**4. Epidemiology of infections**

#### **4.1. Nasal carriage**

**Factors Characteristics**

*Polysaccharide microcapsule* Resist the phagocytosis & killing by polymorphonuclear

*Protein A* It binds to Fc portion of immunoglobulin, prevents

*Panton-Valentine leukocidin (PVL)* PVL is found in most of community-associated MRSA

[17]. *Alpha-toxin (Alpha hemolysin)* It was the first bacterial exotoxin to be identified as a

*Chemotaxis-inhibitory protein of S. aureus (CHIPS):* CHIPS is an extracellular protein which inhibits the

[19].

*Extracellular adherence protein (Eap)* An exoprotein which binds to host cell matrix, plasma

Enterotoxins *S. aureus* produces battery of enterotoxins which are

Toxic shock syndrome toxin -1 (TSST-1) TSST-1 & some of enterotoxins are called as pyrogenic

*Exfoliative toxins A and B* Serine proteases which selectively recognize and

death [18].

phagocyte [14].

immune response [15].

Cell surface proteins which interact with host molecules such as collagen, fibronectin & fibrinogen, thus, facilitate the tissue attachment. Staphylococcal protein A, fibronectin-binding proteins A and B, collagen-binding protein & clumping factor A & B belong to this family. They are also involved in host immune evasion [13].

opsonization, functions as super antigen & limits the host

(CA-MRSA) [16]. PVL belongs to group of membrane pores forming proteins. It consists of two protein components (LukS-PV and LukF-PV) which act together as subunits and form porins on cell membrane of host cells, leading to leakage of cell contents and cell death

cell membrane pore former which causes cell leakage &

chemotaxis functioning of neutrophil and monocytes

proteins & endothelial cell adhesion molecule ICAM-1. In addition to the roles of adhesion and invasion, it also has

These extracellular enzymes cause tissue destruction and, thereby, help in bacterial penetration into tissues.

potent gastrointestinal exotoxins. The Staphylococcal food poisoning is an intoxication which results from consumption of foods containing sufficient amount of

toxin super antigens. TSST-1 causes toxic shock syndrome especially in menstrual women [7].

hydrolyze desmosomal proteins in the skin. ETs cause staphylococca-scalded skin syndrome, a disease

predominantly affecting infants [22].

immune-modulatory activity [20].

preformed enterotoxins [21].

**Helping attachment to host tissues**

**Breaking/evading the host immunity**

*matrix molecules (MSCRAMM)*

**Tissue invasion**

*Staphylokinase*

**Induces toxinosis**

*Proteases, lipases, nucleases, hyaluronatelyase, phospholipase C, metalloproteases (elastase),* &

**Table 1.** Virulence factors of *S. aureus* and its characteristics.

*Microbial Surface Components Recognizing adhesive* 

*S. aureus* is a commensal and opportunistic pathogen. The anterior nares are the principal ecological niche, where the organism colonizes in humans. The nasal carriage of *S. aureus* increases the risk of infection especially in the hospital settings [23]. The average nasal carriage of *S. aureus* could be at 30% of human population [24]. Since, the nasal carriage increases the risk of development of surgical site, lower respiratory and blood stream infections in hospitals, efforts are made to eliminate the carriage using various strategies. Methods such as local application of antibiotics (eg. mupirocin) or disinfectants, administration of systemic antibiotics and use of a harmless *S. aureus* strain (type 502A) which competes for the colonization of nares with existing one are employed to decolonize the *S. aureus* from nares [25–28].

## **4.2. Emergence and evolution of MRSA**

The MRSA are those *S. aureus* strains carrying a *mecA* gene, which codes for additional penicillin-binding protein, PBP2a. The beta-lactam antibiotics exert their antibacterial activity by inactivation of penicillin-binding proteins (PBPs), which are essential enzymes for bacterial cell wall synthesis. However, these antibiotics have only a low affinity towards PBP2a, thus this enzyme evades from inactivation and carry out the role of essential PBPs resulting in cell wall synthesis and survival of bacteria even in presence of beta-lactam antibiotics. Due to the presence of *mecA*, MRSA are resistant to nearly all beta-lactam antibiotics [29].

Penicillin is the first beta-lactam antibiotic discovered in 1928 and found to be effective weapon against *S. aureus* infections. In 1940s, sooner after its introduction into clinics, there were reports of *S. aureus* strains that were resistant to penicillin [30]. These strains produced plasmid-encoded beta-lactamase enzyme (penicillinase) which enzymatically cleaved the beta-lactam ring of penicillin rendering the antibiotic inactive [31, 32]. In 1950s, the penicillin resistance was restricted to hospital isolates of *S. aureus*. By late 1960s, more than 80% *S. aureus* isolates, irrespective of community and hospital origin, were resistant to penicillin due to plasmid transfer of penicillinase gene (*blaZ*) and clonal dissemination of resistant strains [33, 34].

