Epidemiology and Pathogenesis

### **Chapter 3**

## The Molecular Epidemiological Study of MRSA in Mexico

*Miguel Ángel Ortíz Gil and Monica Irasu Cardona Alvarado*

#### **Abstract**

The rapid spread of infections by methicillin-resistant *Staphylococcus aureus* (MRSA) emerged in the early 1960s, and this pathogen is one of the most common agents of nosocomial infections. As a reaction to the appearance and spread of multi-drug-resistant MRSA in Mexico, some hospitals have established molecular epidemiological surveillance, where pandemic clones of MRSA have been detected in different states in the north, the center, and the south of Mexico. The pandemic clones detected in Mexico are the Iberian, the New York/Japan, the pediatric, the EMRSA-16, and the USA-300. The surveillance or evolutionary studies carried out in Mexico, using different molecular methodologies, have shown a predominance of the New York/Japan clone, which has even displaced other MRSA clones. Therefore, it is necessary to continue establishing molecular surveillance and diagnostic programs as a special management for the confirmed MRSA infections, if these measures are not carried out to understand and control the changing lineages of MRSA, in the future, it may become an important public health problem, since the New York/Japan clone, which is the most predominant in our country, clearly demonstrates its great capacity for geographical expansion, multi-resistance, and virulence.

**Keywords:** *Staphylococcus aureus*, MRSA, clones, CA-MRSA, HA-MRSA, Mexico

### **1. Introduction**

In Mexico, la Red Hospitalaria de Vigilancia Epidemiologica (RHVE) reported that mortality rates among patients infected with *S. aureus* show a variability between 5 and 70%, in addition to high attributable mortality rates, approximately 50% [1]. *S. aureus* produces a wide variety of exoproteins that contribute to its ability to colonize and cause disease in humans [2]. MRSA strains are characterized by the presence of a mobile genetic element called the staphylococcal cassette cromosoma *mec (SCCmec*), which includes the *mecA* gene [3]. The structural *mecA* gene codes for penicillin-binding protein (PBP) 2a, which determines resistance to methicillin [4]. Modifications in PBP2a prevent PBP-penicillin binding, causing cell wall synthesis to proceed normally [5].

Nosocomial infections (NI) are considered a public health problem worldwide. For example, in the Latin American region, the SENTRY Surveillance Antimicrobial Program reported an increase in the proportion of MRSA in medical centers from

33.8% in 1997 to 40.2% in 2006. In Mexico, some studies show an increase in the prevalence of MRSA in recent years, and the incidence of NI ranges between 3.8 and 26.1 cases per 100 discharges; mortality associated with nosocomial infections is an average of 5%, and in 2001, it was the seventh leading cause of death for the general population in 2001 [6]. Reports from the Pan American Health Organization (PAHO) for Mexico informed that there was a prevalence of 52% of MRSA in 2004, while the Pan American Association of Infectious Diseases reported 32% in 2006, and data from the study of the TEST program (Tigecycline Evaluation and Surveillance Trial) showed a prevalence of 48% of MRSA in 2008 [7].

### **2. The molecular epidemiology of MRSA**

Monitoring and stopping the intra- and inter-hospital distribution of MRSA clones require the use of efficient and accurate epidemiological typing systems that allow discrimination between unrelated isolates and recognition of isolates that descend from a common ancestor (i.e., that belong to the same clone). Currently, multiple phenotypic and genotypic typing methods have been developed to type MRSA. The choice of a typing method depends on the needs, the level of skills, the resources of the laboratory, and the type of question to be answered (short-term or long-term analysis) [8].

On the other hand, the molecular epidemiological study of MRSA aims to determine the clonal relationship that exists between several isolates of the same species. This information is very useful, especially when epidemic outbreaks caused by multi-resistant strains occur because it makes it possible to determine the number of circulating clones, evaluate the effectiveness of control measures aimed at preventing their spread, and differentiate between infection and reinfection [9]. Identification of MRSA clones is based on a combination of different typing methods, such as DNA hybridization with *mecA* and Tn554 probes, PFGE, RAPD, *SCCmec typing*, *spa typing*, and *MLST* [10].

## **3. International MRSA clones**

At the present time, it has been shown that multiple clonal lineages of MRSA exist because of the successful horizontal transfer of *mecA* [11]. Six types of HA-MRSA hospital-acquired pandemic clones have been reported (Iberian, Brazilian, Hungarian, New York/Japan, Pediatric, and EMRSA-16), and they are scattered in different regions of the world [12]. The Iberian clone was the first one to be identified in 1989, in a massive outbreak of MRSA in a hospital in Barcelona, Spain [13]; but it seems to have already been present in Belgium and France at least since 1984 [14]. The Brazilian clone is widely distributed in Brazilian hospitals and has spread to neighboring countries in South America: Argentina, Uruguay and Chile and in Europe: Portugal, the Czech Republic, and Greece, where it displaced the main local clones [15]. The Hungarian clone has been widely disseminated in Hungarian hospitals since 1993 [16]. The New York/Japan clone was identified as the main clone in different regions in the United States of America [17], and in a hospital in Tokyo [18]. EMRSA-16 clone was found in the United Kingdom hospitals [19]. This clone has been widely spread in Greece, Mexico, and Canada [20]. The Pediatric clone was reported in Portugal in 1991 and since then, it has been found in Poland, France, the United Kingdom, the United States (EU), Argentina, and Colombia [21].

#### *The Molecular Epidemiological Study of MRSA in Mexico DOI: http://dx.doi.org/10.5772/intechopen.107411*

Reports of community-acquired methicillin-resistant *S. aureus* (CA-MRSA) infections in healthy people began to appear in the 1990s. In 2000, it was described that these types of strains were genetically different from bacteria isolated in hospital settings. Currently, CA-MRSA strain types possess more bacterial resistance genes, and more virulence factors, leading to more severe infections [22]. There are multiple clones of CA-MRSA worldwide, as well as a segregation of these clones based on the MLST technique. For example, USA300 (ST8), which predominates in the USA, USA400 (STI), USA1100 (ST30), and USA1000 (ST59), is clone notable for causing CA-MRSA infections. STI and ST30 are the cause of the main CA-MRSA infections in Australia and Oceania, while clone ST80 predominates in Europe [23].

The MRSA clones were once confined to hospitals for the last 20 years. MRSA infections have emerged in the community in people with no previous exposure to hospitals. Genotypically, CA-MRSA is a newer and more virulent strain, emerging in the late 1990s as leading the cause of skin and soft tissue infections in young healthy

**Figure 1.**

*Main differences between HA-MRSA and CA-MRSA strains detected in Mexico. (Modified by Bustos et al. [25]).*

people. CA-MRSA strains typically carry *SCCmec types IV* or *V* and are generally susceptible to β-lactam antimicrobials. In addition, CA-MRSA carries Panton-Valentine Leukocidin (PVL), which is associated with increased pathogenicity. In relationship to HA-MRSA clones, they carry *SCCmec types I*, II, or III and do not have PVLs. HA-MRSA clones are associated with nosocomial infections, for example, endocarditis, urinary tract infections, and surgical infections, and are resistant to β-lactam antibiotics, especially aminoglycosides, macrolides, lincosamides, and fluoroquinolones. Although CA-MRSA has been predicted to replace HA-MRSA in hospitals, mathematical models predict the coexistence between the two strains given hospital-community interactions [24].

The molecular epidemiological study of MRSA clones has been insufficient in Mexico since there is not a systematized surveillance system, where the appearance or distribution of these clones is reported, monitored, and controlled. There have been few studies in Mexico over the years and they have randomly detected HA-MRSA and CA-MRSA clones in different states of the country, which have described their main phenotypic and genotypic characteristics, which are observed in **Figure 1**.

#### **4. MRSA clones in Mexico**

In Mexico, The Instituto Nacional de Salud Pública in México has been confirmed as a network of tertiary hospitals, which have carried out studies aimed at understanding the molecular epidemiology of MRSA [26] and it is coordinated by Dra. Velázquez-Meza and et al. Studies carried out between 1997 and 2003 at the Hospital de Pediatría del Centro Médico Nacional (CMN), Siglo XXI-IMSS (Mexican Institute of Social Security) in Mexico City, 659 strains of *S. aureus* were analyzed, with a variation in the prevalence of MRSA from 17 to 23% until 2001. It subsequently decreased drastically to a prevalence of 4% in 2002, which was due to nosocomial infection control measures. During this investigation, the presence of the clone EMRSA-16 (*SCCmec type IV*) was detected, and the clone New York/Japan (SCCmec *type II*) was introduced into the hospital in 2001, which completely displaced clone EMRSA-16 in 2002 [27].

At the Hospital Civil de Guadalajara, "Fray Antonio Alcalde" between 1999 and 2003, 839 strains of MRSA were isolated from adult and pediatric patients. A total of 216 MRSA strains showed antimicrobial resistance to β-lactams, macrolides, chloramphenicol, and imipenem, and sensitivity to gentamicin, rifampicin, trimethoprimsulfamethoxazole, and vancomycin. The New York/Japan clone was also detected in the 216 MRSA strains studied, like the one found in the Hospital de Pediatría del CMN-Sigloi XXI [28]. The New York/Japan clone may have been transferred from the United States to Mexico.

At the Instituto de Cardiología "Dr. Ignacio Chavez" (ICh), located in Mexico City, which is a 246-bed tertiary teaching hospital between 2002 and 2009, 90 MRSA strains were collected from bronchial secretions, wound secretions, blood, catheter, pleural fluid, peritoneal fluid, and others, from pediatric and adult populations. MRSA isolates were resistant to amoxicillin, cefotaxime, cephalothin, cefazolin, chloramphenicol, imipenem, clindamycin, erythromycin, clarithromycin, penicillin, and oxacillin, while only 94.4% of isolates were also resistant to ciprofloxacin. The New York/Japan clone, which was isolated from a variety of sites of infection, was identified in 50% of MRSA isolates. The studies showed that the New York/Japan clone had *SCCmec type II*. EMRSA-16 was found in 2002 and it presented *SCCmec IV*, and this chromosomal cassette is related to CA-MRSA clones [29].

In the north of Mexico, an investigation was carried out to identify MRSA responsible for nosocomial infection in five medical centers in Monterrey, Nuevo León (NL) Mexico, between 2005 and 2009, and 190 strains of MRSA were isolated from five hospitals affiliated to the Mexican Institute of Social Security. This study clearly documented the high dissemination capacity and persistence of the New York/Japan clone in these centers [30].

At the Hospital San José Tec de Monterrey, Nuevo León, Mexico, the first five cases of a clone of community MRSA were described in 2008, and three of the patients were children. The first patient with a history of retinoblastoma in the left eye was diagnosed in November 2007, when he just started chemotherapy. In 2008, he returned to the hospital with a fever for 2 weeks of evolution. Blood cultures showed MRSA and vancomycin was started for 1 week. Two other children who were considered as healthy ones previously arrived at the hospital with abscesses and with a severe local reaction from where MRSA was isolated. After drainage, both were treated with clindamycin. Two other patients who were considered healthy adults previously had abscesses and because of it, they required hospitalization. The drainage of the lesions showed MRSA in the culture and the patients were treated with linezolid. All patients recovered. This study revealed that the pattern was similar to that observed for the CA-MRSA clone USA300 genotype [31].

On the other hand, at the Hospital Universitario Dr. José Eleuterio González, in Monterrey, between 2012 and 2013, a prophylactic protocol was carried out that consisted of applying a solution with chlorhexidine gluconate (CXG), throughout his body, with the aim of reducing nosocomial infections, where 158 strains of MRSA were collected. During these CXG washouts, antibiotic resistance significantly decreased for clindamycin, levofloxacin, and norfloxacin. During the pre-intervention period, 65.7% of the isolates were resistant to oxacillin, and in the post-intervention period, this percentage was reduced to 32.6%. This result indicates a significant reduction in the frequency of MRSA isolates as a result of the lavage with CXG. The presence of two clones descending from clone ST5-MRSA-II (New York/Japan) and clone ST8-MRSA-IV (USA300) was evidenced. The New York/Japan clone decreased significantly in the intervention period but recovered in the post-intervention period, while the USA300 clone was established under pressure from CXG [32].

Although in Monterrey, Mexico, the first clone of community origin of MRSA with a history in previously hospitalized patients was identified. In another study carried out in 2013 at the Universidad Autónoma Metropolitana-Xochimilco, in which healthy volunteers from schools and factories in Mexico City were recruited, to whom nasal or throat sampling was applied, a total of 131 strains of MRSA are obtained from 1039 strains of *S. aureus*. Considerable diversity in PFGE patterns in CA-MRSA isolates was observed in clonal analysis, allowing only a small number of clones to be detected: USA300 and USA100. This study provides the first description of CA-MRSA in healthy people in Mexico City, suggesting that community MRSA clones could replace hospital MRSA clones in the future [33].

