic resin and disks of silicon. The effective concentrations for *C. candida* biofilm inhibition without cytotoxic effects on mouse fibroblasts (ATCC L929) and human keratinocytes (ATCC HeLa S3) were 156.3 g/ml and 78.1 g/ml, respectively. These results demonstrated the potential use of these biosurfactants for the prevention of *C. albicans* biofilm adhesion to catheter and prosthesis materials.

#### **2.2 Natural antibiofilm agents from endophytic fungi**

Several recent studies have shown the potential of endophytic fungi as producers of biomolecules with antimicrobial activity [58]. Historically, fungi are known for

#### *Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

their diverse production, including penicillin–the first antibiotic discovered [59]. For such reason, over the years researchers have focused on the discovery of new fungal antimicrobials, such as clavatol, sordaricin, jesterone, and javanicin [60]. Based on this, it is evident how interesting endophytic fungi can be in terms of the production of antimicrobial compounds.

May Zin et al. [61] obtained several bioactive metabolites from the endophytic fungus *Eurotium chevalieri* KUFA 0006 isolated from *Rhizophora mucronata*. The new compounds were tested to verify their antibiofilm activity against *E. coli* ATCC 25922, *E. faecalis* ATCC 29212, and *S. aureus* ATCC 25923. Thirteen metabolites effectively inhibited the growth of biofilms, whereas eight inhibited the biofilm formation by *E. coli* ATCC 25922, six by *S. aureus* ATCC 25923 and only one by *E. faecalis* ATCC 29212. This work also highlighted compound 3, which showed antibiofilm activity against *E. coli* ATCC 25922 and *S. aureus* ATCC 25923, causing a reduction of about 80% in the staphylococcal biofilm. The authors also performed tests to evaluate the antibiotic activity of these metabolites against the same pathogenic strains and found a positive result in only one compound. This is a very interesting finding, because even though certain compounds did not present an inhibitory effect against the pathogen alone, they had an inhibitory activity against the biofilm.

Narmani et al. [62] isolated the fungus *Chaetosphaeronema achilleae* from *Taxus baccata* and reported the production of seven compounds from the endophyte. In general, the metabolites were tested at different concentrations against *S. aureus* DSM 1104 biofilms and all of them presented some inhibitory activity even at lower concentrations. Among them, compound 4 stood out, showing strong biofilm inhibitory activity of about 96.82% at a concentration of 256 μg/mL and approximately 91.95% at 128 μg/mL. In addition, compound 7 was able to inhibit about 96.18% at 256 μg/mL of the biofilm, which represents a quite positive result. In the same work, it was observed that not all compounds exhibited antimicrobial activity against *S. aureus* DSM 1104 alone, as only metabolites 2 and 7 were positive.

Kaur et al. [63] isolated the fungus *Alternaria destruens* (AKL-3) from *Calotropis gigantea* and observed antibiofilm activity of the active fractions AF1 and AF2 during biofilm formation and in the preformed biofilm. The test microorganisms were *P. aeruginosa*, *C. albicans*, *E. coli* and *Salmonella enterica*, and two different concentrations of each active fraction were tested. In the case of AF1, all biofilms had their formation relatively inhibited, in addition to having been moderately reduced in the preformed biofilm. With regard to AF2, the same could be observed, that is, all biofilms were inhibited in the initial phase and in the preformed biofilm. Nonetheless, according to the authors AF1 was more promising and showed significantly greater activity than AF2 in all tests with the pathogenic strains.

Kaur et al. [64] evaluated the antibiofilm activity of the chloroform extract of the endophytic *Aspergillus fumigatus* isolated from *Moringa oleifera* against *S. aureus* MTCC 740, *K. pneumoniae* MTCC 109, and *C. albicans* MTCC 227. In this study, the authors performed tests at different stages of the biofilm, namely, the initial cell fixation phase and the preformed biofilm. In the initial fixation tests, the fungal extract was able to inhibit the formation of *S. aureus*, *K. pneumoniae,* and *C. albicans* biofilms by 69.2%, 57.66%, and 55%, respectively, with the standard antimicrobials showing similar results. The authors also argued that the inhibition of the initial fixation of the *C. albicans* biofilm by the fungal extract was better than that of the standard antifungal (amphotericin B) since the value obtained was approximately 53.3%. Regarding the tests against preformed biofilms, the extract reduced by about 51%, 53.4% and 47.6% of the *S. aureus*, *K. pneumoniae* and *C. albicans* biofilms.

Elkhouly et al. [65] studied the metabolism of the endophytic fungus *Aspergillus Tubenginses* ASH4 isolated from *Hyoscyamus muticus* in order to understand the production of antibiofilm compounds. During the study, pathogenic biofilms of *S. aureus* ATCC6538-P, *Bacillus subtilis*, *P. aeruginosa* ATCC27853 and *E. coli* were bioindicators of the extract as well as of the pure compound. The endophytic extract was able to suppress the formation of the *S. aureus*, *B. subtilis*, *P. aeruginosa,* and *E. coli* biofilms by 60.8%, 50.06%, 28.44%, and 37.68%, respectively. Subsequently, the pure compound identified as anophinic acid was tested against the same strains, reaching an inhibition of 61.39%, 54.93%, 69.51%, and 34.45%, respectively. Based on these results, it is possible to observe that the values are similar between them, except in the case of *P. aeruginosa*.

Qader et al. [66] isolated the marine endophytic fungi *Epicoccum nigrum* M13 and *Alternaria alternata* 13A from *Thalassia hemprichii* and tested 16 pure compounds obtained from them. The bioindicators for the antibiofilm activity test were *E. coli*, *S. aureus*, *B. subtilis,* and *P. aeruginosa*, all clinically isolated from hospitals in Egypt. Among the tested compounds of *E. nigrum* M13, three showed antibiofilm activity against pathogenic strains ranging from moderate to weak. The authors pointed out that some compounds such as 1 exhibited moderate activity against *S. aureus* and *B. subtilis*, but weak activity against *E. coli* and *P. aeruginosa*. In addition, compounds 3 and 5 showed moderate activity against Gram-positive bacteria, but weak activity against Gram-negative ones. As for the compounds isolated from *A. alternata* 13A, five of them presented activity against the biofilms of the indicator strains. Unlike what was seen in *E. nigrum* M13, compounds 7, 8, 9 and 10 from *A. alternata* 13A inhibited by 70–80% the *S. aureus* and *B. subtilis* biofilms, indicating an excellent activity. The same compounds also showed moderate activity against *E. coli* and *P. aeruginosa* biofilms. On the other hand, compound 11 exhibited weak activity against *S. aureus*, *E. coli* and *P. aeruginosa* but moderate activity against *B. subtilis*.

#### **2.3 Anti-quorum sensing activity of natural agents from endophytic microorganisms**

Quorum sensing (QS) is a complex density-dependent microbial cell communication system that occurs in single or mixed populations through autoinducers (AIs) or QS molecules. It is a population-dependent signaling mechanism in which microorganisms activate some signaling molecules according to the cell density. This behavior can be observed in several species of fungi and bacteria [67–69], being considered an inter- and intraspecies communication behavior that leads to genetic responses to autoinducers. This allows the microbial community to perceive and respond to various factors, including the presence of threats. The QS activity is responsible for the regulation of several bacterial physiological activities, such as pathogenesis, biofilm formation, swarming motility, bioluminescence, pigment disposal, polysaccharide production, and virulence, transforming the QS molecules into an important target for alternative antimicrobial therapy and antibiofilm activity [70].

After their production, when AIs reach an optimal concentration they bind to receptors on microbial cells, causing an alteration in gene expression. This ability gives biofilms adaptability to the environment as well as greater resistance to elimination, which in turn increases their virulence [71, 72]. In addition, QS molecules are also considered responsible for inhibiting or delaying the growth of other bacteria or fungi that are not part of their biofilm.

*Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

It is known that QS molecules are different for each microbial species. Furthermore, the type of communication in mixed biofilms also differs, that is, it can be either inter or intraspecies. There are four main categories of AIs: AI-1, AI-2, AI-3, and AIP. According to Schauder et al. [73], the molecules AI-2 are responsible for interspecies communication, while Smith et al. [74] argue that the molecules AI-1, AI-3, and AIP are in charge of the intraspecies communication.

**Figure 1** shows the QS mechanism in a fungal cell in a simplified way. AIs (named signal molecules) are synthesized by fungal cells and released to the outside of the cell. Signal receptor proteins detect AIs and stimulate the expression of various genes, such as virulence, growth, and morphogenesis regulators.

Since the QS mechanism is responsible for the survival and increased virulence of biofilms, the development of QS inhibition strategies has been of great importance. Most QS inhibition mechanisms use one of the following strategies: i) degradation and/or inactivation of AIs; ii) inhibition of AI synthesis; iii) inhibition of AI detector; and iv) antibiotics as QS inhibitors [76]. In the context of QS mechanisms of biofilms, endophytic microorganisms–considered to be synthesizers of QS inhibitors–have gained increasing attention. According to Mookherjee et al. [76], as endophytic microorganisms need to constantly produce defenses against competing microbial populations, they become an interesting source of QS inhibitors. QS inhibitor molecules can be produced by either endophytic fungi or bacteria [40, 77, 78].

Since QS can regulate the expression of virulence factors, QS inhibitors (QSIs) appear to be a promising antimicrobial strategy. As they act by imitating the QS autoinducers, they can be used to attenuate bacterial virulence, thus requiring lower doses, being more susceptible to the host immune system and reducing the use of antibiotics [39]. There are several studies reporting the QSI activity of biofilms.

**Figure 1.** *QS mechanism scheme adapted from Sharma et al. [75].*

#### *2.3.1 QSIs produced by endophytes*

It is known that endophytic fungi are responsible for the control and regulation of physiological activities of pathogens in animals and plants. Several studies have identified the production of QS inhibitors by endophytic fungi. Rajesh and Rai [39] isolated the endophytic fungus *Fusarium graminearum* from *Ventilago madraspatana* and measured the enzyme production using spectrophotometric and plate assay methods. Its anti-QS activity was analyzed against *Chromobacterium violaceum* CVO26, yielding strong positive results. Additionally, the extract of the endophytic fungus was able to inhibit the production of violacein pigment in the bacterium tested without any changes in bacterial growth. The authors then concluded that there was production of QS inhibitors by the endophytic fungus from *Ventilago madraspatana*, which in turn can be used for the development of anti-QS drugs–mainly against drugresistant microorganisms.

Anti-QS molecules of *Lasiodiplodia sp.* from marine plants were also tested against *C. violaceum* CVO26 by Martín-Rodríguez et al. [41]. Four strains of the endophytic fungus stood out for their strong anti-QS activity. These strains were identified as belonging to four genera: *Sarocladium* (LAEE06), *Fusarium* (LAEE13), *Epicoccum* (LAEE14), and *Khuskia* (LAEE21). The authors reported that this was the first time that QS inhibitors were found in endophytic fungi extracted from marine plants.

Mishra et al. [70] showed that 2,4-di-tert-butylphenol (2,4-DBP), a component isolated from the endophytic fungus *Daldinia eschscholtzii*, is capable of inhibiting the QS activity of *P. aeruginosa*–one of the top three gram-negative bacteria considered a global threat due to its multiple drug resistance. They noticed that when exposed to 2,4-DBP, *P. aeruginosa* reduced the biofilm production and its virulence factors, as well as the expression of QS-related genes, confirming that 2,4-DBP can be used in combination with antibiotics to combat *P. aeruginosa.*

Zhou et al. [79] conducted a study that identified the QSI activity of 1-(4-amino-2-hydroxyphenyl) ethanone (AHE) isolated from the endophytic fungus *Phomopsis liquidambari* S47 from the leaves of *Punica granatum* against *P. aeruginosa* PAO1. The compound acted by suppressing the expression of genes related to QS, inhibiting the activity of antioxidant enzymes and enhancing oxidative stress. Pellissier et al. [80] explored the QSI activity of endophytic fungi extracted from the tropical palm *Astrocaryum sciophilum* against *P. aeruginosa.* Two pyran derivatives extracted from the endophytic strain *Laccophilus venezuelensis* showed activity affecting QS-regulated virulence factors.

Like endophytic fungi, bacteria are able to interact with each other (intra- and interspecies communication) through AIs. Kusari et al. [77] studied how endophytic bacteria from *Cannabis sativa* plants use QS inhibition as an antivirulence strategy in *C. violaceum*. A total of 13 endophytic bacteria were isolated from *C. sativa*, and their extracts were prepared and tested against *C. violaceum*. Four of them (*Bacillus* sp. strain B3, *Bacillus megaterium* strain B4, *Brevibacillus borstelensis* strain B8, and *Bacillus* sp. strain B11) exhibited the significant potential to weaken *C. violaceum* cell QS signals in a concentration-dependent manner.

Endophytic isolates of the phylum *Actinobacteria* previously isolated from common bean (*Phaseolus vulgaris*) were tested against pathogenic microorganisms by Lopes et al. [81]. Among them, *Microbacterium testaceum* BAC1065 and BAC1093 were found to inhibit QS of *C. violaceum* and *E. coli*. Kiarood et al. [82] found two strains (*Bacillus cereus* Si-Ps1 and *Pseudomonas nitrogenformans* La-Pot3–3) among 64 endophytic bacteria isolated from *Citrus sinensis* able to reduce the detection of QS

*Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

molecules in *Pseudomonas syringae*. The *B. cereus* extract strongly inhibited *P. syringae* biofilm formation. An interesting fact reported by the authors was the increased number of cells in planktonic cultures treated with anti-QS molecules compared to control groups. This demonstrates that the molecules directly affect biofilm formation, but do not interfere with population growth.

#### **3. Metal-based nanoparticles (NPs) synthesized from endophytic microorganisms as antibiofilm agents**

The biosynthesis of metal-based NPs using endophytic microorganisms is a promising green synthetic route, considering the way to obtain these NPs and their final environmental impact [83]. These NPs can be used in many different technology sectors with emphasis on health [84, 85]. The biosynthesis of these NPs can occur intra- and/or extracellularly. The intracellular biosynthesis occurs through electrostatic interaction between positive charges from metal ions in a solution and negative charges from the bacterial/fungal cell wall [86]. In this process, microbial reductases dependent on NADH and NADPH are responsible for the transport of electrons, working as biocatalysts for redox reactions [87, 88]. In contrast, in extracellular synthesis, the culture supernatant, biomass, or cell-free extract is mixed with the metal ion solution, and the NPs are produced outside the microbial cell [89]. This process is performed by reductases produced and secreted into the culture medium by microbial cells and other cofactors [89, 90]. Therefore, biosynthesis through endophytic microorganisms can be used to obtain a series of different NPs, being the most common metallic/metallic oxides.

Noble metal NPs such as Ag has been widely used since ancient times for medicinal purposes due to their antimicrobial action [91]. Thus, it is natural that most of the works in the literature on the production of nanoparticles from endophytic microorganisms for microbial elimination are focused on Ag NPs. When these NPs are used for the inhibition of biofilms, the interaction between the NPs and the biofilm occurs in a succession of steps: first, the NPs are transferred to the biofilm surroundings; then, their superficial fixation occurs, followed by their migration to the biofilm interior [92]. Metal NPs can generate high local oxidative stress as a result of the production of reactive oxygen species (ROS), in addition to releasing M+ ions, which can interact with various functional groups of microorganisms, such as proteins, lipids, and DNA [93]. Furthermore, they can bind to the cell membrane surface by electrostatic interactions and penetrate by endocytosis and direct diffusion [94]. Metal oxide NPs can generate a high concentration of ROS even in the dark, interacting similarly with metal NPs, and cause secondary effects due to both local contact of metal oxide NPs with microorganisms and ionic release (depending on the stability of the oxide in the reaction medium used) [95, 96]. **Figure 2** illustrates the mechanism of action of the nanoparticles on the biofilm.

Bakhtiari-Sardari et al. [97] biosynthesized Ag NPs from the inoculum of two strains of *Streptomyces* sp. (OSIP1 and OSNP14) using the cell-free supernatant from these cultures to inhibit *P. aeruginosa* ATCC 27853 biofilms, resulting in Ag NPs with a spherical shape and an average size of 8 and 15 nm, respectively. The growth of *P. aeruginosa* biofilms was inhibited by up to 85% at a minimum concentration of 125 μg/mL of Ag NPs. The highest activity of the Ag NPs synthesized by the strain of *Streptomyces* sp. OSIP1 was attributed to the smaller size of Ag NPs obtained. Ranjani et al. [98] used the same Ag NP biosynthesis strategy to inhibit the growth of *P. aeruginosa* ATCC

#### **Figure 2.**

*Schematic illustration of antibiofilm effects of metal and metal oxide NPs.*

27853. Using the cell extract of the fungus *L. theobromae* (MK942601), it was possible to obtain agglomerated Ag particles with an average size of 163.3 nm. The result of biofilm growth inhibition was 70% at a concentration of 50 μg/mL of Ag NPs. Bagur et al. [99] biosynthesized Ag NPs with an average size of 16.1 nm through a cell extract of the fungus *E. rostrata* due to its crucial role in the growth inhibition of *P. aeruginosa* and *S. aureus*. It was observed that there was a significant decrease in the growth of both pathogens at a concentration of 5 μg/mL of Ag NPs.

Neethu et al. showed in two different works the effectiveness of Ag NPs against the biofilm growth of the multidrug-resistant bacterium *A. baumanii* [100, 101]. In their first work, the biomass of the fungus *Peridinium polonicum* was used to synthesize spherical Ag NPs with sizes between 10 and 15 nm. It was observed that after 5 hours of exposure to the Ag NPs there was a reduction of more than 99.9% (3 log reduction) in the number of viable bacteria at a concentration of 15.6 μg/mL [100]. In their other work, the authors [101] produced a bionanocomposite coating with biosynthesized Ag NPs for a central venous catheter (CVC) using polydopamine as an adherent film of Ag NPs. Like in their previous work, it was observed that the CVC functionalized with Ag NPs eradicated the *A. baumanii* biofilm.

Ranjani et al. [102] synthesized Ag NPs nanocolloids and used them for the elimination of *E. coli* ATCC 25922 biofilms, commonly present in intensive care units (ICUs). The cell extract of the fungus *L. theobromae* (LtNc's) was able to produce Ag particles with an average size of 436.5 nm. At a concentration of 12.5 μg/mL of these Ag NPs, there was a 50% reduction in *E. coli* biofilm formation. In another work, Chandankere et al. [103] synthesized Ag NPs with sizes between 4 and 26 nm using the fungus *Colletotrichum* sp. DM16.3 to inhibit the growth of biofilms of bacteria *B. cereus* (Gram-positive) and *Vibrio cholerae* (gram-negative). At a concentration of 10 μg/mL of these Ag NPs, it was possible to observe an inhibition of biofilm growth of 45.6% for *B. cereus* and 85.1% for *V. cholerae*. Ibrahim et al. [104] used the cell extract of the bacterium *B. siamensis* to synthesize Ag NPs with sizes between 25 and 50 nm. It was observed that at a concentration of 20 μg/mL these Ag NPs were able

*Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

to inhibit the growth of biofilms of *Xanthomonas oryzae* pv. *oryzae* LND0005 and *Acidovorax oryzae* RS-1 by 86.31 and 80.59%, respectively.

