**2.** *Staphylococcus aureus*

### **2.1 Clinical relevance and virulence factors**

Member of the *Micrococcaceae* family, *S. aureus* is Gram-positive cocci-shape arranged in a grape-like cluster. The cells are anaerobic facultative and catalasepositive with approximately 0.5–1.5 μm in diameter. Overall, 52 species have been described in the staphylococcal genus, *S. aureus* being, by far, the member most clinically relevant [11]. *S. aureus* genome has been completely sequenced and three main components were observed: conserved genes, variable genes, and mobile genetic elements (MGE). More than 97% of the *S. aureus* genome is composed of highly conserved genes found in all staphylococcal strains. More than 700 genes are variable and their distribution defines different lineages [12, 13]. Apart from the core genes, there are several numbers of MGE acquired by horizontal gene transfer by bacteriophages, transposons, and plasmids that contribute to genome plasticity and evolution, such as the antibiotics resistance and virulence gene dissemination [14].

Widely disseminated in nature, *S. aureus* is a commensal component of human cutaneous and mucosal microbiota as well as an adaptive pathogen that leads to numerous invasive and, sometimes, fatal infections [15, 16]. This microorganism can be easily spread by the hands or expelled from the respiratory tract. About 30% of the population is colonized by *S. aureus*, and this increases to 60% when it involves the healthcare environment, implying in either cases high risk of further infection [17]. As a pathogen, this bacterium causes various suppurative diseases, such as boils, carbuncles, folliculitis, and scalded-skin syndrome [18]. Additionally, the lymphatic system and bloodstream contributed to bacterial spread to other parts of the body causing osteomyelitis, medical device infection, endocarditis, and pneumonia [19]. Furthermore, the presence of a variety of antimicrobial resistance mechanisms in some strains leads to treatment failure, increasing healthcare costs and risk of death [20].

Bacterial sepsis confirmed by blood cultures in pediatric hospitals, Grampositive bacteria (62%) were involved in most of the infection cases. Among them, the major reported strains were *S. aureus* (15%), followed by *Staphylococcus* coagulase negative (11%) and *Streptococcus pneumoniae* (10%) [21]. In addition, serious

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**Figure 1.**

*Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*…*

cases of high virulence profile community-associated *S. aureus* (CA-MRSA) infections have been reported globally in recent decades [22, 23]. In Taiwan, for instance, 423 cases of CA-MRSA infections were reported in children, and most of them were associated to bone, joint, and deep soft tissue infections and pneumonia [24]. Despite each disease profile, the staphylococcal species is frequently correlated with both community- and hospital-acquired infections, and it has steadily increased [25, 26]. Thus, it is necessary to look for new therapeutic alternatives to minimize

*S. aureus* can survive in its hosts as a commensal bacterium for a long time; however, it can also be considered one of the most relevant human pathogens [28]. This bacterium has mechanisms to evade the host's immune response through production of a variety of virulence factors, such as adhesins, exotoxins, and hydrolytic enzymes (e.g., coagulase, catalase, and staphylokinase), as summarized in **Figure 1** [29, 30]. The bacterial adherence to extracellular matrix cells in the host is one of the most important steps for colonization. It is mediated mainly by surface-anchored proteins classified as MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family. Among them, two fibronectin binding proteins, FnbA and FnbB, contribute considerably to epithelial tissue colonization in various pathological manifestations and medical device-related infections [31]. Other cell surface protein related in adhesion mechanisms are named clumping factor A and B (ClfA and ClfB). The first one has a highlighted ability to interact with soluble proteins, fibrinogen, and fibrin, present in blood plasma. These surface components aid the microorganism to interact with plasma protein-coated biomaterials and, consequently, make possible the colonization and biofilm formation on medical devices [32]. The ClfB is frequently associated to nasal colonization due to high affinity to cornified envelope of the nostrils, which promotes the formation of skin abscesses by binding to the protein loricrin [33]. It is worth mentioning genes capable of encoding proteins on the cell surface, *cna* (collagen adhesin), *ebp* (elastin-binding protein), *bbp* (bone sialoprotein-binding protein), and *eno* (laminin-binding protein), closely related to pathogenesis of implant infections

*DOI: http://dx.doi.org/10.5772/intechopen.91833*

this public health problem [27].

caused by *S. aureus* [34–36].