At the Hospital de Oncología (INCAN), a tertiary care hospital in Mexico City in 2006 and later in 2010, the New York/Japan clone was isolated as the cause of an outbreak of nosocomial infections that arose from an index case [34, 35].

In relationship to MRSA clones in Veracruz, Veracruz, Mexico, at the Hospital Regional de Alta Especialidad in Veracruz (HRV) in 2010, the presence of two pandemic clones was identified, the New York/Japan clone (ST5-*SCCmec type II*) and the Iberian clone (ST247-*SCCmec type IA*). The IB1 clonal subtype was isolated from the emergency department in a patient with an ear infection, who stated that he had

traveled to the USA, and on his return, he presented the infection, making it probable that the Iberian clone arrived from the USA to the HRV. The strains with IB2 and IB3 patterns were later isolated in two other patients from the same hospital medical service, which reveals the introduction of this clone from an external service to a critical area of the HRV [ 36 ]. Although the New York/Japan clone has been previously identified in other hospitals in Mexico [ 12 , 13 , 31 ], this clearly demonstrates its great capacity for geographical expansion, multi-resistance, and virulence. The importance of this finding lies in the fact that the first strain of MRSA resistant to vancomycin with a minimum inhibitory concentration (MIC) of 1024 μg/ml belongs to the New York/Japan lineage [ 37 , 38 ].

 **Figure 2.**  *Distribution of MRSA clones in Mexico.* 

#### *The Molecular Epidemiological Study of MRSA in Mexico DOI: http://dx.doi.org/10.5772/intechopen.107411*

At the Hospital General "Dr. Manuel Gea González," located in the southern zone of Mexico City, from 2011 to 2012, 109 strains of MRSA were isolated from wound secretions, soft tissues, blood cultures, cerebrospinal fluid, pleural fluid, bone, etc., of hospitalized patients. The most prevalent infection was ventilator-associated pneumonia. The isolated strains were characterized by resistance to β-lactams. A single predominant clone named New York/Japan (NY) was identified [39].

A prospective observational cohort study was carried out and 24 hospitals in Latin America participated from 2011 to 2014 and collected 1346 strains of *S. aureus*. The Hospital Civil de Guadalajara, Fray Antonio Alcalde de Guadalajara, Mexico, participated, and 18% of the MRSA isolates in this hospital showed the typical pattern of USA300, suggesting that this strain is likely circulating in Mexico [40].

In a cross-sectional study carried out at the Hospital Central Dr. Ignacio Morones Prieto, in San Luis Potosí, Mexico, from 2017 to 2018, a total of 191 isolates of *S. aureus* were obtained from different patients in all wards of the hospital, in the pediatric and adult population, coming from the emergency services, surgery, intensive care unit, internal medicine, gynecology, burn unit, and outpatient service. Clinical samples were obtained from skin and soft tissue infections, respiratory tract, blood, bones and joints, and the cerebrospinal fluid. A total of 77% of the strains were considered as coming to the hospital and 23% were classified as community ones. The most frequent *S. aureus* infections were those that affected the skin, soft tissues, and bacteremia. Instead, the type of infection more frequent cause by isolates of MRSA was the infection of the surgical site. The presence of clones ST5-MRSA-II-t895 (clone New York/Japan) and ST1011-MRSA-II-t9364 (clone New York/Japan) was evidenced by the PFGE technique. In addition to the clone mentioned above, the presence of endemic clones of MRSA was evidenced, such as USA300, Irish and Pediatric, these being the ones with the highest prevalence [41].

As seen in previous studies in Mexico, the predominant clone is the New York/ Japan [42], which has the ability to spread, cause outbreaks and replace existing clones [43], and this is due, among other things, to its great virulence, since it presents staphylococcal enterotoxins and it also possesses the toxic shock syndrome toxin 1, which enables it to cause a wide variety of clinical syndromes, including toxic shock syndrome and suppurative infections [44]. In addition to this, it is resistant to β-lactams and a wide range of antibiotics [15].

The epidemiological study of MRSA clones acquired in hospitals is an area of little study, which does not allow knowing exactly the behavior or evolution of MRSA pandemic clones, as shown in the following **Figure 2**, which compiles the reported clones in Mexico.

### **5. Conclusions**

It is necessary to promote and encourage the molecular epidemiological surveillance of HA-MRSA and CA-MRSA clones, to prevent and control this pathogen, which causes outbreaks and high mortality rates in Mexico, due to hospital or community infections. Attention shoulder be paid to the detection, surveillance, and control of CA-MRSA due to the increase in the non-hospitalized population, which could displace HA-MRSA and become a health problem.

The molecular epidemiological surveillance of MRSA clones is essential knowledge for its prevention, control, and possible eradication. This type of research allows the nosocomial infection control committees of each institution to be informed. This

in sum would help to strengthen measures, such as the restriction of prescription of broad-spectrum antibiotics, daily supervision of cultures and results, monthly reports of infections, training aimed at health workers in general, and strengthening of medical practices.

In Mexico, the predominant clone is New York/Japan, which has the ability to spread, cause outbreaks and replace existing clones, this is due, among other things, to its great virulence and antimicrobial multi-resistance. The importance of this clone lies in the fact that the first strain of MRSA resistant to vancomycin belongs to the New York/Japan lineage. Vancomycin is considered one of the latest therapeutic alternatives against infections caused by MRSA and other gram-positive microorganisms.

## **Author details**

Miguel Ángel Ortíz Gil\* and Monica Irasu Cardona Alvarado Department of Medical Science, Division of Health Sciences, Leon Campus, University of Guanajuato, Leon, México

\*Address all correspondence to: maortiz@ugto.mx

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Selvey LA, Whitby M, Johnson B. Nosocomial methicillin-resistant Staphylococcus aureus bacteremia: Is it any worse than nosocomial methicillinsensitive Staphylococcus aureus bacteremia? Infection Control and Hospital Epidemiology. 2000;**21**(10): 645-648. DOI: 10.1086/501707

[2] Dinges MM, Orwin PM, Schlievert PM. Exotoxins of Staphylococcus aureus. Clinical Microbiology Reviews. 2000;**13**(1):16-34. DOI: 10.1128/cmr.13.1.16

[3] Milheiriço C, Oliveira DC, de Lencastre H. Update to the multiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy. 2007;**51**(9):3374-3377. DOI: 10.1128/AAC.00275-07

[4] Chambers HF. Methicillin resistance in staphylococci: Molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews. 1997;**10**(4):781-791. DOI: 10.1128/CMR.10.4.781

[5] Chambers HF. Methicillin-resistant staphylococci. Clinical Microbiology Reviews. 1988;**1**(2):173-186. DOI: 10.1128/CMR.1.2.173

[6] López IKR, Hernández JBBD, Rosas DOP, Camacho MV, Ruiz ÉMF, Novales MGM. Bacterial resistance in isolates from patients with nosocomial infections. Enfermedades Infecciosas y Microbiología. 2007;**27**(1):15-21

[7] Guzmán BM, Mejía C, Isturiz R, Alvarez C, Bavestrello L, Gotuzzo E, et al. Epidemiology of methicillinresistant Staphylococcus aureus (MRSA) in Latin America. International Journal of Antimicrobial Agents. 2009;**34**:304-308

[8] Struelens MJ. Consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems. Clinical Microbiology and Infection. 1996;**2**(1):2-11. DOI: 10.1111/j.1469- 0691.1996.tb00193.x

[9] Fernández-Cuenca F. Aplicaciones de las técnicas de PCR a la epidemiología molecular de las enfermedades infecciosas. Enfermedades Infecciosas y Microbiología Clínica. 2004;**22**(6): 355-360. DOI: 10.1157/13063748

[10] Castellano GM, Perozo MA, Vivas VR, Ginestre PM, Rincón VG. Tipificación molecular y fenotípica de Staphylococcus aureus resistentes a meticilina (SAMR) en un hospital universitario. Revista Chilena de Infectología. 2009;**26**:39-48

[11] Fluit AC, Schmitz FJ. MRSA: Current Perspectives. EEUU: Edit. Horizon Scientific Press; 2003

[12] Lencastre H, Oliveira D, Tomasz A. Antibiotic resistant Staphylococcus aureus: A paradigm of adaptive power. Current Opinion in Microbiology. 2007;**10**:428-435

[13] Oliveira DC, Tomasz A, De Lencastre H. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: Identication of two ancestral genetic backgrounds and the associated mec elements. Microbial Drug Resistance. 2001;**7**:349-361

[14] Deplano A, Witte W, van Leeuwen WJ, Brun Y, Struelens MJ. Clonal dissemination of epidemic methicillin-resistant Staphylococcus aureus in Belgium and neighboring countries. Clinical Microbiology and Infection. 2000;**6**(5):239-245. DOI: 10.1046/j.1469-0691.2000. 00064.x

[15] Teixeira LA, Resende CA, Ormonde LR, Rosenbaum R, Figueiredo AM, de Lencastre H, et al. Geographic spread of epidemic multiresistant Staphylococcus aureus clone in Brazil. Journal of Clinical Microbiology. 1995;**33**(9):2400-2404. DOI: 10.1128/jcm.33.9.2400-2404.1995

[16] Aires de Sousa M, Crisóstomo MI, Sanches IS, Wu JS, Fuzhong J, Tomasz A, et al. Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China. Journal of Clinical Microbiology. 2003;**41**(1):159-163. DOI: 10.1128/ JCM.41.1.159-163.2003

[17] Simor AE, Ofner-Agostini M, Bryce E, Mcgeer A, Paton S, Mulvey MR. Laboratory characterization of methicillin resistant Staphylococcus aureus in Canadian hospitals: Results of 5 years of National Surveillance, 1995-1999. The Journal of Infectious Diseases. 2002;**186**:652-660

[18] Aires de Sousa M, de Lencastre H, Santos Sanches I, Kikuchi K, Totsuka K, Tomasz A. Similarity of antibiotic resistance patterns and molecular typing properties of methicillin-resistant Staphylococcus aureus isolates widely spread in hospitals in new York City and in a hospital in Tokyo, Japan. Microbial Drug Resistance. 2000 Autumn;**6**(3):253-258. DOI: 10.1089/mdr.2000.6.253

[19] Moore PC, Lindsay JA. Molecular characterization of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and

EMRSA-16. Journal of Medical Microbiology. 2002;**51**:516-521

[20] Murchan S, Kaufmann ME, Deplano A, de Ryck R, Struelens M, Zinn CE, et al. Harmonization of pulsed field gel electrophoresis for epidemiological typing of methicillin-resistant Staphylococcus aureus by consensus in 10 European centers and its use to plot the spread of related strains. Journal of Clinical Microbiology. 2003;**41**:1574-1585

[21] Aires de Sousa M, de Lencastre H. Bridges from hospitals to the laboratory: Genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunology and Medical Microbiology. 2004;**40**(2):101-111. DOI: 10.1016/ s0928-8244(03)00370-5

[22] Castañeda-Méndez PF. Frecuencia de infecciones por S. aureus en pacientes hospitalizados en un hospital privado de tercer nivel de la Ciudad de México. Revista Médica MD. 2018;**9**:317-321

[23] García EC, González RG, Schettino PMS. Staphylococcus aureus asociado a la comunidad. Revista Mexicana de Patología Clínica y Medicina de Laboratorio. 2015;**62**:100-111

[24] Kateete DP et al. CA-MRSA and HA-MRSA coexist in community and hospital settings in Uganda. Antimicrobial Resistance & Infection Control. 2019;**8**(1):1-9

[25] Bustos J, Hamdan A, Gutiérrez M. Staphylococcus aureus: la reemergencia de un patógeno en la comunidad. Revista Biomedica. 2006;**17**(4):287-305

[26] Aires De Sousa M, Miragaia M, Sanches IS, Avila S, Adamson I, Casagrande ST, et al. Threeyear assessment of methicillin-resistant Staphylococcus aureus clones in

*The Molecular Epidemiological Study of MRSA in Mexico DOI: http://dx.doi.org/10.5772/intechopen.107411*

Latin America from 1996 to 1998. Journal of Clinical Microbiology. 2001;**39**(6):2197-2205. DOI: 10.1128/ JCM.39.6.2197-2205.2001

[27] Velazquez-Meza ME, Aires de Sousa M, Echaniz-Aviles G, Solórzano-Santos F, Miranda-Novales G, Silva-Sanchez J, et al. Surveillance of methicillin-resistant Staphylococcus aureus in a pediatric hospital in Mexico City during a 7-year period (1997 to 2003): Clonal evolution and impact of infection control. Journal of Clinical Microbiology. 2004;**42**(8):3877-3880. DOI: 10.1128/JCM.42.8.3877-3880.2004

[28] Echániz-Aviles G, Velázquez-Meza ME, Aires-de-Sousa M, Morfín-Otero R, Rodríguez-Noriega E, Carnalla-Barajas N, et al. Molecular characterisation of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999-2003). Clinical Microbiology and Infection. 2006;**12**(1):22-28. DOI: 10.1111/j. 1469-0691.2005.01283.x

[29] Elena V-MM et al. Pulsed field gel electrophoresis in molecular typing and epidemiological detection of methicillin resistant Staphylococcus aureus (MRSA). Gel Electrophoresis–Advanced Techniques. 2012;**10**:179

[30] Velazquez-Meza ME, Hernández-Salgado M, Contreras-Cordero JF, Pérez-Cortes P, Villarreal-Treviño L. Surveillance of methicillin-resistant staphylococcus aureus causing nosocomial infections in five medical centers of Monterrey, Nuevo León, México from 2005-2009. Archives of Medical Research. 2013;**44**(7):570-574. DOI: 10.1016/j.arcmed.2013.09.001

[31] Velazquez-Meza ME, Ayala-Gaytán J, Carnalla-Barajas MN, Soto-Noguerón A, Guajardo-Lara CE, Echaniz-Aviles G.