Metal oxide NPs can also be synthesized by endophytic microorganisms and used to inhibit biofilm growth. Dhandapani et al. [105] synthesized TiO2 NPs (10–30 nm) from the biomass of the bacterium *B. subtilis* (FJ460362). Tests were performed using microorganisms present in local aquatic sources and in the presence of light so that TiO2 produced more ROS, causing high oxidative stress to microorganisms. The Se and SeO2 particles (75–225 nm) were synthesized from the extract of the bacterium *Bacillus* sp. MSh-1 and tested against the biofilms of *P. mirabilis*, *S. aureus,* and *P. aeruginosa*, resulting in inhibitions of 53.4, 48.1, and 55.1%, respectively [106]. Balaji et al. synthesized ZrO2 particles using the bacterium *B. niancini* and used them to remove the biofilms of *E. coli* (91.5%), *Klebsiella aerogenes* (71%), *P. vulgaris* (83.25%), *S. aureus* (92.5%) and *S. mutant* (90.5%) at a concentration of 40 μg/ml [107].

#### **4. Conclusions**

Biofilms are known to be closely linked to the growing resistance of pathogens, posing a threat to public health. Based on this fact, endophytic microorganisms considered as potential and eco-friendly producers of compounds with antibiofilm activity may be a source for the discovery of new biomolecules to combat these pathogens since they can synthesize compounds with anti-adherent properties, being capable of dispersing pre-synthesized biofilms.

These microorganisms also produce QS inhibitors that can harm the communication between pathogens in biofilm and, consequently, interrupt its formation. There are several researches showing the capacity of endophytic production in the prevention and dispersion of biofilms of, for example, ESKAPE pathogens, and this is really relevant because these microorganisms had been causing such a considerable problem to public health.

In addition, the microbial products of endophytes can also be used in the biosynthesis of metal-based nanoparticles, which have been demonstrating an interesting activity against biofilms. Some studies showed that metal-based nanoparticles can allocate on the surface of biofilm and migration to its interior, interacting directly with the pathogens inside, causing their death in different ways.

Thus, endophytic microorganisms deserve a position in the discussion about the development of new antimicrobial and antibiofilm medicines, mainly because several researches described in this review showed the potential of endophytes against harmful pathogens and their biofilms.

#### **Acknowledgements**

This work was supported by grants from the São Paulo Research Foundation (FAPESP) No. 2020/16299-9 to SHR, Nos. 2016/13423-5 and 2017/12905-9 to CPS and No. 2013/07296-2 to EL, and from the Coordination for the Improvement of Higher Education Personnel (CAPES) under finance code (001) to MA.

### **Author details**

Saulo Henrique Rodrigues1,2, Marcelo Assis3 , Camila Cristina de Foggi3 , Andréa Cristina Bogas1,4, Mariana Ottaiano Gonçalves1,4, Lavinia Cipriano1,2, Elson Longo3 , Evandro Leite de Souza5 and Cristina Paiva de Sousa1,4\*

1 Laboratory of Microbiology and Biomolecules - LaMiB, Biotechnology Graduate Program (PPGBiotec), Federal University of São Carlos, São Carlos, Brazil

2 Biological Sciences Undergraduate Program, Federal University of São Carlos, São Carlos, Brazil

3 Center for the Development of Functional Materials (CDMF), Federal University of São Carlos, São Carlos, Brazil

4 Biotechnology Graduate Program – PPGBiotec, Federal University of São Carlos, São Carlos, Brazil

5 Nutritional Sciences Graduate Program, Federal University of Paraíba, João Pessoa, Brazil

\*Address all correspondence to: prokarya@ufscar.br

© 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.

*Development of Antibiofilm Substances by Endophytic Microorganisms with an Emphasis... DOI: http://dx.doi.org/10.5772/intechopen.104522*

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**Chapter 12**

## Natural Products as Antibiofilm Agents

*Cynthia Amaning Danquah, Prince Amankwah Baffour Minkah, Theresa A. Agana, Phanankosi Moyo, Michael Tetteh, Isaiah Osei Duah Junior, Kofi Bonsu Amankwah, Samuel Owusu Somuah, Michael Ofori and Vinesh J. Maharaj*

#### **Abstract**

Biofilms, are vastly structured surface-associated communities of microorganisms, enclosed within a self-produced extracellular matrix. Microorganisms, especially bacteria are able to form complex structures known as biofilms. The presence of biofilms especially in health care settings increases resistance to antimicrobial agents which poses a major health problem. This is because biofilm-associated persistent infections are difficult to treat due to the presence of multidrug-resistant microorganisms. This chapter will give an idea about documented agents including isolated compounds, crude extracts, decoctions, fractions, etc. obtained from natural sources such as plants, bacteria, fungi, sponge and algae with antibiofilm activities. Furthermore, we have done phylogenetic analysis to identify plant families most prolific in producing plant species and compounds with good antibiofilm properties so as to aid in prioritizing plant species to investigate in future studies. The data in this chapter will help serve as valuable information and guidance for future antimicrobial development.

**Keywords:** biofilm, natural products, quorum sensing, anti-biofilm agents, antimicrobials

#### **1. Introduction**

The empirical approach to antimicrobial therapy among health care professionals and the concurrent patronage of over-the-counter antibiotics by patients have together caused an exponential rise in multidrug resistance among clinically relevant antimicrobials and with increasing trends for the past two decades [1]. Different mechanisms of antimicrobial resistance have been proposed, including the (i) alteration of the antibiotic target by genetic mutations or post-translational modification, (ii) deactivation of the antibiotic through hydrolysis or modification, such as phosphorylation by an enzyme, (iii) increased efflux of the antibiotic out of the cell by efflux pumps and porins, (iv) decreased influx/penetration of the antibiotic into the cell, through changes in cell wall structure; and overproduction of the antibiotic target through gene amplification [2]. However, one of bacteria's preferred and commonly deployed strategies to

overcome the effect of antimicrobials is the formation of biofilms. Over 90% of pathogenic bacterial species, including *Staphylococcus aureus* (*S. aureus*) and *Pseudomonas aeruginosa* (*P. aeruginosa)*, possess an inherent ability to produce biofilms, making biofilms the leading cause of multidrug resistance among microorganisms [3–5].

Biofilm is a complex community of sessile microbial communities embedded in a self-producing polymeric matrix comprising exopolysaccharides, proteins, nucleic acids, and cell surface proteins [6–8]. As a community of microorganisms, biofilms constitute either a single microbial species or a combination of a different class of bacteria, fungi, protozoa, archaea, and yeast, with a unique ability to colonize almost any environmental niche, biotic or inert surfaces [9–13]. Biofilm enables microorganisms to withstand harsh environmental conditions such as nutrient deficiencies, high osmotic pressure, the low potential of hydrogen, oxidative stress, and antimicrobial insults [14]. The increased resistance of biofilms to antimicrobials arise from phenotypic cell variation and gene transcription. In particular, there is an exponential growth of microorganisms and genetic transfer of extrachromosomal elements via cell-to-cell communication system called quorum sensing [14–17]. Quorum sensing is critical in the development and survival of biofilms; thus, it regulates the nutritional demands of microorganisms within the biofilm to meet the external supply of resources [18, 19]. In addition quorum sensing is essential for the biosynthesis and secretion of small molecule signals that activate a range of downstream processes including virulence and drug resistance mechanisms as seen in biofilms [20, 21].

The health risks of biofilms are enormous, which underscore their utilization in plant protection, bioremediation, wastewater treatment, and corrosion prevention in agricultural and industrial settings [22–24]. In particular, the biofilm grows on living human tissues such as the lungs and teeth and the surfaces of implanted biomedical devices, including contact lenses, central venous catheters [8, 25], prosthetic joints, pacemakers, and intrauterine devices [7]. Unlike single bacterial plankton cells, the treatment of biofilm-mediated infections is challenging owing to the decreased susceptibility to antimicrobial agents and other chemotherapeutics. The availability of qualitative (such as Congo red agar, microtitre plate, tube methods) and quantitative (including polymerase chain reaction (PCR)) techniques have enabled the detection and measurement of biofilms [26]. Conversely, the evaluation and screening of antimicrobials against biofilms are of great challenge. In particular, standard microdilution testing cannot evaluate the susceptibility of biofilms to antimicrobial drugs because these tests focus on planktonic (suspended) organisms rather than biofilm (surface-associated) organisms [7]. Instead, susceptibility must be determined directly against biofilm-associated organisms, preferably under conditions that mimic *in vitro* and/or *in vivo* conditions. In this light, several biofilm models systems have been developed to permit accurate screening and evaluation of novel agents for their antibiofilm activity [27, 28].

Although nature has provided a plethora of natural products with varying chemotherapeutic properties to fight human infectious diseases, discovering new and effective antimicrobials has been slow. The decline in the efficacy of existing chemotherapy and the surge in drug resistance has triggered an expedient exploration of natural products, especially from plants and microbial origin, for their antibiofilm activity against biofilm-mediated human infections. Plant extracts and plant-derived chemical products, such as essential oils, flavonoids, terpenoids, have been shown in vitro to have antimicrobial and antibiofilm activity [27–31]. Secondary metabolites and other peptidic compounds from microorganisms also exhibit antagonistic effects against biofilms [6, 32]. These chemical constituents exert their action by inhibiting

*Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

critical elements within a biofilm and/or terminating biofilm formation processes [33]. Given the unique nature of plants and microbes, natural products derived from these sources could provide an avenue for developing newly efficacious and clinically desirable chemotherapies against biofilms-mediated infections and their associated health consequences.

This chapter aims to provide a comprehensive summary of natural products from plants and microbial sources as potential sources of antibiofilm agents. Again, it highlights the strategies and model organisms used to identify and evaluate the antibiofilm capacity of these naturally isolated chemical compounds.

#### **2. Biofilm formation**

Biofilm formation represents a survival mechanism deployed by microorganisms in response to unfavorable environmental conditions [34]. Structurally, biofilms are a collection of adherent microorganisms in a milieu of an extracellular matrix consisting of polysaccharides, proteins, nucleic acids, and lipids. This unique architecture enables biofilms to cling firmly to surfaces of implanted body organs and biomedical devices and, more importantly, increase their resistance to antimicrobial therapy. The presence of bacterial secreted glycocalyx and degrading matrix enzymes reduces the antimicrobial concentration of which individual plankton cells within the biofilm are exposed [35, 36].

The morphogenesis of biofilms constitutes five distinct stages; namely, reversible attachment, irreversible adhesion, production of extracellular polymeric substances, biofilm maturation, and dispersal/detachment. As the initial step in biofilm formation, reversible attachment is characterized by the interaction between plankton cells and the conditioned surface. Fewer plankton cells move to the surface of the substrate by convection, pedesis, or sedimentation [37]. Consequently, chemotaxis directs bacterial cells along a nutrient gradient [38]. Upon reaching the surface of the substratum, the interaction between the cell surfaces and the substratum is dependent on the net sum of repulsive or attractive forces generated by the two characters [39, 40]. The presence of fimbriae, flagella, pili, and glycocalyx enables the microorganisms to overcome the repulsive forces (such as electrostatic, hydrophobic, Van der Waals, and hydration interactions) from the substratum and subsequently cling [39, 41, 42]. The rate of biofilms formation is influenced by the substrate's physicochemical properties, including the surface roughness, hydrophobicity, surface charge, and the presence of conditioning films [41, 43, 44].

Furthermore, bacterial cells transition into an irreversible adhesion phase. Irreversible attachment occurs through the combined effect of short-range forces of the substrate (such as dipole-dipole, hydrogen, ionic and covalent interactions) and adhesive structures of the bacterial cells. The flagella and pili, for instance, are critically important in the attachment process of various strains of microorganisms [45–48]. For example, Vatanyoopaisarn et al. demonstrated the firm clinging ability of wild-type *Listeria monocytogens* (*L. monocytogens*) compared to the non-flagellated mutant type [45]. Similarly, Di Martino and colleagues showed the distinctive role of type one and type three fimbriae in initiating the attachment of *Klebsiella pneumonia* (*K. pneumonia*) to abiotic surfaces [46]. Alarcon and coworkers also observed the critical role of pilus in the twitching substrate movement of *P. aeruginosa* [48].

Moreover, the resident plankton cells produce extracellular polymeric substances (EPS), an essential biofilm component. Quorum sensing and cyclic-di-GMP mediated EPS formation [49–52]. The formation of EPS promotes cohesion among bacteria and the adhesion of biofilms via hydrophobic and ionic interactions [49, 53, 54]. In addition, EPS is vital in constructing biofilms, maintaining biofilm architecture, quorum sensing, and genetic transfer among individual organisms within the biofilm [49, 55].

The resident bacterial cells proliferate into microcolonies mediated by autoinducers (AIs). AIs are chemical signaling molecules that permit intra-species and inter-species bacterial cell-to-cell communication [56, 57]. The surge in AIs activates critical enzymatic machinery in bacterial species for regulating the formation of microcolonies and the maturation of biofilms [52]. For example, the increase in AIs causes synchronous activation of the 15 gene-long epsA-O in *Bacillus subtilis* (*B. subtilis)* that causes an increased production of EPS. The proliferation of microcolonies and the increased accumulation of EPS trigger gene expression [52]. This alteration in gene expression reversibly stimulates additional EPS as adhesive molecules to bind individual plankton cells. In addition to EPS production, water channels are created to facilitate the inflow of nutrients to the individual cells within the biofilm [58]. During the maturation stage of biofilm formation, there is restricted motility of the bacterial cells together with characteristic variation in gene and protein expression between biofilm and plankton cells [59, 60].

The terminal phase of biofilm formation, delineated as detachment or dispersal, is regulated by a complex mechanism constituting signal transduction, effector, and environmental factors [61]. Detachment/dispersal represents a unique phase in the life cycle, where plankton cells segregate and escape from biofilms to establish microcolonies on fresh surfaces [62, 63]. Of note, the dispersal phase of a biofilm is characterized by the detachment of plankton cells from hitherto biofilm, seeding or passive movement of plankton to new uncolonized surfaces, and clinging or attachment to substrates [61, 64, 65].

#### **3. Models for assessing antibiofilm activity**

Several methods have been developed to study the antibiofilm activities of various compounds *in vitro*. However, only a few *in vivo* strategies for studying biofilms have been described. Given the importance of bacterial biofilm infections worldwide, we describie some models for assessing the efficacy of antibiofilm compounds *in vivo.*

#### **3.1 The human organoid model**

The human epidermis organoid model has a tough methicillin-resistant *S. aureus* (MRSA) USA300 and *P. aeruginosa* PAO1 biofilm system for studying host-microbe interplay and enable the screening of novel antibiofilm agents. This model allows the screening of synthetic host peptides to reveal their superior antibiofilm activity against MRSA compared to the antibiotic mupirocin. This model provides an exciting tool for elucidating disease pathology and testing novel drugs toxicities and efficacies. It also has the added advantage of reducing the use of animals in pre-clinical testing and replacing in vivo infection models with an ethical alternative that better reflects human disease [27].

This method involves establishing bacterial biofilm by seeding the center of the skin model with 5 µL of 2 × 108 CFU/ml of MRSA or *P. aeruginosa* PA01 or fluorescently-tagged MRSA or PA01-mCherry or luminescent MRSA-lux or PA01-lux resuspended in PBS and cultured at 37 °C and 7.3% CO2. 30 µL of 1–4 mg/ml DJK-5

#### *Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

peptide was then added on top of the biofilm for 4 h, 1–3 days post inoculation. Luminisense signal are monitored daily after the establishment of infection until luminescence are observed in the culture medium underneath the skin. This is to study how long the skin could endure biofilm growth. ChemiDoc imaging system is used to visualize biofilms and bacterial counts quantified by sonicating, votexing and serially diluting excised skin samples on agar plates [27].

#### **3.2 Wound models**

Among the most widely used models to investigate antibiofilm compounds is the skin wound model. It involves either causing damage to the skin (abrasion, burns or surgical excisions) and subsequently infecting the injured region with biofilm-forming bacteria, or inducing the formation of absess or wounds by seeding high-density biofilm forming bacteria subcutaneously. The commonly used clinically relevant organisms are *S. aureus, Stapylococcus epidermidis (Staphylococcus epidermidis)* and *P. aeuruginosa* [66]*.* The inoculum can differ depending on the expected severity of the infection ranging from acute to chronic, with chronic infections mimicking biofilm infection in human more accurately. Recovery and/or healing of the infected wound therefore indicates antibiofilm activity. Effectiveness of antibiofilm compounds can also be assessed by (a) examining the infectious process and recovery via real-time imaging with an in vivo imaging system as well as wound size measurement using calipers and photographs, (b) tissue analysis to assess tissue regeneration process, (c) assessment of genetic fingerprints associated with the formation of biofilms such as *pslD, mucC* and quorum sensing related genes (d) analysis of inflammatory patterns (e) assessmet of underlying organs [67, 68].

#### **3.3 Oral infections model**

Various biofilms from disease and non-disease causing microorganisms results in the formation of dental caries. Dental caries results from the interation between diet and microbiota-matrix that occur on the oral surface [69]. This is mostly replicated in animal models using newly weaned rats. Prior treatmet with antbiotics is essential to elimintate existing microbiome. Subsequently, the animals are fed with cariogenic diet while also receiving the bacteria (e.g. *Streptococcus mutans* (*S. mutans*)) orally daily for period of 5–7 days. The infection is ascertained by sowing oral samples. The topical application of the compounds is carried out on the teeth, daily for 30–45 days and the mandibles and molars excised at the end of the study to evalauate the carious lesions [70].

Periodontitis can as well be replicated in animal models using its associated bacteria (e.g. *Streptococcus gordoni* (*S. gordoni*) and *Porphyromonas gingivalis* (*P. gingivalis*)) and confirmed by oral sowing or PCR analysis [71]. The treatment can be perfomed topically either to prevent or eradicate already formed biofilm infection. The animals are euthanized at the end of the experiment, and the skull excised for alveolar bone loss assay of the maxilla [71, 72].

#### **3.4 Respiratory tract chronic infections model**

The primary organism associated with biofilm lung infection in cystic fibrosis (CF) has been identified to be *P. aeruginosa.* In the cystic fibrosis murine model, bacteria are inoculated either intrathecally, intranasally or by instillation [73]. The inoculum and the frequency of inoculation underscores the severity of infection.

Bacteria carriers such as alginate formed by the bacteria strain itself or by bacteria incorporation on agar beads can be used to establish chronic pulmonary infection. Intrathecal instillation is however, the most preferred route for inoculation of bacteria in this scenario [74].

