**3. Oxacillin resistance**

96 Epidemiology Insights

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

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

Oxacillin resistance in CoNS is a problem in hospitals around the world, and there are reports of oxacillin-resistant samples in all continents (Witte, 1999). The use of methicillin, a semisynthetic penicillin, commenced in 1959, and only two years after its first use, the first report of a methicillin-resistant *Staphylococcus* spp. sample was published (Hiramatsu et al., 2001).

Oxacillin resistance rates vary among various studies, but they are usually high, above 50%. Chaudhury & Kumar (2007) reported that, in a study conducted in a tertiary Indian hospital, 64.6% of the CoNS samples were oxacillin resistant. The most prevalent species was *S. haemolyticus*, isolated from urine samples. Another study also performed in India, described

In the European continent, several reports described high oxacillin resistance levels in hospital wards. In a study analyzing samples collected from various hospitals in Eastern Europe in 2005, Sader et al. (2007) reported oxacillin resistance levels in CoNS which varied from 54.8% in Sweden to 83.3% in Greece. A study conducted at a university hospital in Turkey found 54.4% of resistant samples from a total of 158 isolated samples (Ercis et al., 2008). The rates found by a multi-center study conducted in the USA in 2007 and 2008 were

Nevertheless, in the last few years, resistance levels have stabilized. In Spain, a multi-center study on 146 hospitals detected oxacillin resistance in 61.3% of the samples in 2002 and 66.7% in 2006 (Cuevas et al., 2008). In Brazil, recent studies showed resistance rates of 69% (Caierão et al., 2004), 78.4% (Perez & d'Azevedo 2007) and even 88.1% (Antunes et al., 2007). In the case of non-*epidermidis* CNS, Secchi et al. (2008) reported 71% of resistant samples.

*Staphylococcus* spp.

community infection.

**2. Epidemiological aspects** 

resistance levels of approximately 63% (Jain et al., 2008).

of 74% of CoNS oxacillin-resistant samples (Sader & Jones, 2009).

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

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 (Tomacz et al., 1991).

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 (Swenson, 2002).

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

Epidemiological Aspects of Oxacillin-Resistant

integrated plasmid, pUB110 (Shore et al., 2005).

present in the other types and is referred to as

side of the cassette which is referred to as *orf*35.

smaller than type II (Shore et al., 2005).

al., 2005).

*Staphylococcus* spp.: The Use of Molecular Tools with Emphasis on MLST 99

community samples. Types I, II and III are mainly responsible for nosocomial infections, and they are significantly larger than the last types IV and V. Type-I cassette was described with a size of 34.364 bp, and it is the largest of the three. This cassette does not feature any inserted transposons or plasmids that provide resistance to other drugs besides methicillin or to heavy metals. It has a sub-type known as IA, which differs from type I for having an

The second cassette, referred to as II, has 53.017 bp, and in addition to the *mecA* and *mecRI* genes, which cause methicillin resistance, it contains transposon Tn 554. The latter is responsible for the resistance of this sample type to erythromycin and streptomycin. This cassette has a sub-type that is referred to as IIA, with a size of 40 Kb, thus being a little

Type-III cassette, the largest of the five types, has a size of 66.896 bp and contains genes *mecA*, *mecRI*, transposons Tn 554 and Tn 554 and plasmid pt 181. Transposon Tn 554 induces cadmium resistance, and plasmid pt 181 is responsible for tetracycline and mercury resistance. In addition to the information described above, the authors also mentioned differences between the *ccr* gene types, by describing, in type-III *SCCmec,* a gene that is not

resistance characteristics in type III can be used as selective markers. This cassette type presents two sub-types: IIIA, which does not have plasmid pt 181 and flanks with element IS 431, and IIIB, where there are no copies of plasmids pt 181 or tn 554 (Shore et al., 2005).

Type-IV SCC*mec* is mainly responsible for community infections. It is a small element that does not carry other resistance genes, except for *mecA*. It is also divided into multiple subtypes, which suggests that type-IV SCC*mec* is highly transmittable (Ito et al., 2004). The four sub-types of type-IV, -IVA, -IVB, -IVC and -IVD cassettes differ for presenting different sequences on the left extremity of the *ccr* complex, which is known as L – C Region (Shore et

Type-V SCC*mec* was identified by Ito et al. (2004) in an Australian isolate. It has the size of 27.624 bp, and it is a little larger than SCC*mec* IV although smaller than the other existing types. Similarly to type IV, it has only genes that codify methicillin resistance; however, differently from the other elements in this family, type V has a new *ccr* gene type that is characterized as type c and is individually found, contrarily to other elements, which have a pair of such genes. Another new element found only in type V is a restriction and modification system codified by genes V22 and V23. Recent studies associate the presence of this *mec* cassette type with community infections (O'Brien et al., 2005; Ho et al., 2007).