First report of community-associated methicillin-resistant Staphylococcus aureus (USA300) in Mexico. Journal of Clinical Microbiology. 2011;**49**(8):3099- 3100. DOI: 10.1128/JCM.00533-11

[32] Velázquez-Meza ME, Mendoza-Olazarán S, Echániz-Aviles G, Camacho-Ortiz A, Martínez-Reséndez MF, Valero-Moreno V, et al. Chlorhexidine whole-body washing of patients reduces methicillin-resistant Staphylococcus aureus and has a direct effect on the distribution of the ST5- MRSA-II (New York/Japan) clone. Journal of Medical Microbiology. 2017;**66**(6):721-728. DOI: 10.1099/ jmm.0.000487

[33] Hamdan-Partida A, Sainz-Espuñes T, Bustos-Martínez J. Isolation of community-acquired methicillinresistant Staphylococcus aureus in healthy carriers in a Mexican community. International Journal of Infectious Diseases. 2014;**18**:22-26. DOI: 10.1016/j. ijid.2013.08.010

[34] Cornejo-Juárez P, Velásquez-Acosta C, Díaz-González A, Volkow-Fernández P. Tendencia del perfil de sensibilidad antimicrobiana de los aislamientos de sangre en un hospital oncológico (1998-2003). Salud Pública de México. 2005;**47**(4):288-293. DOI: 10.1590/s0036-36342005000400006

[35] Cornejo-Juárez P, Volkow-Fernández P, Sifuentes-Osornio J, Echániz-Aviles G, Díaz-Gonzalez A, Velázquez-Acosta C, et al. Tracing the source of an outbreak of methicillinresistant Staphylococcus aureus in a tertiary-care oncology hospital by epidemiology and molecular methods. Microbial Drug Resistance. 2010;**16**(3):203- 208. DOI: 10.1089/mdr.2010.0048

[36] Ortíz-Gil MÁ, Velazquez-Meza ME, Echániz-Aviles G, Mora-Domínguez JP,

Carnalla-Barajas MN, Mendiola Del Moral EL. Tracking methicillin-resistant Staphylococcus aureus clones in a hospital in southern Mexico. Salud Pública de México. 2020;**62**(2):186-191. DOI: 10.21149/10786

[37] McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillinresistant Staphylococcus aureus isolates from the United States: Establishing a national database. Journal of Clinical Microbiology. 2003;**41**(11):5113-5120. DOI: 10.1128/JCM.41.11.5113-5120.2003

[38] Weigel LM, Clewell DB, Gill SR, Clark NC, McDougal LK, Flannagan SE, et al. Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science. 2003;**302**(5650):1569-1571. DOI: 10.1126/ science.1090956

[39] Gasca S, De H, Meza V, Avilés E, Castro V, Moreno F. Caracterización molecular de cepas de S. aureus meticilino resistentes (MRSA) aisladas en pacientes del Hospital General "Dr. Manuel Gea González". [Tesis doctoral]. Instituto de Salud Pública: Cuernavaca, Morelos; 2013. https://catalogoinsp.mx/ files/tes/053411.pdf

[40] Arias CA, Reyes J, Carvajal LP, Rincon S, Diaz L, Panesso D, et al. A prospective cohort multicenter study of molecular epidemiology and phylogenomics of Staphylococcus aureus bacteremia in nine Latin American countries. Antimicrobial Agents and Chemotherapy. 2017;**61**(10):e00816-17. DOI: 10.1128/aac.00816-17

[41] Negrete-González C, Turrubiartes-Martínez E, Galicia-Cruz OG, Noyola DE, Martínez-Aguilar G, Pérez-González LF, et al. High prevalence of t895 and t9364

spa types of methicillin-resistant Staphylococcus aureus in a tertiary-care hospital in Mexico: Different lineages of clonal complex 5. BMC Microbiology. 2020;**20**(1):213. DOI: 10.1186/ s12866-020-01881-w

[42] Coombs GW, Van Gessel H, Pearson JC, Godsell M-R, O'Brien FG, Christiansen KJ. Controlling a multicenter outbreak involving the New York/Japan methicillin-resistant Staphylococcus aureus clone. Infection Control and Hospital Epidemiology. 2007;**28**(7):845-852. DOI: 10.1086/518726

[43] Orii KO, Iwao Y, Higuchi W, Takano T, Yamamoto T. Molecular characterization of methicillin-resistant Staphylococcus aureus from a fatal case of necrotizing fasciitis in an extremely low-birth-weight infant. Clinical Microbiology and Infection. 2010;**16**(3):289-292. DOI: 10.1111/j. 1469-0691.2009.02806.x

[44] Durand G, Bes M, Meugnier H, Enright MC, Forey F, Liassine N, et al. Detection of new methicillinresistant Staphylococcus aureus clones containing the toxic shock syndrome toxin 1 gene responsible for hospitaland community-acquired infections in France. Journal of Clinical Microbiology. 2006;**44**(3):847-853

## **Chapter 4**

## Main Factors of *Staphylococcus aureus* Associated with the Interaction to the Cells for Their Colonization and Persistence

*Samuel González-García, Aída Hamdan-Partida, Juan José Valdez-Alarcón, Anaid Bustos-Hamdan and Jaime Bustos-Martínez*

### **Abstract**

*Staphylococcus aureus* is a microorganism that can colonize the nose, pharynx, and other regions of the body. It has also been observed that it can cause persistence. Successful colonization of *S. aureus* depends in the factors that favor the interaction of the bacteria with host cells. The bacterial determinants of *S. aureus* that have the capacity to adhere to human tissues involve adhesion factors such as teichoic acids and cell-wall-anchored proteins (CWA) such as ClfA, IcaA, SdrC, FnBPA, among others. The colonization and persistence process first involve adhesion to the tissue, followed by its reproduction and the possible formation of a biofilm. This review will describe the main virulence factors that allow bacterial adhesion and biofilm formation, including the accessory gene regulator genes (*agr*), related to colonization and persistence of *S. aureus*.

**Keywords:** *S. aureus*, colonization, persistence, adhesins, biofilm, virulence factors, regulation, *agr*

### **1. Introduction**

*Staphylococcus aureus* is a versatile pathogen that can cause infections in several mammal species including human. This is possible because several genetic variants have been associated with the host and the type of infection [1]. *S. aureus* can form a normal part of the human microbiota or act as an opportunistic pathogenic bacterium that produces a wide range of diseases that can be acquired in the hospital or in the community [2].

Several studies of colonization of *S. aureus* in the nose show that it can persist, following three patterns of carriers in the population. Around 20% of people are persistent carriers, around 30% are intermittent carriers, and non-carriers are on average 50% [3]. It has been reported that persistent carriers usually present a single strain over time, shed the bacteria in the environment, and they can be infected more than intermittent carriers and non-carriers. Intermittent carriers may have different strains over time and less colonization [4].

In the adults *S. aureus* can be found apart from the nose at other sites in the body: pharynx (4–64%), abdomen (15%), armpits (8%), intestines (17–31%), perineum (22%), and vagina (5%) [5–7].

Bacterial adhesion to the skin or mucous membranes is usually the initial and fundamental step in colonization and persistence, with the subsequent possibility of producing infections and pathological processes in the host. By attaching, bacteria can also bypass the innate response, allowing access to nutrients, colonization, and possibly subsequent persistence, which is favored by biofilm formation, toxin production, cell invasion, and evasion mechanisms of the immune response [8].

#### **2. Colonization factors of** *S. aureus*

Colonization with *S. aureus* requires direct human contact or contact with contaminated fomites. But this does not guarantee colonization, and some people remain as non-carriers [9]. Once colonizing, permanence is an important trail in persistent infections. Therefore, it is required to study the factors involved in colonization and persistence.

#### **2.1 Initial** *S. aureus* **interaction**

Colonization begins by the interaction of the bacteria with the cells of the host. *S. aureus* has many adhesins that allow it to first adhere to the human cell, multiply, and even persist in the tissue. Next, several components of the bacterium that intervene in the interaction with the host are reviewed.

#### *2.1.1 Wall teichoic acids (WTA)*

Reversible binding of *S. aureus* to host cells is through wall teichoic acids (WTAs) and/or receptor-mediated protein interactions [10]. The surface of *S. aureus* is composed of polysaccharides, such as capsular polysaccharides (PC) and also by WTA*.* Two types of acids have been described: lipoteichoic acids (LTA), which are found in the cytoplasmic membrane, and teichoic acids (WTAs), which are bound to peptidoglycan in the cell wall [11, 12]. WTAs are found on the surface of the cell wall, which are polyanionic cell wall glycopolymers (CWGs). They are made up of approximately 40 repeat units of ribitolphosphate linked with D-alanine and N-acetylglucosamine, which are covalently linked to peptidoglycan [13, 14].

WTAs have been shown to participate in the adhesion and colonization of staphylococci [14, 15], also participate in cell division, as well as in the formation of biofilms, an elevated expression increases the virulence of *S. aureus* [16]. It has also been seen that the D-alanine residues of the WTA participate in resistance against antimicrobial peptides (defensins or cathelicidins), in addition to participating in the resistance of some antibiotics such as teicoplanin or vancomycin [11, 17]. The biosynthesis of these biopolymers in *S. aureus* is mediated by N-acetylglucosaminyltransferase (Tar) enzymes [18].

Weidenmaier et al. [19], using a *S. aureus* model for nasal colonization in cotton rats, found that the proteinaceous adhesins of the bacterium act mainly during the

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

later stages of colonization, while the non-proteinaceous WTA-type adhesin acts in the early stages. This is due to the high expression of the WTA *tagO* and *tarK* genes during the first and last stages of colonization, while the expression of adhesin proteins such as clumping factor B (ClfB) increases in the early stages and decreases in the later stages of colonization [14, 20–22]. Therefore, WTA is not required for the *in vitro* growth of *S. aureus*; however, it is required for establishing infection in animals [18].

The action of WTA in the initial interaction of *S. aureus* to a surface is through non-covalent surface charge interactions (WTA is a polyanionic molecule), with various associated polymeric proteins in the cell membrane (recently its interaction with the Scavenger receptor SREC-1) having been demonstrated, which allows its adhesion to the structural molecules of the cell matrix such as fibronectin, fibrinogen, collagen, etc. [23, 24].

#### *2.1.2 S. aureus cell wall-anchored (CWA) proteins*

*S. aureus* has been shown to produce some 25 different cell-wall-anchored (CWA) proteins, linked to peptidoglycan via transpeptidases. These CWA may function in adhesion, biofilm formation, invasion, and evasion of host immune responses [25].

Five groups have been proposed to classify *S. aureus* CWA proteins (**Table 1**). Where there are many microbial surface components recognizing adhesive matrix molecules (MSCRAMM), including fibronectin-binding proteins (FnBPA and FnBPB), proteins of the Serine-Aspartate repeat family (SdrC, SdrD, and SdrE), clumping factors (ClfA and ClfB), Protein A (Spa), iron-regulated surface determinants (IsdA, IsdB, IsdC, and IsdH), plasmin-responsive protein (Pls), *S. aureus* surface protein G (SasG), and bone sialoprotein-binding protein (Bbp). All of these proteins participate in the initial interaction with the host cell through cell adhesion and/or biofilm formation [26].

#### *2.1.2.1 Microbial surface components recognizing adhesive matrix molecules (MSCRAMM) used to attach to cells*

*S. aureus* reversibly or irreversibly binds to the cell surface via MSCRAMM proteins [25, 27]. During infection, these proteins allow bacteria to bind to host receptors. These proteins are made up of three parts: a binding domain, a domain that spans the entire cell wall, and a third part on the bacterial surface that serves for non-covalent binding of MSCRAMM proteins to the host cell [25, 26].

The main binding factors of *S. aureus* (**Table 1**) are reviewed below.