Clinical isolates of *P. aeuruginosa* has also been used in some models. This model has an advantage of having a shorter time between establishment of infection and end of treatment than that described above. Since the bacteria is directly inoculated, it can result in severe acute respiratory distress (SARS) and eventually death even before treatment has been effective [67].

#### **3.5 Foreign body infection model**

The ability of biofilm forming bacteria to grow and multiply on the surfaces of certain medical devices [75] has led to the discovery of this model. The preformation of biofilm on these surgically implanted foreign bodies affect the activity of defense cells [25]. This model can be executed using two (2) approaches. These are Site Specific Device Model where biofilm forming bacteria are introduced at the injection site after devices are placed in particular organ or region in humans for evaluation of antibiofilm activity, and Subcutaneous Device Model where deliberately colonized foreign bodies are inserted in the subcutaneous layer, mostly at the back of the animals [76]. In Site Specific Device Model, antibiofim activity is measured at the part of the device that made contact with bacteria or measured by bacterial recovery at injection site [75]. In Subcutaneous Device Model, the mobility of antibiofilm peptides can be restricted with the aim of preventing bacterial contact and eventually biofilm development [75]. However other modes of assessment like histological analysis, imaging by IVIS, scanning microscopy, and inflammatory response detection can also be employed in evaluating antibiofilm activity in test organisms [75].

#### **4. Methods used to determine anti-biofilm effects of natural products**

Bacteria undergo an evolutionary mechanism to withstand harsh environmental conditions. The antibacterial agents derived from natural sources may serve as an effective alternative due to the presence of secondary metabolites, which possess selectional advantages against the biofilm-forming microorganisms [77–79]. Several methods have been reported as reliable protocols to investigate the anti-biofilm effects of natural products (**Table 1**) [88, 89]. Crystal violet assay is the widely accepted assay used to identify the anti-biofilm potentials of natural products despite the limitation, including the repeated washing that could lead to loss of cells and biofilm disruption [77, 88, 90, 91]. Other methods used to determine the antibiofilm effects of natural products are the Tissue Culture Plate (TCP) method [82], which exists as the most typical use standard method and is a comparatively reliable method to Congo Red Agar method (CRA) and Tube method [80]. Tube method and Congo red agar methods qualitatively detect biofilm formed, whiles the tissue culture plate method quantitatively determines the amount of biofilm formed [76]. Real time, conventional and multiplex PCR are other techniques used at molecular level to detect biofilm genes [92–94].

In measuring the anti-biofilm activities of natural products, viability and matrix biomass can be assessed, where resazurin and crystal violet staining are performed sequentially in the same plate. Wheat germ agglutinin-Alexa Fluor 488 fluorescent


#### **Table 1.**

*Methods to determine anti-biofilm effects of natural products.*

conjugate is mainly used to stain the matrix, which is essential to measure the biofilm matrix, biomass, and viability to investigate the potencies of anti-biofilm effects of natural products [95, 96].

#### **5. Antibiofilm agents from nature**

#### **5.1 Plant-derived antibiofilm agents**

Plants have since time immemorial served as a source of therapeutics for the treatment and prevention of a plethora of diseases. This practice continues today, with more than 80% of people globally reportedly using various herbal remedies as


*IC, inhibitory concentration; MBIC, minimum biofilm inhibitory concentration; MBEC, minimum biofilm eradication concentration.*

#### **Table 2.**

*Potent antibiofilm plant species.*


#### **Table 3.**

*Potent antibiofilm plant-derived compounds.*

a source of primary healthcare [97]. In mainstream medicine, plants have proven to be a prolific source of novel chemical matter from which essential drugs used to treat various diseases have been developed [98]. Galvanized by the emergence and spread of the antimicrobial drug resistance phenomena, numerous plant species have been

**Figure 1.**

*Chemical structures of some active plant derived antibiofilm compounds.*

#### *Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

thoroughly investigated as novel sources of antibacterial agents. To complement these strategies, the search for agents that can reverse resistance (resistance breakers) or target alternative mechanisms of overcoming antibacterial resistance, including biofilms, is being pursued [99, 100]. Plants have been identified as a potential oasis of such agents, prompting many studies in the last decade inspired towards the search for antibiofilm agents from plants. This section summarizes current studies on the investigation of antibiofilm agents, including crude extracts, fractions thereof, and pure compounds from plants (**Tables 2** and **3**; **Figure 1**).

#### *5.1.1 Apiaceae*

Despite being one of the least investigated, the Apiaceae plant family has produced some of the most prolific antibiofilm plant species. Among them is the annual herb *Trachyspermum ammi* popularly called bishop's weed [114]**.** Investigations on its seed led the isolation of a potent novel naphthalene compound, (4aS, 5R, 8aS) 5, 8a-di-1-propyl-octahydronaphthalen-1-(2H)-one, which remarkably inhibited both adherence (IC50 = 39.06 μg/ml) and formation of *S. mutans* biofilms (~60% inhibition at 78.13 μg/ml) *in vitro* (**Figure 2**). This activity was strikingly more pronounced than its parent compound's bacteriostatic and bactericidal properties (MIC = 156.25 μg/ml; MBC = 312.5 μg/ml) against *S. mutans* [114]. Thymol, a monoterpenoid isolated from *Carum copticum*, showed good activity against three bacterial species, namely *Klebsiella pneumoniae*, *Escherichia coli* (*E. coli*), and *Enterobacter cloacae* (*E. cloacea*), at sub-MIC levels, reducing biofilm formation by 80, 78, and 83%, respectively at 50 μg/ml (**Figure 2**). The compound was approximately fourfold more potent than its parent species [115].

#### *5.1.2 Asteraceae*

The Asteraceae is one of the most prominent species-rich plant families that produce highly active terpenoid compounds. A study on *Helichrysum italicum* led to the isolation of 21 compounds demonstrating varied activity of either inhibiting the formation or eradication of preformed *P. aeruginosa* biofilms. From the 21 compounds screened, chlorogenic acid emerged as the most active inhibiting biofilm formation (45% inhibition at 128 μg/ml). In contrast, biofilm eradication for all compounds was weak (<30%) [116]. Chondrillasterol, a terpenoid isolated from the plant *Vernonia adoensis,* has shown an intriguing activity profile being more potent in disrupting

#### **Figure 2.**

*Chemical structures of (4aS, 5R, 8aS) 5, 8a-di-1-propyl-octahydronaphthalen-1-(2H)-one and thymol.*

*P. aeruginosa* biofilms (complete disruption at 1.6 μg/ml) in comparison to inhibiting biofilm formation (wholly inhibited at 100 μg/ml) (**Figure 3**) [117].

#### *5.1.3 Burseraceae*

An aqueous extract of *Commiphora leptophloeos* showed promising inhibition of *Staphylococcus epidermidis* biofilm formation on different surfaces. At a concentration of 4 mg/ml, an aqueous stem bark extract of *C. leptophloeos* showed equipotent activity on inhibiting *S. epidermis* biofilms on a polystyrene (84% inhibition) and glass surface (82% inhibition) [118]**.** *Boswellia papyrifera (B. papyrifera)* is a deciduous tree 12 m high with a rounded crown, a white to pale brown bark that peels off in large flakes and exudes a fragrant resin [119]. Traditionally, as therapeutics, its leaves and roots are used to manage lymphadenopathy, while the resin serves as a febrifuge. The burnt leaves of *B. papyrifera* act as a mosquito repellent [120]. Essential oils obtained from *B. papyrifera* resin inhibited preformed *S. epidermidis* and *S. aureus* biofilms by 99–71%, and 95.3–59.1% at 217.3–6.8 μg/ml, respectively [121]. At a sub-MIC concentration of 0.27 μg/ml, the essential oil of *B. papyrifera* observed, under fluorescence microscopy, showed to inhibit the adhesion of stained *S. epidermidis* cells [122].

#### *5.1.4 Combretaceae*

The medicinal plant *Terminalia bellerica* (*T. bellerica*) is found predominantly in India, Sri Lanka, Bangladesh, and South-East Asia. Its fruits are traditionally used as a laxative, astringent, and antipyretic in treating menstrual disorder, piles, and leprosy. An investigation by Ahmed et al. [122] showed that the dried fruits of *T. bellerica* ethanol extracts could inhibit *S. mutans* biofilm formation *in vitro* on a glass surface by 92.2% at 250 μg/ml. Another *Terminalia* species, *T. fagifolia*, has been shown to have good antibiofilm properties. The ethanol stem bark extract of *T. fagifolia* inhibited the formation of preformed *S. epidermis* and *S. aureus* strains *in vitro*. It was particularly active against S. *epidermis* by inhibiting biofilm formation by ~70% at a sub-MIC concentration of 12.5 μg/ml compared to ~85% inhibition at 50 μg/ml against *S. aureus* [123]. Similarly, a water fraction of *Combretum elaeagnoides* showed potency against multiple species being able to reduce biofilm formation of *S. aureus*, *Salmonella typhimurium* (*S. typhimurium*), *Salmonella* 

**Figure 3.** *Chemical structure of chondrillasterol.*

*enteritidis* (*S. enteriditis*), *Klebsiella pneumoniae,* and *Enterobacter cloacae* by 80, 73, 63, 54, and 66%, respectively, at 1 mg/ml [124].

#### *5.1.5 Fabaceae*

Along with the Asteraceae, the Fabaceae family is one plant species that has received substantial interest as a source of antibiofilm agents. *Copaifera pauper C. paupera*) is a medicinal tree commonly found in South America that exhibits activity against monospecies and multispecies formed biofilms [125]. For the monospecies (*Aggregatibacter actinomycetemcomitans* and *Porphyromonas gingivalis*) *produced biofilms*, *C. paupera* oleoresins showed marked activity against the individual strains and with IC50 (eradication of biofilm) values of 58.66 μg/ml and 104.9 μg/ml, respectively. Activity against the multispecies biofilms was marginally lower with a measured IC50 (eradication of biofilm) of 594.5 μg/ml. *Copaifera pubiflora* oleoresins have shown a similar pattern of activity against individual *A. actinomycetemcomitans* [IC50 (eradication of biofilm) = 189.4 μg/ml)] and *P. gingivalis* [IC50 (eradication of biofilm) = 94.02 μg/ml)] strains and their combined multispecies biofilm [IC50 (eradication of biofilm) = 556.8 μg/ml)]. Three compounds, namely polylactic acid, hardwikiic acid, and kaurenoic acid, have been isolated from a *Copaifera* spp. and also shown to have potency against both the monospecies and multispecies biofilms of *A. actinomycetemcomitans* and *P. gingivalis* [IC50 (eradication of biofilm) ranging from 55.79 to 462 μg/ml)] [125]. Other species that have shown marked activity against multispecies biofilms include *Pityrocarpa moniliformis*, *Anadenanthera colubrina,* and *Dioclea grandiflora* [125].

*Trigonella foenum-graceum* (*T. foenum-graceum*), commonly called fenugreek, is an annual legume and a traditional spice crop native to the eastern Mediterranean. It has been known for its medicinal properties in the Mediterranean and Asian cultures for many years. Fenugreek seeds are traditionally used as laxative, expectorant, carminative, and demulcent [126]. The methanol extracts of *T. foenum-graceum* seeds inhibited *P. aeruginosa* biofilms in a dose-dependent pattern (24.1–68.7% at 125–1000 μg/ml) without affecting bacterial proliferation [127]. The extract caused a reduction to the exopolysaccharide (EPS) produced by *P. aeruginosa* biofilms. In addition to *P. aeruginosa*, *T. foenum-graceum* showed activity against the aquatic pathogen *Aeromonas hydrophila* reducing EPS production and biofilm formation by 46 and 76.9%, respectively, at 800 μg/ml [127]**.**

#### *5.1.6 Lamiaceae*

The Lamiaceae is a family of flowering plants commonly known as the mint family with a cosmopolitan distribution containing about 236 genera and about 6900–7200 species. Many plants in this family are aromatic and include widely used culinary herbs like basil, mint, rosemary, and sage [128]. Several Lamiaceae species have been interrogated for their antibiofilm activity and have shown pronounced activity against different biofilm stages of various microorganisms. One such species is the plant *Marrubium vulgare* (*M. vulgare*), a perennial herb found right across the globe. The plant is well renowned for its medicinal properties and serves as a therapeutic agent for several ailments, including gastrointestinal disorders, asthma, pulmonary infections, and ulcers. The aqueous decoctions of *M. vulgare* inhibited adherence of methicillin-resistant *S. aureus* biofilms with IC50 of 8 μg/ml and IC90 of 128 μg/ml [129].

However, the plant was less effective in inhibiting *S. aureus* biofilm growth on a plastic surface (31% inhibition at 128 μg/ml). Surprisingly, at the highest test concentration of 128 μg/ml, *M. vulgare* showed no bacteriostatic activity suggesting the species is selectively more potent against biofilm mechanisms. Aqueous extract prepared from the aerial parts of *Ballota nigra*, mirrored this bioactivity profile. Specifically, inhibiting methicillin-resistant *S. aureus* biofilm formation and adherence by 45–90% at 8–128 μg/ml while demonstrating limited bacteriostatic activity at the highest test concentration [129].

The genus *Salvia* is well documented for its bacteriostatic and bactericidal properties. Various species within this genus possess dual antibiofilm properties. Hexane-soluble and dichloromethane soluble fractions and sub-fractions of *Salvia officinalis* (*S. officinalis*) have shown impeccable antibiofilm and bacteriostatic properties with an MBIC50 and MIC values ranging from 3.668 to 200 μg/ml and 25 to 400 μg/ml, respectively, against *P. gingivalis*, *F. nucleatum*, *P. melaninogenica,* and *A. actinomycetemcomitans*. The labdane diterpenoid manool has been isolated and identified as the active principle from *S. officinalis*, showing pronounced activity with MBIC50 and MIC values of 12.5 μg/ml and 3.12 μg/ml, respectively against *A. actinomycetemcomitans* (**Figure 4**) [130].

While *Mentha piperita* oil is considerably active against *Chromobacterium violaceum* (Inhibited biofilm formation by 72.5% at 0.049 μg/ml), it is inactive against *P. aeruginosa* at reasonably higher test concentrations of 6.25, 3.125 and 1.56 μg/ml. In the same study, *Thymus vulgare* essential oil showed marked potency against both species inhibiting their biofilm formation by 70% at 0.049 μg/ml (against *C. violaceum*) and 65% at 3.125 μg/ml (against *P. aeruginosa*) [103]. Equally impressive is the species *Perovskia artemisioides,* which has inhibited biofilm formation of *L. monocytogenes*, *P. aeruginosa, S. aureus, Acinetobacter baumanii (A. baumanii), and Pectobacterium carotovorum* by 92, 95, 71, 35, and 94% at 4 μg/ml. Subsequent work led to the identification of numerous antibiofilm compounds from *P. artemisioides* [110].

#### *5.1.7 Malvaceae*

*Alcea longipedicellata* (*Aulonemia longipedicellata*) is a member of the *Alcea* genus with over 80 flowering plants in the family Malvaceae, commonly known as the hollyhocks and native to Asia and Europe. The compound, malvin, isolated from the flowers of *A. longipedicellata* flower, exhibited about 55% inhibition of *S. mutans* biofilm adherence at 0.1% v/v (**Figure 5**) [131]. *Hibiscus rosa-sinensis* a tropical shrub used in folk medicine to treat respiratory disorders and diarrhea, among other ailments, has shown remarkable activity against drug-resistant strains of *Helicobacter pylori* (*H. pylori*).

**Figure 4.** *Chemical structure of manool.*

*Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

An ethyl acetate fraction of *H. rosa-sinensis* demonstrated strong biofilm formation inhibition against *H. pylori* at sub-MIC concentrations (79% inhibition at 125 μg/ml) [132].

#### *5.1.8 Myristicaceae*

The Myristicaceae are flowering plants native to Africa, Asia, Pacific islands, and the Americas. The family consists of 20 genera and at least 500 species. Fruit of the Myristicaceae, particularly the lipid-rich aril surrounding the seed in some species, are essential as food for birds and mammals of tropical forests [133]. Plants in the

**Figure 6.** *Chemical structure of macelignan.* family Myristicaceae with reported antibiofilm activities include *Myristica fragrans* (*M. fragrans*), *Syzygium aromaticum,* and *Syzygium cumini*. *M. fragrans* has been shown to inhibit *Salmonella enterica* biofilm formation by 88% at 50 μg/ml. Biosynthesised silver nanoparticles of *M. fragrans* showed marginally improved activity inhibiting the formation of *S. enterica* biofilm by 99.1% at 50 μg/ml [134]. Another study on *M. fragrans* led to the isolation of the compound macelignan, which reduced the formation of *S. mutans* and *S. sanguis* biofilm by >50% at 10 μg/ml (**Figure 6**) [113]. The methanol fruit extract of *S. cumini* disrupted *Klebsiella pneumoniae* biofilm biomass in a dose-dependent manner by 35.85, 64.03, and 79.94% at test concentrations of 0.1, 0.5, and 1 mg/ml, respectively [135]. Essential oils from the aerial parts *of S. aromaticum* reduced *Staphylococcus epidermidis* biofilm biomass by 50.3% at 20 μg/ml [136]**.**

#### *5.1.9 Amaryllidaceae*

Extracts of *Crinum asciaticum*, a member of the family Amaryllidaceae, was investigated for its anti-tuberculosis, anti-efflux pump and antibiofilm activity. This study reealed the anti-infective activity of the extracts against *Mycobacterium smegmatis* (*M. smegmatis*) (NCTC 8159) and *Mycobacterium aurum* (*M. aurum*) (NCTC 10437) at MICs of 125 μg/ml and 250 μg/ml respectively. Also, efflux pump inhibition was observed for both *M. smegmatis* and *M. aurum.* Of great importance is the *in vitro* inhibition of *M. smegmatis* and *M. aurum* biofilms which was very significant at p < 0.005 [77].

#### **5.2 Antibiofilm agents obtained from mushrooms**

Research has shown that some species of macrofungi have various chemical components with antibacterial, antifungal, antiviral, antioxidant, anticancer and antiprotozoal properties [137]. The extracts of some species, including *Laetiporus sulphureus*, *Ganoderma lucidum*, and *Lentinus edodes* have demonstrated antibacterial activity [138]. *Fistulina hepatica*, *Ramaria botrytis*, and *Russula delica* extracts had promising antibacterial activity against multi-resistant microorganisms namely MRSA, *E. coli* and *Proteus mirabilis.*

In addition, some of these compounds were found to inhibit biofilm formation [137].

Studies on the aqueous extracts of *Macrolepiota procera*, *Pleurotus ostreatus*, *Auricularia auricula-judae*, *Armillaria mellea*, and *Laetiporus sulphureus* were shown to inhibit *Staphylococcal spp* biofilm formation. These extracts reduced biofilm formation by 47.72–70.87% without affecting bacterial growth [139].