Oliveira et al. (2006) characterized type-VI SCC*mec*, which is found in samples belonging to the pediatric clone of MRSA (Methicillin Resistant *S.aureus*) samples and previously typed as SCC*mec* IV. The authors described differences in the *ccrAB* complex, identified as type 4,

Type-VII SCC*mec* was identified by Higuchi et al. (2008) through a detailed analysis of community *S.aureus* samples isolated in Taiwan. It has a size of 41.347 bp, and its *mec* complex is homologous to that found in type V, but presents substitutions, insertions and rearrangements that differentiate it. The main characteristics of this cassette type are the presence of the *ccrC* complex, of transposon ΨIS431 and of a unique sequence on the right

and the presence of the type-B *mec* complex, which does not have the *mecl* gene.

*ccr*. They also suggested that some of the

resistance mediated by the *mecA* gene as long as the clinical significance of the resistance determined by the *mecA* gene is greater.

The *mecA* gene is inserted in a transposable genetic element known as Staphylococcal Chromosomal Cassette *mec* (*SCCmec*). This element varies in its constitution and is divided into eleven types. The typing of *SCCmec* types is useful as an epidemiological tool (Mombach Pinheiro Machado et al., 2007) given that the different types are more prevalent in hospital and community environments. The SCC*mec* types (IWG-SCC, 2009) differ from one another in relation to the number of genes that they carry in their gene architecture (Hiramatsu et al., 2001). Some of these types are carriers of resistance genes that are determinant for multiple antibacterial drugs. In addition to beta-lactam antibiotics, macrolides, lincosamides, streptogramins, aminoglycosides and tetracycline are noteworthy. Hence, when a bacterial cell acquires such SCC*mec*, it at once acquires a multiple-resistance phenotype (Ito et al., 2003).

The SCC*mec* types have been defined by the combination of two parts: the *ccr* complex and the *mec* complex, with three phylogenetically distinct *ccr* genes classified as: *ccrA, ccrB* and *ccrC*. Additionally, there are five classes of *mec* gene complexes (classes *A*, *B*, *C1*, *C2* and class *D*) (Ito et al., 2004; IWG-SCC, 2009). The different SCC*mec* types are classified as: type-I SCC*mec* (class *B* and *ccrA1B1 mec* gene complex), type-II SCC*mec* (class *A* and *ccrA2B2 mec*  gene complex), type-III SCC*mec* (class *A* and *ccrA3B3 mec* gene complex), type-IV SCC*mec*  (class *B* and *ccrA2B2 mec* gene complex), type-V SCC*mec* (class *C2* and *ccrC mec* gene complex), type-VI SCC*mec* (class *B* and *ccrA4B4 mec* gene complex), type-VII SCC*mec* (class *C1* and *ccrC mec* gene complex) and type-VIII SCC*mec* (class *A* and *ccrA4B4 mec* gene complex). The remaining region of SCC*mec* is called the J region (Joining region), which constitutes non-essential components of the cassette that can carry additional antimicrobial resistance determinants (Hanssen & Sollid, 2006; IWG-SCC, 2009). Recently, types IX (class C2, ccr1 *me*c gene complex), X (class C1, ccr7 *mec* gene complex) and XI (class E, ccr8 *mec* gene complex) have been described (IWG-SCC, 2011) (Table 1).


Source: http://www.sccmec.org/Pages/SCC\_TypesEN.html. Accessed on 06/20/2011

Table 1. SCC*mec* types identified in *S.aureus*

The first three types were detailed by Ito et al. in 2001, and the same author also reported and described type V in 2004. Ma et al. (2002) described type IV, which is mainly found in

resistance mediated by the *mecA* gene as long as the clinical significance of the resistance

The *mecA* gene is inserted in a transposable genetic element known as Staphylococcal Chromosomal Cassette *mec* (*SCCmec*). This element varies in its constitution and is divided into eleven types. The typing of *SCCmec* types is useful as an epidemiological tool (Mombach Pinheiro Machado et al., 2007) given that the different types are more prevalent in hospital and community environments. The SCC*mec* types (IWG-SCC, 2009) differ from one another in relation to the number of genes that they carry in their gene architecture (Hiramatsu et al., 2001). Some of these types are carriers of resistance genes that are determinant for multiple antibacterial drugs. In addition to beta-lactam antibiotics, macrolides, lincosamides, streptogramins, aminoglycosides and tetracycline are noteworthy. Hence, when a bacterial cell acquires such SCC*mec*, it at once acquires a multiple-resistance