#### *2.1.2.1.1 Clumping factor B (ClfB)*

*S. aureus* binds to nostrils during colonization via clumping factor B (ClfB) by highly affine binding to the cornified cell envelope, mainly due to the fibrinogen binding mechanism, which is an important step in colonization by *S. aureus* [28, 29], as well as *in vitro* biofilm formation [30]. Therefore, the union of ClfB with fibrinogen promotes nasal colonization. ClfB expression occurs mainly in the early phase of bacterial exponential growth and is de-expressed in the late growth phase and stationary phase [31]. Most strains of *S. aureus* have the *clfB* gene [21, 32, 33]. The ClfB protein exhibits sequence variations depending on *S. aureus* clonal complexes, but protein variants have about 94% amino acid identity with each other [34].


*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*


*ECM: extracellular matrix; DLL: dock, lock and latch. The shaded rows belong to the main ligands of S. aureus to the host (modified from Foster et al. [25]).*

#### **Table 1.**

*Main cell-wall-anchored (CWA) proteins of Staphylococcus aureus.*

ClfB also binds to cytokeratin 10, in addition to binding fibrinogen, cytokeratin 10 is one of the main components of the interior of squamous cells. ClfB also binds loricrin, one of the most abundant protein in the cornified envelope of squamous cells, and is key in the colonization of *S. aureus* in the nose and human skin [28, 29, 31, 34, 35]. Initially, it was found that the ClfB protein binds to fibrinogen, it can undergo the proteolytic action of the *S. aureus* metalloprotease aureolysin [36]. ClfB protein in digested form cannot bind fibrinogen, but can bind cytokeratin 10 with good affinity. At the ligand level, ClfB interacts with the amino acid sequence Y[GS]nY found in the carboxyl-terminal of cytokeratin 10 [37].

The ClfB binding is carried out using the so-called dock, lock, and latch (DLL) mechanism, where a short peptide of cytokeratin 10 or loricrin binds the N2 and N3 domains of the ClfB protein [38, 39].

#### *2.1.2.1.2 Serine-aspartate repeats (SdrC and SdrD) proteins*

Within the MSCRAMM is the subfamily of serine-aspartate repeat (Sdr) proteins, which have an R region that presents repeats of the serine-aspartate dipeptide and is located in the sdr locus [38, 40]. In *S. aureus*, three members of Sdr are known, and they are SdrC, SdrD, and SdrE, which share a conserved structure [38].

Askarian et al. [41] reported that SdrD is required for survival of *S. aureus* within the host, giving it the ability to evade some processes of the innate immune response, particularly by inactivating the complement system through the lectin pathway. On the other hand, SdrE functions in the recognition of complement binding protein C4b.(C4BP) [42, 43]. On the other hand, SdrC is important for the formation of bacterial biofilms [44]. SdrC can also bind specifically and with high affinity to

β-neurexin [45]. *S. aureus* has at least two of the sdr genes, with the sdrC gene always being found, the other two may or may not be in the *S. aureus* genome [40]. Strains that possess only the sdrC gene are less likely to cause bone infections, because it is related to one of the SdrE variants that has been identified as a bone sialoproteinbinding protein [46]. SdrD is crucial for abscess formation and can interact with desmoglein (desmosomal protein that maintains the structure of the epidermis through its adhesive function) [41, 47, 48].

#### *2.1.2.2 Iron-regulated surface proteins (Isd)*

Iron-regulated surface proteins (Isd) are responsible for transporting the heme group, the system is made up of nine proteins (IsdA-IsdI) and are activated if the bacterium has iron-limited conditions [21, 49, 50]. The heme group binds to a membrane, and from there it passes to the cytoplasm, once at this site, the heme oxygenases release the iron atoms [25]. *S. aureus* requires these hemoproteins for growth and virulence [51, 52].

Isd proteins present domains of the nearby iron transporter (NEAr iron Transporter, NEAT), which participate in the capture of the heme group of hemoglobin, favoring the development of bacteria in the host in places where there is low iron concentration. Isd proteins have NEAT domains, which vary according to the type of Isd, since IsdA only has one, IsdB has two, and IsdH has three, with which it can bind to the heme group, IsdA also has a hydrophilic end C-terminal, which is responsible for decreasing the hydrophobicity of the cell surface, making the bacteria resistant to lipid bactericides and other antimicrobial peptides [25].

Isd proteins are important during bacterial pathogenesis. IsdA can bind to various host proteins in addition to the heme group (fibrinogen, fibronectin, cytokeratin 10, etc.), promoting adherence to cell lines and tissues, and acts together with IsdB to provide resistance to neutrophil killing [53].

#### *2.1.2.3 S. aureus surface proteins (SasG and SasX)*

There is a broad association between *S. aureus* surface protein G (SasG) and accumulation-associated protein (Aap), the latter being required by *Staphylococcus epidermidis* for biofilm formation and promoting intercellular adhesion [54, 55].

SasG binds covalently to the cell wall via homophilic protein-protein interactions through Zn2+-dependent cleaved SasG B domains, resulting in cell-cell adhesion. However, the host cell binding ligand is still unknown [56–59].

*S. aureus* colonizes the nasal epithelium mainly due to ClfB and IsdA proteins, which allow adhesion to desquamated epithelial cells [25]. However, adhesion to epithelial cells is also promoted by SasG and may contribute to colonization [60]. In addition, overexpression of the *sasG* gene can inhibit clumping proteins (ClfA and ClfB) to increase biofilm formation [61, 62].

SasX protein, another CWA protein, seems to have been important in the epidemics caused by MRSA in hospitals on the Asian continent [63]. The *sasX* gene is known to be encoded by a bacteriophage that is in lysogenic form [34], SasX protein increases the formation of biofilms, by increasing cell aggregation it leads to a decrease in phagocytosis of neutrophils [63, 64] and adhesion to desquamated cells [25]. Therefore, the *sasX* gene not only encodes a colonization factor but also helps virulence of *S. aureus* by evading immune response [65]. SasX has also been associated with disease severity in skin and lung infections [63].

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

#### *2.1.3 Adhesins regulation*

The regulation of the virulence factors of *S. aureus* is carried out by a system that integrates signals derived from the host and the environment in a coordinated manner. Two-Component Systems (TCSs) are processes that identify environmental changes and produce regulation. Generally, membrane-associated histidine kinase is activated by an external signal, this induces its autophosphorylation and then phosphorylates a regulatory protein. This phosphorylated protein can bind to a specific DNA sequence, causing altered expression of the target gene. The majority of *S. aureus* strains have 16 different TCSs [66], the WalR/WalS system involved in regulating cell wall metabolism is essential, and some of the other 15 may be inactivated in various strains [67, 68]. Other TCSs such as arlRS, agrAC, and saeRS are implicated in *S. aureus* virulence by regulating many secreted proteins that affect the host [69].

#### *2.1.3.1 Accessory gene regulator (Agr) system*

Among the most studied regulatory systems is the accessory gene regulator (Agr), which is responsible for encoding a *quorum sensing* system that serves as the master regulator of virulence [69].

The Agr system detects a signal given by an autoinducer peptide (AIP), composed of 7–9 amino acids. There are four different alleles for the *agr* locus, each strain presenting only one of them. All four known *S. aureus* AIPs contain a cysteine residue that forms a cyclic thiolactone ring with the carboxylate at the C-terminal end of the peptide, which seems to be essential for its function [70]. Once the peptide AIP reaches the critical concentration or depletion of glucose in the extracellular medium, the system is activated in the quorum cells of the population [71]. This mechanism can induce the production of virulence factors and mechanisms of resistance to antibiotics [72]. Interestingly, AIP with a structure different from that produced by the same strain may excerpt an inhibitory effect on the Agr system, instead of the cognate autoinducing function [70]. *S. aureus* requires the Agr system to be able to adapt to changes in the environment during growth to regulate the bacteria's virulence factors [70]. The Agr system has two adjacent transcriptional regions, named RNAII and RNAIII, its expression is regulated by P2 and P3 promoters. Regarding the RNAII region, it is known that it is an operon of four genes (*agrBDCA*), which is responsible for encoding the mechanism of the *quorum sensing* system [73]. The RNAIII transcript is the main effector molecule, and its function is to regulate the expression of most of the target genes that depend on the Agr system (**Figure 1**) [69].

AgrB is a membrane endopeptidase whose function is to cleave the mature AIP from the AIP precursor (AgrD), to form the macrocyclic thiolactone structure and release it into the cytoplasm [70]. AIP interacts with AgrC, a membrane-bound histidine receptor kinase, which subsequently phosphorylates AgrA in the cytoplasm [74]; once phosphorylated, AgrA joins P2 and P3, regulating RNAII and III transcription [73].

AgrA also acts by inducing the expression of phenol-soluble modulins (PSMs). The RNAIII gene encodes a small RNA molecule that is the main effector molecule of the quorum sensing system that is responsible for increasing the expression of cell surface proteins. Four groups of Agr are known in *S. aureus* called agr I-IV each producing a distinctive AIP structure [73]. The Agr system produces increased expression of enzymes and toxins such as serine proteases, DNase, toxic shock toxin-1 (TSST-1),

#### **Figure 1.**

*The Agr system can regulate the virulence of S. aureus. The activation of the system is carried out by an autoinducing peptide (AIP), which accumulates extracellularly when reaching a critical concentration or depletion of glucose. Agr system has two adjacent transcriptional regions (RNAII and RNAIII), and its expression is mediated by the promoters P2 and P3. The RNAII transcript is encoded by the agrBDCA operon, which is the main part of the system, while the RNAIII transcript is the main effector molecule and is responsible for regulating the expression of most Agr-dependent target genes. RNAIII contains the hld (hemolysin δ) genes and leads to the expression and secretion of virulence factors (hemolysins, proteases, enterotoxins, etc.), it is also responsible for inhibiting the expression of cell surface proteins. (modified from Salam and Quave [73].*

fibrinolysin, and enterotoxin B and also regulates the expression of colonization and biofilm formation factors [75].

During infectious processes, *S. aureus* produces a large number of enzymes, including lipases, proteases, and elastases, which serve to invade and damage host tissues. This bacterium can produce septic shock, and some strains produce superantigens, causing various intoxications, such as toxic shock syndrome and food poisoning. Some strains produce exfoliative toxins and epidermolysins that can cause bullous impetigo or scalded skin syndrome [76].

During the pathogenesis of *S. aureus*, it is essential to carry out the regulation of the expression of virulence factors. This regulation occurs in a coordinated manner during the bacterial infection. MSCRAMM expression generally occurs during the logarithmic phase of growth, whereas toxins are synthesized during the stationary phase. For the infectious process, the early expression of the MSCRAMM proteins is required, which promotes the initial colonization of the tissues, while later the synthesis of toxins that are secreted by the bacteria and that can cause direct damage to the host, this facilitates the spread and persistence of bacteria in the host [76, 77].

Although Agr system is one of the most important studied virulence factor regulation mechanisms, there are several other global regulators of virulence gene transcription that function in a complex network to regulate virulence. Some of these regulatory systems are *sar*, *sae*, *srr*, *sigB*, *rot*, and *mgr loci*, among others, and form a complex regulatory network controlling virulence [78]. With the advent of whole genome sequencing techniques in addition to the accumulating knowledge of virulence gene regulation and functions, attempts have been proposed to construct system biology tools to predict virulence of *S. aureus* strains from genomic sequence [79]. Although there is the great amount of information on *S. aureus* pathotypes and genomic sequence, this goal is still far to be reached due to the complexity of the virulence regulatory network in *S. aureus*.

#### **2.2 Biofilms**

#### *2.2.1 Polysaccharide intercellular adhesion (PIA)*

Polysaccharide of intercellular adhesion (PIA) or poly-N-acetylglucosamine (PNAG) is a fundamental biofilm exopolysaccharide and constitutes most of the extracellular matrix of staphylococcal biofilms [71].

The PIA is constituted by the linear polysaccharide of poly-β(1-6)-Nacetylglucosamine and allows the mediation of bacterial intercellular adhesion; in addition, it forms the structure of the biofilm and bacterial adhesion on surfaces, in addition to protection against host defenses [75]. This is because PIA generates positive charges around the surface of bacteria (which are negatively charged by WTA), triggering electrostatic interactions that allow them to adhere to cells and tissues [71]. PIA is synthesized by the *icaADBC* locus, which is part of the accessory genes on plasmids, and therefore not all *S. aureus* strains have it [75]. However, PIA is so far the only important element involved in biofilm generation in vivo [80], but it does not appear in all isolates from biofilm-associated *S. aureus* infections, so other proteins are involved in its formation (SasG, SpA, Fnbp, among others) [26].