A study by Borges et al demonstrated that ferulic and gallic acid inhibited biofilm formation in *P. aeruginosa* by interfering with cell motility and physico-chemical features on the cell surface. It also inhibited biofilm formation by *E. coli* due to phenolic compounds present therein [140]. Again, wild mushroom extracts had antibiofilm activity against *E. coli*, *Leucopaxillus gigantes* and *Mycenus rosea.* From this same study, extracts from *Sarcodon imbricants,* and *Russula delica* inhibited biofilm formation of *P. mirabilis* that is resistant to fluoroquinolones, ampicillin, and cephalosporins [138].

Extracts from *Lentinus edodes*, one of the mostly cultivated edible mushrooms, reacted negatively to biofilm proliferation by some bacteria in a study conducted by Lingström and colleagues [141]. Upon further fractionation and isolation, the compounds; oxalic acid, quinic acid, inosine and uridine (**Figure 7**) were discovered to be responsible for the various levels of antibiofilm activity against *S. mutans*, *Actinomyces naeslundii*, and *Prevotella intermedia* strains [141].

*Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

#### **Figure 7.**

*Structures of compounds isolated from mushrooms with antibiofilm activities.*

Melanin obtained from *Auricularia aricula*, an edible mushroom, has established antibiofilm properties [142]. This pigment exhibited significant antibiofilm inhibitory activity against *E. coli* K-12, *P. aeruginosa* PA01, and *Pseudomonas fluorescens* P-3 [142].

#### **5.3 Sponges as antibiofilm agents**

Marine sponges produce an array of secondary metabolites such as enzymes, enzyme inhibitors, and antibiotics and represent an untapped reservoir of bioactive compounds [143]. These compounds serve as defense against environmental threats like microbial infection, competition for space, or overgrowth by fouling organisms [144].

Phorbaketals isolated from the Korean marine sponge *Phorbas spp*. had antibiofilm activity against *S. aureus* [143]*.* Moreover, all six phorbaketals (phorbaketal A, phorbaketal B, phorbaketal C, phorbaketal A acetate, phorbaketal B acetate, phorbaketal C acetate, **Figure 8**) assessed for their antibiofilm activities revealed a minimum inhibitory concentration against *S. aureus* 6538 higher than 200 μg/ml. All six compounds significantly inhibited biofilm formation of methicillin-sensitive *S. aureus* in a dose-dependent manner, with Phorbaketal B and Phorbaketal C having the highest inhibitory effects, probably due to the presence of two hydroxyl groups in its structure. Phorbaketal B and C exerts their action via reduction of the expression of alpha-hemolysin (*hla*) and nuclease (*nuc1*). Phorbaketal C further reduced the expression of RNAIII (a regulatory molecule) which stimulates *hla* translation, thereby repressing the expression of *hla* [143].

**Figure 8.** *Chemical structures of phorbiketals isolated from Phorbas sp.*

In addition, natural compounds such as collismycin, hydroxyl flavonoids, hydroxylbipyridine, and hydroxyl anthraquinones exhibited antibiofilm activity depending on the number and positions of hydroxyl groups in the backbone structures [145]. The planktonic cell growth of *S. aureus* was relatively unaffected by the six phorbaketals at <100 µg/ml [143].

In another study by Paul and Puglisi, cell-free supernatants (CFSs) isolated from the sponge-associated bacteria belonging to the genera *Colwellia, Pseudoalteromonas, Shewanella* and *Winogradskyella* were evaluated for antibiofilm activity at 4°C and 25°C against Antarctic strains of *P. aeruginosa* ATCC27853 and *S. aureus* ATCC29213*.* Inhibition of biofilm formation was observed differently among strains which was dependent on the incubation temperature. Significant antibiofilm activity was observed by CFSs at 4 °C and 25 °C respectively against *S. aureus* and *P. auruginosa* without exhibiting cidal activity on bacterial growth [146]. The different physico-chemical nature of exopolymers produced by the *Colwellia sp.* GW185, *Shewanella sp*. CAL 606

and *Winogradsyella* CAL396 is responsible for their antibiofilm activity (**Table 4**). In another study, marine sponge-derived *Strepomyces sp*. SBT343 extracts were investigated for their antibiofilm activity on *Staphylococcal* biofilm formation. Results from *in vitro* biofilm assay of an organic extract showed inhibition of biofilm formation on polysterene, glass and contact lens surfaces. This same extract inhibited biofilm formation of *Staphylococcus epidermidis* and *S. aureus* with no antibiofilm activity against *Pseudomonas* biofilms [147].

#### **5.4 Algal sources of antibiofilm agents**

Existing literature proves the existence of compounds obtained from algae that possess antibiofilm properties against human pathogenic microbes. The scientific


#### **Table 4.**

*Bacterial exopolysaccharide with antibiofilm activity against pathogenic bacteria [143].*

**Figure 9.** *Structure of fucoidan.*

*Natural Products as Antibiofilm Agents DOI: http://dx.doi.org/10.5772/intechopen.104434*

**Figure 10.** *Structure of a halogenated furanones.*

research community however, continues to discover such natural antibiofilm agents. These compounds do not exist in their pure forms but are isolated from crude extracts through a series of processes [148].

Marine algae produce certain sulfated polysaccharides that exhibit antimicrobial and antibiofilm activities [149]. Fucoidan F85 (**Figure 9**), a sulfated polysaccharide extracted from *Fucus vesiculosus* upon observation was found to possess antimicrobial and antibiofilm properties against some dental plaque bacteria [149]. Fucoidans are made up of L-fucose and sulfate esters with other different molecules [150] and are normally extracted from brown algae using acid, solvent or water at a high temperature and a long reaction [151]. According to Yunhai and colleagues, Icelandic local seaweed species (*Ascophyllum nodosum* and *Laminaria digitate*), are sources of fucoidans with antibacterial activity [152].

A study conducted by Maggs et al proves that marine brown algae, *Halidrys siliquosa* produces compounds with antibiofilm activity against *Staphylococcus sp*, *Streptococcus sp*, *Enterococcus sp*, *Pseudomonas sp*, *Stenotrophomonas sp*, and *Chromobacterium sp*. *Halidrys siliquosa* can be found in rock pools and sometimes forests in the shallow subtidal zone [148].

*Delisea pulchra* red alga, produces halogenated furanones which show antibiofilm effects against *B. subtilis*, *E. coli* [153] and *P. aueroginosa* [154]. These furanones oppose the transmission of intracellular signals and speed up LuxR transcription turnover (**Figure 10**) [155].

The algal fronds of *Plocamium magga* has been reported to produce an isolate, KS8 from the *Pseudoalteromonas* genus that shows antibiofilm activity against acyl homoserine lactone base reporter strains (*Chromobacterium violaceum* (*CV*) ATCC 12472 and CV026) [156].

Ethanolic extracts of *Chlorella vulgaris* and *Dunaliella salina* can inhibit biofilm formation by *S. mutans* and *P. aueroginosa* [157]. This antibiofilm characteristic may be associated with the activity of glucotransferases [157].

Methanol extract of *Oscillatoria sp*., green algae containing silver nanoparticles also showed strong antibiofilm activity against all test pathogens in an experiment conducted by Adebayo-Tayo and associates [158].

Silver nanoparticles associated with aqueous extract of *Turbinaria conoides* have been reported to possess antibiofilm activity via adherence inhibition against *Salmonella typhi*, *E. coli* and *Serratialique faciens* [159].

#### **6. Miscellaneous agents with antibiofilm activities**

Several agents from natural products such as essential oils, honey etc. have shown great potential as bacterial biofilm inhibitors. These have been described below;

#### **6.1 Essential oil**

Essential oils from medicinal plants have received attention in recent times for their potential exploitations. This is as a result of the increasing reports of their composition and biochemicals to possess medicinal properties. A number of *in vitro* evidences indicates that essential oils can act as antibacterial and antibiofilm agents against a large spectrum of pathogenic bacterial strains.

The effect of *Lippia alba* (*L. alba*) and *Cymbopogon citratus* (*C. citratus*) (lemon grass) essential oils on biofilms of *S. mutans* was tested by Tofiño-Rivera et al. in an attempt to find new compounds against dental caries using the MBEC-highthroughput (MBEC-HTP) assay. The *L. alba* essential oils demonstrated significant eradication activity against *S. mutans* biofilms of 95.8% in 0.01 mg/dL concentration, and *C. citratus* essential oils showed eradication activity of 95.4% at 0.1 and 0.01 mg/ dL concentrations and of 93.1% in the 0.001 mg/dL concentration [160]. Further, geraniol and citral were later identified as the major components of the essential oils. A similar investigation by Ortega-Cuadros et al., showed 93.0% growth inhibition of *S. mutans* biofilms at a concentration of 1.00 μg/ml of *C. citratus* essential oil [161].

In an investigation to access the ability of *Allium sativum* fermented extract and cannabinol oil extract to inhibit and remove *P. aeruginosa* biofilms on soft contact lenses, the cannabinol oil extract inhibited biofilm formation by about 70% and eradicated preformed biofilms in both *P. aeruginosa* (ATCC 9027 strain) and *P. aeruginosa* clinical isolates from the ocular swabs tested [162]. Cannabigerol, a non-psychoactive cannabinoid which is also naturally present in trace amounts in the Cannabis plant was able to reduced the QS-regulated bioluminescence and biofilm formation of *Vibrio harveyi* (a marine quorum-sensing and biofilm-producing bacterial species) at concentrations not affecting the planktonic bacterial growth [163].

Essential oils from *Cyclamen coam* (*C. coam*) and *Zataria multiflora* (*Zinnia multiflora*) extracts inhibited biofilm formation on *P. aeruginosa* 214, a strong biofilm producing clinical strain [164]. *C. coam* and *Z. multiflora* essential oils inhibited biofilm formation completely at concentrations <0.062 mg/ml and 4 μl/ml, respectively. It is reported that carvacrol, a major constituent of *Z. multiflora* essential oil inhibits biofilm formation by preventing the initial adhesion of biofilm cells to the surface [165, 166].

#### **6.2 Lectin**

A study by Moura et al. reported the antibiofilm activity of a lectin extracted from *Moringa oleifera* (*M. oleifera*) seed. The lectin from this plant exhibited antibiofilm activity against *Bacillus* spp. and *Serratia marcescens* at concentrations of 20.8–41.6 μg/ ml and 0.325–1.3 μg/ml respectively [167]. The antibiofilm activity of the *M. oleifera* seed lectin might be due to the ability of these lectins to damage the cell wall and cell membranes through its interactions with glycoconjugates and polysaccharides constituents within the bacterial cell wall [168].

*Solanum tuberosum* lectins had a varying biofilm inhibitory effect when evaluated against an isolate of *P. aeruginosa* PA01. At a concentration between 2.5 and 15 μg/ml, the lectins inhibited the biofilm formation by 5–20% [169].

Plant lectins are reported to also exhibit antibiofilm activities against pathogenic microorganisms. A typical example are, lectins extracted from *Canavalia ensiformis*, *Calliandra surinamensis*, *Canavalia marítima* and *Alpinia purpurata* [170].

#### **6.3 Chitosan**

Chitosan is a polysaccharide composed of units of glucosamine (2-amino-2-deoxy-d-glucose) and *N*-acetyl glucosamine (2-acetamido-2-deoxy-d-glucose) linked by β (1 → 4) bonds. Chitosan is produced as a result of partial deacetylation of chitin leads. Chitin is found on the shells of crustaceans, arthropods and fungal cell wall [171].

The antibiofilm activity of chitosan from crab and shrimp species indigenous to the Philippines was investigated against *P. aeruginosa* and *S. aureus*. Biofilm inhibitory activity for both crab and shrimp chitosan were not observed against *S. aureus* at the concentration used, but activity was observed for shrimp chitosan at a concentration of 2.5 g/L. A 2.5 g/L mixed (1:1) chitosan solution of the two extracts had the highest percentage antibiofilm formation inhibition in *P. aeruginosa* biofilms. *S. aureus* biofilm formation was sensitive to the 10 g/L mixed (1:1) solution. The same mixed solution produced an inhibition against *P. aeruginosa* [172].

Costa et al. also reported that chitosan demonstrated antibiofilm and biofilm eradication activity against the fungus *Candida albicans* [171].

#### **6.4 Honey**

The exploration of new antibiotics to combat biofilm formation in resistant microbes has led to an increase interest evaluating the antibiofilm properties of honey. Manuka honey have demonstrated good antibiofilm forming activity against a range of bacteria, including S*treptococcus and Staphylococcus* species*, P. mirabilis, A. baumannii, E. coli, E. cloacae* and *P. aeruginosa* [173, 174].

Lu and colleagues studied the antibiofilm properties of four New Zealand based honeys; monofloral manuka honey, Medihoney (a manuka-based medical-grade honey), manuka-kanuka blend, and a clover honey on two *P. aeruginosa* strains PAO1 and PA14 with different biofilm forming abilities. All the different types of honey used in the study were effective at inhibiting both the planktonic cell growth and biofilm formation of both strains. In the study of the biofilm eradication properties of the honey, they concluded that honey used at clinically obtainable concentrations completely eradicated established *P. aeruginosa* biofilms [175]. Similar results were obtained using different strains of *S. aureus*, including methicillin-resistant *S. aureus* (MRSA) strains. In this study, they demonstrated that honey is able to reduce biofilm mass and also to kill cells that remain embedded in the biofilm matrix; and planktonic cells released from biofilms following honey treatment do not have elevated resistance to honey [176].

The biofilm inhibitory effect of Costa Rican Meliponini stingless bee honeys has also been reported against *S. aureus* and *P. aeruginosa* biofilm formation. The meliponini stingless bee honeys in a concentration-dependent manner inhibited the planktonic growth and biofilm formation, and also caused the destruction of *S. aureus* biofilm [177].

Australian honey has also been reported to possess antibacterial and biofilm inhibitory activities. Sindi A and colleagues in their investigation reported that Western Australian honeys from *Eucalyptus marginata* (Jarrah) and *Corymbia calophylla* (Marri) trees exhibited antimicrobial activity against Gram-negative and Gram-positive pathogens. They reduced both the formation of biofilms and the production of bacterial pigments, which are both regulated by quorum sensing. The Western Australian honey when applied to preformed biofilms had biofilm eradication activity by reducing metabolic activity in the biofilms [178].

#### **6.5 Peptides**

Peptides are small molecules made of 10–100 amino acids that are part of the innate immune response, and found among all classes of life contributing to the first line of defense against infections. In the search for an effective agent that can treat chronic infections, antimicrobial peptides (AMPs) have been shown to demonstrate antimicrobial, antibiofilm and biofilm eradication properties. Although there has not been much studies on the biofilm inhibitory action of AMP compared to its antibacterial activity, some naturally occurring AMP's have been reported to exhibit strong antibiofilm activities against multidrug resistant as well as clinically isolated bacterial biofilms [179].

Cathelicidin peptides are one of the most important classes of AMP. Investigation of cathelicidin AMP, indicates that SMAP-29, BMAP-28, and BMAP-27 have antimicrobial activity and are able to significantly reduce biofilm formation by multidrug-resistant (MDR) *P. aeruginosa* strains isolated from patients with cystic fibrosis. In addition, they were bactericidal in preformed biofilms [180]. Blower et al. also demonstrated that the SMAP-29 peptide is able to inhibit biofilm production in *Burkholderia thailandensis* by about 50% at peptide concentrations at or above 3 μg/ml [181].

Hepcidin 20 alters the biofilm architecture of *Staphylococcus epidermidis* by targeting the polysaccharide intercellular adhesin after it has reduced the extracellular matrix mass [182].

The peptides lactoferrin, conjugated lactoferricin, melimine and citropin 1.1 have all shown good anti-biofilm activity against *S. aureus* and *P. aeruginosa* infection in medical devices [183].

#### **7. Conclusion**

Microorganisms, though form biofilms as a defense mechanism for survival, this action poses a threat to the healthcare system by compromising the therapeutic efficacy of antimicrobial agents and causing ascendancies in antimicrobial resistance. Natural products from plants and microorganisms provide a plethora of chemical compounds with antibiofilm properties capable of disrupting pre-formed biofilms or inhibiting the formation of new biofilms. Identifying novel antibiofilm compounds from these sources is essential to mitigate biofilm-mediated infections. Similarly, the exploration of model systems is critical for evaluating the antibiofilm properties of newly identified medicinal compounds. Altogether, understanding the antibiofilm potential of these natural products could serve as an impetus in antimicrobial drug discovery.

### **Author details**

Cynthia Amaning Danquah1 \*, Prince Amankwah Baffour Minkah1,2, Theresa A. Agana<sup>3</sup> , Phanankosi Moyo4 , Michael Tetteh1 , Isaiah Osei Duah Junior5 , Kofi Bonsu Amankwah6 , Samuel Owusu Somuah7 , Michael Ofori1 and Vinesh J. Maharaj4

1 Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

2 Global Health and Infectious Disease Research Group, Kumasi Centre for Collaborative Research in Tropical Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

3 Department of Pharmaceutics, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

4 Department of Chemistry, University of Pretoria, Pretoria, South Africa

5 Department of Optometry and Visual Science, College of Science, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

6 Department of Biomedical Sciences, University of Cape Coast, Cape Coast, Ghana

7 Department of Pharmacy Practice, School of Pharmacy, University of Health and Allied Sciences, Ho, Ghana

\*Address all correspondence to: cadanq@yahoo.com; cadanquah.pharm@knust,edu.gh

© 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 13**

## Efficacy of Radiations against Bacterial Biofilms

*Salma Kloula Ben Ghorbal, Rim Werhani and Abdelwaheb Chatti*

#### **Abstract**

A biofilm has been defined as a community of bacteria living in organized structures at a liquid interface. Biofilms can colonize a wide range of domains, including essentially industrial sectors, different natural environments, and also biomedical environments. Bacteria in biofilms are generally well protected against environmental stresses and, as a consequence, are extremely difficult to eradicate. The current study was to investigate the efficacy of different radiations against bacterial biofilms on different surfaces. It was established that the majority of available treatments have proven less effective against pathogenic biofilms, compared to planktonic bacteria. Therefore, new biofilm treatment strategies are needed, including physical treatments such as radiations. UV LEDs offer new solutions to prevent biofilm formation on inaccessible surfaces, such as medical and food equipment and, potentially, sanitary facilities, to limit nosocomial infections, compared to continuous UV irradiation treatment. Moreover, the antimicrobial effectiveness of gamma irradiation is therefore guaranteed in the treatment of bacteria associated with a biofilm, compared to planktonic bacteria. However, limited studies have been conducted to evaluate the inactivation effect of low-energy X-rays on more resistant biofilm pathogens on foodcontact surfaces.