Meanwhile, scientists who were challenged with penicillinase-mediated resistance in *S. aureus* discovered methicillin, a semi-synthetic penicillin that withstood the enzymatic degradation of penicillinase. Methicillin was introduced into clinics in 1961; however, in less than a year, resistance of *S. aureus* isolates to methicillin (MRSA) was reported [35]. Over the next 10 years, increasing number of MRSA outbreaks was reported in different parts of the world especially from the European countries [36, 37]. The notable feature of these reports is that, the incidences were from hospitals and thus MRSA emerged as a hospital-borne pathogen. The mechanism of resistance to beta-lactam antibiotics in these MRSA isolates was uncovered in 1981 [38].

As mentioned earlier, MRSA isolates carry a gene *mec A* which codes for PBP2a. The gene is part of a 21–60 kb mobile genetic element referred to as staphylococcal cassette chromosome *mecA* (SCC*mecA*). There are two hypotheses that explain the evolutionary origin of MRSA. The single clone hypothesis suggests that the mobile genetic element entered the *S. aureus* population on one occasion and resulted in the formation of a single MRSA clone that has since spread around the world. The second and the most agreed hypothesis is that MRSA strains evolved number of times by means of the horizontal transfer of the mobile genetic element into phylogenetically distinct methicillin-susceptible *S. aureus* (MSSA) precursor strains [39, 40].

SCC*mec* elements are highly diverse in their structural organization and genetic content (**Figure 1**) and have been classified into types based on the combination of *mec* and *ccr*, which share variations (five classes in *mec* and eight in *ccr*). To date, at least 11 types of *SCCmec* elements have been identified [41–43].

#### **4.3. Health care-associated and community MRSA**

#### *4.3.1. Health care-associated MRSA (HA-MRSA)*

Health care-associated MRSA (HA-MRSA) are those *S. aureus* isolates obtained from patients 2 or more days after hospitalization or with the MRSA risk factors (history of recent hospitalization, surgery, dialysis, or residence in a long-term care facility within 1 year before the MRSA-culture date or presence of a permanent indwelling catheter or percutaneous medical device (e.g. tracheostomy tube, gastrostomy tube or Foley catheter) at the time of culture or previous isolation of MRSA [44, 45]. Community-associated MRSA (CA-MRSA) are those *S. aureus* isolates obtained from patients within 2 days of hospitalization and without the above-mentioned MRSA risk factors.

Till 1990s, MRSA isolates were predominantly HA-MRSA and were also resistant to non-betalactam antibiotics. The multi-drug resistant phenotype of HA-MRSA was due to presence of non-beta-lactam antibiotic-resistant determinants in relatively large SCC*mec* [46]. During the period of 1960s to early 1990s, number of clones of HA-MRSA had spread widely across the world and HA-MRSA became endemic in hospitals and emerged as leading nosocomial pathogen [47]. The genetic background of these MRSA clones was characterized initially using phage typing subsequently by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), *spa* typing and SCC*mec* typing. The analysis of the genetic background of HR-MRSA

**Figure 1.** Basic structure of SCC*mec*. SCC*mec* constituted by *mec* gene and *ccr* gene complexes. The *mec* gene complex encodes PBP2a (*mecA*) and resistance regulators (*mecI* and *mcR1*). The *ccr* gene complex encodes the integration and excision of entire SCC element. The gene complexes are flanked by characteristic nucleotide sequences, inverted repeats (IR) and direct repeats (DR), at both ends. J (joining) regions are J1 (between right chromosomal junction and *ccr* complex, J2 (between *ccr* and *mec* complexes) and J3 (between *mec* complex and left chromosomal junctions). Adopted from Ref. [41].

isolates using these methods revealed the spread of early MRSA clone (Archaic clone) which contained type I SCC*mec* and sequence type 250 (ST250) in 1960s and extended into the 1970s in the form of Iberian clone. The Iberian clone was sequence type 247 (ST247) which evolved from ST250-MRSA by a single point mutation [48]. In the mid to late 1970s, Archaic and Iberian MRSA clones declined while, clones with novel SCC*mec* types II and III had emerged marking the on-going worldwide pandemic of HA-MRSA in hospitals and health care facilities [49, 50]. The lineages of common HA-MRSA clones are represented in **Table 2**. The rise in the prevalence of HA-MRSA throughout the world has been dramatic. In the United States, the proportion of MRSA among *S. aureus* isolates from the hospitalized patients was 2.4% in 1975, which increased to 51.6% (ICU patients) and 42% (non-ICU inpatients) by 1998–2003. Similar persistently high or increasing rates of MRSA among *S. aureus* isolates have also been observed for health care settings in many other regions of the world [51].