**Figure 2** shows that the structure of the *icaADBC* locus, *icaA* (N-acetylglucosaminyltransferase) encodes a very important transmembrane protein in the synthesis of the poly-N-acetylglucosamine polymer, being more efficient with polymer residues of more than twenty, and is only synthesized together with the *icaD* gene protein. Both proteins (*icaA* and *icaD*) are essential in the synthesis of exopolysaccharides. The third gene, *icaC*, translocates the poly-N-acetylglucosamine polymer to the cell surface, and the product of the *icaB* gene produces its deacetylation; this is very important for the structural maturation of the exopolysaccharide biofilm and allows the adhesion of the polymer with the surface of the bacteria [75, 82]. *icaR* is the fifth gene of the *icaADBC* locus, and it is transcribed in the opposite direction to the aforementioned genes, the start codon between *icaR* and *icaA* is separated by 163 bp (**Figure 2**). The role of *icaR* is to be a negative regulator of the *icaADBC* locus of *S. aureus*, and it encodes a 22 kDa protein of the TetR family. Otherwise, *icaZ* has only been found in strains of *S. epidermidis*, and its expression depends on the conditions of the medium and the incubation temperature [82, 83].

The *icaR* gene is responsible for the expression of the *ica* locus and in turn is regulated by the SarA and σB stress sigma systems (**Figure 3**). SarA belongs to the family of staphylococcal regular accessory proteins (Sar) and functions as an activator or repressor of the transcription of various *S. aureus* genes involved in its pathogenicity, so SarA is a virulence factor of great importance. The *agr* locus is regulated by SarA [78]. The Agr system regulates the change in expression of cell surface proteins in the early phases of bacterial growth (latency and exponential phase), to the synthesis of degrading proteins and toxins (post-exponential and stationary phase). The ability of *S. aureus* to form biofilms can be reduced by expression of the *agr* locus [75, 80, 82].

The formation of biofilms is generated from a complex production of extracellular polymeric molecules, such as amyloid fibrils, extracellular DNA, and phenol-soluble modulins (PSM), and this is due to the synthesis of nucleases, proteases, and PSM peptides [84]. The presence of PSM is highly regulated by Agr, this could indicate that

#### **Figure 2.**

*Structure of the icaADBC locus. Organization of the locus in S. epidermidis RP62A. Colored arrows indicate the coding regions. icaA encodes the enzyme N-acetylglucosaminyl-transferase (membrane protein), icaC is responsible for the translocation of the poly-N-acetylglucosamine polymer to the surface of the bacteria, in the case of icaB, it deacetylates the polymer. icaR is transcribed in the opposite direction with respect to the mentioned genes, and its function is to regulate the operon and therefore the biofilm. icaZ has only been reported in S. epidermidis under some environmental and temperature conditions (modified from Lerch et al. [81]).*

#### **Figure 3.**

*Diagram of the interactions that favor the formation and degradation of biofilms. The anabolic phase of biofilm formation is shown on the left side of the figure, where several critical extracellular polymeric substances (EPS): PIA, amyloid fibrils, and eDNA, are present. Also shown is the lytSR operon with its lrg/cid target genes. Membrane protein components involved in biofilm formation are shown in the center of the figure, including FnBP adhesins, biofilm-associated protein (Bap), Spa, and SasG. On the right side, the molecules of the catabolic processes of the biofilm are shown, including extracellular proteins and PSM. The Agr system, the σB factor, and SarA are the main regulators, modifying bacterial behavior in response to various environmental stimuli (modified from Arciola et al. [75]).*

the biofilm formation processes that depend on the Agr system are due to the expression of PSM [77]. The mechanisms of sessile and planktonic phenotypes require sensitive coordinated and efficient control during the invasive phase of bacteria [75]. *Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

There is evidence that *S. epidermidis icaA*(+) overexpresses the biofilm formation phenotype under *in vitro* conditions. However, *S. aureus* makes it different since the positive strains of the *ica* locus are not always expressed *in vitro* and do not need anaerobiosis or medium supplementation with other nutrients to express it. In contrast, *S. aureus* strains have higher biofilm production under *in vivo* conditions. Some stress-induced conditions *in vitro*, such as starvation, iron limitation, noninhibitory concentrations of ethanol, heat stress, NaCl, and various antibiotics, have been reported to increase biofilm production [75].

#### *2.2.2 Amyloid proteins*

The stability of the biofilm is due to the presence of amyloid proteins [85]. The amyloid structure is composed of three packed β-fibers that are resistant to denaturing conditions and are not degraded by proteases [86].

Amyloid proteins can bind to eDNA and function as inters fibrils in the biofilm, functioning as a solid bond, which allows the bacteria to wait for the environmental conditions to improve to favor their dissociation and allow the dispersion of the biofilm [85]. PSMs are necessary to increase the volume, roughness, thickness, and channel formation in the biofilm [87]. These surfactant peptides (PSM) play a fundamental function in the three-dimensional structure of the biofilm, in addition to favoring its detachment [87], and are determinants of biofilm maturation in vivo [71, 82]. **Figure 4** shows a diagram of the main components expressed by *S. aureus* in the formation of biofilms.

#### *2.2.3 Fibrin biofilm*

*S. aureus* can survive in the blood due to the production of the enzyme coagulase (CoA), which is regulated by the SaeRS two-component system. Detection of enzyme activity (Coa or staphylocoagulase) is very common in the clinical laboratory to identify strains of *S. aureus* from other staphylococci [89]. Highly relevant in the development of biofilms is *coa* gene, under natural conditions and is present in 100% of *S. aureus* strains. After maturation, fibrin-coated biofilms have increased defense and resistance against antibiotics [88].

Coa function is activated by binding to prothrombin from the blood, allowing the formation of the active staphylothrombin complex that converts soluble fibrinogen to insoluble fibrin, which is used by *S. aureus* to reinforce the biofilm. Whether S*. aureus* can form biofilms mediated by the *coa* gene depends on contact of bacterial cells with the host cell surface, and an important protein for this binding is ClfA [90].

There are indications that the colonization of medical devices by *S. aureus* is due to the production of fibrin biofilms mediated by the *coa* gene; however, over longer periods of time, other adhesins that also form biofilms play a more important role in their maturation [88]. Zapotoczna et al. [91] observe that after 24 h of fibrin biofilm formation, they became weaker in the presence of antibiotics compared with biofilms of another protein composition (e.g., FnBP) in the same period of development; however, with the passage of time, the fibrin biofilms became more resistant.

#### **2.3 Biofilm formation**

Upon initial contact, a planktonic cell can reversibly associate with a surface, and if the cell does not detach, then it will irreversibly bind to it [25, 27].

#### **Figure 4.**

*Main types of biofilms. A: PIA/PNAG polysaccharide biofilm by strains with the icaADBC operon (common in MRSA), B: surface proteins (BAP, FnBP, and SasG), interact between cells during biofilm formation. eDNA and cytoplasmic proteins diffused after lysis participate as elements of the biofilm matrix, C: coagulase-mediated activation of fibrinogen (Fg) into fibrin, which is activated to strengthen the biofilm, which can be dissociated by the plasmin produced post-staphylokinase (SAK) (plasminogen-mediated), D: PSMs have surface-active properties that promote biofilm breakdown and, in turn, can accumulate as amyloid aggregates (modified from Zapotoczna et al. [88]).*

When *S. aureus* adheres to host cells and tissues or to the surface of prosthetic materials, it can reproduce, colonize, and persist in these sites, in a variety of ways [76]. The first of the mechanisms used by bacteria is the formation of biofilms, *S. aureus* can form them on the surface of tissues, thereby colonizing and persisting in tissues, in addition to evading some of the host's immune mechanisms, also to blocking the role of antibiotics [92].

The biofilm is defined as a set of aggregated bacteria and is made up of cells adhered to each other (sessile cells). The cells are located within a matrix with extracellular polymeric substances (proteins, exopolysaccharides, adhesins, eDNA, etc.), which present an altered phenotype of growth, genetic expression, and protein production [92, 93], with respect to normal cells, normal planktonic (free life) [90]. Biofilms can form on biotic and abiotic surfaces, and those bacteria that are coated within the biofilm are 10–1000 times less sensitive to antibiotics than planktonic bacterial cells [71, 94, 95].

The formation of biofilms has been described through a cycle from the study of different bacterial species and is composed of (1) reversible adhesion, (2) irreversible union (formation of microcolonies), (3) maturation, and (4) dispersion [71, 96]. **Figure 5** shows a schematic of the biofilm formation cycle. However, in 2014, Moormeier et al. [23] proposed five stages in the formation of biofilms for *S. aureus*: (1) fixation, (2) multiplication, (3) exodus, (4) maturation, and (5) dispersion. The first stage of biofilm formation was mentioned in the section on adhesins.

#### *2.3.1 Components of the biofilm matrix*

#### *2.3.1.1 Extracellular DNA (eDNA)*

When the biofilm is formed, the extracellular matrix (ECM) is produced, made up of polysaccharides, proteins, and/or extracellular DNA, which confers the threedimensional structure that stabilizes and matures the biofilm [97]. The hypothetical *Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

#### **Figure 5.**

*The five parts of S. aureus biofilm formation. The process of biofilm formation can be explained in five main stages: (1) initial attachment or binding, (2) multiplication, (3) exodus or primary migration, (4) maturation, and (5) dispersal. 1. S. aureus Binds to a surface (abiotic or biotic) via MSCRAMM or nonpolar interactions. 2. Once cells adhere, a biofilm is formed, which is a confluent layer of cells, eDNA, and protein matrix. 3. When confluence is reached, cell exodus occurs, releasing a small number of cells from the biofilm by degradation of nuclease enzymes to eDNA (regulated by Sae), which allows the development of microcolonies in the biofilm space. 4. These microcolonies are formed from cellular sources that remain attached in the exodus stage. This stage consists of accelerated cell division that forms protein aggregates, including eDNA and PSM. 5. Quorum sensing by the Agr system initiates regulation of the biofilm matrix and cell dispersal through activation of proteases and/or PSM (modified from Moormier et al. [26]).*

mechanism of eDNA adhesion postulates that eDNA is adsorbed on the membrane of individual bacteria in long loop structures measuring up to 300 nm [98]. It has also been described that DNA loops interact with rough surfaces at the nanoscale, which increases the bacterial adhesion surface to this type of surface (**Figure 5**) [99].

eDNA favors the hydrophobicity of the bacterial surface, single-stranded DNA has amphiphilic properties, the hydrophilic part for deoxyribose, and the hydrophobic part for nitrogenous bases. Otherwise, double-stranded DNA hybridizes with each other by hydrogen bonds (Watson-Crick bonds) and hydrophobic interactions. Various studies have reported that eDNA increases the hydrophobicity of bacteria. Das et al. [100] reported that the presence of eDNA increases the adhesion of bacterial cells on hydrophobic surfaces (**Figure 6**) [99].

eDNA also favors resistance to antimicrobial drugs by inducing the expression of resistance genes. eDNA can form complexes with divalent metal cations (Mg2+, Ca2+, Mn2+, and Zn2+), which neutralizes the negative charge on the outer part of the bacterial membrane and increases its resistance to host antimicrobial peptides and cationic antibiotics such as aminoglycosides. However, eDNA can induce immune system activation, although the biofilm protects bacteria from some processes such as phagocytosis [99].

How components of the biofilm matrix are externalized is still not fully understood. Mutant strains defective in autolysis have been reported to have poor biofilmforming capacity compared with strains that do not produce PIA biofilms [94]. Phagocytosis-mediated cell death is another mechanism of eDNA release and lysisindependent methods such as specialized secretion or vesicle formation [101, 102].

#### *2.3.2 Biofilm multiplication stage*

After bacterial attachment to a surface and under sufficient nutritional conditions, adherent *S. aureus* cells can multiply and accumulate. However, newly divided cells

#### **Figure 6.**

*Functions of extracellular DNA. (A) eDNA aids adhesion on surfaces by penetrating the electrically repulsive double layer. Acid-base interactions lead to bacterial adhesion. (B) eDNA generates chelating complexes with cationic antimicrobial peptides of the host's innate immune system. (C) eDNA generates complexes with divalent cations, triggering a response in the bacteria that increases pathogenicity and antimicrobial resistance (modified from Okshevsky et al. [99]).*

are very susceptible to detachment, primarily from fluid flow. To maintain immature biofilm stability, *S. aureus* can produce a wide range of molecules that stabilize intracellular interactions. This process is called the multiplication stage [26].

Staphylococci strains can produce a wide range of extracellular proteins (CWA, FnBP, SdrC, and ClfB), which promote biofilm formation by favoring intercellular binding, once they are attached to the surface through a dual role in the stage's union and accumulation. But there is evidence that they are also involved during the multiplication stage of biofilm development [23]. PIA functions as a component of ECM in the early stages of *S. aureus* biofilm formation [26].

Foulston et al. [103] showed that the enzymes enolase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (which is not a biofilm-forming protein) can be activated as a component of the ECM in response to a decrease in pH, around the biofilm (**Figure 7**). This would imply that under acidic conditions, enolase and GAPDH can bind to eDNA [104]. Otherwise, it has been reported that other extracellular proteins such as PSM, β-hemolysin (Hlb), and IsaB (immunodominant surface antigen B) bind to eDNA to stabilize the ECM [26].

