**5. Genetic characteristics of CA-MRSA**

As a result of these observations genetic studies began focusing on molecular mechanisms responsible for the resistance to antibiotics and identification of MRSA strains. These strains had acquired and integrated into their genome harboring resistance genes, called Staphylococcal cassette chromosome *mec* (SCC*mec*). SCC*mec* present the gene responsible for methicillin and other beta-lactamic antibiotic resistance (*mecA*), and can carry genes that determine resistance to other classes of antibiotics. Strains related to community-acquired infections contain the smaller and lighter mobile element, the types and subtypes of SCC*mec*  IV or V (21 to 25 kb) (Zhang et al., 2005).

Strains of HA-MRSA carry heavier mobile elements (SCC*mec* I to III) because they have genes that encode for resistance to several antimicrobial classes (Okuma et al., 2002). The mobile element SCC*mec* is characterized by the presence of essential genetic elements: the *mec* complex (classes A to E), the ccr complex (Hanssen & Sollid, 2007), junkyard regions (J) (Zhang et al., 2009) and end 3' is connected to the open reading frame (*ORF*), *orfX*. The SCC*mec* is integrated into the chromosome of *Staphylococcus* called attBscc a specific site located downstream of *orfX* (Zhang et al., 2008). There are several types of SCC*mec* (I to XI) (IWG-SCCmec, 2011) in addition to subtypes IIA to E and the IVg to IVa (Hanssen & Sollid, 2007). The *ccr* complex has five different allotypes for the *ccr*A and the *ccr*B: *ccr*A1 to *ccr*A5, the *ccr*B1 to *ccr*B4 and *ccr*B6 and *ccr*C1 (IWG-SCC*mec*, 2011). The allotypes of the SCC*mec* complexes are characterized by the presence of certain *ccr* genes (Ito et al., 2004). The *ccr*A

FNB expression of genes that encode the fibronectin binding protein, but the mechanisms have not yet been defined. This may explain why patients who had prior use of

The colonization by CA-MRSA in adults and children differs in the profiles of resistance to beta-lactam antibiotics, in which it is more typical that multi-sensitive strains colonize and affect children. This is mainly due to different environments they attend and hygienic practices follow. In addition, the antibiotics used in children may differ from those administered to adults, thus providing a different selective pressure in the community. Among adults CA-MRSA strains are more frequently resistant to gentamicin, tetracycline, ciprofloxacin, clindamycin and erythromycin than those observed in isolates from children

One study evaluated *S. aureus* strains isolated from blood cultures as the type of SCC*mec*, the presence of PVL and analyzed the clonal profile by PFGE. In this study, they mainly included cases of HA-MRSA defined by the following isolation criterion for MRSA: must be isolated for 48 hours after hospital admission, previous isolation of MRSA colonized or infected patient during hospitalization or surgical procedures in the 12 months preceding the isolation, patients who underwent installation of a catheter or invasive devices. Of all the samples analyzed, 65% had the cassette type IV and that PVL was present in 92% of these samples. The clonal profile of 92% of samples with SCC*mec* type IV was USA300-ST8 (Gonzalez et al., 2006). The isolation of *S. aureus* resistant to methicillin with the cassette type IV suggests that these strains, particularly the USA300, presents an adaptation that expands beyond the community environment which may cause a change in the

As a result of these observations genetic studies began focusing on molecular mechanisms responsible for the resistance to antibiotics and identification of MRSA strains. These strains had acquired and integrated into their genome harboring resistance genes, called Staphylococcal cassette chromosome *mec* (SCC*mec*). SCC*mec* present the gene responsible for methicillin and other beta-lactamic antibiotic resistance (*mecA*), and can carry genes that determine resistance to other classes of antibiotics. Strains related to community-acquired infections contain the smaller and lighter mobile element, the types and subtypes of SCC*mec* 

Strains of HA-MRSA carry heavier mobile elements (SCC*mec* I to III) because they have genes that encode for resistance to several antimicrobial classes (Okuma et al., 2002). The mobile element SCC*mec* is characterized by the presence of essential genetic elements: the *mec* complex (classes A to E), the ccr complex (Hanssen & Sollid, 2007), junkyard regions (J) (Zhang et al., 2009) and end 3' is connected to the open reading frame (*ORF*), *orfX*. The SCC*mec* is integrated into the chromosome of *Staphylococcus* called attBscc a specific site located downstream of *orfX* (Zhang et al., 2008). There are several types of SCC*mec* (I to XI) (IWG-SCCmec, 2011) in addition to subtypes IIA to E and the IVg to IVa (Hanssen & Sollid, 2007). The *ccr* complex has five different allotypes for the *ccr*A and the *ccr*B: *ccr*A1 to *ccr*A5, the *ccr*B1 to *ccr*B4 and *ccr*B6 and *ccr*C1 (IWG-SCC*mec*, 2011). The allotypes of the SCC*mec* complexes are characterized by the presence of certain *ccr* genes (Ito et al., 2004). The *ccr*A

ciprofloxacin have a higher likelihood of MRSA colonization.