#### *4.3.2. Community-associated MRSA (CA-MRSA)*

single clone hypothesis suggests that the mobile genetic element entered the *S. aureus* population on one occasion and resulted in the formation of a single MRSA clone that has since spread around the world. The second and the most agreed hypothesis is that MRSA strains evolved number of times by means of the horizontal transfer of the mobile genetic element into phylo-

SCC*mec* elements are highly diverse in their structural organization and genetic content (**Figure 1**) and have been classified into types based on the combination of *mec* and *ccr*, which share variations (five classes in *mec* and eight in *ccr*). To date, at least 11 types of *SCCmec* ele-

Health care-associated MRSA (HA-MRSA) are those *S. aureus* isolates obtained from patients 2 or more days after hospitalization or with the MRSA risk factors (history of recent hospitalization, surgery, dialysis, or residence in a long-term care facility within 1 year before the MRSA-culture date or presence of a permanent indwelling catheter or percutaneous medical device (e.g. tracheostomy tube, gastrostomy tube or Foley catheter) at the time of culture or previous isolation of MRSA [44, 45]. Community-associated MRSA (CA-MRSA) are those *S. aureus* isolates obtained from patients within 2 days of hospitalization and without the

Till 1990s, MRSA isolates were predominantly HA-MRSA and were also resistant to non-betalactam antibiotics. The multi-drug resistant phenotype of HA-MRSA was due to presence of non-beta-lactam antibiotic-resistant determinants in relatively large SCC*mec* [46]. During the period of 1960s to early 1990s, number of clones of HA-MRSA had spread widely across the world and HA-MRSA became endemic in hospitals and emerged as leading nosocomial pathogen [47]. The genetic background of these MRSA clones was characterized initially using phage typing subsequently by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), *spa* typing and SCC*mec* typing. The analysis of the genetic background of HR-MRSA

**Figure 1.** Basic structure of SCC*mec*. SCC*mec* constituted by *mec* gene and *ccr* gene complexes. The *mec* gene complex encodes PBP2a (*mecA*) and resistance regulators (*mecI* and *mcR1*). The *ccr* gene complex encodes the integration and excision of entire SCC element. The gene complexes are flanked by characteristic nucleotide sequences, inverted repeats (IR) and direct repeats (DR), at both ends. J (joining) regions are J1 (between right chromosomal junction and *ccr* complex, J2 (between *ccr* and *mec* complexes) and J3 (between *mec* complex and left chromosomal junctions). Adopted

genetically distinct methicillin-susceptible *S. aureus* (MSSA) precursor strains [39, 40].

ments have been identified [41–43].

above-mentioned MRSA risk factors.

from Ref. [41].

**4.3. Health care-associated and community MRSA**

*4.3.1. Health care-associated MRSA (HA-MRSA)*

MRSA isolates obtained from outpatients or from patients within 48 h of hospitalization and if they lack HA-MRSA risk factors mentioned earlier are referred to as CA-MRSA [52]. Scattered case reports of MRSA infections in healthy population whom had no exposure to health care facilities were published in the 1980s and mid-1990s. Beginning in 1993, case series of MRSA infection and colonization of patients lacking health care-associated risk factors were reported from six continents, in diverse states, nations and regions [51, 53]. The phenotypic and genotypic characterization of CA-MRSA isolates revealed the differences between CA-MRSA and HA-MRSA strains. While HA-MRSA strains carried a relatively large SCC*mec*, belonging to type I, II or III, CA-MRSA strains carried smaller SCC*mec* elements, most commonly type IV or type V. HA-MRSA strains were resistant to many classes of non-beta-lactam antibiotics, thus display multi-drug resistant phenotypes. CA-MRSA strains were often sensitive to non-beta-lactam antibiotics. Another notable feature of CA-MRSA strains was presence of genes for the PVL, which was rare among the HA-MRSAs. With respect to clinical cases, CA-MRSA infections were prevalent in previously healthy younger patients in contrast to HA-MRSA, which cause infections in hospitalized patients. CA-MRSA was often associated with skin and skin structure infections while HA-MRSA was implicated in wide range of infections such as pneumonia, bacteraemia, and invasive infections [48, 51]. Compared to infections caused by HA-MRSA, CA-MRSA infections had been associated with fulminant and lethal infections and worse clinical outcomes [49, 53].

Among the various clones of CA-MRSA, ST93, ST80 and ST8 are presently the predominant clones in Australia, Europe and the United States, respectively. In the United States, ST8-USA 300 is the most wide spread CA-MRSA clone [54], which harbour SCC*mec* type IV and genes encoding PVL. The concern about this clone is high virulence and increase in resistance to non-beta-lactam antibiotics [50, 53]. In United Kingdom, EMRSA-15 (ST22) and EMRSA-16 (ST36) are the dominant clones [49]. In Europe, ST80-IV, ST8-IV, ST398-V and ST152-V were commonly reported [55]. In Mediterranean countries, the dominant clones are ST80-IV and ST5-IV/V [55, 56].

In the last 10 years, there is a dramatic change in epidemiology of CA-MRSA as they invaded the health care settings. In 2008, first case of MRSA isolated from hospitalized patient turned out to


**Table 2.** The lineages of common HA-MRSA (based on Ref. [49]).

be a CA-MRSA which marked the arrival of CA-MRSA into nosocomial settings [57]. Since then, hospital outbreaks of *S. aureus* strains which are phenotypically and genotypically CA-MRSA, have been reported many parts of the world [55]. Entry of CA-MRSA into hospitals blurred the differences between CA-MRSA and HA-MRSA. The increased reports of CA-MRSA outbreaks in hospital suggest that CA-MRSA may eventually displace HA-MRSA in hospitals [58].