**2.2 Antibiotic resistance and biofilm formation**

*Illustration of virulence factors produced by* S. aureus*.*

Historically, infections caused by MDR *S. aureus* strains have been often reported worldwide. This microorganism has a notable ability to acquire systems of antibiotics inactivation. Production of reduced-affinity penicillin binding,

### *Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*… DOI: http://dx.doi.org/10.5772/intechopen.91833*

cases of high virulence profile community-associated *S. aureus* (CA-MRSA) infections have been reported globally in recent decades [22, 23]. In Taiwan, for instance, 423 cases of CA-MRSA infections were reported in children, and most of them were associated to bone, joint, and deep soft tissue infections and pneumonia [24]. Despite each disease profile, the staphylococcal species is frequently correlated with both community- and hospital-acquired infections, and it has steadily increased [25, 26]. Thus, it is necessary to look for new therapeutic alternatives to minimize this public health problem [27].

*S. aureus* can survive in its hosts as a commensal bacterium for a long time; however, it can also be considered one of the most relevant human pathogens [28]. This bacterium has mechanisms to evade the host's immune response through production of a variety of virulence factors, such as adhesins, exotoxins, and hydrolytic enzymes (e.g., coagulase, catalase, and staphylokinase), as summarized in **Figure 1** [29, 30].

The bacterial adherence to extracellular matrix cells in the host is one of the most important steps for colonization. It is mediated mainly by surface-anchored proteins classified as MSCRAMM (microbial surface components recognizing adhesive matrix molecules) family. Among them, two fibronectin binding proteins, FnbA and FnbB, contribute considerably to epithelial tissue colonization in various pathological manifestations and medical device-related infections [31]. Other cell surface protein related in adhesion mechanisms are named clumping factor A and B (ClfA and ClfB). The first one has a highlighted ability to interact with soluble proteins, fibrinogen, and fibrin, present in blood plasma. These surface components aid the microorganism to interact with plasma protein-coated biomaterials and, consequently, make possible the colonization and biofilm formation on medical devices [32]. The ClfB is frequently associated to nasal colonization due to high affinity to cornified envelope of the nostrils, which promotes the formation of skin abscesses by binding to the protein loricrin [33]. It is worth mentioning genes capable of encoding proteins on the cell surface, *cna* (collagen adhesin), *ebp* (elastin-binding protein), *bbp* (bone sialoprotein-binding protein), and *eno* (laminin-binding protein), closely related to pathogenesis of implant infections caused by *S. aureus* [34–36].

### **2.2 Antibiotic resistance and biofilm formation**

Historically, infections caused by MDR *S. aureus* strains have been often reported worldwide. This microorganism has a notable ability to acquire systems of antibiotics inactivation. Production of reduced-affinity penicillin binding,

**Figure 1.** *Illustration of virulence factors produced by* S. aureus*.*

*Bacterial Biofilms*

the major cause of treatment failure [5–7].

against biofilm of multidrug-resistant *S. aureus*.

**2.1 Clinical relevance and virulence factors**

**2.** *Staphylococcus aureus*

therapeutic options available [1]. Consequently, it leads to a serious public health problem frequently associated with increase of healthcare costs and high morbimortality rates [2]. One worldwide recognized bacterial pathogen with the ability to develop severe clinical conditions such as pneumonia and septicemia is *Staphylococcus aureus* [3]. Historically, this bacterium has shown a great ability to become resistant to several antibiotics [4]. Furthermore, *S. aureus* has a highlighted ability to build surface-associated bacterial communities, called biofilm, being one of the most determinant factors for the development of chronic infections, and it is

Recently, the use of natural compounds, such as EOs obtained from different parts of the plants, is receiving attention for their biological activities, including antioxidant, anti-inflammatory, and anticancer effect [8]. Moreover, EOs have been frequently mentioned on scientific literature as a promising antimicrobial agent, being effective against a wide range of pathogenic bacteria and yeast [9, 10]. Thus, this chapter will present a comprehensive overview about general features of *S. aureus*, including virulence factors, antibiotic resistance, and biofilm formation. Additionally, it will introduce the EOs used as potential therapeutic approaches