**Keywords:** biofilm, bacteria, UV, X-rays, gamma irradiation, efficacy

#### **1. Introduction**

Biofilms consist of structured communities of bacteria, embedded in a self-produced polymeric matrix and adherent to inert or living surfaces [1–3]. Biofilm mode of growth is an approach in microorganisms to survive harsh growth conditions. Most microorganisms such as *Pseudomonas aeruginosa* [4], *Staphylococcus aureus* [5], and *Escherichia coli* [6] favor a way of life where the bacterial population is attached to a support, named sessile state, rather than free and isolated in the environment, named planktonic state. The attachment to a surface is a "survival strategy" that allows the bacteria to settle and colonize an environment. This structure represents the normal way of life of a bacterium [7]. This way of life is of great interest for the bacteria since it gives them a resistance to different sources of stress to which planktonic bacteria are sensitive [1]. In effect, bacteria in biofilms are generally well protected against

environmental stresses, antibiotics [8], disinfectants, and the host immune system [9] and as a consequence are extremely difficult to eradicate [10]. Therefore, biofilms constitute a protected mode of growth that allows survival in a hostile environment. This strength is essentially due to the biofilm matrix composed of numerous polysaccharides, proteins, and extracellular DNA (eDNA) which is crucial in biofilm structural integrity [11]. Although it is widely accepted that eDNA is released primarily by cell lysis, several studies have shown that other mechanisms of active secretion may coexist [12]. This implies that eDNA is an interesting target in the control of biofilms. Numerous studies have demonstrated that biofilm formation can be prevented by enzymatic degradation of eDNA by DNase [11]. It was reported for *Campylobacter. jejuni* biofilm-attached to stainless steel surfaces that degradation of eDNA by exogenous addition of DNase led to rapid biofilm removal and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments, like radiations, antibiotics [13]. For UVC radiation, they target genomic DNA by forming thymidine dimers in RNA and DNA, which can interfere with transcription and replication and thus induce bacterial death [14]. For the extracellular DNA, the formation of thymidine dimers, following exposure to UVC, has no consequence on bacterial multiplication. Therefore, the presence of eDNA in the matrix can only increase the viscosity of the matrix and therefore continues to block the passage of radiation through the biofilm which limits the effectiveness of the radiations.

#### **2. Surfaces colonized by biofilms**

Biofilms can colonize a wide range of domains, including essentially industrial sectors, different natural environments (soil, sediment, etc.), and biomedical environments [15]. Many bacteria form clumps at the bottom of the containers. Then, they reach the surface of the liquid-type media. However, some bacteria such as *Salmonella* [16], *E. coli, P. fluorescens,* and *Vibrio cholera* produce rigid or fragile pellicle structures at air-liquid interfaces [17]. Biofilm production by the colonization of the air interface can facilitate and contribute to gas exchange while enabling the acquisition of nutrients and water from the liquid phase. The biofilms at air-liquid interfaces can cause severe problems in industrial water systems [18].

In the medical sector, microbial adhesion resulting in biofilm formation on implanted medical devices is a common occurrence and can lead to serious illness and death [19]. Implanted medical devices like intravascular catheters, urinary catheters, pacemakers, heart valves, stents, and orthopedic implants, normally used for therapeutic purposes, can also be the source of real infectious risks when colonized by bacterial biofilms [20].

#### **3. Biofilms treatment**

The majority of available antibacterial treatments have shown their effectiveness against planktonic bacteria. However, these treatments have proven to be ineffective against pathogenic biofilms [21, 22], which are thousands of times more resistant to this type of treatment [23–25]. It is therefore difficult to eradicate biofilms effectively because of the phenomenon of biofilm recalcitrance [22]. Despite the importance of biofilm treatment either in the medical or environmental sectors, studies into the effectiveness of irradiation on biofilm-associated cells are lacking. Therefore, new

biofilm treatment strategies are needed, including physical treatments such as radiations. This review presents an overview of bacterial biofilm development and seeks to highlight the efficacy of radiations against bacterial biofilms.

#### **3.1 Continuous UVC irradiation treatment efficiency on biofilms**

Though germicidal UV radiation is widely applied for disinfection of water and food from planktonic bacteria, it may also be used to prevent bacterial growth and colonization on surfaces, as biofilms, within engineered systems [26]. Moreover, the UVC-based method is to be of practical use for disinfection of catheters in the clinic, as they are the major sources of infection [27]. However, higher UV doses would be required to inactivate biofilm-bound bacteria than planktonic bacteria because the biofilm would provide some degree of protection from the effects of UVC irradiation [28].

Torkzadeh et al. [26] have developed an experimental device and method to ensure the growth of biofilms in the presence of UV radiation and to measure the resulting reduction in surface biofilm growth. Under optimal growth conditions and after 48 h of growth, the reduction of the bio-volume of the *E. coli* surface is about 95% by a UV intensity of 50.5 μW/cm2 at 254 nm, compared to the control biofilms. The UV intensity required for biofilm prevention was greater than that expected due to the UV dose–response of tested bacteria and the cumulative doses applied to the tested surfaces. This results indicate that biofilms can form even under irradiation conditions that should inactivate planktonic cells completely. This is probably due to the protective effects of colloidal material and microbial exudates, that form biofilm matrix.

In water and wastewater infrastructure, biofilms pose a real problem for disinfection. Until now, the majority of ultraviolet (UV) disinfection studies focus on planktonic bacteria, with limited attention given to UV irradiation of biofilms. Among the few outstanding studies, the study of Myriam et al. [29] focused on the study of UVC dose/biofilm production relationship for five *P.aeruginosa* strains, isolated from wastewater. The aim was to evaluate the impact of incremental UVC doses, up to 100 mJ.cm−2, on the ability of *Pseudomonas* strains to produce biofilm, knowing that the UV dose equal to 40 mJ.cm−2 is the dose recommended for the disinfection of water in Europe and America. The results of this study showed that biofilm production presents a progressive increase in function of an increasing of exposure UVC dose until a threshold UV dose. Moreover, the values of threshold UV doses were different in relation with the response of each bacteria strain to UVC dose (dose/response). This may be explained by the fact that intraspecific difference showed in the UV dose/response relationship is probably dependent on several factors: the degree of DNA damage induced by UV, the speed of induction of DNA repair mechanisms for each tested bacteria. On the other hand, beyond the threshold, a progressive decrease in the production of biofilm correlated with the increase of UV dose was noticed. This decrease in biofilm production can be explained by the fact that the bacterial strains have received a lethal UV dose reducing bacterial sustainability by the accumulation of photoproducts surpassing the capability of bacteria DNA repair mechanisms allowing for consequent, a decrease of biofilm formation and the weakening of this resistant structure.

The UV treatment has evolved a lot since the development of UV light sources from the conventional mercury lamp to the light-emitting diode (LED). It was established that pulsed UV can be more effective than a continuous emitting mode to control biofilms. Moreover, adaptable UV LED is promising to control biofilms in the water distribution system, according to the review of [30]. Luo et al. [30] have, recently, demonstrated that pulsed UV can be more effective than a continuous emitting mode to control biofilms, on one side and that a selective combination of UV LED wavelengths allows targeting damaged biofilm components, on the other hand.

In the medical sector, an application of radiation treatment on catheters looks promising. In this context, the study of Jimmy Bak et al. [31], who proposed a method for disinfecting the inner surface of catheters biofilm, has demonstrated that mean killing rates were 89.6% for 0.5 min exposure, 98% for 2 min exposure, and 99% for 60 min exposure. About 99% of the cells were killed with a UVC dose of 15 kJ m−2. This dose, which is 100 to 1000 times higher than the lethal dose required for planktonic cells, is assumed to be the maximum dose necessary to avoid contamination of newly inserted catheters. The need for high doses to kill mature biofilm and the limited effect of currently available UVC light sources result in a relatively long treatment time of about 60 minutes, hence the need for new UV sources like UV LED.

Recently, Jimmy Bak et al. [31] have tested a newly developed UVC disinfection device, which can be connected to a Luer catheter hub, on polymer tubes contaminated with a wide range of either bacterium, including *S. aureus*, *E. coli,* and *P. aeruginosa* and fungi like *Candida albicans*. Their results have shown no viable counts after 2 min of radiation for bacteria. Whereas, Killing of *C. albicans* needs more than 20 minutes to be obtained in a UVC absorbing suspension.

On any type of surface contaminated by biofilm, the effectiveness of UVC light in inactivating biofilm-forming microorganisms is mainly due to the ability of DNA molecules to absorb UV photons between 200 and 300 nm, with an absorption peak at 260 nm, at first. Then, this uptake causes damage to the DNA by altering the pairing of nucleotide bases, creating new bonds between adjacent nucleotides on the same DNA strand. This damage occurs particularly between pyrimidine bases [32].

#### *Efficacy of Radiations against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.103653*

Therefore, to limit UV damages, bacteria generally possess molecular mechanisms to restore DNA lesions [33], which preserve the irradiated biofilm, from damage due to UVC exposure. This repair mechanism has been shown to be effective up to a threshold dose-related to a maximum accumulation of photoproducts and of reactive oxygen species, which can no longer be managed by this mechanism [29]. Our study in 2016, confirmed the oxidative stress through ROS accumulation, following UVC exposure, and has demonstrated that, in the enzymatic ROS-scavenging pathways, catalase and peroxidase enhancement improved the resistance of *P. aeruginosa* treated with incremental UV-C doses. However, longer exposure to UV-C rays inhibited SOD activity. This result confirms that SOD cannot efficiently remove superoxide radicals that accumulated in cells of *P. aeruginosa* at longer irradiation time and further confirms the inability of the repair system besides the ROS-scavenging pathways to deal with photoproducts and ROS accumulation, respectively [34, 35].

We can then conclude that the resistance of bacteria to UVC treatment remains at dose limits. Beyond these doses, there is an exhaustion of the repair system and a sure bacterial death. Hence the need to exceed the dose limits in order to escape bacterial resistance (**Figure 1**).

#### **3.2 UV LED irradiation treatment efficiency on biofilms**

UV LEDs are emerging as competitive light sources because of advantages such as the possible selection of combined-wavelength UV LED [30], adjustable emitting mode, and the designable configuration that facilitate their incorporation into confined spaces. Therefore, UV LEDs offer new solutions to prevent biofilm formation on inaccessible surfaces, such as medical and food equipment and, potentially, sanitary facilities, to limit nosocomial infections. These results imply that surfaces more exposed to bacterial colonization require adequate UVC irradiation to prevent biofilm establishment. Furthermore, continuous surface irradiation may be insufficient as a sole source for biofilm prevention in many circumstances [26]. However, problems with low wall plugs and reliable power supplies still limit the effectiveness of UV LEDs, which further enlightens the prospective of UV in dealing with the biofilm issue in water infrastructure and also in the medical sector.

In this context, the study of Aikaterini et al. [36] on *P. aeruginosa* biofilms at different growth stages, within 24, 48, and 72 h of growth, was conducted to judge the effectiveness of ultraviolet B (UVB), at 296 nm and ultraviolet C (UVC) irradiation, with central wavelength at 266 nm, two different light-based treatments. The effectiveness of the UVB and UVC irradiations was quantified by counting colony-forming units. For UV exposure, a type of AlGaN light-emitting diodes (LEDs) was used to distribute UV irradiation on the biofilms. For *P.aeruginosa* biofilms, it appears that UVB irradiation is much more effective than UVC radiation for the inactivation of mature biofilms. The fact that UVB at 296 nm is present in daylight and has such a disinfecting capacity on biofilms opens the way to the treatment of infectious pathologies [36].

In parallel, the study of Gora et al. [37] has demonstrated that UV LED irradiation at 265 nm achieved 1.3 log inactivation of biofilm-bound *P. aeruginosa* at a UV dose of 8 mJ/cm2 . This inactivation level is lower than those that have been reported by researchers using UVC LEDs to inactivate planktonic *P. aeruginosa*, a finding that can be explained by the higher resistance of biofilm-bound bacteria to UV inactivation.

Moreover, the combination of UV LED and Blue laser was tested on *S. aureus* biofilm and gave the highest biofilm reduction of about 80.57%. It was then demonstrated that it to be the best choice to eradicate more biofilm [38].

Concerning the effect of radiations on biofilm matrix, it is well established that bacteria enclosed in a layer of exopolysaccharides are protected by 13% from UVC radiation. It was also confirmed that absorption of UV light by the alginate, an important matrix molecule, translated into a higher survival rate than observed with planktonic cells, for the same UV dose [39]. In effect, alginate water retention seems to be at the origin of the obvious ability to survive severe environments, like UVC exposure. On the other hand, the effect of UV LED on exopolysaccharides (EPS) has not been extensively studied, but it is predicted to be similar to the effect of continuous UVC on EPS. It is then assumed that following the prolonged exposure to UVC radiation, the production of EPS is stimulated [34]. Moreover, in the framework of the development of a profitable strategy to improve the EPS yield, UV irradiation mutagenesis of *Bacillus licheniformis* significantly improved the EPS yield. Significantly enhanced yield (>3-folds) of EPS after UVC exposure can only confirm the stimulating effect of UVC radiation on the production of EPS, to ensure better protection against UVC rays and then bacterial survival [40] (**Table 1**).

#### **3.3 Ionizing radiation treatment efficiency on biofilms**

Ionizing radiation is a non-thermal destruction technique that inactivates pathogens that may contaminate certain food products, by exposing them to irradiation sources such as high-energy X-rays at about 5 MeV, gamma rays at about 2.5 MeV, or electron beams at about 10 MeV [41]. Compared to these conventional high-energy irradiation techniques, low-energy X-rays have a higher linear energy transfer (LET) value, resulting in a greater relative biological effect (RBE)[42]. Some previous studies have shown that low-energy X-rays is effective in destroying certain planktonic germs such as *E. coli* O157:H7, *Salmonella*, *Listeria monocytogenes,* and *Shigella flexneri* [43–45]. However, few studies have investigated the effect of low-energy X-rays on more resistant pathogens in mono-microbial or poly-microbial cultured biofilms and on food contact surfaces.

Despite of this, we could not simply conclude that low-energy X-rays destroyed EPS in biofilm. Therefore, we could at least postulate that low-energy X-rays irradiation weakened EPS structure in biofilm. Typical EPS mainly comprises


#### **Table 1.**

*UV doses required for the treatment of biofilms for different microorganisms.*

*Efficacy of Radiations against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.103653*

homopolymers like cellulose and dextran and heteropolymers of alginate, emulsan, gellan, and xanthan, which maintain the stability of the biofilm matrix [46]. Ionizing irradiation can break down glycosidic bonds and consequently degrade polysaccharides and destabilize the biofilm [47].

Similarly, some in vitro studies also showed that the direct effect of radiation on oral *C. albicans* cells leads to a rapid proliferation ability, increase of virulent factors, and resistance to drugs [48]. Moreover, irradiated *Klebsiella oxytoca* strains of oral origin were more virulent than non-irradiated ones [49]. All of these results indicated that direct exposure of X-rays can affect the virulence of oral bacteria microbes even at therapeutic doses [50].

Concerning gamma irradiation, it is an established technology of well-documented safety and efficacy for the inactivation of pathogenic microorganisms such as *Salmonella* [51, 52]. Recently, gamma-ray sterilization was proven to be a viable method of sterilization of conducting polymer-based biomaterials for biomedical applications [53].

The study of [54] has demonstrated that in bacterial biofilms attached to stainless steel, gamma irradiation at a dose of 10.0 kGy reduced the counts of *S. aureus* attached for 1 hr. and overnight by ≥5.1 and 5.0 log CFU/cm2 , respectively. Gamma irradiation at a dose of 1.0 kGy reduced the counts of *P. aeruginosa* counts to below the limit of detection (<2logCFU/cm2 ).

Concerning food sterilization, *Salmonella* is a problematic bacterium due to its biofilm resistance to chemical sanitizing treatments. Ionizing radiation is known to be used to inactivate *Salmonella* on a variety of foods and contact surfaces in the food industry. The relative efficacy of the process against biofilm-associated cells versus free-living planktonic cells was tested for three food-borne-illness-associated isolates of *Salmonella*, by by Niemira and Solomonet [55]. They demonstrated that the dose of radiation required to reduce 90% (D10 values) of *Salmonella enterica* serovar *Anatum* was not significantly different between biofilm-forming bacteria (0.645 kGy) and planktonic cells (0.677 kGy). In contrast, biofilm-forming cells of *S. enterica* serovar *Stanley* were significantly more sensitive to ionizing radiation, with a D10 of 0.531, than planktonic cells, with a D10 of 0.591 kGy. D10 values of *S. enterica* serovar *Enteritidis* were similarly decreased for biofilm-associated cells (0.436 kGy) in comparison to planktonic cells (0.535 kGy). The anti-microbial efficiency of ionizing


#### **Table 2.**

*Gamma irradiation and doses required for the treatment of biofilms for different microorganisms.*

radiation is therefore guaranteed in the treatment of bacteria associated with a biofilm. Ben Miloud YahiaYahia [52] proposed that the biofilm-forming abilities could be reduced with temperature decrease and increasing gamma radiation doses (**Table 2**).

### **4. Conclusion**

This study has demonstrated that ionizing and non-ionizing radiation effectively reduces the populations of both planktonic and biofilm-associated bacteria. However, biofilms are confirmed to be more difficult to eradicate and require enhanced doses for their eradication. It was also confirmed that radiation sensitivity is microorganism specific. Likewise, the influence on radiation sensitivity of the cultured state of the organism, between planktonic and biofilm-associated, is also isolate specific, confirmed for gamma-treated *Salmonella*. But also, the stage of biofilm growth seems to affect the effectiveness of radiations treatment, as confirmed for *Pseudomonas* and *Staphylococcus* biofilms. In general, these results show that, in contrast to chemical antimicrobial treatments, the antimicrobial efficacy of radiation is preserved or enhanced when treating biofilm-associated bacteria, compared to planktonic cells.

### **Author details**

Salma Kloula Ben Ghorbal1 \*, Rim Werhani1 and Abdelwaheb Chatti<sup>2</sup>

1 Laboratoire de Traitement et Valorisation de Rejets Hydriques, Center des Recherches et des Technologies des Eaux (CERTE), Université de Carthage, Tunis, Tunisia

2 Faculté des Sciences de Bizerte, Unité de Biochimie des Lipides et interaction des macromolécules en Biologie, Laboratoire de Biochimie et Biologie Moléculaire, Université de Carthage, Tunis, Tunisia

\*Address all correspondence to: salmakloula@yahoo.fr

© 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.

