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## **Meet the editorss**

Dr. Shymaa Enany is an assistant professor of Microbiology and Immunology at the Suez Canal University, Egypt. She received her PhD degree from the School of Medical and Dental Sciences, Niigata University, Japan. Her dissertation focused on the molecular characterization of community-acquired methicillin-resistant *Staphylococcus aureus* (CA-MRSA), the leading cause of many life-threat-

ening illnesses. For her postdoctoral work, she collaborated with many laboratories in San Diego, California, USA, and in Niigata, Japan, analyzing the proteome of *Staphylococcus aureus* and monitoring the reactions of innate immunity and airway cytokines in response to MRSA infections.

Dr. Laura E. Crotty Alexander is a Pulmonary Critical Care physician at the University of California, San Diego, and VA San Diego Healthcare System. As a biomedical basic science researcher, she studies host-pathogen interactions at molecular, cellular, organ, and organism levels and recently defined pro-virulent effects of inhalants such as cigarette smoke and e-cigarette vapor on Staphylo-

coccal virulence. Dr. Crotty Alexander's clinical interests include bacterial pneumonia and sepsis, e-cigarette effects on innate immunity, and moderate-to-severe asthma.

## Contents

## **Preface XI**

Chapter 6 **SOS Response and Staphylococcus aureus: Implications for Drug Development 95** Luís Cláudio Nascimento da Silva, Roseane Costa Diniz, Isana Maria de Souza Feitosa Lima, Camilla Itapary dos Santos, Matheus Silva Alves, Larissa Isabela Oliveira de Souza and Andrea de Souza Monteiro

## Chapter 7 **Antimicrobial Activity of Chitosan Membranes against Staphylococcus aureus of Clinical Origin 109**

Ana A. Escárcega-Galaz, Jaime López-Cervantes, Dalia I. Sánchez-Machado, Olga R. Brito-Zurita and Olga N. Campas-Baypoli

#### **Section 2 Staphylococcus aureus Virulence Factors 125**

Alžbeta Medved'ová, Adriana Havlíková and Ľubomír Valík

Fany Reffuveille, Jérôme Josse, Quentin Vallé, Céline Mongaret and Sophie C. Gangloff

## Preface

Chapter 7 **Antimicrobial Activity of Chitosan Membranes against Staphylococcus aureus of Clinical Origin 109**

**Section 2 Staphylococcus aureus Virulence Factors 125**

Loir and Koji Nishifuji

**VI** Contents

**Medical Field 187**

Sophie C. Gangloff

**Environmental Factors 145**

Chapter 10 **Surface Proteins of Staphylococcus aureus 169**

Chapter 11 **Staphylococcus aureus Biofilms and their Impact on the**

Chapter 8 **Exfoliative Toxins of Staphylococcus aureus 127**

Ana A. Escárcega-Galaz, Jaime López-Cervantes, Dalia I. Sánchez-Machado, Olga R. Brito-Zurita and Olga N. Campas-Baypoli

Ricardo B. Mariutti, Natayme R. Tartaglia, Núbia Seyffert, Thiago Luiz de Paula Castro, Raghuvir K. Arni, Vasco A. Azevedo, Yves Le

Chapter 9 **Staphylococcus aureus Enterotoxin Production in Relation to**

Alžbeta Medved'ová, Adriana Havlíková and Ľubomír Valík

Janet Jan-Roblero, Elizabeth García-Gómez, Sandra Rodríguez-Martínez, Mario E. Cancino-Diaz and Juan C. Cancino-Diaz

Fany Reffuveille, Jérôme Josse, Quentin Vallé, Céline Mongaret and

Today, *Staphylococcus aureus* (*S. aureus*) is a growing issue both within hospitals and com‐ munities because of virulence determinants, including surface proteins, exotoxins, entero‐ toxins, and biofilm formation. Antibiotic resistance in *S. aureus*, either inherent or acquired, is considered a potential virulence factor—increasing the pathogenesis and dissemination of the bacteria. There has been rapid emergence of drug resistance in *S. aureus,* leading first to methicillin-resistant *S. aureus* (MRSA), which is causing serious public health concerns, and more recently to vancomycin-resistant *S. aureus* (VRSA), which is being reported with in‐ creasing frequency among isolates identified all over the world. The pace of antibiotic resist‐ ance development in *S. aureus*, and its extensive presence worldwide, demands that we work to obtain a comprehensive understanding of the molecular mechanisms of both *S. aur‐ eus* drug resistance and bacterial virulence.

The aim of writing "The Rise of Virulence and Antibiotic Resistance in *Staphylococcus aur‐ eus"* is to provide an unprecedented and comprehensive collection of up-to-date research about the evolution, dissemination, and mechanisms of staphylococcal antimicrobial resist‐ ance alongside bacterial virulence determinants and their impact on the medical field. This book consists of eleven review chapters, written by international leaders in their respective fields. Each chapter starts with a brief introduction, including its aim, and then goes on to provide detailed information about current research relevant to the field. Importantly, we include several review chapters to allow the readers to better understand the mechanisms of methicillin resistance, glycopeptide resistance, and horizontal gene transfer, as well as the effects of alterations in *S. aureus* membranes and cell walls on drug resistance, and induction of an SOS response by application of antibiotics. Additional chapters unveil further details of *S. aureus* pathogenicity by introducing recent research on *S. aureus* exfoliative toxins, en‐ terotoxins, surface proteins, and biofilm.

Our timely book presents the state of the art of *S. aureus* virulence and antibiotic resistance. It is aimed at a wide range of informed readership, including clinicians, researchers, techni‐ cians, scientists, and students. We would like to thank all the authors who contributed chap‐ ters, without whose dedication, brilliant research, and keen support this book would not have been accomplished.

> **Dr. Shymaa Enany** Department of Microbiology and Immunology, Faculty of Pharmacy Suez Canal University, Ismailia, Egypt

> > **Dr. Laura E. Crotty Alexander** Department of Medicine, University of California, San Diego, and VA San Diego Healthcare System, San Diego, CA, USA

**Resistance in Staphylococcus aureus**

#### **The Evolution and Dissemination of Methicillin Resistance Determinant in** *Staphylococcus aureus* **The Evolution and Dissemination of Methicillin Resistance Determinant in** *Staphylococcus aureus*

Abdul Rahim Abdul Rachman, Zarizal Suhaili and Mohd Nasir Mohd Desa Abdul Rahim Abdul Rachman, Zarizal Suhaili and Mohd Nasir Mohd Desa

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65514

#### **Abstract**

*Staphylococcus aureus* is an opportunistic pathogen and is frequently associated with the antimicrobial resistance. There has been horizontal gene transfer of *Staphylococcus* chromosome cassette *mec* (SCC*mec*) among the staphylococcal species that colonize a similar colonization niche, which eventually results in emergence of new variant with enhanced survival ability in terms of antimicrobial resistance and virulence level in *S. aureus*. Evolution and dissemination of SCC*mec* structure resulted in the emergence of methicillin-resistant *S. aureus* (MRSA) clones around the world covering hospital, community, and livestock settings. MRSA also has the ability to resist different antibiotic profiles known as multidrug-resistant *S. aureus* (MDR *S. aureus*).

**Keywords:** *Staphylococcus aureus*, SCC*mec*, MRSA clones, multi-drug-resistant *S. aur‐ eus*

## **1. Introduction**

*Staphylococcus aureus* is an opportunistic pathogen and lives as part of the animal normal flora of skin and nasopharynx. Favorably, it resides in the nasal mucosal environment posing infection threat to human as well as in domestic animals [1, 2]. In human, it is the leading agent of infection involving bloodstream, skin, and soft tissue to the lower respiratory tract [3–5]. *S. aureus* can easily colonize certain part of the body, especially the exposed area on skin due to ulcers, burns, and surgical wounds [6].

© 2017 The Author(s). Licensee InTech. 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. © 2017 The Author(s). Licensee InTech. 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.

Methicillin-resistant *S. aureus* (MRSA) has been well known for being resistant to β-lactam antibiotics, which are the most common antimicrobial agents used to fight staphylococcal infection. Previous studies reported that methicillin resistance in staphylococci was carried by a specific mobile genetic element (MGE) called staphylococcal chromosome cassette *mec* (SCC*mec*), which carries with it several virulence factors as well [7]. SCC*mec* contains *mecA* gene which encodes for a low affinity penicillin-binding protein (pbp2a or pbp2′), which is currently exploited as the methicillin resistance marker in Staphylococcus species including *S. aureus* [8]. SCC*mec* contains several elements that can be categorized into several types. Genetic events such as point mutation, recombination, acquisition, and deletion, coupled with host and environmental selective pressures, make the structure evolve and disseminate in the population [9]. The emergence of certain MRSA clones, which have been disseminating worldwide since 1960, was closely related to the continuous evolution of SCC*mec* structure in *S. aureus*.

Multidrug-resistant MRSAs have also been reported that make the antibiotic regiment limited. Prevalence of MRSA is of a growing concern, particularly due to the more recent increased frequency of community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA). Thus, this pathogen becomes a major concern in public health as well as livestock industry [10–13].

Many studies have been looking at the mechanism and dissemination pattern of MRSA and its genetic characteristics, but due to the potential, geographical, and temporal differences, a comprehensive review is needed to put the whole picture connected.

## **2.** *Staphylococcus aureus*

*S. aureus* is a Gram-positive bacterium with a grape-like cluster morphology and can usually be found in skin or mucous membrane, especially in nasal of healthy person [14]. Kluytmans et al. reported that approximately 20–30% of human population carries *S. aureus* [15]. Morphologically, *S. aureus* can be observed as a 'golden' medium-size colony on solid media such as nutrient agar and can cause β-hemolysis on sheep blood agar [16].

The production of golden pigmentation of *S. aureus* colonies is closely related to the presence of carotenoids which is previously reported as virulence factor protecting *S. aureus* from the immune system [17]. Among Staphylococcus species, only *S. aureus* has the ability to ferment mannitol leading to the production of lactic acid on mannitol salt agar with yellow zones around the colonies [18]. *S. aureus* is also classified as a halophilic bacterium for being able to live in the presence of salt (sodium chloride) up to 1.7 molar. It also produces coagulase that causes blood to clot [14].

Generally, 20–30% of individuals are persistent carriers of *S. aureus* and 30% are transient or intermittent carriers [19]. *S. aureus* may live in human without any clinical symptoms, but it may infect the host when the host defense system is compromised. Individuals may acquire infection by *S. aureus* that they previously carry as commensal [15].

Immunocompromised patients with *S. aureus* infection may suffer several diseases such as bacteremia, ventilator-assisted pneumonia (VAP), endocarditis, and osteomyelitis, especially when the patients are frequently exposed to injections and catheter insertions [20, 21]. *S. aureus* can also cause toxin-mediated disease such as toxic shock syndrome, scalded skin syndrome, and Staphylococcal foodborne diseases (SFD) [21]. Frequently, *S. aureus* is the main cause of skin and soft tissue infection (SSTI) in human [22].