Member of the *Micrococcaceae* family, *S. aureus* is Gram-positive cocci-shape arranged in a grape-like cluster. The cells are anaerobic facultative and catalasepositive with approximately 0.5–1.5 μm in diameter. Overall, 52 species have been described in the staphylococcal genus, *S. aureus* being, by far, the member most clinically relevant [11]. *S. aureus* genome has been completely sequenced and three main components were observed: conserved genes, variable genes, and mobile genetic elements (MGE). More than 97% of the *S. aureus* genome is composed of highly conserved genes found in all staphylococcal strains. More than 700 genes are variable and their distribution defines different lineages [12, 13]. Apart from the core genes, there are several numbers of MGE acquired by horizontal gene transfer by bacteriophages, transposons, and plasmids that contribute to genome plasticity and evolution, such as the antibiotics resistance and virulence gene dissemination [14]. Widely disseminated in nature, *S. aureus* is a commensal component of human cutaneous and mucosal microbiota as well as an adaptive pathogen that leads to numerous invasive and, sometimes, fatal infections [15, 16]. This microorganism can be easily spread by the hands or expelled from the respiratory tract. About 30% of the population is colonized by *S. aureus*, and this increases to 60% when it involves the healthcare environment, implying in either cases high risk of further infection [17]. As a pathogen, this bacterium causes various suppurative diseases, such as boils, carbuncles, folliculitis, and scalded-skin syndrome [18]. Additionally, the lymphatic system and bloodstream contributed to bacterial spread to other parts of the body causing osteomyelitis, medical device infection, endocarditis, and pneumonia [19]. Furthermore, the presence of a variety of antimicrobial resistance mechanisms in some strains leads to treatment failure, increasing healthcare costs

Bacterial sepsis confirmed by blood cultures in pediatric hospitals, Grampositive bacteria (62%) were involved in most of the infection cases. Among them, the major reported strains were *S. aureus* (15%), followed by *Staphylococcus* coagulase negative (11%) and *Streptococcus pneumoniae* (10%) [21]. In addition, serious

**164**

and risk of death [20].

ribosomal active site methylation, and efflux pumps that remove the antibiotic from the bacterial cell are the most cited mechanisms of antibiotic resistance developed by *S. aureus* cells [37]. The isolation of antibiotic-resistant strains began after the introduction of penicillin and methicillin into clinical practice, when resistant lineages, known as methicillin-resistant *S. aureus* (MRSA), were reported in 1950s and 1960s, respectively [38]. This resistance profile is mediated by mecA and mecC genes, which encode penicillin-binding protein 2a (PBP2a) in cell-wall synthesis. Those mec gene complexes are carried in an MGE known as the staphylococcal cassette chromosome mec (SCCmec), which can be acquired by horizontal gene transfer among related species [39].

Subsequently, vancomycin was used as an alternative to cases of MRSA infection [40]. However, the constant use of this antibiotic leads to the emergence of vancomycin-resistant *S. aureus* (VRSA) strains, first detected in 2002 [41]. Due to the fact that VRSA strains are generally also resistant to teicoplanin, the use of other abbreviations has been suggested: GISA (*S. aureus* of intermediate sensitivity to glycopeptides) and GRSA (*S. aureus* glycopeptide resistant) [42]. Moreover, some strains presented a relevant phenomenon known as heterogeneous resistance (heteroresistance) to vancomycin, where they have a mechanism of tolerance against this antibiotic. These strains, called hVISA, display a vancomycin-susceptible profile by microdilution assay; however, some individual cells into bacterial community might exhibit VRSA features [43].

Furthermore, the ability of some microorganisms to form cellular agglomerates, such as biofilms, contributes way more for antibiotic resistance. In summary, biofilm is a three-dimensional community of microorganisms covered and embedded in a self-produced matrix of extracellular polymeric substances (EPS) [44]. Such multicellular structure provides intrinsic protection for biofilm-embedded cells against hostile environments, for instance extreme temperature and pH, high salinity and pressure, poor nutrients, and antibiotics [45–47]. Microorganisms that grow on biofilms often exhibited different physiology profile from planktonic cells, especially in terms of their response to antibiotic treatment [48]. Although biofilm lifestyle can arise from a single cell, differential environmental conditions throughout the community can potentiate the development of distinct subpopulations. Gradients in oxygen, nutrients, and electron acceptors can cause heterogeneous gene expression throughout a biofilm. This communication between these bacterial cells, called quorum sensing, mediated the genes expression and activate virulence factors [49].