*Efficacy of Radiations against Bacterial Biofilms DOI: http://dx.doi.org/10.5772/intechopen.103653*

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#### **Chapter 14**

## Antifouling Strategies-Interference with Bacterial Adhesion

*Zhen Jia*

#### **Abstract**

Biofilm refers to a viable bacterial community wrapped in self-produced extracellular polymeric substances (EPS) matrix. As bacteria shielded by EPS are viable and can resist broad hostile environments and antimicrobial agents, biofilm poses a massive challenge to industries and human health. Currently, biofilm has accounted for widespread and severe safety issues, infections, and economic loss. Various antifouling strategies have been designed and developed to prevent biofilm formation. As bacterial biofilm is perceived as a dynamic multistage process in which bacterial attachment on solid surfaces is the prerequisite for biofilm formation, the interference with the attachment is the most promising environmentally benign option to antifouling. The chapter summarizes and discusses the antifouling strategies that interfere with the adhesion between bacteria and substrate surfaces. These strategies primarily focus on modifying the substrate surface's topographical and physicochemical properties.

**Keywords:** biofilm, antifouling, modification, topography, physicochemical property

#### **1. Introduction**

Bacterial biofilm is a structured community of bacterial cells within a selfgenerated hydrated extracellular polymetric substance (EPS) matrix anchored to a surface [1]. The physical channels formed during biofilm formation facilitate nutrients, air, and water to penetrate and distribute to cells [2], promoting microbial reproduction, metabolism, and EPS secretion. EPS is a biopolymer produced by bacterial cells following surface attachment, serving as a house or shelter for cells [3, 4]. It mainly consists of a wide variety of exopolysaccharides (40–95%), proteins (1–60%), nucleic acids (1–10%), and lipids (1–40%) [2, 5], which are critical factors to enhance bacterial adhesion behavior. On the one hand, EPS possesses mechanical stability, protecting cells from mechanical damages and shear and providing a functional microenvironment for bacterial growth [6]. On the other hand, EPS creates a physical barrier that enables bacteria inside to survive under harsh conditions and to resist antibiotics and antimicrobial agents [7].

Biofilm, different from planktonic cells, is a self-protection growth pattern of bacteria. Over 99% of the world's bacteria present as a form of biofilm [8], broadly distributing on broad infrastructure elements, systems, and devices. Due to strong self-protection ability and resistance to harsh conditions, the unwanted biofilms pose severe threats and challenges to human health and industries, such as the transmissions of disease and infections and interferences of system functions and decreases in the endurance of surfaces and devices [9]. In the medical system, bacteria can form biofilm in healthcare settings (such as sinks, drains, and showers) and medical devices (such as surgical instruments and implantable biomedical devices). Up to 80% of hospital-acquired infections (HAIs) contribute to biofilm infections [10]. Such HAIs affect about 10% of all hospital patients in the United States and lead to nearly 100,000 deaths annually [11, 12]. In the food industry, biofilms have been widely reported on food surfaces, food contact surfaces, and processing systems, leading to product contamination, cross-contamination, food withdrawal, and disease outbreaks [13–15]. In the marine system, biofilm accumulation accelerates corrosion on marine vehicles, resulting in equipment clogging, damage, and roughness [16]. In addition, biofilm increases hydrodynamic drag, which adversely interferes with equipment performance and increases fuel expenditure up to 45% [17]. The economic losses caused due to biofilm are also enormous. For US Navy alone, the estimated fuel cost per annum is around \$500 million, of which \$75–100 million account for drag induced by fouling organisms [18]. Therefore, it is critical and urgent to prevent biofilm formation.

Biofilm formation is a dynamic process, typically containing five stages: initial reversible attachment, irreversible attachment, micro-colony formation, biofilm formation and maturation, and dispersion. Among them, initial reversible attachment is critical. In this stage, bacteria actively seek and anchor to surfaces relying on the motility of planktonic cells using extracellular organelles and proteins (such as pili, curli fibers, flagella, and outer membrane proteins), cells' gravitational transportation, physical forces between cells and surfaces (such as van der Waals forces, steric interactions, and electrostatic interactions), and hydrodynamic forces of the surrounding environment [19, 20]. Additionally, other forces include acid-base interactions at a very short range, around 5 nm range, responsible for bond formation and hydrophobic forces [21] and divalent cations responsible for crosslinking between bacterial surface polymers that aid in matrix stabilization [22]. The attachment of a microbial cell to a surface is called adhesion [23]. The adhesion is reversible as bacteria are loosely attached. The attached cells still exhibit Brownian motion and can easily dissociate back to planktonic forms. The adhesion of bacteria is primarily influenced by various factors, including surface properties, environmental conditions (like pressure and temperature), and bacterial orientation [24].

Based on the process of biofilm formation, it is worth noting that bacterial adhesion is an initial prerequisite for biofilm formation. After being attached to surfaces, bacterial cells initiate to reproduce and ultimately grow into a biofilm, demonstrating that bacterial adhesion is the fundamental and critical step responsible for biofilm formation. Therefore, inhibiting bacterial adhesion is the desirable and key antifouling approach to prevent biofilm formation. The adhesion of bacteria is mainly affected by various factors, including surface properties of substrates, physicochemical properties of microbes, and environmental conditions [25]. As the properties of substrate surfaces are changeable and can be manipulated depending on the purpose, antifouling approaches to control biofilm formation mostly focus on modifying surface properties, including surface topography and physicochemistry (**Table 1**).

The purpose of this chapter is to provide insights into antifouling strategies related to the topographical and physicochemical properties of substrate surfaces in the prevention of cell adhesion and to elucidate corresponding theoretical mechanisms. This chapter also covers the main challenges and future trends of antifouling materials.


#### **Table 1.**

*Surface modification techniques.*

#### **2. Physicochemical modification strategy**

It is well documented that bacterial adhesion can be effectively tuned and reduced by altering surface physicochemical properties using chemically active antifouling coatings [26, 27]. Currently, various coatings have been extensively reported for their effectiveness in preventing bacterial initial adhesion.

#### **2.1 Surface energy**

Surface energy is the binding or interfacial attractive force between materials and solid substrates [28]. It is an essential physicochemical property of a solid surface. Many studies have demonstrated that changing surface energy was related to affecting bacterial adhesion [29, 30]. Baier analyzed the relationship between surface energy and bacterial adhesion, known as the Baier curve depicted in **Figure 1** [31]. According to the curve, bacterial adhesion is minimized when the surface energy of a substrate is in the range of 20–30 mN/m (the lowest values), while antifouling occurs when surface energy is higher than 70 mN/m [32, 33].

Surface energy represents the degree to which water can bind on the surface [34] and can be determined by contact angle (*θ*) [35]. *θ* characterizes the ability of water to maintain contact with a solid surface. 'Hydrophilic surface (*θ* < 90°)' and 'hydrophobic surface (*θ* ≥ 90°)' are two common terms to describe the incongruous behavior of water on solid surfaces [36]. Hydrophilic surfaces are surfaces with high surface energy, while hydrophobic surfaces are surfaces with low surface energy [37].

**Figure 1.** *Correlation between bacterial adhesion and surface energy (Baier curve).*

#### *2.1.1 Hydrophilic surfaces*

Hydrophilic surfaces can be successfully fabricated by functionalizing with polymers or nanoparticles. Polymers include poly (ethylene oxide) (PEO), poly (ethylene glycol) (PEG), oligo (ethylene glycol) (OEG), dextran, phosphatidylcholines, poly (acrylic acid), and poly-(2-hydroxyethyl methacrylate) (poly-HEMA), etc. [38]. Nanoparticles cover TiO2, SiO2, ZnO, Fe3O4, and silver nanoparticles [39–42]. The presence of ∙CH2∙ CH2∙O∙ structure and C∙C∙C linkage enable PEG to be highly water-soluble [43].

Many researchers have reported the antiadhesion ability of hydrophilic surfaces. An increase in surface hydrophilicity can reduce bacterial adhesion [44]. Dong et al. indicated that PEG-modified SS exhibited higher hydrophilicity than bare stainless steel (SS), leading to a 96% reduction in *Listeria monocytogenes* attachment [45]. ZnO nanoparticles, composed of hydrophilic groups like ∙OH, ∙SO3H, and ∙COOH, possess strong hydrophilicity [46]. The increased hydrophilicity derived from ZnO nanoparticles promoted antifouling properties of poly (ether sulfone) surface towards *Escherichia coli* (*E*. *coli*) and *Staphylococcus aureus* (*S. aureus*) [47].

Superhydrophilic surfaces hold near-zero water contact angles (*θ* < 5°) and exhibit outstanding antifouling properties. Superhydrophilic surfaces can be developed by hydrophilic functionalities, such as metal oxides including TiO2, ZnO, SiO2, SnO2, CuO, and WO3, by applying various fabrication methods (e. g. UV irradiation, plasma, sol-gel self-assembly, etching, and spay/spin/dip-coating) [48–50]. TiO2 and ZnO are the primary metal oxides to create superhydrophilic film due to their photoinduced self-cleaning property [51, 52]. SiO2 is also frequently used due to its low price and easy to reach [53]. The adhesion number of *E. coli* cells on superhydrophilic TiO2 coated surface was approximately 45% lower than the surface without coating [54]. Qian et al. prepared superhydrophilic film on the 316L stainless steel surface using methoxy-polyethylene-glycol thiol [55]. The surface showed excellent superhydrophilicity with a water contact angle of zero and exhibited enhanced and more durable antibacterial performances against *E. coli* and *S. aureus* [55].

The antiadhesion mechanism of the (super)hydrophilic surface contributes to forming a highly hydrated layer. Hydrophilic compounds on substrate surfaces, such as PEG or OEG, can strongly bond water molecules, connecting each chain through ether oxygen and generating a thin water film (a highly hydrated layer) between bacteria and surface, which physically blocks bacterial adhesion (as shown in **Figure 2A**) [56, 57]. In addition,

the number of anchor sites can be effectively diminished by the water layer [58]. The more hydrophilic surface is, the more resistant it is to the adhesion of bacteria [59].

#### *2.1.2 Hydrophobic surfaces*

Hydrophobic coatings, such as silicone- or fluorine-based coatings, polydimethylsiloxane (PDMS), and sol-gel, enable the surface to be more hydrophobic [60]. Besides, some biosurfactants, like surfactin secreted by genus *Bacillus* strains and pseudofactin produced by *Pseudomonas fluorescens*, have also been verified to successfully promote surface hydrophobicity [61, 62]. Extensive studies demonstrated that hydrophobicity was closely associated with the antiadhesive ability of surfaces. The adhesion-resistant ability of hydrophobic surfaces is attributed to low surface energy. Microbial adhesion is less to low-energy surfaces and more accessible to clean because of weaker binding at the interface [63]. Zhao et al. compared bacterial adhesion behavior on hydrophobic surfaces with various surface energy, indicating that the number of *E. coli* attachments was significantly reduced when surface energy ranged between 20 and 30 mJ/m2 [64]. By spraying hydrophobic perfluoroalkoxy/nano-silver coatings onto aluminum substrates, Zhai et al. found that besides contact killing of silver ions, the hydrophobic surface property could synergistically prevent the adhesion of *E. coli* [65]. With the presence of surfactin coating (surface energy is roughly 27 mN/m), stainless steel, polypropylene, and polyvinyl chloride could effectively prohibit adhesion of *Enterobacter sakazakii, Listeria monocytogenes* (*L. monocytogenes*), and *Salmonella* Typhimurium [66, 67].

A superhydrophobic surface is a surface having a water contact angle greater than 150°, a sliding grade lower than 5°, and high stability of the Cassie model state [68, 69]. In general, superhydrophobic surfaces can be acquired by rendering with fluorocarbon materials containing ∙CF3 and ∙CF2∙ groups, silicones, organic materials (for example, polyethylene, polystyrene, and polyalkylpyrrole), and inorganic materials (like ZnO and TiO2) [68, 70–72]. The remarkable and well-known property of superhydrophobic surfaces is that an air layer known as air plastron is physically entrapped between liquid and surface (as shown in **Figure 2B**) when a substrate is immersed in liquid or bacterial suspension [73]. The air plastron exhibits a great potency in antifouling and corrosive resistance [74]. The contact area between bacteria and the superhydrophobic surface is reduced by the air plastron, resulting in significant mitigation of adherent bacteria [74, 75]. In addition, due to the higher contact angle and low sliding angle of a superhydrophobic surface, droplets cannot stay on the superhydrophobic surface and roll off immediately, known as the 'lotus effect,' accounting for the low-adhesion or self-cleaning property of the superhydrophobic surface [76, 77]. An approximately 80% reduction in the adhesion

**Figure 2.** *Mechanism of superhydrophilic (A) and superhydrophobic (B) surfaces.*

of *E. coli* K-12 was achieved on a superhydrophobic surface [54]. Freschauf et al. demonstrated low initial concentration (~2%) of *E. coli* could attach to the superhydrophobic polystyrene, polycarbonate, and polyethylene surfaces [78]. Compared to bare glass, poly-pyrene-F6 coated glass showed a significant impact against bacterial attachment: bacterial adhesion could be diminished by about 65% for *Pseudomonas aeruginosa* (*P. aeruginosa*) and *S. aureus* [79].

#### **2.2 Chemical properties**

#### *2.2.1 Metal ions and their compounds*

Metals in various forms, coated on substrate surfaces, are well known for their antibacterial effects [80, 81]. The main metals applied include silver, gold, copper, zinc, magnesium, calcium, cerium, strontium, nickel, titanium, europium, yttrium ions, and anions (such as selenium and fluoride) [82]. Silver can deactivate protein activities by interacting with thiol groups in proteins and interfere with transmembrane energy generation and ion transport by generating stable S-Ag bonds in the cell membrane [81]. Moreover, the silver ion can bind to nucleic acid, affecting replication ability and denaturing them [81, 83]. The antibacterial capability of silver has been utilized to prevent bacterial infection for decades, and nearly 650 types of bacteria are associated [84, 85]. Copper exhibits contact-killing properties by damaging cell membranes, inducing the formation of reactive oxygen species (ROS), inhibiting enzymes' activities, and denaturing nucleic acid [81]. Estimated 90 types of bacteria have been reported to be killed using contacting copper [81]. In hospitals, copper alloys, used in doorknobs and other surfaces, exerted an antimicrobial effect against *E. coli* O157, methicillin-resistant *S. aureus* (MRSA), and *Clostridium difficile* while equivalent stainless-steel surfaces did not [86].

Metal oxides such as zinc oxide (ZnO), copper oxide (CuO), Fe2O3, MgO, and titanium oxide (TiO2) have been implemented to prevent biofilm formation in recent years since they are stable under harsh conditions and generally safe for humans and animals [87]. Among metal oxide antibacterial agents, ZnO and TiO2 aroused increasing attention due to their efficient antibacterial activities on a broad spectrum of bacteria [88, 89]. The antibacterial ability of ZnO may contribute to its destruction of bacterial cell integrity and the formation of ROS [90]. ZnO is a photocatalytic material that can respond to UV light and induce ROS creation [91]. TiO2, also known as a photocatalyst, has received more attention because of its strong antiadhesion and antibacterial properties [92]. Moreover, TiO2 is abundant in nature, biologically and chemically stable, non-toxic, corrosion-resistive, and inexpensive [93]. When illuminated by ultraviolet light with paper energy under aerobic conditions, TiO2 can induce the generation of electrons and holes that react with organic substance and dioxygen molecules to form hydroxyl radicals and superoxide ions, preventing bacteria from adhering to substrate surfaces [94–96] by penetrating cell walls, rupturing membrane, and discomposing organic substances [97, 98]. Many studies have reported TiO2-coated surfaces exhibited antiadhesion properties against both Gram-negative and Gram-positive bacteria, such as *S. aureus* and *Streptococcus mutants* [99], *E. coli* [93], *L. monocytogenes* [100], and *Salmonella* [101].

Besides, metal/metal oxide nanoparticles and metal-organic frameworks (MOFs) are porous materials with nanostructures, acting as reservoirs of metal ions. They also possessed significant antibacterial ability, inhibiting biofilm formation and acting as antiadhesion agents [102]. The mechanisms of their actions are similar to those at the molecular level [103].

#### *2.2.2 Biomacromolecules*

Surfaces modified by natural/synthetic proteins/peptides exhibit effective ability to prevent/reduce bacterial adhesion [104]. Proteins/peptides are low toxicity, assembly, and biocompatibility and can be coated on the surfaces of various materials, such as metals, oxides, and polymers [105]. Proteins/peptides avoid bacterial attachment by shifting the hydrophobicity of surfaces and providing hydration [106]. Binding between proteins and bacterial cells is also responsible for adhesion resistance [107]. In addition, due to zwitterionic charges and high hydrogen bond-donor/acceptor abilities of polar functional groups, proteins can interact with negative charged groups on the bacterial cell membrane, destructing cells' integrity [108–110] and exhibiting non-fouling characteristics [111]. Albumins, such as human serum albumin (HSA) and bovine serum albumin, are remarkable proteins that can prevent bacterial adherence to implant surfaces. Eighty-two to ninety-five percent of *S. aureus* was significantly inhibited from binding to HAS-coated titanium surfaces [112]. The antibody is a 'Y-shaped' protein. Its opsonization can impede the adhesion of bacterial cells to implant surfaces by blocking the way of cell-surface attachment and phagocytizing cells [113]. With the presence of antibodies, the adhesion of *E. coli* was markedly reduced on polymer substrates [114].

Probiotic microorganisms, such as *Lactobacillus* and Lactic acid bacteria (LAB), play an important role in antiadhesion. Due to their high adherence capability, probiotics exhibited vigorous antiadhesion activity by competing with bacteria for attachment sites [115]. In addition, antimicrobial substances (such as bacteriocins and hydrogen peroxide) produced by probiotics can also inhibit bacterial adhesion [116]. Studies on the antiadhesion ability of LAB and *Lactobacillus* strains have been largely reported, including *Lactobacillus fermentum (L. fermentum)* in the prevent adhesion of *S. aureus* [117], antiadhesion effects of *L. Plantarum*, *L. crustorum*, *L. coryniformis*, and *L. rhamnosus* on *E. coli* [118], and antiadhesion activity of *L. crispatus* against *Enterococcus faecalis* [115].

Bioactive materials present effective possibilities of resisting biofilm formation. Polysaccharides are a crucial bioactive substance [119], like chitosan, hyaluronic, and alginic acid. The mechanism of the antiadhesion capability of polysaccharides might be that polysaccharides could dissolve biofilms by interacting with the EPS layer and distort biofilm formation and kill cells by inhibiting the metabolic activity of bacterial cells [120]. Chitosan possesses significant antibacterial and antibiofilm activities, making it widely used in medical and food fields, such as food preservation, scaffolds, and bandages [121–123]. The positive-charge property of chitosan enables it to bind with negatively charged cell membranes, inducing the leakage of proteinaceous and other intracellular constituents [124]. Moreover, chitosan can cross through the membrane, bind with DNA, and interfere with the synthesis of mRNA and protein [113]. It was found that chitosan with quaternary ammonium groups could eradicate biofilm formation of *Staphylococcus aureus* [125], and carboxymethyl chitosan could restrain *S. aureus* or *P. aeruginosa* from adhering to surfaces with an efficiency of >90% [126].