## **3. Methicillin-resistant** *S. aureus* **(MRSA)**

Methicillin-resistant *S. aureus* (MRSA) has been well known for being resistant to β-lactam antibiotics, which are the most common antimicrobial agents used to fight staphylococcal infection. Previous studies reported that methicillin resistance in staphylococci was carried by a specific mobile genetic element (MGE) called staphylococcal chromosome cassette *mec* (SCC*mec*), which carries with it several virulence factors as well [7]. SCC*mec* contains *mecA* gene which encodes for a low affinity penicillin-binding protein (pbp2a or pbp2′), which is currently exploited as the methicillin resistance marker in Staphylococcus species including *S. aureus* [8]. SCC*mec* contains several elements that can be categorized into several types. Genetic events such as point mutation, recombination, acquisition, and deletion, coupled with host and environmental selective pressures, make the structure evolve and disseminate in the population [9]. The emergence of certain MRSA clones, which have been disseminating worldwide since 1960, was closely related to the continuous evolution of SCC*mec* structure in

4 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

Multidrug-resistant MRSAs have also been reported that make the antibiotic regiment limited. Prevalence of MRSA is of a growing concern, particularly due to the more recent increased frequency of community-associated MRSA (CA-MRSA) and livestock-associated MRSA (LA-MRSA). Thus, this pathogen becomes a major concern in public health as well as livestock

Many studies have been looking at the mechanism and dissemination pattern of MRSA and its genetic characteristics, but due to the potential, geographical, and temporal differences, a

*S. aureus* is a Gram-positive bacterium with a grape-like cluster morphology and can usually be found in skin or mucous membrane, especially in nasal of healthy person [14]. Kluytmans et al. reported that approximately 20–30% of human population carries *S. aureus* [15]. Morphologically, *S. aureus* can be observed as a 'golden' medium-size colony on solid media such

The production of golden pigmentation of *S. aureus* colonies is closely related to the presence of carotenoids which is previously reported as virulence factor protecting *S. aureus* from the immune system [17]. Among Staphylococcus species, only *S. aureus* has the ability to ferment mannitol leading to the production of lactic acid on mannitol salt agar with yellow zones around the colonies [18]. *S. aureus* is also classified as a halophilic bacterium for being able to live in the presence of salt (sodium chloride) up to 1.7 molar. It also produces coagulase that

Generally, 20–30% of individuals are persistent carriers of *S. aureus* and 30% are transient or intermittent carriers [19]. *S. aureus* may live in human without any clinical symptoms, but it may infect the host when the host defense system is compromised. Individuals may acquire

comprehensive review is needed to put the whole picture connected.

as nutrient agar and can cause β-hemolysis on sheep blood agar [16].

infection by *S. aureus* that they previously carry as commensal [15].

*S. aureus*.

industry [10–13].

**2.** *Staphylococcus aureus*

causes blood to clot [14].

MRSA has the ability to resist almost all available β-lactam antibiotics. Statistics showed about 40–70% of *S. aureus* nosocomial infections worldwide are caused by MRSA. MRSA was first reported in a hospital in the United Kingdom in 1961 after the introduction of methicillin to treat patient with penicillin-resistant Staphylococcus infection [23].

Generally, MRSA can be categorized into two major groups known as hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). Globally, the majority of MRSA infections are HA-MRSA that are acquired from healthcare facilities. Currently, MRSA isolates are subdivided into three major groups known as hospital-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-MRSA). Previous reports revealed that both HA-MRSA and CA-MRSA isolates differ distinctly from each other, with HA-MRSA showing high antimicrobial resistance but less virulence and lack of capabilities as colonizers [23–26]. Meanwhile, CA-MRSA isolates exhibit a low antimicrobial resistance but a high virulence harboring PVL gene and numerous pathogenicity factors, as well as good colonizers [26–29].

MRSA spread in population since 1990 and become the major cause of community-associated infection [27]. The scenario worsens when multidrug MRSA emerges, in which it can resist more than two antibiotics of different classes that reduce the option for available treatment of Staphylococcal infection [30, 31].

Methicillin resistance characteristic in *S. aureus* is due to the presence of altered penicillinbinding protein (PBP2a) in the cell wall that has a reduced binding affinity to β-lactam antibiotics. PBP2a is encoded by *mecA* gene that is located in the large chromosomal cassette called staphylococcal chromosome cassette *mec* element (SCC*mec*) [32–35]. The *mecA* gene expression is controlled by *mecI*-*mecRI* regulatory genes encoding repressor and inducer protein, respectively [36].

## **4. SCC***mec* **structure**

Staphylococcal cassette chromosome *mec* (SCC*mec*) has a size of about 20–60 kb. The structure is unique as it carries various mobile genetic elements that are integrated in it [37]. To date, more than 80 SCC*mec* elements have been identified in several staphylococci species [38]. SCC*mec* disseminates among Staphylococcal species by horizontal gene transfer and integrates at a specific site called *att*B or ISS (integration site sequence) at the 3′ end of *orf*X gene that encodes for unknown function [39].

A single SCC*mec* carries *mec* complex and cassette chromosome recombinase (*ccr*) flanked by direct inverted repeat (DR) and inverted repeat (IR) sequences; *mec* complex consists of *mecA* gene (methicillin resistance determinant), *mecRI* (sensor inducer), and *mecI* (*mec* repressor). Both *mecRI* and *mecI* are recognized as *mec* regulator elements, while *ccr* genes encode serine recombinases (*ccr*A, *ccr*B, *ccr*C) responsible for site and orientation of specific integration and excision of SCC*mec*. In addition, SCC*mec* also harbors other elements such as insertion sequences (IS), plasmids, and transposons [24, 40, 41].

## **4.1. Complete SCC***mec*

To date, the International Working Group-SCC*mec* (IWG-SCC*mec*) identified eleven SCC*mec* types based on complete nucleotide sequences in Staphylococcal databases, and each SCC*mec* type is named using a roman numeral based on the unique combination of *ccr* complex and *mec* complex [40–42]. A complete SCC*mec* structure in *S. aureus* contains a *mec* complex (*mecA, mecRI* and *mecI*), a *ccr* complex (*ccrA, ccrB, ccrC*), and a J region (region other than *mec* and *ccr* complexes) [40].

Furthermore, many different SCC*mec* subtypes have also been described containing the same *ccr* and *mec* gene combination but vary in the J regions [40]. Among the eleven SCC*mec* types (I–XI) that have been reported so far, five of them (SCC*mec* I, II, III IV, and V) are globally distributed, while others only distributed in certain countries [38, 43]. Three (SCC*mec* IVa, SCC*mec* IVc, SCC*mec* V) from the 11 SCC*mec* types have been detected in MRSA isolated from animals called LA-MRSA [42]. In general, SCC*mec* type IV and V are more widely found among CA-MRSA, and the other three types (SCC*mec* I, II, III) are frequently found among HA-MRSA [44, 45, 46]. An early study by Ito et al. detected only three types of SCC*mec* structures (SCC*mec* type I, II, III) isolated from human [37], and a recent finding showed that MRSA with SCC*mec* type I, II, III is originated from animals [41].

Different types of SCC*mec* in MRSA are also observed to be geographically distributed. For example, SCC*mec* type III or IIIA was most commonly found in Asian countries, but Korea and Japan had more type II while Taiwan had more type IV [47]. SCC*mec* type IV was also commonly found in Latin and European countries [48, 49]. Similarly, in African countries, SCC*mec* type III was also predominant with SCC*mec* types II, IV, and V found in selected countries such as Egypt, Niger, Nigeria, Algeria, Tunisia, and South Africa [50].

#### **4.2. Pseudo-SCC***mec*

Pseudo-SCC*mec* is recognized as SCC*mec* that does not carry *ccr* complexes but has *mecA* gene. Although this element is different from the complete SCC*mec* in terms of gene or operon organization, it still has some similarities in certain parts in both pseudo-SCC*mec* and complete SCC*mec* structure. Deletion is the major event as inferred by the absence of certain genes or operon in pseudo-SCC*mec* structure. For example, regions within *mec* complex and J region are absent in both pseudo-SCC*mec* II.5 and pseudo-SCC*mec*16691. It was observed that pseudo-SCC*mec*16691 lacks J1, J2 regions, and *ccr* genes, whereas missing parts were detected in pseudo-SCC*mec* II.5 and replaced by transposable elements called *Tn6012* [51, 52].

However, certain pseudo-SCC*mec* does not carry both *mec* and *ccr* complexes. An example is the arginine catabolic mobile element (ACME) for having SCC-like elements but lack in *mecA* and *ccr* genes. This could be the remnant of SCC*mec* structure that had gone through multiple mutational events. Lindqvist et al. discovered first remnant of pseudo-SCC*mec* structure in methicillin susceptible *S. aureus* (MSSA) that caused clonal outbreak in Sweden. They suggested that this pseudo-SCC*mec* structure could be derived from SCC*mec* type II [52].

ACME is found in both MRSA and MSSA, especially with sequence type ST8 (ST8), and has been disseminated in virulent *S. aureus* by horizontal gene transfer [3, 53]. Nevertheless, ACME was frequently associated with MRSA-IVa with sequence type 8 (ST8-MRSA-IVa), which was also known as CA-MRSA USA300 [3, 53]. ACME has been associated with the ability of CA-MRSA to colonize on other parts of human body such as skin and mucosal membranes rather than limited to only nostril. The acquisition of ACME may enhance the ability of CA-MRSA to survive in acidic environment of human skin by driving production of polyamine-resistant enzyme that combats excess host polyamine (toxic compound on human skin for *S. aureus*) [54].

## **5. Origin of SCC***mec* **structure**

SCC*mec* disseminates among Staphylococcal species by horizontal gene transfer and integrates at a specific site called *att*B or ISS (integration site sequence) at the 3′ end of *orf*X gene that

A single SCC*mec* carries *mec* complex and cassette chromosome recombinase (*ccr*) flanked by direct inverted repeat (DR) and inverted repeat (IR) sequences; *mec* complex consists of *mecA* gene (methicillin resistance determinant), *mecRI* (sensor inducer), and *mecI* (*mec* repressor). Both *mecRI* and *mecI* are recognized as *mec* regulator elements, while *ccr* genes encode serine recombinases (*ccr*A, *ccr*B, *ccr*C) responsible for site and orientation of specific integration and excision of SCC*mec*. In addition, SCC*mec* also harbors other elements such as insertion

To date, the International Working Group-SCC*mec* (IWG-SCC*mec*) identified eleven SCC*mec* types based on complete nucleotide sequences in Staphylococcal databases, and each SCC*mec* type is named using a roman numeral based on the unique combination of *ccr* complex and *mec* complex [40–42]. A complete SCC*mec* structure in *S. aureus* contains a *mec* complex (*mecA, mecRI* and *mecI*), a *ccr* complex (*ccrA, ccrB, ccrC*), and a J region (region other than *mec* and *ccr*

Furthermore, many different SCC*mec* subtypes have also been described containing the same *ccr* and *mec* gene combination but vary in the J regions [40]. Among the eleven SCC*mec* types (I–XI) that have been reported so far, five of them (SCC*mec* I, II, III IV, and V) are globally distributed, while others only distributed in certain countries [38, 43]. Three (SCC*mec* IVa, SCC*mec* IVc, SCC*mec* V) from the 11 SCC*mec* types have been detected in MRSA isolated from animals called LA-MRSA [42]. In general, SCC*mec* type IV and V are more widely found among CA-MRSA, and the other three types (SCC*mec* I, II, III) are frequently found among HA-MRSA [44, 45, 46]. An early study by Ito et al. detected only three types of SCC*mec* structures (SCC*mec* type I, II, III) isolated from human [37], and a recent finding showed that MRSA with SCC*mec*

Different types of SCC*mec* in MRSA are also observed to be geographically distributed. For example, SCC*mec* type III or IIIA was most commonly found in Asian countries, but Korea and Japan had more type II while Taiwan had more type IV [47]. SCC*mec* type IV was also commonly found in Latin and European countries [48, 49]. Similarly, in African countries, SCC*mec* type III was also predominant with SCC*mec* types II, IV, and V found in selected

Pseudo-SCC*mec* is recognized as SCC*mec* that does not carry *ccr* complexes but has *mecA* gene. Although this element is different from the complete SCC*mec* in terms of gene or operon organization, it still has some similarities in certain parts in both pseudo-SCC*mec* and complete SCC*mec* structure. Deletion is the major event as inferred by the absence of certain genes or operon in pseudo-SCC*mec* structure. For example, regions within *mec* complex and J region

countries such as Egypt, Niger, Nigeria, Algeria, Tunisia, and South Africa [50].

encodes for unknown function [39].

