**1. Introduction**

94 Epidemiology Insights

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prevalence of methicillin-resistant *Staphylococcus aureus* colonization in feedlot

A. (2008). Prevalence of methicillin-resistant *Staphylococcus aureus* among veterinarians: an international study. *Clinical Microbiology and Infection*, Vol.14, Studying infections caused by the *Staphylococcus* spp. genus is highly important for human health given that such organisms are causal agents of superficial infections, such as abscesses and impetigo, as well as of systemic infections, namely bacteremia and osteomyelitis. This genus is divided into two large groups. The first group is characterized by the production of enzyme coagulase, and its main representative is *S. aureus*, which is frequently associated with a large variety of infections. The second group, known as coagulase-negative staphylococci (CoNS), is usually associated with immunocompromised patients or those who use catheters. (Kloos & Bannerman, 1995). The main CoNS species associated with infection in humans are *S. epidermidis, S. haemolyticus, S. saprophyticus, S. cohnii, S. xylosus, S. capitis, S. warneri, S. hominis, S. simulans, S. saccharolyticus, S. auricularis, S. caprae, S. lugdunensis* and *S. schleiferi* (Kloos & Bannerman, 1995).

Several studies have reported increased prevalence of CoNS infection in hospitals, which is usually associated with resistance to the antibiotic of choice for treatment. Hence, this is a serious clinical and epidemiological problem (Jain et. al., 2008).

The use of methicillin and other semi-synthetic penicillins, such as oxacillin and penicillinase-resistant methicillin, which began in 1959, represented a significant phase in anti-staphylococcal therapy. The first report on methicillin resistance was in 1961, a short time after its use was implemented (Hiramatsu et al, 2001). In Brazil, it is estimated that the frequency of oxacillin resistance is high among *S. aureus* samples, particularly in large and in university hospitals. Gales (2009) described an oxacillin resistance rate of 31% in a multicenter study involving four Brazilian hospitals. As regards CoNS, 78.7% of the samples were resistant. At the Botucatu School of Medicine University Hospital – SP, approximately 45% of the *S. aureus* samples from hemocultures were positive for the *mecA* gene (Martins et al., 2010). In a study conducted at the neonatal intensive care unit of the same hospital, a total of 18% of MRSA samples was found (Pereira et al., 2009). According to a study by Sader et al. (2004) in Latin America and Brazil, respectively, 80.4% and 84.6% of the CoNS samples from hemocultures were oxacillin resistant.

Oxacillin resistance in CoNS samples varies significantly among the different species in the genus, a fact that reinforces the importance of their correct identification (Secchi et al., 2008).

Epidemiological Aspects of Oxacillin-Resistant

of infection control measures in hospitals.

preventing its binding to oxacillin (Zhang et al., 2009).

**3. Oxacillin resistance** 

(Tomacz et al., 1991).

(Swenson, 2002).

*Staphylococcus* spp.: The Use of Molecular Tools with Emphasis on MLST 97

Based on the described reports, it is clear that oxacillin resistance is prevalent in hospital in all continents, a fact that reinforces the importance of good antibiotic therapy practices and

Oxacillin resistance is associated with the drug's reduced capacity to adhere to the penicillin-binding protein (PBP), thus also losing its capacity to lyse the bacterial cell. There are three mechanisms of resistance to semi-synthetic penicillinases, a group of drugs in which oxacillin is included. The first is related to the hyperproduction of β-lactamases, enzymes that cleave the drug's β-lactam ring, thus inactivating it (McDougal & Thornsberry, 1986). The second mechanism, known as MOD-SA, occurs when normal PBPs have reduced affinity with oxacillin (Tomasz et al., 1989). The third and most important mechanism is the presence of the *mecA* gene. This gene codifies a changed PBP, known as PBP 2a, thus

Although resistance mediated by the *mecA* gene is present in all cells of the population with intrinsic resistance, it can only be expressed by a small percentage of such cells, thus leading to the so-called heterogeneous resistance. Resistance expression in lineages with intrinsic resistance has been categorized into four phenotypic classes; classes 1 to 4, in which class 1 is the most heterogeneous and class 4 is the homogeneous one (Tomacz et al., 1991). The majority of cells (99.9 or 99.99%) in the culture of lineages with class-1 heterogeneous resistance show minimum inhibitory concentration (MIC) of 1.5 to 3 g/ml, but such culture also contains a small number of bacteria (10-7 to 10-8) that could form colonies even in the presence of 25 g/ml or more of oxacillin. In class-2 lineage cultures, the majority of cells ( 99.9%) show MIC of 6 to 12 g/ml, and in these cultures, the frequency of highly resistant cells (capable of growing in the presence of 25 g/ml) is higher (10-5) than in class-1 lineages (Tomacz et al. , 1991). Class-3 lineage cultures consist of bacteria (99 to 99,9%) that show high levels of oxacillin resistance (MIC = 50 to 200 g/ml), but they usually have a subpopulation (10-3) of highly resistant cells that are capable of forming colonies even in the presence of 300 to 400 g de oxacillin/ml. Class-4 cultures comprise cells with homogeneous resistance, with all cells showing high resistance levels and MIC of 400 to 1,000 g/ml

The phenotypic expression codified by the *mecA* gene is affected by various factors, including pH, temperature and osmolarity (Swenson, 2002). When proper conditions are used for laboratory MRSA detection, including Mueller-Hinton agar supplementation with NaCl and adequate temperature and time, as recommended by CLSI (Clinical and Laboratory Standards Institute), detection is achieved without much difficulty. However, for more heterogeneous lineages, detection can be more difficult, even with reference methods

Adequate detection of oxacillin resistance mediated by the *mecA* gene is important for clinical laboratories. Although the recommended methods detect most of the oxacillinresistant lineages, there are two situations that require additional phases to confirm sensitivity or resistance. The first is the occurrence of extremely heterogeneous lineages that are found to be sensitive by reference methods. The second is the occurrence of borderline resistance (MIC close to the sensitivity breakpoint), which must be differentiated from

Although *S. epidermidis* is the most frequently found species (Vuong & Otto, 2002), others are also associated with human infection, such as *S. haemolyticus*, which can be multiresistant and present intermediate resistance to vancomycin (Secchi et al., 2008). The main resistance mechanism is the presence of the *mecA* gene, inserted in the chromosomal cassette, known as staphylococcal chromosomal cassette mec (SCC*mec*). The detection of this gene and the typing of the chromosomal cassette by means of various molecular methods are important tools for the diagnosis and epidemiology of oxacillin-resistant *Staphylococcus* spp.

There are eleven SCC*mec* types, with subtypes, which are characterized by molecular tools, such as Multilocus Sequence Typing (MLST), pulsed-field gel electrophoresis (PFGE), *spa* typing and Multiplex PCR for SCC*mec* detection. They are useful in the characterization and detection of alterations in molecular structure. By using these methods, it was possible to identify pandemic clones as well as to characterize strains causing outbreaks in hospitals. Among these methods, the MLST is noteworthy due to its high reproducibility and capacity of detecting pandemic clones. Given the fact that vancomycin is a therapeutic option for oxacillin-resistant samples, with the emergence of vancomycin-resistant *Staphylococcus* spp., the characterization of circulating strains and clones is highly important. This chapter aimed at addressing aspects related to the molecular epidemiology in *Staphylococcus* spp. since these microorganisms have been increasingly frequent as agents of nosocomial and community infection.