#### **3. Topographic modification strategy**

Topographical features of substratum surfaces can modulate bacterial attachment and biofilm formation as surface morphology dominates surface roughness and wettability [127]. Typically, the topographical surface can be classified into three different scales: macro-, micro-, and nano-scale [128]. Roughness is a critical factor affecting bacterial attachment by reducing the attachment area between a particle and a surface [129]. Since most microbes are approximately 0.2–2 μm in diameter [130] which is much smaller than the groove distance of macro-roughness, cells can swim and entrap into the grooves of macro-roughness surfaces, suggesting that macro-scale roughness surfaces are not related to antifouling [127]. Therefore, micro-and nanoscale topography surfaces are crucial for preventing bacterial adhesion. Many studies have investigated how micro/nano-scale topographies affect bacterial adhesion. Discrete, ordered, and hierarchical surface structures from nano-scale to micro-scale were self-assembled, designed, or bioinspired by mimicking natural surfaces (such as skins of marine mammals and sharks, shells of mollusks and crabs, wings of insects and birds, and leaves of plants) [131, 132].

#### **3.1 Micro-scale topography surfaces**

Micro-structure can be fabricated on surfaces of metals, plastics, and polymer films, like stainless steel [133], polyethylene terephthalate (PET) [134], and PDMS [135]. The micro-patterned topographies exhibited positive influences on preventing the adhesion of various bacteria strains while being non-toxic [135]. Wang et al. designed and fabricated micro-patterned PET surfaces, which simultaneously include curved and straight edges, flat plateaus (top of pillars), and flat surfaces between pillars [134]. The results indicated that PET surfaces with pillars could significantly reduce the attachment of *E. coli* cells under both static and dynamic (shaking at 200 r/min) conditions in nutritious media and oligotrophic solution at 37°C. The Sharklet diamond-shaped micropattern, inspired by shark surface architecture, was widely reported due to its impressive ability to prevent colonization and biofilm formation of various bacteria strains, including *Mycobacterium abscessus* [136], *E. coli* [137], *S. aureus* [138], and *P. aeruginosa* [139].

Features of micropatterns, including pattern shape, size, and groove distance, affect antifouling efficiency [140]. Varied topographical pattern shapes have been created and presented antiadhesion ability. Pattern shapes cover ordered geometric shapes (i.e. line [26], pyramid [141], and cross [142]), pillar [143], pit [144], brush [145], wrinkle [17], and biomimetic shapes (like Sharklet diamond shape [136], lotus-like shape [146], rice leaf [147], rose petals [148], and mytilid shells [149]). In general, with the increase in pattern size, the antiadhesion ability of micropatterns decreased. Lu et al. studied the adhesion of *E. coli*, *P. aeruginosa*, and *S. aureus* on micro-patterned PDMS films with three different pattern sizes [135]. It was found that when pattern size was smaller than bacteria size, the surface was effective in preventing bacteria adhesion; however, as the pattern size was comparable to or larger than bacteria size, the antiadhesion capability of the surface decreased markedly, with more bacteria attachment but still less compared with the flat surface. Similar results were reported by other researchers [150]. This phenomenon might be attributed to the contact area between microorganisms and the surfaces. The available cell-surface contact area reduces with a smaller pattern size than bacterial cell size [151]. The groove between patterns provides anchor sites for cell contact, creates vortices under

#### *Antifouling Strategies-Interference with Bacterial Adhesion DOI: http://dx.doi.org/10.5772/intechopen.102965*

dynamic conditions, and acts as dead zones for cells sheltered from sanitation treatment [152, 153]. It was also reported that bacteria prefer to distribute in the grooves rather than the top of protruding patterns [135]. As groove distance is smaller than bacteria size, less bacterial cells are entrapped [154]. Similar results were obtained by Lu et al. and Romero et al. [135, 155]. However, the attachment of bacteria can be enhanced when the groove distance is equal to bacteria size because microorganism cells can fit between grooves, and binding energy can be increased [135].

Besides, the effectiveness of surface microstructures on antifouling is also affected by surface energy and hydrophobicity [156]. According to Wenzel and Cassie and Baxter, surface topography can alter the surface to be hydrophobic and superhydrophobic [157]. Carman et al. demonstrated that hexagons could increase the hydrophobicity of the polydimethylsiloxane elastomer [158]. Micro-scale structure could enhance surface hydrophobic ability, allowing more air bubbles to effectively form between surface and liquid [159]. Since a large portion of surfaces was occupied by air, the contact area between bacteria and surfaces was significantly reduced, leading to less cell attachment [160]. Additionally, due to the effect of surface tension, bacteria cannot cross the air-water interface, thereby inhibiting bacterial adhesion [157].

#### **3.2 Nano-scale topography surfaces**

Nano-topography provides an effective way to repel bacterial adhesion and prohibit biofouling. Like the micro-scale patterns, the topographical features such as shape, size, density, and groove width can markedly affect cell adhesion onto surfaces [161, 162]. Compared to low-density patterns, nanostructures with highly dense patterns greatly improve the reduction rate of bacterial attachment [163, 164]. Adhesion numbers of *E. coli* and *S. aureus* were significantly reduced by 55.6 and 40.5% on a nanoscale (6 nm) titanium surface with a low density of 213 peaks/μm<sup>2</sup> compared to 2 nm with a high density of 2240 peaks/μm2 [165].

Numerous shapes of nano-patterns with varying size, depth, and groove width have been reported as excellent impeders of bacterial adhesion and biofilm formation [166–169]. A topographical surface characterized by nanometer-size pores (approximately 0.20 μm2 ) surrounded by nano ridges, mimicking the pilot whale skin, exhibited antifouling activity based on reduced available space for bacterial attachment [170]. The more the topography resembled the size and shape of features on bio-skins, the better the antifouling activity was [16, 171]. Bhadra et al. fabricated a nanowire array (average size is approximately 40.2 nm) on titanium and estimated its antifouling ability [172]. It was revealed that the nanowire arrays could render titanium as a moderately effective bactericidal surface, with more excellent bactericidal activity, eliminating almost 50% of *P. aeruginosa* cells and about 20% of *S. aureus* cells. The surfaces of cicada and dragonfly wings exhibit bactericidal properties towards some bacteria strains due to their nano-scale pillar structure [173, 174]. Cicada-inspired fluoridated hydroxyapatite with nanopillars has been successfully fabricated using electrochemical additive manufacturing (ECAM) by Ge et al. [175]. Different types of nanopillar array were obtained: with diameters, heights, and aspect ratios of ~65–95 nm, ~380–510 nm, and ~4.5–7.5 nm, respectively. It was demonstrated that the nanopillars with diameters of ~80 nm were lethal to both Gram-negative and Gram-positive bacteria when the nanopillar density is proper [176, 177].

The cell-nanostructure adhesion mechanisms are still poorly understood. Currently, there are three mechanisms proposed to elucidate the antifouling behavior of nano-textured surfaces. (1) nanostructures induce the formation of the superhydrophobic surface [178]. As explained by the Cassie-Baxter state, the nanostructure can promote air pockets generating in the solid/liquid interface and increase the surface contact angle [179]. As a result, the available contact area for bacteria on the surface is reduced, thus preventing bacterial adhesion [180]. (2) Bacterial membrane can be ruptured and stretched by the nanostructure, leading to cell disruption and eventually cell death, known as the biophysical model, developed by Pogodin et al. [181]. This occurs because the size of most bacterial cells is in the micrometer range, while the structured surfaces are in the nanometer range [182]. Based on the model, bacterial cells absorbed on pattern surface may lead to a drastic increase of contact area, accompanied by stretching the cell membrane between the pillars, which induces membrane disruption and cell death. Furthermore, in terms of the model, the rigidity of cell membranes plays a crucial role in bacterial attachment behavior: the more rigid cells are, the more resistant they are. This may be the reason why nano-pillared surfaces were less effective against gram-positive bacteria strains (*Bacillus subtilis*, *Planococcus maritimus*, and *S. aureus*) when compared to less rigid gram-negative bacteria strains (*P. aeruginosa*) [173, 183–185]. (3) Since the nano-structured topography is unfavorable for bacterial cells, the immobilized cells push and pull the structure while attempting to move away, imposing fatal shear force on the membrane, which initiates bacterial membrane damage [174]. In addition, the solid adhesive force between bacteria and nanostructure also facilitates membrane deformation and cell membrane rupture [174].

#### **4. Conclusions, challenges, and future trends**

Bacterial biofilm is a universal and ubiquitous phenomenon. It can directly cause severe problems on public health, the environment, and industries and subsequently lead to economic losses. Consequently, various strategies have been developed and implemented to control biofilm formation. As bacterial adhesion on a surface is the prerequisite for biofilm formation, much attention has been paid to the antifouling strategies that utilize topography and physicochemistry modification to prevent bacterial adhesion to surfaces. This chapter only summarizes the positive effect of surface topographical and physicochemical properties on preventing bacterial adhesion. However, inconsistent and even conflicting impacts could be found in various reported studies. No one particular surface structure or physicochemical property has demonstrated universal antiadhesion ability against all types of microorganisms. Therefore, it is needed to continue the development of strategies that are truly and broadly effective. Furthermore, though surface topographical and physicochemical properties exhibited significant and effective ability to resist the adhesion of specific bacteria strains, the surface structures and physicochemical properties are easily destroyed by various forces, thus decreasing their antifouling capabilities. Therefore, developing a long-term and durable surface with effective antifouling properties remains a huge challenge for the future.

*Antifouling Strategies-Interference with Bacterial Adhesion DOI: http://dx.doi.org/10.5772/intechopen.102965*

### **Author details**

Zhen Jia Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida, United States

\*Address all correspondence to: janejia1988@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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#### **Chapter 15**

## *Curcuma Xanthorrhiza* Roxb. An Indonesia Native Medicinal Plant with Potential Antioral Biofilm Effect

*Dewi F. Suniarti, Ria Puspitawati, Rezon Yanuar and Ranny R. Herdiantoputri*

#### **Abstract**

Most common oral diseases are directly related to oral biofilm, a complex community of microorganisms inhibiting the oral cavity. Recent studies provide deeper knowledge on how free-floating bacteria form a structurally organized microecosystem and on its pathogenicity and its self-defense mechanisms; thus, creating an understanding of the challenges in eliminating oral biofilm and maintaining the balance of oral ecosystem. Chlorhexidine has been the standard oral antimicrobial agent for decades. However, studies showed that it is less effective against bacteria in the form of biofilm that leads to an ongoing search of another method to fight against biofilm, including the use of plant-derived compounds. Medicinal plants are known to contain secondary metabolites, which are not only important in protecting the plant from any harmful environment but also potential as antimicroorganism and antioral biofilm for humans. *Curcuma xanthorrhiza* Roxb., containing xanthorrhizol (XNT), an essential bioactive compound, is an Indonesian native medicinal plant proven to have antibacterial and antibiofilm activities by several in vitro studies. The understanding of biofilm formation, its resistance to common drugs, and the potential role of *C. xanthorrhiza*-derived compounds as antibacterial and antibiofilm may contribute to developing *C. xanthorrhiza* into the alternative weapon against oral biofilm-related diseases.

**Keywords:** *Curcuma xanthorrhiza* Roxb., xanthorrhizol, oral biofilm, antibacterial

#### **1. Introduction**

Oral biofilm or dental plaque is the complex community of microorganisms that can be found on the surfaces of various orodental tissues, especially on tooth surfaces. It had become a common knowledge that oral biofilm directly causes several oral diseases such as dental caries, periodontal disease, i.e., gingivitis and periodontitis, and many other oral diseases [1]. Compared with the planktonic microorganism, oral biofilm is masses of bacteria that form structure known as extracellular matrix

(ECM), that allows microorganism to persist under environmental conditions, and able to resist antimicrobial drugs [2]. In biofilm, there is a unique cell-to-cell communication system, namely quorum sensing (QS) that allows bacteria to detect and respond to cell population density mediating gene expression [3, 4]. It has been reported that QS is also responsible in antimicrobial resistance through regulating bacteria multidrug resistance (MDR) efflux pumps, regulating biofilm formation, and regulating bacterial secretion systems [5–9].

For many decades, antimicrobial agents, i.e., chlorhexidine (CHX) have become the best weapon against bacteria in oral cavity. However, CHX is less effective against biofilm bacteria because of the drug resistance properties of biofilm [10, 11]. This condition led researchers to develop another method to fight against biofilm, including use of alternative drugs, such as plant-derived compounds or essential oils. On the other hand, medicinal plants or herbs have been proved empirically and scientifically to have some important biological activities. As antibacterial and antibiofilm, medicinal plant-derived compounds and essential oils could inhibit biofilm formation by inhibiting peptidoglycan synthesis, modulating QS, and damaging bacteria membrane structures [12, 13]. Nowadays the use of natural products and their derivatives in dentistry, especially to prevent dental caries, is receiving large attention [14]. Moreover, many studies have reported the effect of various medicinal plant extracts on inhibiting biofilm formation and inhibiting bacterial adhesion. These suggest that medicinal plant-derived compounds might become promising alternative therapy in dental care.

*Curcuma xanthorrhiza* Roxb., known as Javanese turmeric or "temulawak," is a native Indonesian medicinal plant, which has been utilized traditionally as an ingredient of *jamu* (Indonesia herbal supplement and medicine) [15]. Most people use the rhizome as they believe it has medicinal effect for stomach illness, liver ailments, constipation, bloody diarrhea, dysentery, arthritis, children's fevers, hypotriglyceridemic, hemorrhoids, vaginal discharge, rheumatism, and skin eruptions empirically [16, 17]. Furthermore, the beneficial medicinal effect of *C. xanthorrhiza* has been proven in scientific studies. *C. xanthorrhiza* has been confirmed to have pharmacology effects such as anti-inflammatory, antibacterial, antioxidative, neuroprotective, nephroprotective, antitumor, and hepatoprotective activities [18–22]. Recently, in dentistry scope, the development of *C. xanthorrhiza*-derived compound as antibacterial drug has been extensively studied especially in East Asia and Southeast Asia countries. Xanthorrhizol (XNT) is the one of main active compound isolated from the essential oil of the rhizomes of *C. xanthorrhizza*, has a variety of pharmacological activities, one of that is antibacterial effects [23]. The bactericidal and bacteriostatic activity of xanthorrhizol against several oral bacteria has been reported using planktonic or biofilm models and showed promising result.

Thus, the use of *C. xanthorrhiza*-derived compounds as antibacterial and antibiofilm agent could be advantageous because natural-based medicines have fewer side effects. In this chapter, we will outline and summarize about inhibition of biofilm formation, mechanism action, and potential roles of *C. xanthorrhiza*-derived compounds as antioralbacterial and antioralbiofilm.

#### **2. Oral biofilm and the most common oral infectious disease**

The human oral cavity is a dynamic environment, which houses the most diverse microbiota, inhabited by more than 700 species of bacteria that colonize in the

#### Curcuma Xanthorrhiza *Roxb. An Indonesia Native Medicinal Plant with Potential Antioral… DOI: http://dx.doi.org/10.5772/intechopen.104521*

surfaces of both hard and soft tissues [24]. Inside the oral cavity there are two types of bacteria: a single free-living cell known as planktonic bacteria mostly found in saliva, and multicellular-living, where the cells are sessile and live in biofilm. Oral biofilm is a complex community of microorganisms, which are attached on the oral surface and embedded in an extracellular matrix. Thus, the biofilm-associated bacteria differ compared with the planktonic bacteria in many ways, for example, growth rate, gene expression, transcription, and translation because bacteria biofilm lives in different complex microenvironments due to higher cell density of heterogeneous bacteria community [25]. The formation of the three-dimensional structure of biofilm causes the bacteria to be protected from the various environmental stresses, such as antimicrobial drugs.

The development of oral biofilm is a multistep process. The initial stage is pellicle formation on tissue surface, which is composed of a variety of host-derived molecules and source of receptors such as mucins, agglutinins, proline-rich proteins, phosphaterich proteins, and enzymes such as α-amylase that could be recognized by early colonizer. These receptors allow various planktonic bacteria, which have been classified as early colonizer, such as *Streptococci* species that constitute around 60–90% of the bacteria that first colonize the teeth, and other bacteria include *Actinomyces* sp., *Capnocytophaga* sp., *Eikenella* sp., *Haemophilus* sp., *Prevotella* sp., *Propionibacterium* sp., and *Veillonella* sp. [26]. However, at this stage, the bacteria are still susceptible against antimicrobial drugs, because the biofilm matrix structure is not completely formed.

The interaction between the early-colonizing bacteria has been shown to regulate many gene expression in response to the environment and provide specific direct binding sites (not through salivary glycoprotein for various other bacteria to colonize) and promote the development of biofilm. The bacteria that bind to this initial layer of biofilm are known as known as late colonizers such as *Fusobacterium nucleatum*, *Treponema* sp., *Tannerella forsythensis*, *Porphyromonas gingivalis*, *Aggregatibacter actinomycetemcomitans*, etc. They recognize polysaccharide or protein receptors on the pioneer bacteria cell surface and then attach on them [26]. The presence of late colonizer bacteria causes the change of environment and proportional shift, for example, relative amount of *Streptococci* sp. and *Neisseria* sp. is decreased, while the amount of *Actinomyces* sp., *Corynebacterium* sp., *Fusobacterium* sp., and *Veillonella* sp. increases [27]. The proportional shift occurs due to the interaction between bacteria in the community and the change of environment in biofilm. The competitive and cooperative interaction in biofilm may be essential to develop a successful mixedspecies colonization.

During biofilm formation, there's cell-to-cell communication in the biofilm called QS. This phenomenon is mediated through production and release of chemical signals by bacteria termed autoinducer (AI), as response to changes in bacterial density and environment in biofilm. This mechanism initiates modification in gene expression to regulate cell or group behavior. During the maturation biofilm phase, QS also plays an essential role in extracellular matrix (ECM) production [28]. The ECM is a mixture of secreted high-molecular-weight polymers produced by bacteria, consisting of three major components: extracellular polysaccharides (EPS), proteins, and extracellular DNA, which form a cross-linked meshwork that serves as a shield [29]. At this stage, the biofilms show maximum resistance to antimicrobial drugs. The presence of biofilm ECM represents a strong barrier. The molecules of antimicrobial drugs must diffuse through the biofilm matrix to inactivate the bacterial cells. The biofilm ECM contains numerous anionic and cationic molecules that can bind charged molecules

of antimicrobial drugs [30]. The resistance provided by ECM may be discouraged by longer exposure and higher concentration of antimicrobial drugs; however, the toxicity for oral application should be the main consideration.

The drug resistance of oral biofilm against antimicrobial drugs becomes the main problem in eliminating oral biofilm. Other mechanisms that have been proposed to explain how bacteria protect itself from the effects of antimicrobials such the ability to adapt to various stress responses; the decrease of growth rate and metabolism; efflux pump mechanism; and QS [7, 10, 31].