The SCC*mec* types have been defined by the combination of two parts: the *ccr* complex and the *mec* complex, with three phylogenetically distinct *ccr* genes classified as: *ccrA, ccrB* and *ccrC*. Additionally, there are five classes of *mec* gene complexes (classes *A*, *B*, *C1*, *C2* and class *D*) (Ito et al., 2004; IWG-SCC, 2009). The different SCC*mec* types are classified as: type-I SCC*mec* (class *B* and *ccrA1B1 mec* gene complex), type-II SCC*mec* (class *A* and *ccrA2B2 mec*  gene complex), type-III SCC*mec* (class *A* and *ccrA3B3 mec* gene complex), type-IV SCC*mec*  (class *B* and *ccrA2B2 mec* gene complex), type-V SCC*mec* (class *C2* and *ccrC mec* gene complex), type-VI SCC*mec* (class *B* and *ccrA4B4 mec* gene complex), type-VII SCC*mec* (class *C1* and *ccrC mec* gene complex) and type-VIII SCC*mec* (class *A* and *ccrA4B4 mec* gene complex). The remaining region of SCC*mec* is called the J region (Joining region), which constitutes non-essential components of the cassette that can carry additional antimicrobial resistance determinants (Hanssen & Sollid, 2006; IWG-SCC, 2009). Recently, types IX (class C2, ccr1 *me*c gene complex), X (class C1, ccr7 *mec* gene complex) and XI (class E, ccr8 *mec*

SCC*mec* type *mec* gene complex *ccr* gene complex

1 (A1B1) 2 (A2B2) 3 (A3B3) 2 (A2B2) 5 (C1) 4 (A4B4) 5 (C1) 4 (A4B4) 1 (A1B1) 7 (A1B6) 8 (A1B3)

B A A B C2 B C1 A C2 C1 E

Source: http://www.sccmec.org/Pages/SCC\_TypesEN.html. Accessed on 06/20/2011

The first three types were detailed by Ito et al. in 2001, and the same author also reported and described type V in 2004. Ma et al. (2002) described type IV, which is mainly found in

gene complex) have been described (IWG-SCC, 2011) (Table 1).

determined by the *mecA* gene is greater.

phenotype (Ito et al., 2003).

I II III IV V VI VII VIII IX X XI

Table 1. SCC*mec* types identified in *S.aureus*

community samples. Types I, II and III are mainly responsible for nosocomial infections, and they are significantly larger than the last types IV and V. Type-I cassette was described with a size of 34.364 bp, and it is the largest of the three. This cassette does not feature any inserted transposons or plasmids that provide resistance to other drugs besides methicillin or to heavy metals. It has a sub-type known as IA, which differs from type I for having an integrated plasmid, pUB110 (Shore et al., 2005).

The second cassette, referred to as II, has 53.017 bp, and in addition to the *mecA* and *mecRI* genes, which cause methicillin resistance, it contains transposon Tn 554. The latter is responsible for the resistance of this sample type to erythromycin and streptomycin. This cassette has a sub-type that is referred to as IIA, with a size of 40 Kb, thus being a little smaller than type II (Shore et al., 2005).

Type-III cassette, the largest of the five types, has a size of 66.896 bp and contains genes *mecA*, *mecRI*, transposons Tn 554 and Tn 554 and plasmid pt 181. Transposon Tn 554 induces cadmium resistance, and plasmid pt 181 is responsible for tetracycline and mercury resistance. In addition to the information described above, the authors also mentioned differences between the *ccr* gene types, by describing, in type-III *SCCmec,* a gene that is not present in the other types and is referred to as *ccr*. They also suggested that some of the resistance characteristics in type III can be used as selective markers. This cassette type presents two sub-types: IIIA, which does not have plasmid pt 181 and flanks with element IS 431, and IIIB, where there are no copies of plasmids pt 181 or tn 554 (Shore et al., 2005).

Type-IV SCC*mec* is mainly responsible for community infections. It is a small element that does not carry other resistance genes, except for *mecA*. It is also divided into multiple subtypes, which suggests that type-IV SCC*mec* is highly transmittable (Ito et al., 2004). The four sub-types of type-IV, -IVA, -IVB, -IVC and -IVD cassettes differ for presenting different sequences on the left extremity of the *ccr* complex, which is known as L – C Region (Shore et al., 2005).

Type-V SCC*mec* was identified by Ito et al. (2004) in an Australian isolate. It has the size of 27.624 bp, and it is a little larger than SCC*mec* IV although smaller than the other existing types. Similarly to type IV, it has only genes that codify methicillin resistance; however, differently from the other elements in this family, type V has a new *ccr* gene type that is characterized as type c and is individually found, contrarily to other elements, which have a pair of such genes. Another new element found only in type V is a restriction and modification system codified by genes V22 and V23. Recent studies associate the presence of this *mec* cassette type with community infections (O'Brien et al., 2005; Ho et al., 2007).