*S. aureus* has a great capacity to form biofilms on human body tissues and medical devices, increasing the risk of invasive infections [50]. It is estimated that *S. aureus* causes about 40–50% of prosthetic heart valve infections, 50–70% of catheter biofilm infections, and 87% of bloodstream infections [24]. The main stages of biofilm formation consist of four sequential steps: attachment, formation of microcolonies, accumulation or maturation, and detachment or dispersal (**Figure 2**) [51]. Firstly, planktonic cells adhere to biotic or abiotic surfaces and further proliferate into sticky aggregations called microcolonies. The EPS produced by bacterial cells during biofilm maturation serves as scaffold for establishing this three-dimensional architecture, also known as mushroom-like structures. Upon reaching a specific cell density, a mechanism is triggered to initiate EPS degradation that releases cells embedded into biofilm to disperse and reinitiate the biofilm formation at distal sites [7].

*S. aureus* shows a variety of adhesins that mediate attachment to host factors, essential for biofilm formation [48]. These proteins are surface-associated by different means, such as ionic or hydrophobic interactions, such as autolysin, SERAM (secretable expanded repertoire adhesive molecules) proteins, membrane-spanning proteins, and the polysaccharide intercellular adhesin (PIA) [52]. It is worth to

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**3. Essential oils**

**Figure 2.**

**3.1 General aspect**

ics, insecticides, and food additives [60].

tion of artifacts and modifying their biological activity [62].

*Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*…*

highlight that the presence of the ica gene located in the icaADBC (intercellular adhesion [ica]) operon works like a genetic determinant that contributes for biofilm establishment [53]. This genetic element can mediate the production of an extracellular mucopolysaccharide composed mainly of N-acetylglucosamine, which is

as target to antibiofilm approaches. As many conventional antimicrobial agents have no satisfactory effect against mature biofilms, EOs already used for hundreds of years as a natural medicine to combat a variety of infections became a great antimicrobial alternative. The EOs are made up of various compounds, and it is further believed that this makes it difficult to develop bacterial resistance compared to antibiotics that have only one target action, making it attractive to fight MDR biofilm-forming bacteria [56]. Then, such attributes qualify the EOs as an important product from natural source to be explored by pharmaceutical industry [57, 58].

Thus, several steps regarding biofilm formation of *S. aureus* might be considered

Essential oils are compounds obtained from the secondary metabolism of the plants. They are characterized as complex mixtures of volatile compounds abundant in aromatic plants found in different parts of the plant, including leaves, flowers, stem, roots, seeds, and fruits [59]. There is a diversity of these substances described in the literature in commercial use, such as in perfumes, pharmaceuticals, cosmet-

Generally, they are oily-looking liquids at room temperature of complex mixtures of volatile lipophilic substances, usually with pleasant scent. In water, EOs have a limited solubility, which allows their separation by steam or water distillation. Other methods to obtain EOs include cold-press extraction used for citrus peels, separation by solubility using organic solvents, and through supercritical fluid extraction [61]. They are usually unstable against environmental factors such as light, temperature, water activity, and salinity, affecting their constitution, contributing to the appearance of chemotypes with particular compositions. Depending on the technique used in the course of a separation, reactions such as ester hydrolysis, autoxidation, and rearrangements may occur, leading to the forma-

associated to adhesion and colonization of several surfaces [54, 55].

*DOI: http://dx.doi.org/10.5772/intechopen.91833*

*General steps of* S. aureus *biofilm formation.*

*Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*… DOI: http://dx.doi.org/10.5772/intechopen.91833*

**Figure 2.** *General steps of* S. aureus *biofilm formation.*

*Bacterial Biofilms*

transfer among related species [39].

munity might exhibit VRSA features [43].