**4.1. Complete SCC***mec*

complexes) [40].

**4.2. Pseudo-SCC***mec*

sequences (IS), plasmids, and transposons [24, 40, 41].

6 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

type I, II, III is originated from animals [41].

The origin of SCC*mec* in MRSA is still in debate; *mecA* gene was believed to be originated from *Staphylococcus fleurettii* due to a high sequence similarity (>99%) with *mecA* gene of a MRSA strain N315. It was proposed that SCC*mec* is a combination of SCC elements without *mec* complex, and the *mec* gene complex was derived from *S. fleurettii* since no evidence showed that *S. fleurettii* contained SCC*mec* structure in its chromosome [55].

Several studies described coagulase-negative staphylococci (CoNS) as the primary reservoir of the SCC*mec* structure in *S. aureus*, which was considered as the recipient strain due to some reasons; a very similar SCC*mec* structure and organization was observed in both *S. aureus* and CoNS [56, 57], and the prevalence of methicillin-resistant coagulase-negative staphylococci (MRCoNS) in human is higher as compared to MRSA [35, 56–59]. Although a study discovered other non-staphylococci species called *Macrococcus* to also carry SCC*mec*-like elements, those were different with SCC*mec* in MRSA in terms of nucleotide sequences and genetic organization of the *mec* complex [55].

#### **5.1. From coagulase-negative staphylococci species to MRSA**

The existence of various forms of SCC*mec* in MRCoNS as compared to MRSA becomes the main argument why MRCoNS is suggested as the main reservoir of SCC*mec* for *S. aureus* leading to the emergence of MRSA [40, 57]. In a rapid genetic typing, polymerase chain reaction (PCR) technique is used to characterize the SCC*mec* types instead of nucleotide sequencing analysis. Consequently, SCC*mec* from MRCoNS is frequently defined as non-typeable due to a diverse combination of *ccr* and *mec* complexes that could not be assigned based on current SCC*mec* structure databases used against *S. aureus* [7]. Nevertheless, Zong et al. successfully assigned 10 SCC*mec* elements with a new combination of *ccr* and *mec* complexes in various species of MRCoNS. They assigned these untypeable SCC*mec* elements as UT1–UT10 [35]. In addition, another study also described new SCC*mec* types in *Staphylococcus hominis* and described those as NT1till NT4 [60].

*Staphylococcus epidermidis, Staphylococcus haemolyticus*, and *S. hominis* were found to carry a diverse SCC*mec* structure among CoNS. SCC*mec* type IV is the common structure found in *S. epidermidis*, while other SCC*mec* types I, II, III, V, VI and non-typeable SCC*mec* were also detected at a lower rate [61, 62]. For *S. haemolyticus*, SCC*mec* type V predominated in combination with other novel SCC*mec* types [60, 63]. In *S. hominis*, SCC*mec* types contained a combination of novel non-typeable SCC*mec*, SCC*mec* types VI, VIII, III, and other elements [35, 61].

#### **5.2. From MSSA to MRSA**

MRSA emerges when MSSA receives SCC*mec* structure elements from other MRSA or MRCoNS via horizontal gene transfer [64]. In a specific condition (high vancomycin concentration), SCC*mec* is unstable in certain MRSA that can lead to complete or partial deletion of SCC*mec* structure, which may result in the presence of certain SCC*mec* DNA fragment to remain in *S. aureus* chromosome [64–67].

Wong et al. [64] identified SCC*mec* type II with internal deletion in MSSA isolates from different geographical areas. This happened during *in vitro* exposure to vancomycin [64]. Furthermore, Vandendriessche et al. [67] described MSSA CC398 as the precursor for emergence of MRSA CC398 in livestock. They found non-SCC*mec* elements in MSSA CC398 harboring *czrC* and *tet*(K) genes generated during partial excision of SCC*mec* elements [67].

## **6. Clonal dissemination of MRSA**

Nowadays, the dissemination of MRSA has become a major global problem that threatens human health [27]. However, only limited clones of MRSAs could be inferred to disseminate in different countries and continents through genotypic analysis using several DNA typing methods such as SCC*mec* typing, PFGE, MLST, and spa typing [27, 68, 69]. For example, more than 3000 MRSA isolates from certain continents (Europe, USA, and South America) were described to belong to only five major pandemic clones or clonal complexes (CC5, CC8, CC22, CC30, and CC45) [70]. To date, 11 clonal complexes (CC1, CC5, CC8, CC12, CC15, CC22, CC25, CC30, CC45, CC51, and CC121) have been detected in which 5 of them (CC8, CC15, CC22, CC30, and CC45) were isolated from human [71, 72]. These successful clones may transmit their genetic elements into other *S. aureus*, which are well adapted to hospital environment [73]. MRSA strain COL was the first MRSA clone detected carrying SCC*mec* type I with sequence type 250 (ST 250) and belonged to clonal complex 8 (CC8). Then, other MRSA clones with SCC*mec* type II and III were reported and recognized as EMRSA-1 (ST239), EMRSA-5 (ST247), and New York/Japan clone (ST5, USA100) [74]. Certain MRSA clones were originated from community setting. For example, Wang and co-workers (2007) detected the spread of community-associated SCC*mec* type IV and V MRSA in hospital setting in Taiwan between 1999 and 2005. They concluded that SCC*mec* types IV and V are carried by both CA-MRSA and HA-MRSA [75–77].

analysis. Consequently, SCC*mec* from MRCoNS is frequently defined as non-typeable due to a diverse combination of *ccr* and *mec* complexes that could not be assigned based on current SCC*mec* structure databases used against *S. aureus* [7]. Nevertheless, Zong et al. successfully assigned 10 SCC*mec* elements with a new combination of *ccr* and *mec* complexes in various species of MRCoNS. They assigned these untypeable SCC*mec* elements as UT1–UT10 [35]. In addition, another study also described new SCC*mec* types in *Staphylococcus hominis* and

*Staphylococcus epidermidis, Staphylococcus haemolyticus*, and *S. hominis* were found to carry a diverse SCC*mec* structure among CoNS. SCC*mec* type IV is the common structure found in *S. epidermidis*, while other SCC*mec* types I, II, III, V, VI and non-typeable SCC*mec* were also detected at a lower rate [61, 62]. For *S. haemolyticus*, SCC*mec* type V predominated in combination with other novel SCC*mec* types [60, 63]. In *S. hominis*, SCC*mec* types contained a combination of novel non-typeable SCC*mec*, SCC*mec* types VI, VIII, III, and other elements [35,

MRSA emerges when MSSA receives SCC*mec* structure elements from other MRSA or MRCoNS via horizontal gene transfer [64]. In a specific condition (high vancomycin concentration), SCC*mec* is unstable in certain MRSA that can lead to complete or partial deletion of SCC*mec* structure, which may result in the presence of certain SCC*mec* DNA fragment to

Wong et al. [64] identified SCC*mec* type II with internal deletion in MSSA isolates from different geographical areas. This happened during *in vitro* exposure to vancomycin [64]. Furthermore, Vandendriessche et al. [67] described MSSA CC398 as the precursor for emergence of MRSA CC398 in livestock. They found non-SCC*mec* elements in MSSA CC398 harboring *czrC* and

Nowadays, the dissemination of MRSA has become a major global problem that threatens human health [27]. However, only limited clones of MRSAs could be inferred to disseminate in different countries and continents through genotypic analysis using several DNA typing methods such as SCC*mec* typing, PFGE, MLST, and spa typing [27, 68, 69]. For example, more than 3000 MRSA isolates from certain continents (Europe, USA, and South America) were described to belong to only five major pandemic clones or clonal complexes (CC5, CC8, CC22, CC30, and CC45) [70]. To date, 11 clonal complexes (CC1, CC5, CC8, CC12, CC15, CC22, CC25, CC30, CC45, CC51, and CC121) have been detected in which 5 of them (CC8, CC15, CC22, CC30, and CC45) were isolated from human [71, 72]. These successful clones may transmit their genetic elements into other *S. aureus*, which are well adapted to hospital environment [73].

*tet*(K) genes generated during partial excision of SCC*mec* elements [67].

described those as NT1till NT4 [60].

8 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

**5.2. From MSSA to MRSA**

remain in *S. aureus* chromosome [64–67].

**6. Clonal dissemination of MRSA**

61].

The popular human MRSA pandemic clones, the EMRSA-15 and EMRSA-16, were identified in the United Kingdom (UK) around early 1990s. Since then, the clones become predominant healthcare-associated MRSA in UK [78, 79] and several European countries such as Denmark [80], Sweden [81], Belgium [79], and Spain [82]. Studies in Kuwait [83] and USA [84] also reported the spread of EMRSA-15 and 16 clones in hospital setting in the countries. To date, these clones have already been widespread in 15 countries around the world [85]. Both MRSAs belong to SCC*mec* type IV with sequence type 22 (ST 22) for EMRSA-15 and sequence type 30 (ST 30) for EMRSA-16 and originated from hospital setting. EMRSA-15 and 16 have high surviving and spreading rate in hospital compared to other EMRSA in UK [78]. In 2013, MRSA clone with a rare sequence type, ST 779, was identified in eleven Irish hospitals from 2006 until 2011 harboring a novel pseudo (SCC*mec*)-SCC-SCC*CRISPR* composite element. This clone contained novel *mec* class region, a fusidic acid resistance gene (*fusC*), and two copper resistance genes (*copB* and *copC*) but lacking *ccr* genes [86].

CA-MRSA clones have also been observed to disseminate worldwide particularly with sequence types ST80 and ST30. MRSA clone with ST80 is the most common CA-MRSA clone in European countries and usually carries PVL genes. Moreover, ST80 clone also showed resistance toward fluoroquinolones, tetracyclines, and fusidic acid [87]. CA-MRSA clone with sequence type ST30 was observed to disseminate in Asian and Oceanic countries. An example is the multidrug USA300 clone, known as West Pacific clone. It was first identified in the USA and carried plasmid that encodes several antibiotic resistance genes [88]. Enany et al. identified novel clones with sequence types ST1010 (121)c and ST1009 (1153)c isolated from Egypt after they analyzed different genetic patterns of PVL+CA-MRSA isolates from different countries [89].