#### *2.3.3 Biofilm exodus stage*

In time-lapse microscopic observations of biofilms, a phase was found that was termed "Exodus," due to a clear coordinated cell release around 6 h after the start of the multiplication stage, which is an early dispersal event that occurs at the same time as the formation of the microcolony and produces the restructuring of the biofilm (**Figure 7**). The exodus phase is determined by the degradation of eDNA by nucleases and does not depend on the Agr system, which is produced after the development of the microcolony. The degradation of eDNA in the ECM by endogenous nucleases decreases the total biomass of the biofilm [23, 24, 83, 105]. The exodus phase is highly regulated, since only a part of the bacterial cells in the biofilm presents the expression of the *nuc* gene (which encodes a thermonuclease, used as an identification criterion for *S. aureus*), which favors the shedding of most of the cell population of the biofilm formed [36]. Also, Moormeier et al. [23] noted changes important in ECM as the biofilm structure advances, initially only consisting of membrane protein components (binding and multiplication phase), to relying on eDNA and proteins released to the outside (exodus stage). Therefore, a biofilm is only composed of PIA, protein, and eDNA must be replaced by a more complex model of biofilm development and ECM composition over time as the biofilm forms [26]. Therefore, the reduction of the *Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

#### **Figure 7.**

*Scheme of intercellular interactions in the biofilm multiplication stage. Early in biofilm development, free-living (planktonic) cells adhere to surfaces through electrostatic interactions mediated by teichoic acids, PSM, autolysin A, etc. As the multiplication stage progresses, some cells die or lyse (blue circles) releasing cytoplasmic proteins (green circles) and eDNA (red lines) into the extracellular medium, enveloping living bacteria (yellow circles) in a mixture of DNA and proteins cytoplasmic (modified from Moormeier et al. [23]).*

bacterial population at the beginning of biofilm formation (by death or exodus) is an important requirement for its maturation. It has been observed that when there is no exodus phase, as is the case with *S. aureus* strains that have mutations in the *nuc* gene, the formation of microcolonies does not occur [23].

#### *2.3.4 Biofilm maturation stage*

The formation of microcolony structures is essential in the biofilm maturation process, since they provide a larger contact surface for obtaining nutrients and eliminating waste, in addition to favoring the dispersion of bacterial cells within the biofilm. Research carried out on other species of bacteria has reported the development of microcolony-like structures during the biofilm formation stages of *S. aureus* [23, 87, 106], the mechanism of its formation is not known.

A previously described model [87] mentions that the formation of microcolonies in the development of the biofilm is a subtractive process, in which channels are formed in it due to the dispersion caused by the PSM. However, in microscopy observations at different times, it has been described that microcolonies are formed from different cell foci of the basal layer once the exodus phase begins (**Figure 5**).

After the maturation stage, the release of bacteria from the interior of the biofilm occurs through dispersion, which reactivates the free-living state of the bacterial cell (planktonic state) [93, 107]. DNase I has been reported to be an inhibitor of PIAindependent biofilm development in MRSA strains of clinical isolates; however, it does not inhibit PIA-dependent MSSA strains [104]. In the same investigation, DNase I effectively inhibited biofilm development of MRSA strains, but failed to destroy already formed biofilms [108, 109].

#### *2.3.5 Biofilm dispersion stage*

Dispersion processes are fundamental in the composition of the biofilm, since through these the cells are released from the biofilm individually or in large groups of bacteria, if there are favorable environmental conditions. This is very important in biofilm-associated infections, as they facilitate systemic spread, and it has been shown that cells shed from biofilms from medical devices and catheters can cause endocarditis or sepsis [71, 80].

Mechanisms influencing the control of biofilm scattering have been studied and reported to be mediated by Agr quorum sensing control [84]. In the dispersion stage, the bacteria of the outermost layers of the biofilm are responsible for the expression of the *agr* genes, which leads to the detachment of the cells, and at the same time the renewal of the biofilm; however, *agr* genes are also expressed by bacteria in the inner part of the biofilm, where it is used for channel formation [70, 87, 110].

Some toxins influence the development of biofilms. For example, α-hemolysin (Hla) and leukocidin AB (LukAB) are involved in biofilm persistence [111]; Hla and LukAB are also synergistically involved in promoting macrophage dysfunction and death. Dastgheyb et al. [112] showed that PSMs block biofilm formation by disrupting interactions between ECM molecules with the bacterial surface. Perasamy et al. [87] reported similar results regarding the influence of the PSMs of *S. aureus* with the development of the biofilm, and that PSM degraded it, which produced its early dispersion due to the surfactant properties of the toxin [113].

The importance of the Agr system is essential for cell communication within the biofilm formed, to form and establish the three-dimensional structure by controlling cell dispersion. However, Agr system does not regulate other adhesive molecules of biofilm formation, as is the case with PIA [75].

## **3. Conclusions**

*S. aureus* is a highly relevant pathogenic bacterium for humans and other mammals, since it can bind very intensely to different components of the extracellular matrix and thus infect cells. It also has mechanisms that allow it to colonize, persist, and survive in unfavorable environmental conditions for growth, such is the case of the formation of biofilms, which allows it to evade various human immune mechanisms very efficiently. The complex and dynamic composition of *S. aureus* biofilms, as well as the existence of a complex genetic regulatory network driving biofilm formation and maturation, offer a wide variety of potential pharmacological targets for the control of *S. aureus* infections.

## **Conflict of interest**

The authors declare no conflict of interest.

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

## **Author details**

Samuel González-García1 , Aída Hamdan-Partida2 , Juan José Valdez-Alarcón3 , Anaid Bustos-Hamdan4 and Jaime Bustos-Martínez<sup>2</sup> \*

1 Doctorate in Biological and Health Sciences, Autonomous Metropolitan University, Mexico City, Mexico

2 Department of Health Care, Autonomous Metropolitan University-Xochimilco, Mexico City, Mexico

3 Faculty of Veterinary Medicine and Zootechnics, Multidisciplinary Center for Studies in Biotechnology, Universidad Michoacana de San Nicolás de Hidalgo, Michoacán, Mexico

4 National Institute of Perinatology "Isidro Espinosa de los Reyes", Mexico City, Mexico

\*Address all correspondence to: jbustos@correo.xoc.uam.mx

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

[1] Barrera-Rivas CI, Valle-

Hurtado NA, González-Luego GM, Baizabal-Aguirre VM, Bravo-Patiño A, Cajero-Juárez M, et al. Bacteriophage therapy: An alternative for the treatment of *Staphylococcus aureus* infections in animals and animal models. In: Frontiers in *Staphylococcus aureus*. London, UK: IntechOpen; 2017. pp. 179-201. DOI: 10.5772/65761

[2] Danelli T, Duarte FC, de Oliveira TA, da Silva RS, Frizon-Alfieri D, Goncalves GB, et al. Nasal carriage by *Staphylococcus aureus* among healthcare workers and students attending a University Hospital in Southern Brazil: Prevalence, phenotypic, and molecular characteristics. Interdisciplinary Perspectives on Infectious Diseases. 2020;**2020**:3808036. DOI: 10.1155/ 2020/3808036

[3] Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in *Staphylococcus aureus* infections. The Lancet Infectious Diseases. 2005;**5**:751-762. DOI: 10.1016/ S1473-3099(05)70295-4

[4] Roghmann MC, Johnson JK, Stine OC, Lydecker AD, Ryan KA, Mitchell BD, et al. Persistent *Staphylococcus aureus* colonization is not a strongly heritable trait in Amish families. PLoS One. 2011;**6**:e17368. DOI: 10.1371/journal. pone.0017368

[5] Sollid JUE, Furberg AS, Hanssen AM, Johannessen M. *Staphylococcus aureus*: Determinants of human carriage. Infection, Genetics and Evolution. 2014;**21**:531-541. DOI: 10.1016/j. meegid.2013.03.020

[6] Mertz D, Frei R, Jaussi B, Tietz A, Stebler C, Flückiger U, et al. Throat

swabs are necessary to reliably detect carriers of *Staphylococcus aureus*. Clinical Infectious Diseases. 2007;**4**:475-477. DOI: 10.1086/520016

[7] González-García S, Hamdan-Partida A, Bustos-Hamdan A, Bustos-Martínez J. Factors of nasopharynx that favor the colonization and persistence of *Staphylococcus aureus*. In: Zhou X, Zhang Z, editors. Pharynx—Diagnosis and Treatment. London: IntechOpen; 2021. pp. 1-21. DOI: 10.5772/intechopen.95843

[8] Leonard AC, Petrie LE, Cox G. Bacterial anti-adhesives: Inhibition of *Staphylococcus aureus* nasal colonization. ACS Infectious Diseases. 2019;**5**:1668- 1681. DOI: 10.1021/acsinfecdis.9b00193

[9] Brown AF, Leech JM, Rogers TR, McLoughlin RM. *Staphylococcus aureus* colonization: Modulation of host immune response and impact on human vaccine design. Frontiers in Immunology. 2014;**4**:507. DOI: 10.3389/ fimmu.2013.00507

[10] Kim SJ, Chang J, Rimal B, Yang H, Schaefer J. Surface proteins and the formation of biofilms by *Staphylococcus aureus*. Biochimica et Biophysica Acta— Biomembranes. 2018;**3**:749-756. DOI: 10.1016/j.bbamem.2017.12.003

[11] Mistretta N, Brossaud M, Telles F, Sanchez V, Talaga P, Rokbi B. Glycosylation of *Staphylococcus aureus* cell wall teichoic acid is influenced by environmental conditions. Scientific Reports. 2019;**9**:1-11. DOI: 10.1038/ s41598-019-39929-1

[12] Winstel V, Kühner P, Salomon F, Larsen J, Skov R, Hoffmann W, et al. Wall teichoic acid glycosylation governs *Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

*Staphylococcus aureus* nasal colonization. mBio. 2015;**4**:e00632-e00615. DOI: 10.1128/mBio.00632-15

[13] Brown S, Santa Maria Jr JP, Walker S. Wall teichoic acids of grampositive bacteria. Annual Review of Microbiology. 2013;**67**:313-336. DOI: 10.1146/annurev-micro-092412-155620

[14] Baur S, Rautenberg M, Faulstich M, Grau T, Severin Y, Unger C, et al. A nasal epithelial receptor for *Staphylococcus aureus* WTA governs adhesion to epithelial cells and modulates nasal colonization. PLoS Pathogens. 2014;**5**:e1004089. DOI: 10.1371/journal. ppat.1004089

[15] Misawa Y, Kelley KA, Wang X, Wang L, Park WB, Birtel J, et al. *Staphylococcus aureus* colonization of the mouse gastrointestinal tract is modulated by wall teichoic acid, capsule, and surface proteins. PLoS Pathogens. 2015;**7**:e1005061. DOI: 10.1371/journal. ppat.1005061

[16] Wanner S, Schade J, Keinhörster D, Weller N, George SE, Kull L, et al. Wall teichoic acids mediate increased virulence in *Staphylococcus aureus*. Nature Microbiology. 2017;**4**:1-12. DOI: 10.1038/ nmicrobiol.2016.257

[17] Winstel V, Sanchez-Carballo P, Holst O, Xia G, Peschel A. Biosynthesis of the unique wall teichoic acid of *Staphylococcus aureus* lineage ST395. mBio. 2014;**2**:e00869-e00814. DOI: 10.1128/mBio.00869-14

[18] Suzuki T, Swoboda JG, Campbell J, Walker S, Gilmore MS. In vitro antimicrobial activity of wall teichoic acid biosynthesis inhibitors against *Staphylococcus aureus* isolates. Antimicrobial Agents and Chemotherapy. 2011;**2**:767-774. DOI: 10.1128/AAC.00879-10

[19] Weidenmaier C, Kokai-Kun JF, Kulauzovic E, Kohler T, Thumm G, Stoll H, et al. Differential roles of sortaseanchored surface proteins and wall teichoic acid in *Staphylococcus aureus* nasal colonization. International Journal of Medical Microbiology. 2008;**5-6**:505- 513. DOI: 10.1016/j.ijmm.2007.11.006

[20] Weidenmaier C, Goerke C, Wolz C. *Staphylococcus aureus* determinants for nasal colonization. Trends in Microbiology. 2012;**5**:243-250. DOI: 10.1016/j.tim.2012.03.004

[21] Burian M, Wolz C, Goerke C. Regulatory adaptation of *Staphylococcus aureus* during nasal colonization of humans. PLoS One. 2010;**4**:e10040. DOI: 10.1371/journal.pone.0010040

[22] Burian M, Rautenberg M, Kohler T, Fritz M, Krismer B, Unger C, et al. Temporal expression of adhesion factors and activity of global regulators during establishment of *Staphylococcus aureus* nasal colonization. The Journal of Infectious Diseases. 2010;**9**:1414-1421. DOI: 10.1086/651619

[23] Moormeier DE, Bose JL, Horswill AR, Bayles KW. Temporal and stochastic control of *Staphylococcus aureus* biofilm development. mBio. 2014;**5**:e01341-e01314. DOI: 10.1128/ mBio.01341-14