ribosomal active site methylation, and efflux pumps that remove the antibiotic from the bacterial cell are the most cited mechanisms of antibiotic resistance developed by *S. aureus* cells [37]. The isolation of antibiotic-resistant strains began after the introduction of penicillin and methicillin into clinical practice, when resistant lineages, known as methicillin-resistant *S. aureus* (MRSA), were reported in 1950s and 1960s, respectively [38]. This resistance profile is mediated by mecA and mecC genes, which encode penicillin-binding protein 2a (PBP2a) in cell-wall synthesis. Those mec gene complexes are carried in an MGE known as the staphylococcal cassette chromosome mec (SCCmec), which can be acquired by horizontal gene

Subsequently, vancomycin was used as an alternative to cases of MRSA infection [40]. However, the constant use of this antibiotic leads to the emergence of vancomycin-resistant *S. aureus* (VRSA) strains, first detected in 2002 [41]. Due to the fact that VRSA strains are generally also resistant to teicoplanin, the use of other abbreviations has been suggested: GISA (*S. aureus* of intermediate sensitivity to glycopeptides) and GRSA (*S. aureus* glycopeptide resistant) [42]. Moreover, some strains presented a relevant phenomenon known as heterogeneous resistance (heteroresistance) to vancomycin, where they have a mechanism of tolerance against this antibiotic. These strains, called hVISA, display a vancomycin-susceptible profile by microdilution assay; however, some individual cells into bacterial com-

Furthermore, the ability of some microorganisms to form cellular agglomerates, such as biofilms, contributes way more for antibiotic resistance. In summary, biofilm is a three-dimensional community of microorganisms covered and embedded in a self-produced matrix of extracellular polymeric substances (EPS) [44]. Such multicellular structure provides intrinsic protection for biofilm-embedded cells against hostile environments, for instance extreme temperature and pH, high salinity and pressure, poor nutrients, and antibiotics [45–47]. Microorganisms that grow on biofilms often exhibited different physiology profile from planktonic cells, especially in terms of their response to antibiotic treatment [48]. Although biofilm lifestyle can arise from a single cell, differential environmental conditions throughout the community can potentiate the development of distinct subpopulations. Gradients in oxygen, nutrients, and electron acceptors can cause heterogeneous gene expression throughout a biofilm. This communication between these bacterial cells, called quorum sensing, mediated the genes expression and activate virulence factors [49]. *S. aureus* has a great capacity to form biofilms on human body tissues and medical devices, increasing the risk of invasive infections [50]. It is estimated that *S. aureus* causes about 40–50% of prosthetic heart valve infections, 50–70% of catheter biofilm infections, and 87% of bloodstream infections [24]. The main stages of biofilm formation consist of four sequential steps: attachment, formation of microcolonies, accumulation or maturation, and detachment or dispersal (**Figure 2**) [51]. Firstly, planktonic cells adhere to biotic or abiotic surfaces and further proliferate into sticky aggregations called microcolonies. The EPS produced by bacterial cells during biofilm maturation serves as scaffold for establishing this three-dimensional architecture, also known as mushroom-like structures. Upon reaching a specific cell density, a mechanism is triggered to initiate EPS degradation that releases cells embedded into biofilm to disperse and reinitiate the biofilm

*S. aureus* shows a variety of adhesins that mediate attachment to host factors, essential for biofilm formation [48]. These proteins are surface-associated by different means, such as ionic or hydrophobic interactions, such as autolysin, SERAM (secretable expanded repertoire adhesive molecules) proteins, membrane-spanning proteins, and the polysaccharide intercellular adhesin (PIA) [52]. It is worth to

**166**

formation at distal sites [7].

highlight that the presence of the ica gene located in the icaADBC (intercellular adhesion [ica]) operon works like a genetic determinant that contributes for biofilm establishment [53]. This genetic element can mediate the production of an extracellular mucopolysaccharide composed mainly of N-acetylglucosamine, which is associated to adhesion and colonization of several surfaces [54, 55].

Thus, several steps regarding biofilm formation of *S. aureus* might be considered as target to antibiofilm approaches. As many conventional antimicrobial agents have no satisfactory effect against mature biofilms, EOs already used for hundreds of years as a natural medicine to combat a variety of infections became a great antimicrobial alternative. The EOs are made up of various compounds, and it is further believed that this makes it difficult to develop bacterial resistance compared to antibiotics that have only one target action, making it attractive to fight MDR biofilm-forming bacteria [56]. Then, such attributes qualify the EOs as an important product from natural source to be explored by pharmaceutical industry [57, 58].