In certain countries, it was found that MRSA can also spread among livestock, known as LA-MRSA. LA-MRSA CC398 is the popular clonal complex among livestock and has already been reported to spread in several European farms in Netherland, Denmark, Germany, France, and Italy [90]. MRSA CC398 was originated from pigs and spread among dairy cattle and turkey [91, 92]. In Netherlands, MRSA contamination on meat was reported after 2217 meat samples were analyzed covering 35.3% turkey, 15.2% beef, 15.2% veal, 10.7% pork, and 6.2% lamb meat [93]. LA-MRSA can be transmitted to human by physical contact with livestock contaminated with MRSA [94]. LA-MRSA may have equal virulence ability as compared to CA-MRSA and HA-MRSA toward human. Therefore, persons with continuous exposure to livestock carrying LA-MRSA are at high risk [95]. Other than meat, LA-MRSA can also be found in dairy milk. Recently, 11 sequence types were detected from LA-MRSA isolated from 15 Brazilian dairy farms (n = 552) with four of them contain novel sequence types (ST1622, ST1623, ST1624, and ST1625) [96].

## **7. Multidrug-resistant (MDR) MRSA**

Antibiotic or antimicrobial drugs are the most effective therapeutic agents used in treating microbial infections through either one or both bactericidal and bacteriostatic effects. Nevertheless, antibiotic or antimicrobial drug resistance has been a major problem worldwide, with incidence of MRSA reported in healthcare facilities in Asia to reach its peak in late 1990s, and stayed at plateau level during 2000s [97]. The heavy usage of drugs in treatment hastens the selection of bacteria that harbor multidrug resistance genes particularly *S. aureus* to proliferate and dominate [98, 99]. Moreover, over-crowded community creates environment that is suitable for the rapid spread of numerous multidrug-resistant pathogens, particularly the airborne organisms such as *S. aureus*.

The emergence of multidrug-resistant *S. aureus* in both hospitals and community invokes a tremendous financial burden due to the persistence of hard-to-treat infections [97, 100–102]. Until present, it was reported that <90% of *S. aureus* strains are resistant to penicillin as well as ordinary antimicrobial agents such as drug from categories of aminoglycosides, ansamycins, anti-staphylococcal β-lactams (or cephamycins), chloramphenicols, fusidanases, fluoroquinolones, glycopeptides, lincosamides, macrolides, phenicols, and tetracyclines [103–105]. We are now observing the emergence of multidrug-resistant *S. aureus* and MDR-MRSA with broad spectrum of resistance with a distinct ability to survive and spread in the hospital environment, community setting, as well as livestock sectors.

There has been a dramatic increase in the incidence of nosocomial infections as well as community-associated MRSA and livestock-associated MRSA caused by strains of *S. aureus* that are resistant to multiple antibiotics [106]. At present, there have been reports that some strains demonstrate resistance to as many as 20 antimicrobial compound types, including antiseptics and disinfectants [107, 108]. Central Asian surveillance studies found that the prevalence of MRSA infection in tertiary hospital was reported in 10 among 1000 hospital admissions [109] and incidence reported previously in Japan was between 0.7 and 0.8 per 100 admission from 1999 to 2003 with a total rate among hospitalized patients in the Asia-Pacific region at 45.9% [110, 111]. Previous surveillance also reported that Asia is among the highest for the incidence of MRSA in the world, and interestingly a novel MRSA strain with glycopeptides resistance had spread in livestock animals making it as a potential human pathogen in this region [112].

Several studies attempted to profile all possible multidrug-resistant MRSA since 1987, encompassing samples from hospitals, community, as well as veterinary settings [113, 114]. Lim et al. (2013) carried out temporal comparative surveillance of antibiograms from clinical samples in 2003–2008 and showed a significant increase in resistance rates (from 1 to 96%), as well as multidrug-resistant phenotypes (96%). This study also indicated the prevalence of multidrug-resistant MRSA with SCC*mec* type III and ST239 [99]. Another study also reported the prevalence of resistance against other important antibiotics such as mupirocin, whose resistance rate in Malaysia is still low, but still higher than previous reports in Malaysia [107]. Another cross-sectional studies at a few major medical centers in Malaysia found that the occurrence of MRSA infection increased gradually with years, from 25.7 to 28.7% in 1996, 27.9% in 1998, and 33% in 2000 [115, 117, 118]. Meanwhile, a study done at a single Malaysian hospital found a gradual reduction in MRSA prevalence from 2002 to 2006, most likely due to the improvement in the quality of healthcare systems [103, 109, 116 118].

The first international surveillance study on epidemiology of CA-MRSA in Asian countries revealed important findings with regard to the current epidemiology of MRSA infections in the community and hospitals within Asia with multidrug-resistance rates at 73.1 and 83.7% for CA-MRSA and HA-MRSA, respectively [119]. At least, 357 isolates of CA-MRSA were analyzed with resistance rates of gentamicin, ciprofloxacin, and trimethoprim/sulfamethoxazole being significantly lower than those of HA-MRSA isolates, whereas resistance rates of clindamycin, erythromycin, and tetracycline were similarly high in both CA-MRSA and HA-MRSA [119, 120].

## **8. Conclusion**

farms (n = 552) with four of them contain novel sequence types (ST1622, ST1623, ST1624, and

Antibiotic or antimicrobial drugs are the most effective therapeutic agents used in treating microbial infections through either one or both bactericidal and bacteriostatic effects. Nevertheless, antibiotic or antimicrobial drug resistance has been a major problem worldwide, with incidence of MRSA reported in healthcare facilities in Asia to reach its peak in late 1990s, and stayed at plateau level during 2000s [97]. The heavy usage of drugs in treatment hastens the selection of bacteria that harbor multidrug resistance genes particularly *S. aureus* to proliferate and dominate [98, 99]. Moreover, over-crowded community creates environment that is suitable for the rapid spread of numerous multidrug-resistant pathogens, particularly the

The emergence of multidrug-resistant *S. aureus* in both hospitals and community invokes a tremendous financial burden due to the persistence of hard-to-treat infections [97, 100–102]. Until present, it was reported that <90% of *S. aureus* strains are resistant to penicillin as well as ordinary antimicrobial agents such as drug from categories of aminoglycosides, ansamycins, anti-staphylococcal β-lactams (or cephamycins), chloramphenicols, fusidanases, fluoroquinolones, glycopeptides, lincosamides, macrolides, phenicols, and tetracyclines [103–105]. We are now observing the emergence of multidrug-resistant *S. aureus* and MDR-MRSA with broad spectrum of resistance with a distinct ability to survive and spread in the hospital environment,

There has been a dramatic increase in the incidence of nosocomial infections as well as community-associated MRSA and livestock-associated MRSA caused by strains of *S. aureus* that are resistant to multiple antibiotics [106]. At present, there have been reports that some strains demonstrate resistance to as many as 20 antimicrobial compound types, including antiseptics and disinfectants [107, 108]. Central Asian surveillance studies found that the prevalence of MRSA infection in tertiary hospital was reported in 10 among 1000 hospital admissions [109] and incidence reported previously in Japan was between 0.7 and 0.8 per 100 admission from 1999 to 2003 with a total rate among hospitalized patients in the Asia-Pacific region at 45.9% [110, 111]. Previous surveillance also reported that Asia is among the highest for the incidence of MRSA in the world, and interestingly a novel MRSA strain with glycopeptides resistance had spread in livestock animals making it as a potential human pathogen

Several studies attempted to profile all possible multidrug-resistant MRSA since 1987, encompassing samples from hospitals, community, as well as veterinary settings [113, 114]. Lim et al. (2013) carried out temporal comparative surveillance of antibiograms from clinical samples in 2003–2008 and showed a significant increase in resistance rates (from 1 to 96%), as well as multidrug-resistant phenotypes (96%). This study also indicated the prevalence of multidrug-resistant MRSA with SCC*mec* type III and ST239 [99]. Another study also reported

ST1625) [96].

**7. Multidrug-resistant (MDR) MRSA**

10 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

airborne organisms such as *S. aureus*.

community setting, as well as livestock sectors.

in this region [112].

*S. aureus* and MRSA evolve and adapt the changing environment. Therefore, dissemination of MRSA should be continuously monitored for the antibiotic susceptibility pattern and molecular epidemiology comprising hospital, community, and livestock settings. The origin and dissemination of SCC*mec* are also important to be tracked in the diverse staphylococcal population. With the advancement in molecular methods such as next-generation sequencing, the pattern of the genetic evolution, spread of the bacteria, and the resistance determinants can be further explored and understood.

## **Author details**

Abdul Rahim Abdul Rachman1 , Zarizal Suhaili1,2 and Mohd Nasir Mohd Desa1,3\*

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

2 School of Animal Sciences, Faculty of Bioresources and Food Industry, Universiti Sultan Zainal Abidin, Besut, Terengganu, Malaysia

3 Halal Products Research Institute, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

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#### **MRSA and MSSA: The Mechanism of Methicillin Resistance and the Influence of Methicillin Resistance on Biofilm Phenotype of** *Staphylococcus aureus* **MRSA and MSSA: The Mechanism of Methicillin Resistance and the Influence of Methicillin Resistance on Biofilm Phenotype of** *Staphylococcus aureus*

Sahra Kırmusaoğlu Sahra Kırmusaoğlu

Carlos CC, Perera JA, Chiu CH, Liu JW, Chongthaleong A, Thamlikitkul V, Van PH; ansorp study group. Spread of methicillin-resistant *Staphylococcus aureus* between the community and the hospitals in Asian countries: An ANSORP study. Journal of

Antimicrobial Chemotherapy, 2011; 66(5), 1061–1069. doi:10.1093/jac/dkr024

48.12.4926-4928.2004

24 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

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> Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/65452

#### **Abstract**

*Staphylococcus aureus* (*S. aureus*), which is one of the most common causes of indwelling device–associated, nosocomial, and community-acquired infections, can produce biofilm as a virulence factor. Methicillin-resistant *S. aureus* (MRSA) that is resistant to β-lactam antibiotics causes life-threatening infections. Biofilm producer strains of *S. aureus* that causes indwelling device–associated infections resist to antimicrobials and immune system. The combination of methicillin resistance and the ability of biofilm formation of *S. aureus* makes treatment difficult. Methicillin resistance of *S. aureus* can affect biofilm phenotype of *S. aureus*; the *mec*A gene of MRSA increases biofilm production by inactivating accessory gene regulator (agr) quorum sensing regulator system, which is a two-component regulator system of virulence factor production. The aim of this review is to determine virulence factors of *S. aureus*, resistance mechanisms of methicillin, and the influence of methicillin resistance on biofilm phenotype of *S. aureus*.

**Keywords:** *Staphylococcus aureus*, MRSA, MSSA, biofilm, methicillin resistance, virulence, influence of methicillin resistance on biofilm

## **1. Introduction**

The biofilm has an important role in the pathogenesis of certain bacterial infections such as staphylococcal indwelling device–associated infections, wound infections, chronic urinary tract infections (UTI), cystic fibrosis pneumonia, chronic otitis media (OM), chronic rhinosinusitis, periodontitis, and recurrent tonsillitis [1].

© 2017 The Author(s). Licensee InTech. 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. © 2017 The Author(s). Licensee InTech. 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 biofilm infections such as *Staphylococcus aureus* (*S. aureus*) infections are the important problems in hospitalized and immunosuppressed patients worldwide due to their tough and nonresponsive treatment by antibiotics. Biofilm-producing bacteria are resistant to immune defense, antibiotics, and many antimicrobial agents [2, 3].