[24] Kurokawa K, Takahashi K, Lee BL. The staphylococcal surfaceglycopolymer wall teichoic acid (WTA) is crucial for complement activation and immunological defense against *Staphylococcus aureus* infection. Immunobiology. 2016;**10**:1091-1101. DOI: 10.1016/j.imbio.2016.06.003

[25] Foster TJ, Geoghegan JA, Ganesh VK, Höök M. Adhesion, invasion and evasion: The many functions of the surface proteins of *Staphylococcus aureus*. Nature

Reviews. Microbiology. 2014;**1**:49-62. DOI: 10.1038/nrmicro3161

[26] Moormeier DE, Bayles KW. *Staphylococcus aureus* biofilm: A complex developmental organism. Molecular Microbiology. 2017;**3**:365-376. DOI: 10.1111/mmi.13634

[27] Kong C, Chee CF, Richter K, Thomas N, Rahman NA, Nathan S. Suppression of *Staphylococcus aureus* biofilm formation and virulence by a benzimidazole derivative, UM-C162. Scientific Reports. 2018;**1**:1-16. DOI: 10.1038/s41598-018-21141-2

[28] Ganesh VK, Barbu EM, Deivanayagam CC, Le B, Anderson AS, Matsuka YV, et al. Structural and biochemical characterization of *Staphylococcus aureus* clumping factor B/ligand interactions. The Journal of Biological Chemistry. 2011;**29**:25963- 25972. DOI: 10.1074/jbc.M110.217414

[29] Lacey KA, Mulcahy ME, Towell AM, Geoghegan JA, McLoughlin RM. Clumping factor B is an important virulence factor during *Staphylococcus aureus* skin infection and a promising vaccine target. PLoS Pathogens. 2019;**4**:e1007713. DOI: 10.1371/journal. ppat.1007713

[30] Abraham NM, Jefferson KK. *Staphylococcus aureus* clumping factor B mediates biofilm formation in the absence of calcium. Microbiology. 2012;**158**(Pt 6):1504. DOI: 10.1099/ mic.0.057018-0

[31] Mulcahy ME, Geoghegan JA, Monk IR, O'Keeffe KM, Walsh EJ, Foster TJ, et al. Nasal colonisation by *Staphylococcus aureus* depends upon clumping factor B binding to the squamous epithelial cell envelope protein loricrin. PLoS Pathogens. 2012;**12**:e1003092. DOI: 10.1371/journal. ppat.1003092

[32] Hait JM, Cao G, Kastanis G, Yin L, Pettengill JB, Tallent SM. Evaluation of virulence determinants using wholegenome sequencing and phenotypic biofilm analysis of outbreak-linked *Staphylococcus aureus* isolates. Frontiers in Microbiology. 2021;**12**:687625. DOI: 10.3389/fmicb.2021.687625

[33] Hamdan-Partida A, González-García S, de la Rosa GE, Bustos-Martínez J. Community-acquired methicillin-resistant *Staphylococcus aureus* can persist in the throat. International Journal of Medical Microbiology. 2018;**4**:469-475. DOI: 10.1016/j.ijmm.2018.04.002

[34] Lacey KA, Geoghegan JA, McLoughlin RM. The role of *Staphylococcus aureus* virulence factors in skin infection and their potential as vaccine antigens. Pathogens. 2016;**1**:22. DOI: 10.3390/pathogens5010022

[35] Fleury OM, McAleer MA, Feuillie C, Formosa-Dague C, Sansevere E, Bennett DE, et al. Clumping factor B promotes adherence of *Staphylococcus aureus* to corneocytes in atopic dermatitis. Infection and Immunity. 2017;**6**:e00994-e00916. DOI: 10.1128/ IAI.00994-16

[36] McAleese FM, Walsh EJ, Sieprawska M, Potempa J, Foster TJ. Loss of clumping factor B fibrinogen binding activity by *Staphylococcus aureus* involves cessation of transcription, shedding and cleavage by metalloprotease. The Journal of Biological Chemistry. 2001;**32**:29969- 29978. DOI: 10.1074/jbc.M102389200

[37] Ngo QV, Faass L, Sähr A, Hildebrand D, Eigenbrod T, Heeg K, et al. Inflammatory response against *Staphylococcus aureus* via intracellular sensing of nucleic acids in keratinocytes. Frontiers in Immunology. 2022;**13**:1-12. DOI: 10.3389/fimmu.2022.828626

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

[38] Pi Y, Chen W, Ji Q. Structural basis of *Staphylococcus aureus* surface protein SdrC. Biochemistry. 2020;**15**:1465-1469. DOI: 10.1021/acs.biochem.0c00124

[39] Vitry P, Valotteau C, Feuillie C, Bernard S, Alsteens D, Geoghegan JA, et al. Force-induced strengthening of the interaction between *Staphylococcus aureus* clumping factor B and loricrin. mBio. 2017;**6**:e01748-e01717. DOI: 10.1128/ mBio.01748-17

[40] Liu H, Lv J, Qi X, Ding Y, Li D, Hu L, et al. The carriage of the serine-aspartate repeat protein-encoding *sdr* genes among *Staphylococcus aureus* lineages. The Brazilian Journal of Infectious Diseases. 2015;**5**:498-502. DOI: 10.1016/j. bjid.2015.07.003

[41] Askarian F, Ajayi C, Hanssen AM, Van Sorge NM, Pettersen I, Diep DB, et al. The interaction between *Staphylococcus aureus* SdrD and desmoglein 1 is important for adhesion to host cells. Scientific Reports. 2016;**1**:1-11. DOI: 10.1038/srep22134

[42] Sharp JA, Echague CG, Hair PS, Ward MD, Nyalwidhe JO, Geoghegan JA, et al. *Staphylococcus aureus* surface protein SdrE binds complement regulator factor H as an immune evasion tactic. PLoS One. 2012;**5**:e38407. DOI: 10.1371/ journal.pone.0038407

[43] Hair PS, Foley CK, Krishna NK, Nyalwidhe JO, Geoghegan JA, Foster TJ, et al. Complement regulator C4BP binds to *Staphylococcus aureus* surface proteins SdrE and Bbp inhibiting bacterial opsonization and killing. Results in Immunology. 2013;**3**:114-121. DOI: 10.1016/j.rinim.2013.10.004

[44] Barbu EM, Mackenzie C, Foster TJ, Höök M. SdrC induces staphylococcal biofilm formation through a homophilic interaction. Molecular Microbiology. 2014;**1**:172-185. DOI: 10.1111/mmi.12750

[45] Barbu EM, Ganesh VK, Gurusiddappa S, Mackenzie RC, Foster TJ, Sudhof TC, et al. β-Neurexin is a ligand for the *Staphylococcus aureus* MSCRAMM SdrC. PLoS Pathogens. 2010;**1**:e1000726. DOI: 10.1371/journal.ppat.1000726

[46] Sitkiewicz I, Babiak I, Hryniewicz W. Characterization of transcription within sdr region of *Staphylococcus aureus*. Antonie Van Leeuwenhoek. 2011;**2**:409- 416. DOI: 10.1007/s10482-010-9476-7

[47] Cheng AG, Kim HK, Burts ML, Krausz T, Schneewind O, Missiakas DM. Genetic requirements for *Staphylococcus aureus* abscess formation and persistence in host tissues. The FASEB Journal. 2009;**10**:3393-3404. DOI: 10.1096/ fj.09-135467

[48] Hammers CM, Stanley JR. Desmoglein-1, differentiation, and disease. The Journal of Clinical Investigation. 2013;**4**:1419-1422. DOI: 10.1172/JCI69071

[49] Gaudin CF, Grigg JC, Arrieta AL, Murphy ME. Unique heme-iron coordination by the hemoglobin receptor IsdB of *Staphylococcus aureus*. Biochemistry. 2011;**24**:5443-5452. DOI: 10.1021/bi200369p

[50] Farrand AJ, Reniere ML, Ingmer H, Frees D, Skaar EP. Regulation of host hemoglobin binding by the *Staphylococcus aureus* Clp proteolytic system. Journal of Bacteriology. 2013;**22**:5041-5050. DOI: 10.1128/JB.00505-13

[51] Pishchany G, Dickey SE, Skaar EP. Subcellular localization of the *Staphylococcus aureus* heme iron transport components IsdA and IsdB. Infection and Immunity. 2009;**7**:2624-2634. DOI: 10.1128/IAI.01531-08

[52] Contreras H, Chim N, Credali A, Goulding CW. Heme uptake in bacterial pathogens. Current Opinion in Chemical Biology. 2014;**19**:34-41. DOI: 10.1016/j. cbpa.2013.12.014

[53] Hammer ND, Skaar EP. Molecular mechanisms of *Staphylococcus aureus* iron acquisition. Annual Review of Microbiology. 2011;**65**:129-147. DOI: 10.1146/annurev-micro-090110-102851

[54] Yoshii Y, Okuda KI, Yamada S, Nagakura M, Sugimoto S, Nagano T, et al. Norgestimate inhibits staphylococcal biofilm formation and resensitizes methicillinresistant *Staphylococcus aureus* to β-lactam antibiotics. NPJ Biofilms Microbiomes. 2017;**1**:1-9. DOI: 10.1038/ s41522-017-0026-1

[55] Yonemoto K, Chiba A, Sugimoto S, Sato C, Saito M, Kinjo Y, et al. Redundant and distinct roles of secreted protein Eap and cell wall-anchored protein SasG in biofilm formation and pathogenicity of *Staphylococcus aureus*. Infection and Immunity. 2019;**4**:e00894-e00818. DOI: 10.1128/IAI.00894-18

[56] Geoghegan JA, Corrigan RM, Gruszka DT, Speziale P, O'Gara JP, Potts JR, et al. Role of surface protein SasG in biofilm formation by *Staphylococcus aureus*. Journal of Bacteriology. 2010;**21**:5663-5673. DOI: 10.1128/JB.00628-10

[57] Formosa-Dague C, Speziale P, Foster TJ, Geoghegan JA, Dufrêne YF. Zinc-dependent mechanical properties of *Staphylococcus aureus* biofilmforming surface protein SasG. Proceedings of the National Academy of Sciences of the United States of America. 2016;**2**:410-415. DOI: 10.1073/ pnas.1519265113

[58] Paharik AE, Horswill AR. The staphylococcal biofilm: Adhesins, regulation, and host response.

Microbiology Spectrum. 2016;**2**:529- 566. DOI: 10.1128/microbiolspec. VMBF-0022-2015

[59] Sakr A, Brégeon F, Mège JL, Rolain JM, Blin O. *Staphylococcus aureus* nasal colonization: An update on mechanisms, epidemiology, risk factors, and subsequent infections. Frontiers in Microbiology. 2018;**9**:2419. DOI: 10.3389/ fmicb.2018.02419

[60] Corrigan RM, Rigby D, Handley P, Foster TJ. The role of *Staphylococcus aureus* surface protein SasG in adherence and biofilm formation. Microbiology. 2007;**8**:2435-2446. DOI: 10.1099/ mic.0.2007/006676-0

[61] Crosby HA, Kwiecinski J, Horswill AR. *Staphylococcus aureus* aggregation and coagulation mechanisms, and their function in host–pathogen interactions. Advances in Applied Microbiology. 2016;**96**:1-41. DOI: 10.1016/bs.aambs.2016.07.018

[62] Kwiecinski JM, Crosby HA, Valotteau C, Hippensteel JA, Nayak MK, Chauhan AK, et al. *Staphylococcus aureus* adhesion in endovascular infections is controlled by the ArlRS–MgrA signaling cascade. PLoS Pathogens. 2019;**5**:e1007800. DOI: 10.1371/journal.ppat.1007800

[63] Li M, Du X, Villaruz AE, Diep BA, Wang D, Song Y, et al. MRSA epidemic linked to a quickly spreading colonization and virulence determinant. Nature Medicine. 2012;**5**:816-819. DOI: 10.1038/nm.2692

[64] Otto M. MRSA virulence and spread. Cellular Microbiology. 2012;**10**:1513-1521. DOI: 10.1111/j.1462-5822.2012.01832.x

[65] Nakaminami H, Ito T, Han X, Ito A, Matsuo M, Uehara Y, et al. First report of sasX-positive methicillin-resistant *Staphylococcus aureus* in Japan. FEMS

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

Microbiology Letters. 2017;**16**:fnx171. DOI: 10.1093/femsle/fnx171

[66] Villanueva San Martín M, García Martínez B, Valle Turrillas J, Rapún Araiz B, Ruiz de los Mozos Aliaga I, Solano Goñi C, et al. Sensory deprivation in *Staphylococcus aureus*. Nature Communications. 2018;**9**:523. DOI: 10.1038/s41467-018-02949-y

[67] Kawada-Matsuo M, Yoshida Y, Nakamura N, Komatsuzawa H. Role of two-component systems in the resistance of *Staphylococcus aureus* to antibacterial agents. Virulence. 2011;**5**:427-430. DOI: 10.4161/viru.2.5.17711