Dental caries is the most common oral infectious disease characterized by acidic damage on the tooth surface due to a localized structural demineralization that leads to cavitation [32]. The bacteria that are responsible for the initiation of such cavitation process are the acidogenic, Gram-positive, facultative anaerobic bacteria, *Streptococcus mutans*. *S. mutans* along with other species from the same genus, *S. mitis,* are some of the early colonizers of oral biofilm that provide adherence for other microorganisms promoting the growth and maturation of the biofilm. Recent study by Dongyeop Kim et al. [33] showed that the rotund-shaped biofilm with corona-like cell segregation where *S. mutans* located at the very core created a highly acidic region at the interface between the biofilm and enamel, resulting in the characteristics of localized demineralized surface as commonly seen in clinical setting [33]. Therefore, not only dental caries is a diet-dependent disease but also a biofilm-dependent disease [32]. As the understanding of the nature of dental caries grows, the approach of caries management has been shifted from the previously popular approach that focused more on the symptomatic treatment and removal of carious tissue to be replaced by artificial structure, to the current approach that emphasizes the preventive measures: restriction of dietary sugar consumption, removal of bulk bacterial mass through brushing, and reduction of cariogenic bacteria in dental biofilm through chemotherapeutic methods [34].

While dental caries is a result of a chronic destruction of the tooth hard tissue itself, periodontal disease on the other hand is an inflammatory disease of the surrounding tissue of tooth, which may result in loss of attachment, and induced and maintain by the resident of oral biofilm, especially the biofilm located in the gingival crevices that stay in contact with the gingival epithelium [35, 36]. Different from the microbes of the dental caries-related biofilm located on the tooth surface whose ability is to transform carbohydrate into damaging acidic substrates, the microbes of the biofilm in the gingival crevices gain their source of nutrient mainly from the proteinrich gingival cervicular fluid (GCF) accommodating the growth of Gram-negative bacteria, some of which are responsible for the progression of periodontal diseases [35]. Gram-negative, anaerobic, proteolytic bacteria, namely *P. gingivalis*, *Prevotella intermedia*, and *A. actinomycetemcomitans,* are mostly found in the periodontal biofilm and linked to periodontal diseases due to their ability to release toxins that induce host proinflammatory response, which in turn creates an ecological shift to a dysbiosis and causes damage to the periodontal structure [37, 38].

#### **2.1 Current treatment and challenges using CHX and other antibacterial agents/mouth rinse**

The general treatments of periodontal disease are mechanical debridement and ensuring that the proper oral hygiene is maintained by the patient. The use of antibiotics for periodontal disease other than aggressive periodontitis is still controversial to date [36]. Concern has been raised toward drug tolerance and resistance of periodontal bacteria. A study done in Colombia showed that bacterial isolates from subgingival

Curcuma Xanthorrhiza *Roxb. An Indonesia Native Medicinal Plant with Potential Antioral… DOI: http://dx.doi.org/10.5772/intechopen.104521*

biofilm of patient with aggressive periodontitis (*A. actinomycetemcomitans, P. gingivalis,* and *Tannerella forsythia)* were resistant to amoxicillin, azithromycin, and metronidazole [39]. Considering the nature of periodontal biofilm, mechanical disruption of the biofilm's integrity and reduction of the biofilm mass prior to the administration of antibiotics are considered essential [40].

Although CHX is considered as the gold standard antimicrobial agent in the oral cavity, there are some drawbacks of its usage: the risk of extrinsic staining on tooth surface, alteration in taste perception, and increase in calculus formation [41, 42]. Moreover, the effectiveness of CHX for biofilm eradication is also questioned. Due to the fact that *S. mutans* is the early colonizer of dental biofilm and that it inhibits the lowest strata, administration of CHX results in a concentration gradient from the outermost surface of the biofilm toward its innermost area that in turn exposes the *S. mutans* to only subinhibitory concentration of CHX [33, 43]. This suggestion is supported by another research conducted by spatially mapping the architecture of dental biofilm, which found that the intact corona structure of biofilm that conceals *S. mutans* cells in the core beneath layers of other microbes provides enhanced antimicrobial tolerance against CHX [33]. On the other hand, increasing the concentration of CHX in the aim to eliminate the dental caries-related biofilm is not recommended because the wide spectrum nature of CHX will disturb the balance of the oral environment by perturbing the commensal microbiome. As a prevention of periodontal diseases, several studies found its benefit to prevent bacterial surface adhesion, thus preventing the biofilm formation [44]. However, when the biofilm has formed, Gramnegative bacteria such as *P. gingivalis* are able to secrete outer membrane vesicles to bind CHX and provide protection to the bacteria in the biofilm community [43].

To avoid the aforementioned side effects and concerns, treatment and prevention alternatives from many natural products, herbs, and medicinal plants, in the form of extracts and essential oils, have been developed. Medicinal plant's extract from *Acacia arabica*, *Tamarix aphylla* L., and *Melia azadirachta* L. showed evidence of reducing oral biofilm formation and cleaning the well-developed oral biofilm [45]. Medicinal plant from South East Asia, *C. xanthorrhiza* Roxb*.*, has also been proven through several studies to have eradication and inhibition effects against oral bacteria and candida biofilm [23, 46–53].

#### **3.** *Curcuma xanthorrhiza* **Roxb**

*Curcuma xanthorrhiza* Roxb., known as Java turmeric or "temulawak," is a native Indonesian medicinal plant that is mainly cultivated in Southeast Asian countries such as Indonesia, Malaysia, Thailand, Vietnam, and Philippines. For a long time, it has been used to enhance the flavor and color of food. Moreover, this plant has been believed and utilized as medication and supplement [15, 17]. In a few decades, turmeric plants including *C. xanthorrhiza* became the main subject of interest in research because many of its biological activities have been confirmed by experimental scientific studies. In addition, *C. xanthorrhiza may* be used as a treatment for COVID-19 because of its ability to inhibit proinflammatory cytokines [54]. However, it's still requiring more evaluation, especially in the clinical trial setting. Thus, recently market demand for *C. xanthorrhiza* rhizome has increased globally.

*C. xanthorrhiza* is a low-growing plant (2–2.5 m) with a root known as rhizome that looks like ginger. This plant can grow in the lowlands to an altitude of 1500 meters above sea level and has a habitat in tropical forests. The main part of

*C. xanthorrhiza* that has been proved to have beneficial medicinal activity is rhizome [15]. The rhizome of *C. xanthorrhiza* contains terpenoid and curcuminoid compounds, which reportedly have beneficial properties such as antioxidant, anti-inflammatory, antitumor, and anticancer effects [18, 20–22, 55]. The shape of the rhizome of *C. xanthorrhiza* is oval round shape, 3–4 branched, and reddish brown, dark yellow, or dark green in skin color (**Figure 1**). The rhizome flesh is dark, orange or brown in color, has a sharp pungent aroma and tastes bitter.

#### **3.1 Phytochemical properties of** *C. xanthorrhiza* **Roxb**

The rhizome of *C. xanthorrhiza* contains curcuminoids (1–2%), essential oil (3–12%), xanthorrhizol (44.5%), and camphor (1.39%). Moreover, xanthorrhizol (XNT), a bisabolene-type sesquiterpenoid compound isolated from essential oil of rhizome's *C. xanthorrhiza*, had been well established to possess various medicinal effects XNT is one of the most explored and studied phytochemicals, especially its antibacterial, antifungal, and antibiofilm activity. The major group of secondary metabolites has been identified in the rhizome of *C. xanthorrhiza* and can be seen in **Figure 2** [17]. However, the variation of active metabolite of *C. xanthorrhiza* might be influenced by several external factors, such as climate, sun intensity, altitude, and temperature of cultivation. For example, the high percentage of starch is influenced by the altitude of cultivation. The bioactive compound XNT and curcuminoid also reported higher in low altitude, high temperature, and low rainfall [56]. Thus, these are the challenges for development standardization phytomedicine, because of the vast variation of external factor and the different method of cultivation in each site.

#### *3.1.1* C. xanthorrhiza Roxb. *Extraction preparation*

The *C. xanthorrhiza*-derived products, such as extract or as pure compounds, have provided unlimited opportunities for new drug discovery. However, to take advantage of the beneficial effect of the medicinal plant, an extraction process is carried out to obtain the active secondary metabolite. The extraction solvent selection is very essential because it affects the stability and metabolite profiles that implicate the efficacy of medicinal plant extract. Several commonly used solvents are ethanol, methanol, dichloromethane, acetone, and water [57, 58]. Proper actions must be taken to assure that potential compound is not lost or destroyed during the extraction process.

Curcuma Xanthorrhiza *Roxb. An Indonesia Native Medicinal Plant with Potential Antioral… DOI: http://dx.doi.org/10.5772/intechopen.104521*

**Figure 2.**

*Secondary metabolite compound of rhizome* C. xanthorrhiza *Roxb.*

#### *3.1.2 Xanthorrhizol isolate*

XNT is an essential bioactive compound isolated from essential oil of rhizome *C. xanthorrhiza*. There are several methods used to extract the essential oil and XNT, i.e., supercritical fluid carbon dioxide extraction (SCFE-CO2), Soxhlet extraction, and percolation process [59]. According to Salea et.al (2014), extraction using SCFE-CO2 method will result in higher XNT compared with Soxhlet or percolation extraction method. Besides that, the conventional method to isolate XNT, which costs less, is still applicable and more efficient, while SCFE-CO2 method is more applicable in largescale production in the industry [59].

The interest in XNT as an antibacterial has attracted some researchers to develop as plant-derived drugs. The molecular weight and solubility of XNT are 218.33 g/ mol and 28.90 μg/ml, respectively. This makes XNT have lower molecular weight and higher solubility compared with bioactive compound curcumin [60, 61]. Thus, it was expected that XNT might easily penetrate the surface of biofilm. According to the chemical structure, XNT and curcuminoid contain phenolic compounds and hydrocarbons.

#### **4. Antibacterial and antibiofilm activity**

#### **4.1 Antibacterial**

The antibacterial activities of *C. xanthorrhiza* have been studied using various preparations such as extract or fraction preparation and XNT isolation. *C. xanthorrhiza* extract and XNT have been reported to be effective against a variety of oral bacteria. They have been evaluated by standard in vitro susceptibility tests such as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Our studies have shown that the effectiveness of *C. xanthorrhiza* ethanol extract against Gram-positive bacteria was superior compared with its effect against Gram-negative bacteria. In addition, the efficacy of *C. xanthorrhiza* extract and XNT against Gram-positive bacteria is comparable to CHX [48, 49, 52].

The antibacterial activity of C. *xanthorrhiza* is believed to emerge from XNT and curcuminoid compounds. The mechanism of action of phenol compounds, through interaction between the hydroxyl group (-OH) of the phenol compound with bacterial cells wall to facilitate hydrogen bonds subsequently causes alteration of bacterial membrane permeability. The high concentration of phenol can penetrate into cells subsequently leading to protein coagulation on the cell membrane and cell lysis [62, 63].

The Gram-negative bacteria are more resistant to phenol due to the complexity of their cell wall. Gram-positive bacteria possess thick cell walls containing many layers of peptidoglycan and teichoic acids. In contrast, Gram-negative bacteria possess thinner cell walls, but consist of a few layers of peptidoglycan surrounded by lipid membrane (lipopolysaccharides and lipoprotein). The complex cell wall of Gramnegative bacteria has been predicted to slow down the passage of chemicals. This was supported by a previous study by Inouye et al. [64], which concluded that the antibacterial effect of polyphenols was generally more effective against Gram-positive bacteria than Gram-negative [64].

XNT isolate is more effective against bacteria compared with the extract form. Since the crude extract contains various types of bioactive compounds or phytochemicals, usually unnecessary components are still carried away during the extraction process, for example, starch found in *C. xanthorrhiza* extract. Moreover, that unnecessary component has been suggested can affect the bioactive compounds activity. The XNT has been reported effective against several Gram-negative bacteria such as *Fusobacterium nucleatum* and *Enterococcus faecalis* [65, 66].

In addition, a clinical study evaluated the effectiveness of XNT, neem, cetylpyridinium chloride, and 0.2% CHX to decontaminate 60 children's toothbrushes after being used. Their result showed that the antimicrobial effect of XNT on *S. mutans* (78% reduction in *S. mutans*) was higher compared with CHX, but lesser than neem and cetylpyridinium chloride [67].

#### **4.2 Antibiofilm**

The *C. xanthorrhiza* extract and XNT also have been reported to have activity as antibiofilm against several oral bacteria in single species biofilm models. The antibiofilm activity of *C. xanthorrhiza* has been reported in various phases of biofilm formation. Rukayadi study reported that the activity of XNT as an antibiofilm was dependent on the concentration, exposure time, and the phase growth of biofilm. XNT is more effective in the early phase of biofilm formation [68]. Consistent with that, our study also demonstrated that the antibiofilm activity of *C. xanthorrhiza* ethanol extract is more effective in the early phase of biofilm formation. These indicate that the EPS matrix of mature biofilm implicates the resistance [46, 50, 51]. Although high concentration of XNT (1000 μg/mL) reportedly completely killed the biofilm, toxicity should be a major concern.

*C. xanthorrhiza* extract and XNT have been reported to inhibit several single species biofilm formations in in vitro study. Although not completely eliminated, bacteria were removed in the adhesion phase and early accumulation phase of biofilm development. The mechanism of inhibition biofilm formation is still not clear

#### Curcuma Xanthorrhiza *Roxb. An Indonesia Native Medicinal Plant with Potential Antioral… DOI: http://dx.doi.org/10.5772/intechopen.104521*

yet. However, it has been reported that *C. xanthorrhiza* extract has shown to inhibit acid production of *S. mutans* biofilm [53]. Moreover, *C. xanthorrhiza* extract is also reported to have anti-QS or quorum quenching activity [69]. The high level of tannin, phenol, phenolic compound in *C. xanthorrhiza* is suggested to precipitate the proteins that are vital for *rhl* system in *Pseudomonas aeruginosa.* By inhibiting the *rhl* system, the swarming activity of *P. aeruginosa* is inhibited, thus the QS will not take place [69, 70]. Besides that, killing the cells by cell lysis will also degrade and detach the biofilm.

Besides inhibiting the biofilm formation, *C. xanthorrhiza* extract and XNT also reportedly can eradicate the mature biofilm. The in vitro study against single species 72-hour *S. mutans* biofilm model, treated with *C. xanthorrhiza* methanol extract, showed significant fewer colony forming unit (CFU). The TEM and SEM observation showed changes of peptidoglycan layer of *S.mutans* and fewer intact bacteria after treatment [53].

Because the biofilm matrix can limit the penetration of antimicrobial agents, Cho et al. [71] explored the nanoemulsion form of *C. xanthorrhiza* oil in order to facilitate the ease of penetration. The single species *S. mutans* biofilm model, which was treated with nanoemulsion of *C. xanthorrhiza* oil, showed higher dead cells compared with the live cells. Furthermore, quantitative analysis of live/dead biomass and biofilm thickness based on the CLSM images showed that the live/dead ratio with nanoemulsion treatment was 50% less compared with control. It was also reported that nanoemulsions, which were prepared using sonication, are more suitable to be used as antibiofilm materials than emulsions without sonication [71]. These results indicate that *C. xanthorrhiza* extract can penetrate the *S. mutans* biofilm and kill that cell.

Another in vitro study against root canal biofilm *F. nucleatum* presented that XNT at concentrations 1.25% and 1.5% reported similar eradication activity compared with 2.5% NaOCl [72].

The antibiofilm activity of *C. xanthorrhiza* extract and XNT has also been demonstrated in multispecies biofilm models. CLSM analysis demonstrated that biofilm treated with XNT at 2 and 10 μg/ml for 30 min results in reduced bacterial viability in a dose-dependent manner against saliva and multispecies oral biofilm. Moreover, when exposed to 1000 μg/mL XNT, all biofilm cells were completely killed. These results indicate that XNT provides antibiofilm properties by eradicating bacteria viability [73].

Generally, multispecies biofilms were considered to be more resistant to antibiofilm agent compared with single species biofilms. To evaluate this notion, we tested dual species biofilm models (combination Gram-positive and Gram-negative bacteria) treated with *C. xanthorrhiza* ethanol extract, then measured the minimum of biofilm eradication (MBEC) using MTT-assay to assess the viability cell (**Table 1**). Our study demonstrated that *C. xanthorrhiza* ethanol extract was better eradicating dual-species biofilm (for example, *S. sanguinis* with *Porphyromonas gingivalis*; or *S. mutans* with *A*. *actinomycetemcomitans*), whereas not effective against single-species *P. gingivalis* biofilm nor single-species *A*. *actinomycetemcomitans* [46, 51]. This result may be possible due to the antagonist interaction between *S. sanguinis* and *P. gingivalis* that causes an incomplete formation of the EPS matrix surrounding the biofilm. It is supported by a clinical study by Stingu et al. [74], who reported that the presence of *S. sanguinis* has an influence on the presence of *P. gingivalis*, where *S. sanguinis* was found more in healthy gingival sulcus [74], while *P. gingivalis* vice versa. *S. sanguinis* also can produce bacteriocin called streptomycin and hydrogen peroxide, which can inhibit the growth of *P. gingivalis* [75].


#### **Table 1.**

*Effect of* C. xanthorrhiza ethanol extract *against single and dual species biofilm.*

#### **5. Conclusion**

A fight against oral infectious disease is a fight against an adaptive, highly advanced, multispecies, pathogenic oral microbial community comprising oral biofilm. Inhibition and elimination of oral biofilm by means of preventing and treating oral diseases require pharmacological developments in finding alternative therapies that are able to dodge the defensive nature of oral biofilm and avoid cytotoxicity to the host while maintaining the homeostasis of the oral environment. *Curcuma xanthorrhiza* Roxb.–derived compounds such as XNT have been repeatedly proven to be a promising alternative therapy in dental care for its antimicrobial and antibiofilm activity. The phenolic compound of XNT has been proven to alter the permeability of the bacterial cell wall that leads to cell lysis. It is also proposed to prevent QS by inhibiting the swarming activity of bacteria. Further research to obtain the most effective form of compound and research in clinical settings are still needed to fully harness its potential.

Curcuma Xanthorrhiza *Roxb. An Indonesia Native Medicinal Plant with Potential Antioral… DOI: http://dx.doi.org/10.5772/intechopen.104521*

#### **Acknowledgements**

The authors would like to thank the Universitas Indonesia for providing grants to our studies.

#### **Author details**

Dewi F. Suniarti1 \*, Ria Puspitawati1 , Rezon Yanuar2 and Ranny R. Herdiantoputri3

1 Faculty of Dentistry, Department of Oral Biology, Universitas Indonesia, Jakarta, Indonesia

2 Faculty of Dentistry, Department of Pharmacology, Health Sciences University of Hokkaido, Tōbetsu, Japan

3 Department of Oral Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan

\*Address all correspondence to: dewisuniarti17@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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### Section 4