The *mecA* gene, which is located in the staphylococcal chromosomes, enhances virulence of *Staphylococcus* by causing resistant to methicillin antibiotics. Methicillin-resistant *S. aureus* (MRSA) causes hospital-associated (HA-MRSA) and community-associated (CA-MRSA) infections. Methicillin resistance of *S. aureus* causes treatment of *S. aureus* tough by antibiotics due to its resistance to all β-lactam antibiotics. Mechanisms of resistance to β-lactam antibiotics such as methicillin are regulated by regulatory genes in the presence of such antibiotics. *S. aureus* biofilm formation is regulated globally by the accessory gene regulator (agr) quorum sensing system that is also inactivated by the *mec*A gene of MRSA [2–4]. The virulence of *S. aureus*, mechanisms of methicillin resistance, role of methicillin resistance on biofilm, and alteration of biofilm formation of *S. aureus* in methicillin resistance are discussed in this review.

## **2.** *Staphylococcus aureus* **and virulence**

#### **2.1. Staphylococcus aureus**

*S. aureus*, a Gram-positive coccus, produces catalase enzyme and coagulase enzyme, which coagulates blood by reacting with prothrombin, which converts fibrinogen to fibrin [5]. While *S. aureus* is a commensal bacterium and colonizes primary anterior nares of healthy staphylococcal nasal carrier individuals, *S. aureus* causes a wide range of infections such as skin infections, including abscesses, impetigo, and necrotizing fasciitis; tissue infections, including osteomyelitis and endocarditis; and toxinoses, including toxic shock syndrome, when immunity of the staphylococcal nasal carrier is suppressed [6, 7]. If MRSA is colonized in nares of healthy person, 29% potential risk appears for MRSA infections [8].

While antibiotics such as methicillin are used frequently in patients, antibiotic-resistant strains may develop. After penicillin usage had become widespread to treat infections, penicillinresistant *S. aureus* strains arose. Only a few years after following the usage of penicillin, penicillin-resistant *S. aureus* strains had arisen, and penicillinase-resistant methicillin usage had introduced for the treatment of penicillin-resistant *S. aureus* strains. After methicillin usage was introduced in 1961, MRSA strains that were also multidrug-resistant arose within a year. Methicillin that has been providing widespread of MRSA and becomes useless drug has not being used in recent years [7].

MRSA has become epidemic not only in nosocomial infections but also in communityassociated infections [9]. MRSA that has been a common cause of nosocomial infections worldwide also has been arising in the community in recent years [10]. Invasive infections of MRSA have high morbidity and mortality rates [11]. Most of invasive staphylococcal and community-acquired MRSA (CA-MRSA) infections are related to the nasal carriage of *Staphylococcus* [6].

## **2.2. Biofilm and pathogenesis**

The biofilm infections such as *Staphylococcus aureus* (*S. aureus*) infections are the important problems in hospitalized and immunosuppressed patients worldwide due to their tough and nonresponsive treatment by antibiotics. Biofilm-producing bacteria are resistant to immune

The *mecA* gene, which is located in the staphylococcal chromosomes, enhances virulence of *Staphylococcus* by causing resistant to methicillin antibiotics. Methicillin-resistant *S. aureus* (MRSA) causes hospital-associated (HA-MRSA) and community-associated (CA-MRSA) infections. Methicillin resistance of *S. aureus* causes treatment of *S. aureus* tough by antibiotics due to its resistance to all β-lactam antibiotics. Mechanisms of resistance to β-lactam antibiotics such as methicillin are regulated by regulatory genes in the presence of such antibiotics. *S. aureus* biofilm formation is regulated globally by the accessory gene regulator (agr) quorum sensing system that is also inactivated by the *mec*A gene of MRSA [2–4]. The virulence of *S. aureus*, mechanisms of methicillin resistance, role of methicillin resistance on biofilm, and alteration of biofilm formation of *S. aureus* in methicillin resistance are discussed

*S. aureus*, a Gram-positive coccus, produces catalase enzyme and coagulase enzyme, which coagulates blood by reacting with prothrombin, which converts fibrinogen to fibrin [5]. While *S. aureus* is a commensal bacterium and colonizes primary anterior nares of healthy staphylococcal nasal carrier individuals, *S. aureus* causes a wide range of infections such as skin infections, including abscesses, impetigo, and necrotizing fasciitis; tissue infections, including osteomyelitis and endocarditis; and toxinoses, including toxic shock syndrome, when immunity of the staphylococcal nasal carrier is suppressed [6, 7]. If MRSA is colonized in nares

While antibiotics such as methicillin are used frequently in patients, antibiotic-resistant strains may develop. After penicillin usage had become widespread to treat infections, penicillinresistant *S. aureus* strains arose. Only a few years after following the usage of penicillin, penicillin-resistant *S. aureus* strains had arisen, and penicillinase-resistant methicillin usage had introduced for the treatment of penicillin-resistant *S. aureus* strains. After methicillin usage was introduced in 1961, MRSA strains that were also multidrug-resistant arose within a year. Methicillin that has been providing widespread of MRSA and becomes useless drug has not

MRSA has become epidemic not only in nosocomial infections but also in communityassociated infections [9]. MRSA that has been a common cause of nosocomial infections worldwide also has been arising in the community in recent years [10]. Invasive infections of MRSA have high morbidity and mortality rates [11]. Most of invasive staphylococcal and community-acquired MRSA (CA-MRSA) infections are related to the nasal carriage of

of healthy person, 29% potential risk appears for MRSA infections [8].

defense, antibiotics, and many antimicrobial agents [2, 3].

26 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

**2.** *Staphylococcus aureus* **and virulence**

in this review.

**2.1. Staphylococcus aureus**

being used in recent years [7].

*Staphylococcus* [6].

Biofilm plays a role in the pathogenesis of staphylococcal infections. When microorganisms exposed to stress conditions, gene expression of biofilm is induced as a stress response. The biofilm that is a slime-like glycocalyx causes bacteria to survive in the stress conditions, causes bacterial attachment and colonization on biotic or abiotic surfaces such as prosthetic surfaces that may act as a substrate for microbial adhesion, and causes bacterial spread to whole body [12–14]. The biofilm producer *S. aureus* causes chronic infections such as indwelling devicerelated infections and chronic wound infections. Indwelling device-associated infections are mainly caused by biofilm producer *Staphylococci* including *S. aureus* and *Staphylococcus epidermidis*. The treatment of biofilm-embedded bacteria that are not eliminated completely by antimicrobials even at the high doses is tough and irresponsive. The patients whose indwelling device is infected by biofilm producers have higher risk of mortality. Infected implants that cannot be treated by antibiotics are removed out of the body to prevent biofilm-related infections [14].

## **2.3. Virulence of** *S. aureus* **Biofilm**

Biofilm that is a slime-like glycocalyx embedded sessile community of microorganism inside. Polysaccharide matrix, staphylococcal surface proteins, extracellular DNA (eDNA), and teichoic acids construct biofilm of *S. aureus* that is an extracellular polymeric substance. Surface proteins of *S. aureus* also contribute biofilm formation, whereas polysaccharide intracellular adhesin (PIA) is the main component of biofilm formation in *S. aureus*. Extracellular DNA (eDNA) that plays a role in resistance and channels that store antibiotic-degrading enzymes such as β-lactamases construct extracellular polysaccharide matrix [14].

## **3. Mechanisms of biofilm formation and regulation by MRSA and MSSA**

Biofilm is produced by distinct mechanisms in MRSA and Methicillin-sensitive Staphylococcus aureus (MSSA). Fitzpatrick et al. revealed that biofilm formation of the *icaADBC* operon deleted MRSA mutants was not affected, whereas biofilm formation of the *icaADBC* operon deleted MSSA mutants was impaired. This study showed that *ica*-independent biofilm formation is strain specific [15].

Biofilm is constructed not only by polysaccharide intracellular adhesin (PIA) but also by surface proteins. In the catheter infection, biofilm formation of clinical isolates of *S. aureus* of which *ica* operon is mutated is not reduced [13]. Biofilm of MSSA is formed in *ica*-dependent manner (PIA-dependent) by PIA that is encoded by *icaADBC* gene, whereas biofilm of MRSA is formed in *ica*-independent manner (PIA-independent) by surface proteins containing LPXTG anchoring domain that are anchored to peptidoglycan by sortase as a transpeptidase coded by *srtA* gene. Adherence to surfaces and intercellular aggregations of MSSA and MRSA cells are contributed by PIA in *ica*-dependent manner and surface proteins in *ica*-independent manner, respectively [4, 14]. Initial adherence of *S. aureus* to surfaces is contributed by Autolysin Atl that lyses cell causes release of eDNA and accumulation of cells in *ica*-independent manner [14, 16]. Especially, clinical MRSA adheres to polystyrene abiotic surfaces with Atl [16]. Intercellular accumulation of HA-MRSA and CA-MRSA is formed by FnBPA, FnBPB, Bap proteins, SasG, and protein A [4, 17, 18].

Three stages of *ica*-dependent and *ica*-independent biofilm formation that are adherence (adhesion, attachment), aggregation (maturation, accumulation), and detachment (dispersal) are regulated by *ica* operon and accessory gene regulator (agr) quorum sensing two-component signal transduction system, respectively [14].

Not only biofilm formation but also virulence factors such as phenol-soluble modulins (PSMs), toxins, and degradation enzymes production are regulated by agr quorum sensing twocomponent regulatory system [14, 19, 20]. Activation of *agr* system causes reduction in biofilm production due to the production of phenol-soluble modulins (PSMs) as surfactants, proteases, and nucleases that disperse microorganisms embedded in biofilm by enzymatic degradation of the biofilm matrix [14, 21, 22].

Accessory gene regulator (*agr*) system, which includes *agr* locus, regulates cell density, virulence, and biofilm formation of bacteria. RNAII and RNAIII are transcribed by binding of activated AgrA to P2 and P3 promoters in *agr* operon (*agr*BDCA), respectively. RNAII transcript that contains *agrB*, *D*, C, *A* genes encodes AgrB, D, C, A as a component of agr system, whereas RNAIII transcript that contains the *hld* gene encodes the δ-PSM (δ-phenol-soluble modulins or termed δ-hemolysin). RNAIII regulates virulence factors such as surface proteins that cause biofilm formation and exotoxins (RNAIII dependent control). In RNAIII independent control of *S. aureus*, synthesis of α-PSMs and β-PSMs is regulated by binding of AgrA to promoters of α-PSMs and β-PSMs in *psm* operon [14].

Supplementations of certain chemicals to growth media affect biofilm formation of *S. aureus* strains by regulating of gene expressions or breaking bonds that construct biofilm. Sodium chloride (NaCI) that induces expression of *ica* operon increases biofilm formation of MSSA [4, 23, 24]. Sodium metaperiodate that degrades polysaccharide bonds decreases biofilm formation of MSSA, whereas biofilms of MRSA are not affected. Proteinase A does not affect biofilm formation of MSSA [24, 4], whereas biofilm formation of MRSA is affected. pH of growth media that is decreased by glucose degradation represses agr regulator system. So, glucose supplementation of growth media that represses agr regulator system increases biofilm formation [4, 25]. Phenyl-methylsulfonyl fluoride (PMSF) that is a serine protease inhibitor increases biofilm formation by preventing *agr*-related biofilm detachment [21] and enhancing secretion of autolytic enzymes [26]. In early biofilm formation of HA-MRSA, biofilm formation is inhibited by polyanethole sodium sulfanate of which effect is not only preventing autolytic activity but also maintaining growth [16].