(David et al., 2006).

epidemiology of CA-MRSA.

**5. Genetic characteristics of CA-MRSA** 

IV or V (21 to 25 kb) (Zhang et al., 2005).

and *ccr*B genes encode for recombinases of the family "invertase/resolvases." These enzymes mediate the integration within and outside of the SCC*mec* thus giving mobility to the chromosome cassette (Zhang et al., 2005).

The J regions encode several pseudogenes apparently with functions related to the bacterial metabolism (Zhang et al., 2005), and also contain genes for resistance mediated by plasmids or transposons to non beta-lactam antibiotics and heavy metals (Zhang et al., 2009). They are divided into three segments: J1, which is the region between the *ccr* complex the right chromosomal junction and the *ccr* gene complex; J2, between the *ccr* and *mec* regions, and J3 which is located between *orfX* and *mec*. Variations in the J regions within the same *mec-*ccr gene complex are used for defining SCC*mec* subtypes.

The SCC*mec* can be found in several species of *Staphylococcus* spp., such as *S. aureus*, *S. epidermidis, S. haemolyticus, S. hominis* and *S. warneri* (Hanssen & Sollid, 2007). The origin of SCC*mec* is unknown, and there have been no reports that any other genus than *Staphylococcus* carries the Staphylococal cassette chromosome. The presence of SCC*mec* type IV in *S. epidermidis* in healthy people suggests that this can be responsible for the conversion of CA-MSSA for CA-MRSA (Hanssen et al., 2004) where the transmission of the mobile element occurs mainly by transduction mediated by bacteriophages (Ito et al., 1999).

The *mecA* gene codifies a penicillin binding protein PBP 2 'or 2A (Menegoto & Picoli, 2007) present on the outer surface of the cytoplasmic membrane (Ricardo, 2004). In susceptible strains, conventional PBPs have a high affinity with the beta-lactam antibiotics which prevents the proper formation of cell walls. However, the second PBP has low affinity to this class of antimicrobials, which explains resistance to antimicrobials of the group of methicillin (Menegoto & Picoli, 2007; Ito et al., 2001).

The *mecA* gene is regulated by two genes *mecI* and *mecR1* that have similar functions of the *blaR1* and *blaI* mechanism that regulates the production of beta-lactamase (Chambers, 1997). The *mecA* gene is regulated by a repressor *mecI*, a signal transducer and trans-membrane sensitive to the beta-lactam *mecRI*; both are divergently transcribed. In the absence of betalactam antimicrobial, *mecI* represses the expression of *mecA* and *mecRI-mecI*. However in the presence of beta-lactam antibiotics, the *mecI* is cleaved autocatalytically, and a metalloprotease domain, located in the cytoplasmic portion *mecRI*, becomes active. What allows the *mecA* gene transcription and subsequent synthesis of PBP2a is the cleavage of the *mecI* by the metalloprotease and its connection on the operative region of the *mecA* gene (Berger-Bachi & Rohrer, 2002). The presence of insertion sequences IS431 and IS1272 results in the induction of the *mecA* gene (Katayama et al., 2001).

Other resistance mechanisms have been identified in strains that lack the *mecA* gene, for example, the overproduction of beta-lactamase responsible for the inactivation of oxacillin or modified resistance (MOD-SA) mediated by different types of PBPs with changed affinities to this antibiotic. Strains with this profile are called borderline resistant (Wey et al., 1990).