[68] White MJ, Boyd JM, Horswill AR, Nauseef WM. Phosphatidylinositolspecific phospholipase C contributes to survival of *Staphylococcus aureus* USA300 in human blood and neutrophils. Infection and Immunity. 2014;**4**:1559- 1571. DOI: 10.1128/IAI.01168-13

[69] Jenul C, Horswill AR. Regulation of *Staphylococcus aureus* virulence. Microbiology Spectrum. 2019;**1**:669- 686. DOI: 10.1128/microbiolspec. GPP3-0031-2018

[70] Thoendel M, Horswill AR. Identification of *Staphylococcus aureus* AgrD residues required for autoinducing peptide biosynthesis. The Journal of Biological Chemistry. 2009;**33**:21828- 21838. DOI: 10.1074/jbc.M109.031757

[71] Reffuveille F, Josse J, Vallé Q, Gangloff CM, Gangloff SC. *Staphylococcus aureus* biofilms and their impact on the medical field. In: The Rise of Virulence and Antibiotic Resistance in *Staphylococcus aureus*. Vol. 11. 2017. p. 187. DOI: 10.5772/66380

[72] Reuter K, Steinbach A, Helms V. Interfering with bacterial quorum sensing. Perspectives in Medicinal Chemistry. 2016;**8**:1-15. DOI: 10.4137/PMC.S13209

[73] Salam AM. Quave CL, Targeting virulence in *Staphylococcus aureus* by chemical inhibition of the accessory gene regulator system in vivo. mSphere. 2018;**1**:e00500-e00517. DOI: 10.1128/ mSphere.00500-17

[74] Wang B, Muir TW. Regulation of virulence in *Staphylococcus aureus*: Molecular mechanisms and remaining puzzles. Cell Chemical Biology. 2016;**2**:214-224. DOI: 10.1016/j. chembiol.2016.01.004

[75] Arciola CR, Campoccia D, Ravaioli S, Montanaro L. Polysaccharide intercellular adhesin in biofilm: Structural and regulatory aspects. Frontiers in Cellular and Infection Microbiology. 2015;**5**:7. DOI: 10.3389/ fcimb.2015.00007

[76] Gordon RJ, Lowy FD. Pathogenesis of methicillin-resistant *Staphylococcus aureus* infection. Clinical Infectious Diseases. 2008;**46**(Suppl 5):350-359. DOI: 10.1086/533591

[77] Otto M. *Staphylococcus aureus* toxins. Current Opinion in Microbiology. 2014;**17**:32-37. DOI: 10.1016/j. mib.2013.11.004

[78] Jenul C, Horswill AR. Regulation of *Staphylococcus aureus* virulence. Microbiology Spectrum. 2018;**6**(1):GPP30031-GPP32018. DOI: 10.1128/microbiolspec.GPP3-0031-2018

[79] Priest NK, Rudkin JK, Feil EJ, van den Elsen JMH, Cheung A, Peacock SJ, et al. From genotype to phenotype: Can systems biology be used to predict Staphylococcus aureus virulence? Nature Reviews Microbiology. 2012;**10**(11):791- 797. DOI: 10.1038/nrmicro2880

[80] Otto M. Staphylococcal infections: Mechanisms of biofilm maturation and detachment as critical determinants

of pathogenicity. Annual Review of Medicine. 2013;**64**:175-188. DOI: 10.1146/ annurev-med-042711-140023

[81] Lerch MF, Schoenfelder SMK, Marincola G, Wencker FDR, Eckart M, Förstner KU, et al. A non-coding RNA from the intercellular adhesion (*ica*) locus of *Staphylococcus epidermidis* controls polysaccharide intercellular adhesion (PIA)-mediated biofilm formation. Molecular Microbiology. 2019;**6**:1571-1591. DOI: 10.1111/ mmi.14238

[82] Cue DR, Lei MG, Lee C. Genetic regulation of the intercellular adhesion locus in staphylococci. Frontiers in Cellular and Infection Microbiology. 2012;**2**:38. DOI: 10.3389/ fcimb.2012.00038

[83] Beenken KE, Mrak LN, Griffin LM, Zielinska AK, Shaw LN, Rice KC, et al. Epistatic relationships between *sarA* and *agr* in *Staphylococcus aureus* biofilm formation. PLoS One. 2010;**5**:e10790. DOI: 10.1371/journal.pone.0010790

[84] Schwartz K, Syed AK, Stephenson RE, Rickard AH, Boles BR. Functional amyloids composed of phenol soluble modulins stabilize *Staphylococcus aureus* biofilms. PLoS Pathogens. 2012;**6**:e1002744. DOI: 10.1371/journal. ppat.1002744

[85] Taglialegna A, Navarro S, Ventura S, Garnett JA, Matthews S, Penades JR, et al. Staphylococcal bap proteins build amyloid scaffold biofilm matrices in response to environmental signals. PLoS Pathogens. 2016;**6**:e1005711. DOI: 10.1371/journal.ppat.1005711

[86] Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR. Diversity, biogenesis and function of microbial amyloids. Trends in Microbiology.

2012;**2**:66-73. DOI: 10.1016/j.tim.2011. 11.005

[87] Periasamy S, Joo HS, Duong AC, Bach THL, Tan VY, Chatterjee SS, et al. How *Staphylococcus aureus* biofilms develop their characteristic structure. Proceedings of the National Academy of Sciences of the United States of America. 2012;**4**:1281-1286. DOI: 10.1073/ pnas.1115006109

[88] Zapotoczna M, O'Neill E, O'Gara JP. Untangling the diverse and redundant mechanisms of *Staphylococcus aureus* biofilm formation. PLoS Pathogens. 2016;**7**:e1005671. DOI: 10.1371/journal. ppat.1005671

[89] Bautista-Trujillo GU, Solorio-Rivera JL, Rentería-Solórzano I, Carranza-Germán SI, Bustos-Martínez JA, Arteaga-Garibay RI, et al. Performance of culture media useful for the isolation and presumptive identification of *Staphylococcus aureus* from bovine mastitis. Journal of Medical Microbiology. 2013;**63**:369-376. DOI: 10.1099/jmm.0.046284-0

[90] Thomer L, Emolo C, Thammavongsa V, Kim HK, McAdow ME, Yu W, et al. Antibodies against a secreted product of *Staphylococcus aureus* trigger phagocytic killing. The Journal of Experimental Medicine. 2016;**3**:293-301. DOI: 10.1084/ jem.20150074

[91] Zapotoczna M, McCarthy H, Rudkin JK, O'Gara JP, O'Neill E. An essential role for coagulase in *Staphylococcus aureus* biofilm development reveals new therapeutic possibilities for device-related infections. The Journal of Infectious Diseases. 2015;**12**:1883-1893. DOI: 10.1093/infdis/ jiv319

*Main Factors of* Staphylococcus aureus *Associated with the Interaction to the Cells… DOI: http://dx.doi.org/10.5772/intechopen.107974*

[92] Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. *Staphylococcus aureus* biofilms: Properties, regulation, and roles in human disease. Virulence. 2011;**5**:445- 459. DOI: 10.4161/viru.2.5.17724

[93] Lister JL, Horswill AR. *Staphylococcus aureus* biofilms: Recent developments in biofilm dispersal. Frontiers in Cellular and Infection Microbiology. 2014;**4**:178. DOI: 10.3389/fcimb.2014.00178

[94] Boles BR, Thoendel M, Roth AJ, Horswill AR. Identification of genes involved in polysaccharide-independent *Staphylococcus aureus* biofilm formation. PLoS One. 2010;**4**:e10146. DOI: 10.1371/ journal.pone.0010146

[95] Neopane P, Nepal HP, Shrestha R, Uehara O, Abiko Y. In vitro biofilm formation by *Staphylococcus aureus* isolated from wounds of hospitaladmitted patients and their association with antimicrobial resistance. International Journal of General Medicine. 2018;**11**:25-32. DOI: 10.2147/ IJGM.S153268

[96] Miao J, Lin S, Soteyome T, Peters BM, Li Y, Chen H, et al. Biofilm formation of *Staphylococcus aureus* under food heat processing conditions: First report on CML production within biofilm. Scientific Reports. 2019;**9**:1312. DOI: 10.1038/s41598-018-35558-2

[97] Flemming HC, Wingender J. The biofilm matrix. Nature Reviews Microbiology. 2010;**9**:623-633. DOI: 10.1038/nrmicro2415

[98] Das T, Sharma PK, Krom BP, van der Mei HC, Busscher HJ. Role of eDNA on the adhesion forces between *Streptococcus mutans* and substratum surfaces: Influence of ionic strength and substratum hydrophobicity. Langmuir.

2011;**16**:10113-10118. DOI: 10.1021/ la202013m

[99] Okshevsky M, Meyer RL. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Critical Reviews in Microbiology. 2013;**3**:341-352. DOI: 10.3109/1040841X.2013.841639

[100] Das T, Krom BP, van der Mei HC, Busscher HJ, Sharma PK. DNA-mediated bacterial aggregation is dictated by acid–base interactions. Soft Matter. 2011;**6**:2927-2935. DOI: 10.1039/ C0SM01142H

[101] Salgado-Pabón W, Du Y, Hackett KT, Lyons KM, Arvidson CG, Dillard JP. Increased expression of the type IV secretion system in piliated *Neisseria gonorrhoeae* variants. Journal of Bacteriology. 2010;**7**:1912-1920. DOI: 10.1128/JB.01357-09

[102] DeFrancesco AS, Masloboeva N, Syed AK, DeLoughery A, Bradshaw N, Li GW, et al. Genome-wide screen for genes involved in eDNA release during biofilm formation by *Staphylococcus aureus*. Proceedings of the National Academy of Sciences of the United States of America. 2017;**29**:5969-5978. DOI: 10.1073/pnas.1704544114

[103] Foulston L, Elsholz AK, DeFrancesco AS, Losick R. The extracellular matrix of *Staphylococcus aureus* biofilms comprises cytoplasmic proteins that associate with the cell surface in response to decreasing pH. mBio. 2014;**5**:e01667-e01614. DOI: 10.1128/mBio.01667-14

[104] Dengler V, Foulston L, DeFrancesco AS, Losick R. An electrostatic net model for the role of extracellular DNA in biofilm formation by *Staphylococcus aureus*. Journal of

Bacteriology. 2015;**24**:3779-3787. DOI: 10.1128/JB.00726-15

[105] Kiedrowski MR, Crosby HA, Hernandez FJ, Malone CL, McNamara JO. *Staphylococcus aureus* Nuc2 is a functional, surface-attached extracellular nuclease. PLoS One. 2014;**4**:e95574. DOI: 10.1371/journal. pone.0095574

[106] Moormeier DE, Endres JL, Mann EE, Sadykov MR, Horswill AR, Rice KC, et al. Use of microfluidic technology to analyze gene expression during *Staphylococcus aureus* biofilm formation reveals distinct physiological niches. Applied and Environmental Microbiology. 2013;**11**:3413-3424. DOI: 10.1128/AEM.00395-13

[107] Boles BR, Horswill AR. Staphylococcal biofilm disassembly. Trends in Microbiology. 2011;**9**:449-455. DOI: 10.1016/j.tim.2011.06.004

[108] Houston P, Rowe SE, Pozzi C, Waters EM, O'Gara JP. Essential role for the major autolysin in the fibronectinbinding protein-mediated *Staphylococcus aureus* biofilm phenotype. Infection and Immunity. 2011;**3**:1153-1165. DOI: 10.1128/IAI.00364-10

[109] Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated methicillin-resistant and-sensitive strains of *Staphylococcus aureus*. Scientific Reports. 2018;**1**:1-11. DOI: 10.1038/ s41598-018-20485-z

[110] Kiedrowski MR, Kavanaugh JS, Malone CL, Mootz JM, Voyich JM, Smeltzer MS, et al. Nuclease modulates biofilm formation in communityassociated methicillin-resistant *Staphylococcus aureus*. PLoS One. 2011;**11**:e26714. DOI: 10.1371/journal. pone.0026714

[111] Scherr TD, Hanke ML, Huang O, James DB, Horswill AR, Bayles KW, et al. *Staphylococcus aureus* biofilms induce macrophage dysfunction through leukocidin AB and alpha-toxin. mBio. 2015;**4**:e01021-e01015. DOI: 10.1128/ mBio.01021-15

[112] Dastgheyb SS, Villaruz AE, Le KY, Tan VY, Duong AC, Chatterjee SS, et al. Role of phenol-soluble modulins in formation of *Staphylococcus aureus* biofilms in synovial fluid. Infection and Immunity. 2015;**7**:2966-2975. DOI: 10.1128/IAI.00394-15

[113] Oliveira D, Borges A, Simões M. *Staphylococcus aureus* toxins and their molecular activity in infectious diseases. Toxins. 2018;**6**:252. DOI: 10.3390/ toxins10060252