## **4.** *Staphylococcus aureus* **genome**

*Staphylococcus aureus* genome contains core genome, accessory component, and foreign genes. Core genome that constructs backbone of genome has main metabolic function. Core genome is highly conserved, and similarity of genes among isolates is ∼98–100%. Accessory component that constructs 25% of *S. aureus* genome contains mobile genetic elements (MGEs) such as transposons (Tn), chromosomal cassettes, pathogenicity islands (PIs), genomic islands, and prophages acquired horizontally between strains [5] (**Figure 1**). MGEs carry virulence genes that are acquired horizontally by other strains (bacterial horizontal gene transfer (HGT)) [7, 27].

**Figure 1.** Staphylococcal genome.

pendent manner [14, 16]. Especially, clinical MRSA adheres to polystyrene abiotic surfaces with Atl [16]. Intercellular accumulation of HA-MRSA and CA-MRSA is formed by FnBPA,

Three stages of *ica*-dependent and *ica*-independent biofilm formation that are adherence (adhesion, attachment), aggregation (maturation, accumulation), and detachment (dispersal) are regulated by *ica* operon and accessory gene regulator (agr) quorum sensing two-compo-

Not only biofilm formation but also virulence factors such as phenol-soluble modulins (PSMs), toxins, and degradation enzymes production are regulated by agr quorum sensing twocomponent regulatory system [14, 19, 20]. Activation of *agr* system causes reduction in biofilm production due to the production of phenol-soluble modulins (PSMs) as surfactants, proteases, and nucleases that disperse microorganisms embedded in biofilm by enzymatic degradation

Accessory gene regulator (*agr*) system, which includes *agr* locus, regulates cell density, virulence, and biofilm formation of bacteria. RNAII and RNAIII are transcribed by binding of activated AgrA to P2 and P3 promoters in *agr* operon (*agr*BDCA), respectively. RNAII transcript that contains *agrB*, *D*, C, *A* genes encodes AgrB, D, C, A as a component of agr system, whereas RNAIII transcript that contains the *hld* gene encodes the δ-PSM (δ-phenol-soluble modulins or termed δ-hemolysin). RNAIII regulates virulence factors such as surface proteins that cause biofilm formation and exotoxins (RNAIII dependent control). In RNAIII independent control of *S. aureus*, synthesis of α-PSMs and β-PSMs is regulated by binding of AgrA to

Supplementations of certain chemicals to growth media affect biofilm formation of *S. aureus* strains by regulating of gene expressions or breaking bonds that construct biofilm. Sodium chloride (NaCI) that induces expression of *ica* operon increases biofilm formation of MSSA [4, 23, 24]. Sodium metaperiodate that degrades polysaccharide bonds decreases biofilm formation of MSSA, whereas biofilms of MRSA are not affected. Proteinase A does not affect biofilm formation of MSSA [24, 4], whereas biofilm formation of MRSA is affected. pH of growth media that is decreased by glucose degradation represses agr regulator system. So, glucose supplementation of growth media that represses agr regulator system increases biofilm formation [4, 25]. Phenyl-methylsulfonyl fluoride (PMSF) that is a serine protease inhibitor increases biofilm formation by preventing *agr*-related biofilm detachment [21] and enhancing secretion of autolytic enzymes [26]. In early biofilm formation of HA-MRSA, biofilm formation is inhibited by polyanethole sodium sulfanate of which effect is not only preventing autolytic activity but

*Staphylococcus aureus* genome contains core genome, accessory component, and foreign genes. Core genome that constructs backbone of genome has main metabolic function. Core genome

FnBPB, Bap proteins, SasG, and protein A [4, 17, 18].

28 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

nent signal transduction system, respectively [14].

promoters of α-PSMs and β-PSMs in *psm* operon [14].

of the biofilm matrix [14, 21, 22].

also maintaining growth [16].

**4.** *Staphylococcus aureus* **genome**

Each strain of *S. aureus* has virulence varied according to having mobile genetic elements (MGEs) of which genes encode for varied virulence factors and toxins [9]. Genes of many secreted virulence factors such as exfoliative toxin A and B, superantigen toxins (SaPIs), toxic shock syndrome toxin (TSST), and enterotoxins are located in accessory genetic elements such as transposons, plasmids, prophages, and pathogenicity islands (PIs) that are also referred as MGEs, whereas genes that encode toxins such as α-toxins present in whole *S. aureus* strains and are located in core genome [7, 9]. Phenol-soluble modulins (PSMs) that are surfactant and encoded in core genome lyse immune cells such as neutrophils in inflammation and disperse biofilm [9, 14]. Cytolytic activity is present in shorter PSM-α type, while longer PSM-β type does not have cytolytic activity. It is seen that virulence of *S. aureus* is reduced by removing *psm-α* operon [7].

## **4.1. Prophages**

Prophages have an effective role in pathogenicity of *S. aureus* due to causing horizontal gene transfer (HGT) by transferring virulence genes of which products are staphylokinase, enterotoxin A, G, K, Panton-Valentine leukocidin (PVL), and exfoliative toxin [5].

## **4.2. Pathogenicity islands (PIs)**

The gene of superantigen toxins (SaPIs), which is one of the secreted virulence factors of *S. aureus*, is located in pathogenicity islands (PIs) that is also located in chromosome. SaPIs contain bacteriophage-associated genes that encode helicases and terminases involved in replication, integrases involved in integration, recombination and excition of MGE, and certain direct repeats [5, 28, 29].

The most known PI of *S. aureus* is SaPI1 that contains the *tst* gene encoding for TSST [7]. Highfrequency transduction of SaPI1 is mediated by encapsulating SaPI1 by staphylococcal phage 80α that is own phage of *S. aureus* to transfer its genes in transduction process. Enterotoxin B is encoded by SaPI3 that is one of the SaPIs and encapsulated by phage 29 that is phage of *S. aureus* to transfer its genes in transduction process [5, 28].

*S. aureus* not only carry SaPIs but also carry vSa family genomic islands that encode ∼50% of toxin and virulence factors of *S. aureus*. Conserved genes are present in this family. Among this family, vSa1 contains genes encoding for enterotoxin such as *seb*, *tsst*, and *ear,* whereas vSa2 contains genes encoding for enterotoxin such as *sec* and TSST (*tss*t). vSaα and vSaβ that are also present among vSa family genomic islands contain leukocidin genes [5].

#### **4.3. Insertion sequence (IS) and transposons (Tn)**

Insertion sequences (ISs) contain inverted repeats at their terminals and the integrases gene that causes transposition. Transposons (Tn) not only contain the transposase gene but also may contain ISs that induce movement of Tn and certain genes such as antibiotic resistance genes [5]. These elements provide a mechanism to transfer of virulence and resistance genes such as antibiotic resistance genes from place to place within the same cell or to other cell. These movable elements are excised from paired inverted repeats by transposase enzyme. While these elements are excised and inserted to new location such as within a gene that may be located within the same cell or other cell, the gene is disrupted [30].

#### **4.4. Plasmids**

Plasmids that are extrachromosomal genetic elements carry resistance genes causing antibiotic or heavy metal resistance, and virulence genes encoding for virulence factors, rather than genes involved in metabolic processes having vital functions [5]. There are three types of plasmids of *S. aureus* according to their size. Type I plasmids that are the smallest plasmids contain just one antibiotic-resistant determinant. Type II plasmids of which sizes are intermediate contain β-lactamase gene. The largest one is type III plasmids containing multiple resistant determinants such as gentamycin, trimethoprim, and ethidium bromide resistance [31]. Conjugative plasmids that are also type III plasmids are transferred horizontally to other cell by their own *tra* genes [5].

#### **4.5. SCCmec**

MGEs contain the *mecA* gene causing methicillin and other β-lactam resistance and occur in chromosome of methicillin-resistant *Staphylococcus* such as MRSA, and methicillin-resistant *S. epidermidis* MRSE is called staphylococcal cassette chromosome *mec* (*SCCmec*) [7, 9]. Inverted repeats that are localized at both terminals of *SCCmec* are the recognizing sequences for *SCCmec*-specific recombinase in the processes of excision of *SCCmec* from chromosome and integration of *SCCmec* to either other parts of chromosome or chromosome of other strain (**Figure 1**) [32].

*SCCmec* is composed of variable and conserved genetic elements. *SCCmec* carries *mec* operon that contains *mecA,* and regulatory genes such as *mecI* and *mecRI*, and cassette chromosome recombinase genes *ccrA*, *ccrB*, and *ccrC* that are localized in *ccr* locus and contribute excision from *SCCmec* and integration to chromosome. All these elements are highly conserved among *Staphylococcus*. J-region that is a variable region of *SCCmec* composed of genetic elements integrated such as ISs, Tns, and plasmids. In addition to methicillin resistance that is caused by *mecA* in a strain, if these integrated elements include additional genes encoding for antibiotic resistance, rather than methicillin, multiple resistance arises in this strain [5]. Just a year later on the first usage of methicillin for treatment of MRSA, clinical MRSA isolates that have multiple resistant to antibiotics were reported [33].

The most known PI of *S. aureus* is SaPI1 that contains the *tst* gene encoding for TSST [7]. Highfrequency transduction of SaPI1 is mediated by encapsulating SaPI1 by staphylococcal phage 80α that is own phage of *S. aureus* to transfer its genes in transduction process. Enterotoxin B is encoded by SaPI3 that is one of the SaPIs and encapsulated by phage 29 that is phage of *S.*

*S. aureus* not only carry SaPIs but also carry vSa family genomic islands that encode ∼50% of toxin and virulence factors of *S. aureus*. Conserved genes are present in this family. Among this family, vSa1 contains genes encoding for enterotoxin such as *seb*, *tsst*, and *ear,* whereas vSa2 contains genes encoding for enterotoxin such as *sec* and TSST (*tss*t). vSaα and vSaβ that are

Insertion sequences (ISs) contain inverted repeats at their terminals and the integrases gene that causes transposition. Transposons (Tn) not only contain the transposase gene but also may contain ISs that induce movement of Tn and certain genes such as antibiotic resistance genes [5]. These elements provide a mechanism to transfer of virulence and resistance genes such as antibiotic resistance genes from place to place within the same cell or to other cell. These movable elements are excised from paired inverted repeats by transposase enzyme. While these elements are excised and inserted to new location such as within a gene that may be

Plasmids that are extrachromosomal genetic elements carry resistance genes causing antibiotic or heavy metal resistance, and virulence genes encoding for virulence factors, rather than genes involved in metabolic processes having vital functions [5]. There are three types of plasmids of *S. aureus* according to their size. Type I plasmids that are the smallest plasmids contain just one antibiotic-resistant determinant. Type II plasmids of which sizes are intermediate contain β-lactamase gene. The largest one is type III plasmids containing multiple resistant determinants such as gentamycin, trimethoprim, and ethidium bromide resistance [31]. Conjugative plasmids that are also type III plasmids are transferred horizontally to other cell by their own

MGEs contain the *mecA* gene causing methicillin and other β-lactam resistance and occur in chromosome of methicillin-resistant *Staphylococcus* such as MRSA, and methicillin-resistant *S. epidermidis* MRSE is called staphylococcal cassette chromosome *mec* (*SCCmec*) [7, 9]. Inverted repeats that are localized at both terminals of *SCCmec* are the recognizing sequences for *SCCmec*-specific recombinase in the processes of excision of *SCCmec* from chromosome and integration of *SCCmec* to either other parts of chromosome or chromosome of other strain

also present among vSa family genomic islands contain leukocidin genes [5].

located within the same cell or other cell, the gene is disrupted [30].

*aureus* to transfer its genes in transduction process [5, 28].

