**6. Detection of phenotypic resistance to oxacillin**

There are several tests that detect phenotypic resistance to oxacillin, such as the E-test with oxacillin agar screening test, or Kirb-Bauer disk diffusion test, cefoxitin disk diffusion, determining the minimum inhibitory concentration (MIC) (Felten et al., 2002), latex agglutination test to detect PBP2a (Bressler et al., 2005; Martins et al. 2010).

CHROMagar is used for the cultivation of *S. aureus* resulting in pink-colored colonies. Carricajo et al. (2001) tested the sensitivity and specificity of this medium compared with conventional (Columbia agar with 5% horse blood and chocolate agar). The CHROMagar allowed 22 strains of *S. aureus* grown from polymicrobial samples unlike conventional methods. Despite the high cost, this method had a sensitivity of 98% and specificity of 100% (Carricajo et al., 2001).

The latex agglutination test detects the PBP2a protein in the cell wall of staphylococci. To assess the effectiveness of this test, Bressler et al. (2005) compared it with PCR for *mecA* and determined the MIC of the MicroScan PC20 samples tested. The latex agglutination test had a 99% agreement with MicroScan; three strains that were false positive in the *mecA* agglutination had two strains that were resistant, and one presented sensitivity. The latter had a different aminoacid in PBP2a, the M483I that conferred susceptibile to oxacillin. This amino acid can increase the affinity for this antibiotic or eliminate the transpeptidase activity of PBP2a (Bressler et al., 2005).

The disk diffusion test in mannitol salt agar also showed favorable results in the detection of oxacillin resistance and a method almost as effective as the method of disk diffusion in Mueller-Hinton agar (Kampf et al., 1998).

The presence of heteroresistant strains can complicate the detection of the resistance by these methods due to the false-negative results. The disk diffusion test with cefoxitin is widely used due to the induction of *mecA* gene expression with the production of PBP2a, which promotes antibiotics in strains apparently sensitive to oxacillin (Feltenet al., 2002). Factors such as lower incubation temperature (30 to 35 º C), osmolarity of the medium (2-4% NaCl), extended incubation period and inoculum density favor the detection of heteroresistant strains (Camarena & Sanchez, 2009).

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

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

There are several tests that detect phenotypic resistance to oxacillin, such as the E-test with oxacillin agar screening test, or Kirb-Bauer disk diffusion test, cefoxitin disk diffusion, determining the minimum inhibitory concentration (MIC) (Felten et al., 2002), latex

CHROMagar is used for the cultivation of *S. aureus* resulting in pink-colored colonies. Carricajo et al. (2001) tested the sensitivity and specificity of this medium compared with conventional (Columbia agar with 5% horse blood and chocolate agar). The CHROMagar allowed 22 strains of *S. aureus* grown from polymicrobial samples unlike conventional methods. Despite the high cost, this method had a sensitivity of 98% and specificity of 100%

The latex agglutination test detects the PBP2a protein in the cell wall of staphylococci. To assess the effectiveness of this test, Bressler et al. (2005) compared it with PCR for *mecA* and determined the MIC of the MicroScan PC20 samples tested. The latex agglutination test had a 99% agreement with MicroScan; three strains that were false positive in the *mecA* agglutination had two strains that were resistant, and one presented sensitivity. The latter had a different aminoacid in PBP2a, the M483I that conferred susceptibile to oxacillin. This amino acid can increase the affinity for this antibiotic or eliminate the transpeptidase

The disk diffusion test in mannitol salt agar also showed favorable results in the detection of oxacillin resistance and a method almost as effective as the method of disk diffusion in

The presence of heteroresistant strains can complicate the detection of the resistance by these methods due to the false-negative results. The disk diffusion test with cefoxitin is widely used due to the induction of *mecA* gene expression with the production of PBP2a, which promotes antibiotics in strains apparently sensitive to oxacillin (Feltenet al., 2002). Factors such as lower incubation temperature (30 to 35 º C), osmolarity of the medium (2-4% NaCl), extended incubation period and inoculum density favor the detection of

the expression of the inducible clindamycin resistance (Patel et al., 2006).

agglutination test to detect PBP2a (Bressler et al., 2005; Martins et al. 2010).