Oliveira et al. (2006) characterized type-VI SCC*mec*, which is found in samples belonging to the pediatric clone of MRSA (Methicillin Resistant *S.aureus*) samples and previously typed as SCC*mec* IV. The authors described differences in the *ccrAB* complex, identified as type 4, and the presence of the type-B *mec* complex, which does not have the *mecl* gene.

Type-VII SCC*mec* was identified by Higuchi et al. (2008) through a detailed analysis of community *S.aureus* samples isolated in Taiwan. It has a size of 41.347 bp, and its *mec* complex is homologous to that found in type V, but presents substitutions, insertions and rearrangements that differentiate it. The main characteristics of this cassette type are the presence of the *ccrC* complex, of transposon ΨIS431 and of a unique sequence on the right side of the cassette which is referred to as *orf*35.

Epidemiological Aspects of Oxacillin-Resistant

Hungarian clone (*SCCmec III*) (Arakere et al., 2005)

infections (Velasquez-Meza et al., 2004).

**6. Epidemiology and MLST** 

*epidermidis*.

Poland in 1994-98 and 1990-98 (Oliveira et al., 2002).

Sweden from 1997 to 2000 (Aires de Souza & De Lencastre, 2004).

clones from circulating *S. aureus* and *S. epidermidis* samples.

83.3% of samples belonging to clone ST 239 *SCCmec* III (Neela et al., 2010).

April to June 2001 and 31 MSSA samples isolated from 1999 to 2002 were used.

*Staphylococcus* spp.: The Use of Molecular Tools with Emphasis on MLST 101

in Brazil. The samples were characterized as belonging to the same clone by methods such as PFGE and by showing patterns of transposon *Tn554* and the polymorphism of the *mecA* gene (Teixeira et al., 1995). In other countries, the Brazilian clone is also disseminated, as is the case of the Czech Republic, where the isolation of this clone represented 80% of the MRSA samples found in 1996-1997 (Melter et al., 2003). The Brazilian clone was also described in India, in two hospitals in the region of Bengalore, conjointly with the

Other clones are distributed in several parts of the world. The Iberian clone was firstly described in samples from hospitals in Barcelona and Madrid, Spain, and in Lisbon, Portugal. These samples were typed by the PFGE method and probe hybridization, producing a pattern that characterized them as belonging to the same clone. This clone is also described in other countries, such as the Czech Republic (Melter et al., 2003). The clone known as New York – Japan, firstly isolated in the USA in 1994-98 and in Japan in 1997-98 (Oliveira et al., 2002), was also predominant in Mexico during a study on 98 MRSA samples, thus replacing the local clone, known as Mexican (PFGE M, type-IV SCC*mec*), in nosocomial

The Hungarian clone, firstly identified in Hungary in 1993-94, was characterized by the same methods used for the clones described above in studies on MRSA samples from hospitals in 06 provinces in that country (De Lencastre, 1997). The pediatric clone was isolated in large numbers in 1996-98 in Colombia, and it is also found in Argentina and

The last of the large pandemic clones, referred to as EMRSA-16, is prevalent in hospitals in the United Kingdom, Mexico and Greece, in addition to being responsible for an outbreak in

The MLST method was developed by Maiden et al. (1998) by the sequencing and analysis of the loci of eleven constitutive genes of *Neisseria meningitidis*, and it is presently applied in molecular epidemiology studies on various pathogens, among which are *S. aureus* and *S.* 

This methodology has been used in numerous studies on molecular epidemiology with good results due its good reproducibility, thus allowing for the detection of pandemic

Geng et al. (2010) reported the presence of clone ST59-MRSA-IV in China in community samples isolated from 47 children with impetigo and abscesses. In Malaysia, a study on 36 samples isolated from a hospital in Klang Valley for a five-month period reported a rate of

MLST has been used for studies in which circulating clones are identified in replacement of another already established clone. Sola et al. (2006) reported the emergence of a new clone identified as ST5 in hospitals in Cordoba, Argentina, in replacement of the Brazilian clone (ST239), which circulates in that region. In that study, 103 MRSA samples isolated from

Recently, Zhang et al. (2009) have described a new SCC*mec* type which has been denominated as SCC*mec* VIII and was found in an MRSA sample from a hospital. This new cassette has a size of 32.168 base pairs, and genes *mecA mecR1 mecI* of the *mec* complex and *ccrA4 ccrB4* of the *ccr* complex are present in addition to transposon Tn554, which is also present in type II.