30 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

**4.3. Insertion sequence (IS) and transposons (Tn)**

**4.4. Plasmids**

*tra* genes [5].

**4.5. SCCmec**

(**Figure 1**) [32].

Variants of *mec* operon that are located in *SCCmec* are present according to whether *mecI* and *mecRI* genes are intact or having deletions. The variants of *mec* complex are class A, B, C, D, and E *mec*. IS431 that is related to the *mecA* gene are present in all *mec* operon classes. All classes except A consist of deleted portions that are happened in *mecI* and may run through to a portion of *mecRI* gene. Eight types of *SCCmec* were found according to having combinations of distinct variants of *mec* and *ccr* [5]. Multidrug-resistant strains have *SCCmec* type II and III that contain additional resistance genes. While MRSA is characterized by containing *SCCmec* type I or III and II in recent years, CA-MRSA strains are characterized by containing *SCCmec* type IV. Other *SCCmec* types are seen in strains very rare [7].

Methicillin-resistant strains of *Staphylococcus* have *mec* operon, whereas methicillin-sensitive strains of *Staphylococcus* do not have *mec* operon [34]. HGT of *mecA* from one to another strain is proved by researchers; the *mecA* gene of MRSA and *mecA* homolog of *Staphylococcus sciuri* revealed 88% identity. But *S. sciuri* containing *mecA* homolog is susceptible to methicillin. This supported that MRSA strains are descendents of ancestral strains in evolutionary process [35, 36]. *Staphylococcus haemolyticus* (*S. haemolyticus*) genome carries intact IS*1272* element, whereas the gene of *S. aureus* and *S. epidermidis* carries IS*1272* element deleted [37]. This revealed that horizontal gene transfer (HGT) is happened by acquisition of IS1272 from *S. haemolyticus* to *S. aureus* and *S. epidermidis*. HGT of *mecA* that is happened from *S. epidermidis* to *S. aureus* causes arising of MRSA during treatment with antibiotic [38]; *mecA* is transferred to methicillin-resistant *Staphylococcus* by the way that having inverted repeats at terminals of the *mec* gene complex and IS*431* of which location is especially within gene complexes encoding various resistance factors such as the *mec* gene complex.

Methicillin resistance is not only seen in isolates of *S. aureus* but also seen more common in isolates of *S. epidermidis*. Approximately, 70% of whole hospital-acquired methicillin-resistant *Staphylococcus* isolates is S. *epidermidis* [5].

## **5. The relationship between methicillin resistance and biofilm formation**

The association between methicillin resistance and biofilm phenotype is taken attention according to studies executed [39–41]. Researchers determined that biofilm formation of HA- MRSA BH1CC strain is decreased by removing *SCCmec* that results up-regulation of protease activity [4, 42, 43].

Biofilm formation of MRSA is enhanced by both phenol-soluble modulin mec (PSMmec) encoded by *psm-mec* and penicillin-binding protein 2a (PBP2a) encoded by *mecA* that also repress virulence of MRSA [42].

## **5.1. psm-mec**

*SCCmec* not only contains genes encoding methicillin resistance and recombination but also contains genes encoding other antibiotics and heavy metal resistance; the *psm-mec* gene that is located near *mecA* in *SCCmec* especially type II, III, and VIII encodes PSMmec peptide. The PSMmec that is a cytolysin is the only staphylococcal toxin of which the gene is colocated with antibiotic-resistant determinant in MGEs of *S. aureus* rather than core genome; the *psm-mec* gene is conserved region of class A *mec* gene complex (**Figure 1**) [9].

Like many virulence toxins of *S. aureus* such as α-toxin and other PSMs, expression of PSMmec is also regulated by Agr two-component signal transport system [14, 44]. Many virulence toxins are regulated by RNAIII-dependent manner, whereas other PSMs and *psm-mec* are regulated by RNAIII independent manner (**Figure 2**) [14].

**Figure 2.** Mechanisms of *PSMmec*, PBP2a, and β-lactamase regulations. (a) Up-regulation of *psm-mec* by RNAIII independent Agr regulator system. (b) Repression of *mecA* and *blaZ* genes: In lack of β-lactams no transcription occurs. (c) Induction of genes: PBP2a and β-lactamase are transcribed by expression of *mecA* and *blaZ* in the presence of β-lactams, respectively.

Biofilm formation is increased by the repression of Agr system that downregulates *psm-mec* in MRSA [4] and PSMs [14]; the *psm-mec* gene of MRSA also has pleiotropic effect by changing biofilm phenotype and regulation of *psm* gene, decreasing toxin production, and the way decreasing virulence, and the *psm-mec* gene that is up-regulated by Agr regulator system promotes biofilm formation of MRSA by reducing expression of PSMα toxin that is encoded in chromosome. As a result of reduced expression of PSMα toxin, virulence of *S. aureus* is reduced by PSMmec (**Figure 3**) [45, 46].

**Figure 3.** The effect of *mecA* and *psm-mec* induction of Staphylococcus aureus on biofilm formation and virulence.

Biofilm formation (adherence to surfaces and intercellular aggregations) of MSSA and MRSA strains is contributed by PIA in *ica*-dependent manner and surface proteins in *ica*-independent manner, respectively [4, 14]. Interestingly, in spite of the biofilm of MSSA that is formed by *ica*-independent manner is not seen or seen less prevalent, the *psm-mec* gene of type II *SCCmec* enhances expression of Atl and FnBPA in MSSA isolates [42].

## **5.2. mecA**

MRSA BH1CC strain is decreased by removing *SCCmec* that results up-regulation of protease

Biofilm formation of MRSA is enhanced by both phenol-soluble modulin mec (PSMmec) encoded by *psm-mec* and penicillin-binding protein 2a (PBP2a) encoded by *mecA* that also

*SCCmec* not only contains genes encoding methicillin resistance and recombination but also contains genes encoding other antibiotics and heavy metal resistance; the *psm-mec* gene that is located near *mecA* in *SCCmec* especially type II, III, and VIII encodes PSMmec peptide. The PSMmec that is a cytolysin is the only staphylococcal toxin of which the gene is colocated with antibiotic-resistant determinant in MGEs of *S. aureus* rather than core genome; the *psm-mec*

Like many virulence toxins of *S. aureus* such as α-toxin and other PSMs, expression of PSMmec is also regulated by Agr two-component signal transport system [14, 44]. Many virulence toxins are regulated by RNAIII-dependent manner, whereas other PSMs and *psm-mec* are regulated

**Figure 2.** Mechanisms of *PSMmec*, PBP2a, and β-lactamase regulations. (a) Up-regulation of *psm-mec* by RNAIII independent Agr regulator system. (b) Repression of *mecA* and *blaZ* genes: In lack of β-lactams no transcription occurs. (c) Induction of genes: PBP2a and β-lactamase are transcribed by expression of *mecA* and *blaZ* in the presence of β-lac-

Biofilm formation is increased by the repression of Agr system that downregulates *psm-mec* in MRSA [4] and PSMs [14]; the *psm-mec* gene of MRSA also has pleiotropic effect by changing biofilm phenotype and regulation of *psm* gene, decreasing toxin production, and the way

gene is conserved region of class A *mec* gene complex (**Figure 1**) [9].

by RNAIII independent manner (**Figure 2**) [14].

activity [4, 42, 43].

**5.1. psm-mec**

tams, respectively.

repress virulence of MRSA [42].

32 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

Agr system is repressed by expression of PBP2a that is encoded by *mecA,* as a result of oxacillin usage [10]. PSMs are downregulated, proteases and virulence are decreased, and PBP2a promoted biofilm formation enhanced by repressed Agr regulator system. In contrary to this, *ica*-dependent biofilm formation is decreased by *ica* that is repressed by PBP2a (**Figure 3**) [42].

## **6. β-Lactam, methicillin, and multidrug resistance**

## **6.1. Peptidoglycan biosynthesis of** *S. aureus*

Peptidoglycan, surface proteins such as protein A, clumping factor A, fibronectin-binding protein (FnBP), collagen-binding protein, and teichoic acids construct the cell wall of *S. aureus*. Peptidoglycan is constructed by polypeptides containing L-alanine, D-glutamic acid, L-lysin and D-alanine, respectively, and glycan polysaccharide strands [5].

At the beginning of peptidoglycan synthesis, UDP-*N*-acetylmuramyl-pentapeptide (UDP-NAM-pentapeptide) and UDP-*N*-acetylglucosamine (UDP-NAG) that are nucleotide sugarlinked precursors are synthesized in cytoplasm of *S. aureus*. Pentapeptide with the sequence of L-alanine, D-glutamic acid, L-lysin, D-alanine, and D-alanine, respectively, is linked to NAM in cytoplasm. Then, bactoprenol that is a membrane-bound lipophilic acceptor transfers UDP-NAM-pentapeptide and UDP-NAG that are hydrophilic precursors from cytoplasm to the outer surface of cell membrane, respectively [5].

Then, transglycosylation and transpeptidation reactions are catalyzed by penicillin-binding proteins (PBPs) of which 4 types (PBP1, PBP2, PBP3, PBP4) are present in *S. aureus* [5]. PBPs that are DD-peptidases are bound to membrane [36]. *N*-acetylglucosamines (NAG) and *N*acetylmuramic acids (NAM) that are bound by β(1-4) glycosidic bond catalyzed by PBPs in transglycosylation process construct glycan strands that form backbone of peptidoglycan. Transglycosylation reaction is catalyzed by PBPs, especially penicillin-binding protein 2 (PBP2) and glycosyltransferase Mtg. In transpeptidation reaction that is catalyzed by PBPs, L-lysine that is the amino acid of polypeptide linked to NAM of one glycan strand is cross-linked to Dalanine that is the amino acid of polypeptide linked to NAM of other glycan strand by pentaglycine cross bridge synthesized by family of FemABX non-ribosomal peptide. The last D-alanine of pentapeptide of UDP-NAM-pentapeptide is cleaved during transpeptidation reaction that cross-links peptidoglycan (**Figure 4**) [5].

**Figure 4.** Biosynthesis of staphylococcal peptidoglycan. Peptidoglycan is constructed by transglycosylation and transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs).

Teichoic acids that are polymers of glycerol phosphate or ribitol residues give negative feature to cell membrane and act as receptor of *S. aureus* phage [5].

## **6.2. Effect of β-lactam antibiotics against cell wall**

At the beginning of peptidoglycan synthesis, UDP-*N*-acetylmuramyl-pentapeptide (UDP-NAM-pentapeptide) and UDP-*N*-acetylglucosamine (UDP-NAG) that are nucleotide sugarlinked precursors are synthesized in cytoplasm of *S. aureus*. Pentapeptide with the sequence of L-alanine, D-glutamic acid, L-lysin, D-alanine, and D-alanine, respectively, is linked to NAM in cytoplasm. Then, bactoprenol that is a membrane-bound lipophilic acceptor transfers UDP-NAM-pentapeptide and UDP-NAG that are hydrophilic precursors from cytoplasm to the

Then, transglycosylation and transpeptidation reactions are catalyzed by penicillin-binding proteins (PBPs) of which 4 types (PBP1, PBP2, PBP3, PBP4) are present in *S. aureus* [5]. PBPs that are DD-peptidases are bound to membrane [36]. *N*-acetylglucosamines (NAG) and *N*acetylmuramic acids (NAM) that are bound by β(1-4) glycosidic bond catalyzed by PBPs in transglycosylation process construct glycan strands that form backbone of peptidoglycan. Transglycosylation reaction is catalyzed by PBPs, especially penicillin-binding protein 2 (PBP2) and glycosyltransferase Mtg. In transpeptidation reaction that is catalyzed by PBPs, L-lysine that is the amino acid of polypeptide linked to NAM of one glycan strand is cross-linked to Dalanine that is the amino acid of polypeptide linked to NAM of other glycan strand by pentaglycine cross bridge synthesized by family of FemABX non-ribosomal peptide. The last D-alanine of pentapeptide of UDP-NAM-pentapeptide is cleaved during transpeptidation

**Figure 4.** Biosynthesis of staphylococcal peptidoglycan. Peptidoglycan is constructed by transglycosylation and trans-

Teichoic acids that are polymers of glycerol phosphate or ribitol residues give negative feature

outer surface of cell membrane, respectively [5].