SCC*mec* called SCCmec VT (Boyle-Vavra et al., 2005).

(Carricajo et al., 2001).

activity of PBP2a (Bressler et al., 2005).

Mueller-Hinton agar (Kampf et al., 1998).

heteroresistant strains (Camarena & Sanchez, 2009).

**6. Detection of phenotypic resistance to oxacillin** 

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* (Swenson et al., 2009).

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 heterogenenous strains were tested (Swenson, 2002).

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 (Cauwelier et al. 2004).

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

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

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 the criteria used for its execution.

CA-MRSA: Epidemiology of a Pathogen of a Great Concern 65

mucosa of the trachea (Lina et al., 1999). Due to these facts, studies have proposed that the propensity of CA-MRSA infections cause severe skin and soft tissue lesions, and possibly necrotizing pneumonia, is due to the presence of the gene encoding the production of PVL

PVL was first described as a "substance leukocidin" by Van deVelde in 1894 but was first associated with skin and soft tissue infections by Panton and Valentine in 1932. The acquisition of genes encoding PVL is made by transduction of a specific type of bacteriophage, phiSLT, which causes cytolysis in carrier gene cells and transport this gene to another cell. From its transcription two exoproteins , the Luks-PV and LukF-PV are produced, acting through the synergistic action of both subunits (Melles et al., 2006, Saïd-Salim et al., 2005). When secreted, LukS-PV initiates a connection to the membrane of the polymorphonuclear leukocyte (PMN) and is dimerized with LukF-PV, alternating one and another until the complete formation of a heptamer. Calcium channels are formed by inducing the production of interleukins and inflammatory mediators. Because of this evidence, probably the PVL is not directly associated with tissue necrosis, but related to lysosomal granules released by cytotoxic lysis of PMN, the release of granulocyte reactive

The main target of PVL is human and rabbit neutrophils, having little or no effect on nonhuman primates and mice (Löffler et al., 2010). The reason for differences in sensitivities to PVL is not yet fully known but may be related to receptor/signal transducers that are species-specific (Löffler et al., 2010). Its action is directly related to the concentration: at high concentrations, it causes cell lysis; at low concentrations, it mediates caspase dependent apoptosis by forming pores in the membrane of mitochondria (Boyle-Vavra & Daum, 2007; Lo & Wang 2011). Sub-lytic concentrations induce apoptosis of human neutrophils within 6 hours, and at high concentrations leads to cell death in only 1 hour (Lo & Wang, 2011).

The pathogenicity of *S. aureus* depends on several determinants, among them, the production of toxins and extracellular membrane components (Jarraud et al., 2002; Gandhinagar & Silva, 2004). The molecular basis of pathogenicity of *S. aureus* depends on the expression of broad classes of accessory genes producing components of the cell wall and extracellular proteins. The expression of these virulence factors is regulated by genes in the operon *agr* (accessory gene regulator), which regulates the expression of genes for toxins and adhesins (Purcell & Fergie, 2005). Enzymes such as coagulase and catalase are

The toxins are related to staphylococcal toxic shock syndrome (TSS), staphylococcal scarlet fever (both due to the toxin of toxic shock syndrome 1 [TSST-1] and staphylococcal enterotoxins), scalded skin syndrome (SSS due to exfoliatins) and food poisoning (SE's, staphylococcal enterotoxins) (Santos et al., 2007; Jarraud et al., 2002; Johnson et al., 1991). Genetic sequencing of a strain called MW2, CA-MRSA, revealed the presence of genes responsible for specific virulence factors such as the PVL toxin, staphylococcal enterotoxin H

Toxic shock syndrome was related primarily to the use of a particular brand of tampons in 1980. The TSS can occur in patients of any age, with the main presenting symptoms being

responsible for its evasion of the immune system (Gandhinagar & Silva, 2004).

(*seh*) and staphylococcal enterotoxin C (*sec*) (Saïd-Salim et al., 2005).

oxygen or even the inflammatory cascade (Boyle-Vavra & Daum, 2007).

**9. Other virulence factors of MRSA** 

(Saïd-Salim et al. 2005).