CA-MRSA can express resistance inducible clindamycin resistance, an option for treating both MSSA and MRSA in particular in cases of toxic shock syndrome. In MLSBi positive strains (macrolide-lincosamides-streptogramin B resistance), an inducer promotes methylase production expressed by the gene *emr* and leading to the subsequent methylation of the 23S

CA-MRSA: Epidemiology of a Pathogen of a Great Concern 63

The use of cefoxitin disk diffusion to detect strains carrying the *mecA* gene is widely applied, but there are laboratories that use only the broth dilution test to determine the MIC of a sample. One study showed correlation of the MIC of cefoxitin in the presence of the *mecA* gene for both *Staphylococcus aureus* and coagulase-negative *Staphylococcus* testing three brands of Mueller-Hinton broth. After testing, the strains were sent to the CDC for molecular detection of the *mecA* gene by PCR. For *mecA* negative *S. aureus* strains the MIC of cefoxitin was <4ug/ml and for *mecA* positive the MIC was >6 or 8 µg/ml when read in 18 hours of incubation, the result was highly sensitive and specific (99.7 and 100%). However, for coagulase-negative levels of cefoxitin were sensitive (at 24 hours, 94 to 99% for *S. epidermidis* and 91 to 100% non *S. epidermidis*), but not specific (24 hours, 85 to 91 % for *S. epidermidis* and 54 to 69% non *S. epidermidis*) to detect the presence of *mecA*

Screening with oxacillin agar supplemented with 4% NaCl proposed by CLSI, as well as the oxacillin agar dilution and broth microdilution with 2% NaCl is used with results close to 100% sensitivity for the detection of the *mecA* gene. However, when it comes to very diverse strains, detection of oxacillin resistance and the *mecA* gene are compromised, especially in disk diffusion testing, decreasing its specificity (classifying *mecA* negative strains as resistant) and agar screening, where low sensitivity values are obtained when more

Experiments aimed at improving identification and detection MRSA strains, particularly heterogeneous strains, which are more difficult to detect by conventional methods. The sensitivity of disk diffusion testing using 1g oxacillin disk increased from 83.5% at 35 ° C to 91.7% when incubated at 30° C. Similarly, the sensitivity and specificity of the cefoxitin disk diffusion of 30 µg were 100% at a temperature of 30°C. Tests with cefoxitin are effective, because this antibiotic is able to detect strains inducing heterogeneous subpopulations expressing the *mec*A gene better than oxacillin. The sensitivity of the test screening at 6 µl oxacillin on Mueller-Hinton agar supplemented with 4% NaCl was 91.7% and specificity of 100% in 24 hours of incubation at 35°C. The latex agglutination test for detection of PBP2a can reach a sensitivity of 100%, being able to identify strains with low levels of PBP2a

Pereira et al. (2009) analyzed the sensitivity of the method of disk diffusion with oxacillin and cefoxitin disks, incubated for 24 h at 35°C in 100 samples of *S. aureus* isolated from pediatric and neonatal ICUs. They reported oxacillin disk sensitivity of 94.4% and specificity of 98%, while the cefoxitin disk presented 98% sensitivity and 100% specificity. The same authors also tested the screening method on Mueller Hinton agar with 6µg/ml of oxacillin

Martins et al. (2010) compared the screening methods of disk diffusion and E-test for detection of oxacillin resistance. They found that approximately 45% of samples were positive for the *mecA* gene, and the disk diffusion method with oxacillin disk showed a sensitivity of 86.9% and specificity 91.1%, respectively. The screening method showed the same sensitivity and specificity of 91.3%, while the E-test showed the same specificity of

The results found in several studies to determine the accuracy of these methods show that the results can vary depending on a number of factors especially the origin of the samples

and 4% NaCl and found a sensitivity of 98% and specificity of 100%.

other methods and a sensitivity of 97.8% (Martin et al., 2010).

and the criteria used for its execution.

(Swenson et al., 2009).

(Cauwelier et al. 2004).

heterogenenous strains were tested (Swenson, 2002).

ribosome unit causing an expression of the resistance to lincosamides (e.g. clindamycin). Phenotypically the strains are resistant to erythromycin and clindamycin sensitive, but when the disk of erythromycin is set at 15mm from the clindamycin disk, the clindamycininduced resistant strain expressed the resistance forming a D zone near clindamycin. The presence of the *mecA* gene in CA-MRSA does not fulfill the identification criteria in itself for the expression of the inducible clindamycin resistance (Patel et al., 2006).

Boyle-Vavra et al. (2005) conducted a study with patients who had skin and soft tissue infections and another with colonized individuals with CA-MRSA, in order to test the resistance to various antibiotics and isolate those carrying the *mecA* gene. The results showed that 94% of the strains recovered from these infections and 85.3% of the strains that colonized healthy individuals showed resistance to three or more non-beta-lactam antibiotics. The *SCCmec* IV was present in 34% of the samples from individuals who had at least one risk factor for acquisition of MRSA and 14.7% of the isolates showed a different SCC*mec* called SCCmec VT (Boyle-Vavra et al., 2005).