34 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

reaction that cross-links peptidoglycan (**Figure 4**) [5].

peptidation reactions catalyzed by penicillin-binding proteins (PBPs).

to cell membrane and act as receptor of *S. aureus* phage [5].

Binding of β-lactams to PBPs that have high affinity to β-lactams is lethal for *Staphylococcus* [36]. Transpeptidase domain of PBPs in peptidoglycan is inactivated by β-lactam agents such as penicillins, cephalosporins, and methicillin and oxacillin that are both penicillinaseinsensitive β-lactams acting as substrate of PBPs, rather than D-alanyl-D-alanine. Before enzyme substrate complex of β-lactam and PBP is formed completely, they can be dissociated by disrupting noncovalent association between them at the beginning of this complex. Later on, irreversible complex is formed by covalently binding of β-lactam that is a structural analog of D-alanyl-D-alanine substrate of PBP to active site of PBP complex that is the site for the binding of D-alanyl-D-alanine as a substrate during transpeptidation reaction (**Figure 4**). By this way, transpeptidation reaction that is the last step of peptidoglycan biosynthesis is blocked by β-lactam antibiotics that inactivate PBP. *Staphylococcus* undergoes to death due to the inhibition of peptidoglycan biosynthesis [5, 47].

## **6.3. Mechanism of β-lactam resistance of** *Staphylococcus aureus*

β-lactamase enzymes cause resistance of cell to β-lactam antibiotics by inactivating β-lactam antibiotics. β-lactamase inactivates β-lactam antibiotics by disrupting amide bond of βlactam ring [5].

Expression of the *bla*Z gene that is located in plasmid or transposon and encodes β-lactamase is regulated by *bla*I and *bla*RI that are own regulators. In the lack of β-lactam antibiotic, BlaI that bound to promoter-operator region repress *blaZ* gene, *bla*I-*bla*RI operon, so transcription of *bla*Z is not happen. In the usage in treatment or supplementation of β-lactam antibiotic to growth media, β-lactam binds to BlaRI that is a β-lactam-sensing signal transducer, and then, intracellular zinc metalloprotease domain of BlaRI is separated and cleaves BlaI that is already bound to operator. By this way, in the presence of β-lactam, *blaZ* is transcribed to β-lactamase that permits MRSA to grow by inactivating β-lactam (**Figure 2**) [5].

A study that showed the association between the antibiotic susceptibility patterns and the antibiotic resistance genes in staphylococcal isolates obtained from various clinical samples of patients revealed that 93.5% of *S. aureus* clinical isolates and 86.8% coagulase negative *Staphylococci* strains carry the blaZ gene [48].

#### **6.4. Mechanism of methicillin resistance of** *Staphylococcus aureus*

Resistance to methicillin, oxacillin, and nafcillin that are semisynthetic β-lactamase-insensitive β-lactams has developed by acquiring of the *mecA* gene [5]. MRSA is not only resistant to methicillin, but also resistant to all β-lactams [5, 36].

*mec*A gene expression is regulated by *mec*I and *mec*RI that are own regulators. In the lack of β-lactam antibiotic, MecI that bound to promoter-operator region repress *mecA*, and *mec*I*mec*RI operon, so transcription of *mec*A is not happen. In the usage or supplementation of βlactam antibiotic to growth media, β-lactam binds to MecRI that is a β-lactam-sensing signal transducer, and then, metallo-protease domain of MecRI that is placed in cytoplasmic site is separated and cleaves MecI that is already bound to operator. By this way, *mecA* is transcribed to PBP2a of which affinity is low to β-lactams [49]. Low affinity of PBP2a to β-lactams permits MRSA to grow as a result of peptidoglycan synthesis in the presence of β-lactams concentrations that can inactivate transpeptidase activity of PBPs. PBP2a that belongs to PBPs contains transpeptidase domain and non-penicillin-binding protein (**Figure 2**) [5].

Structure, function, mechanism, and molecular organization of *mec*I and *mec*RI are similar to *bla*I and *bla*RI, respectively [36]. Expression of *mecA* is regulated by both MecI and BlaI. When MecI and BlaI are both present at the same time, *mec*A is repressed even stronger; the *mec*I gene of most of the clinical MRSA isolates has deletions; due to this, expression of *mec*A is regulated by BlaI [5].

#### **6.5. Multidrug resistance**

There are eight types of *SCCmec* (I-VIII). *SCCmec* type II and III that demonstrate multiresistance also contain additional antibiotic resistance genes such as erythromycin and tetracycline as well as methicillin. *SCCmec* type IV that is essential for community-acquired MRSA strains (CA-MRSA) that is one of the virulent strains and infect healthy person in community rather than hospital arised. The other types of *SCCmec* are rare [7].

IS*431* is mainly found in chromosome and plasmids of *Staphylococcus* and is also related to encoding various resistance factors such as tetracycline, mercury, and cadmium resistance. If other additional resistance genes such as *aadD* encoding an enzyme for tobramycin resistance are integrated within *SCCmec* cassette (IS*431mec*), multiple drug resistance is developed in methicillin-resistant *Staphylococcus* [36]. Plasmids pUB110, pI258, and pT181 integrated in *SCCmec* have additional resistance genes encoding kanamycin, tobramycin and bleomycin resistance (*ant(4′)*), penicillin and heavy metal resistance, and tetracyclin resistance, respectively. Tn554 integrated in *SCCmec* have additional resistance gene *erm*A encoding inducible macrolide, lincosamide, and streptogramin resistance [49].

#### **6.6. Homogeneous and heterogeneous resistance of MRSA**

Heterogeneity is a characteristic of MRSA of which resistance level varies according to contents and ingredients of culture medium in which MRSA is grown and β-lactam antibiotic used. Most of the cells of heterogeneous methicillin resistance (HeR) strains (∼99.9% or above) are susceptible to β-lactam of which concentration is low that is about 1–5 μg/mL of methicillin, whereas just a few subpopulations (such as 1 in 106 cfu/mL) grow in 50 μg/mL or above of methicillin by expressing high-level resistance. Homogeneous strains (HoR) are resistant to low concentration of β-lactam and can grow in higher concentrations of methicillin that is about 5 μg/mL or above [36].

Heterogeneity of MRSA is unstable and changeable according to growth conditions. HeR strains become homogeneous strains (HoR) by growth media supplemented with NaCI or sucrose for providing hypertonicity of media, or supplemented with higher concentrations of β-lactam antibiotic, or incubated at 30°C in incubator. Supplementation of growth media with EDTA or incubation at 37–43°C leads to conversion of HoR strains to HeR [36]. This conversion of HeR and HoR in distinct culture conditions is due to the regulation of gene expression by Agr regulator system [42]. These conversions of MRSA can be repeated by repeated culturing in changed media that have different supplementations.

Most clinical isolates of MRSA grow as HeR in routine growth conditions, and most of them show low or moderate level of resistance, whereas a few subpopulations show high-level resistance [36].

## **Author details**

separated and cleaves MecI that is already bound to operator. By this way, *mecA* is transcribed to PBP2a of which affinity is low to β-lactams [49]. Low affinity of PBP2a to β-lactams permits MRSA to grow as a result of peptidoglycan synthesis in the presence of β-lactams concentrations that can inactivate transpeptidase activity of PBPs. PBP2a that belongs to PBPs contains

Structure, function, mechanism, and molecular organization of *mec*I and *mec*RI are similar to *bla*I and *bla*RI, respectively [36]. Expression of *mecA* is regulated by both MecI and BlaI. When MecI and BlaI are both present at the same time, *mec*A is repressed even stronger; the *mec*I gene of most of the clinical MRSA isolates has deletions; due to this, expression of *mec*A is regulated

There are eight types of *SCCmec* (I-VIII). *SCCmec* type II and III that demonstrate multiresistance also contain additional antibiotic resistance genes such as erythromycin and tetracycline as well as methicillin. *SCCmec* type IV that is essential for community-acquired MRSA strains (CA-MRSA) that is one of the virulent strains and infect healthy person in

IS*431* is mainly found in chromosome and plasmids of *Staphylococcus* and is also related to encoding various resistance factors such as tetracycline, mercury, and cadmium resistance. If other additional resistance genes such as *aadD* encoding an enzyme for tobramycin resistance are integrated within *SCCmec* cassette (IS*431mec*), multiple drug resistance is developed in methicillin-resistant *Staphylococcus* [36]. Plasmids pUB110, pI258, and pT181 integrated in *SCCmec* have additional resistance genes encoding kanamycin, tobramycin and bleomycin resistance (*ant(4′)*), penicillin and heavy metal resistance, and tetracyclin resistance, respectively. Tn554 integrated in *SCCmec* have additional resistance gene *erm*A encoding inducible

Heterogeneity is a characteristic of MRSA of which resistance level varies according to contents and ingredients of culture medium in which MRSA is grown and β-lactam antibiotic used. Most of the cells of heterogeneous methicillin resistance (HeR) strains (∼99.9% or above) are susceptible to β-lactam of which concentration is low that is about 1–5 μg/mL of methicillin,

methicillin by expressing high-level resistance. Homogeneous strains (HoR) are resistant to low concentration of β-lactam and can grow in higher concentrations of methicillin that is

Heterogeneity of MRSA is unstable and changeable according to growth conditions. HeR strains become homogeneous strains (HoR) by growth media supplemented with NaCI or sucrose for providing hypertonicity of media, or supplemented with higher concentrations of β-lactam antibiotic, or incubated at 30°C in incubator. Supplementation of growth media with EDTA or incubation at 37–43°C leads to conversion of HoR strains to HeR [36]. This conversion

cfu/mL) grow in 50 μg/mL or above of

community rather than hospital arised. The other types of *SCCmec* are rare [7].

macrolide, lincosamide, and streptogramin resistance [49].

**6.6. Homogeneous and heterogeneous resistance of MRSA**

whereas just a few subpopulations (such as 1 in 106

about 5 μg/mL or above [36].

transpeptidase domain and non-penicillin-binding protein (**Figure 2**) [5].

36 The Rise of Virulence and Antibiotic Resistance in The Rise of Virulence and Antibiotic Resistance in Staphylococcus aureus *Staphylococcus aureus*

by BlaI [5].

**6.5. Multidrug resistance**

Sahra Kırmusaoğlu

Address all correspondence to<EMAIL_ADDRESS>
Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, T.C. Haliç University, Istanbul, Turkey

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#### **Chapter 3 Provisional chapter**

#### **Glycopeptide Resistance in** *S. aureus* **Glycopeptide Resistance in** *S. aureus*

#### Hasan Cenk Mirza Hasan Cenk Mirza