**2. Material and methods**

#### **2.1 Hospital setting**

The Instituto Nacional de Cardiologia "Dr. Ignacio Chavez" (CAR) is a tertiary-care cardiology hospital located, in Mexico City with 246 beds, distributed 10 wards: surgery, adults and pediatric cardiology, neumology, nephrology, coronary unit and others. In addition the hospital has 17 external services. The microbiology laboratory receives an average of 18,000 samples annually. The hospital has 5,800 admission and 5,700 discharges per year.

#### **2.2 Bacterial isolates**

We studied a total of ninety single-patient clinical MRSA isolates, between January 2002 and December 2009. The strains were collected from several clinical sources: bronchial secretions (n=34); wound secretions (n= 25), blood (n= 16); catheter (n=3); pleural liquid (n=3); peritoneal fluid (n=1) and others (n=13). MRSA strains were collected from different wards: pediatric surgery, adult surgery, coronary unit, nephrology, surgery and cardiology. Of the 90 MRSA isolates, 24 were from children and 66 were from adults.

#### **2.3 Antimicrobial susceptibility testing**

Antimicrobial susceptibility testing for MRSA isolates was performed using the automated method of MicroScan® (DADE-BEHRING, Sacramento, CA) for: penicillin, oxacillin,

amoxicillin, cefotaxime, cephalothin, cefazolin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, clarithromycin, gentamicin, rifampin, tetracycline and vancomycin, following the Clinical Laboratory Standards Institute guidelines (Clinical Laboratory Standards Institute [CLSI], 2009).

### **2.4 Molecular typing**

180 Gel Electrophoresis – Advanced Techniques

(Enright et al., 2000) The combination of these methods allows the unambiguous assignment

The prevalence of MRSA in Mexico differs widely from one hospital to another and according to different studies performed; an increasing frequency of MRSA (7% in 1989, 14% in 2001 and 36% in 2004) are documented by reports of routine oxacillin disk diffusion tests only (Alpuche et al., 1989; Calderón et al., 2002; Chávez, 2004). This is of great concern, because it is a common experience that once MRSA is introduced in a hospital it is difficult to eradicate it (Creamer et al., 2011; Rebmann & Aureden, 2011). However, reports from Mexico documenting the clonality of MRSA isolates are very scarce, Aires de Sousa *at al*. in 2001 (Aires de Sousa et al., 2001) reported dominant and unique MRSA clone designated the Mexican clone (I::NH::M), identified by PFGE among isolates collected in 1997, 1998 from a pediatric hospital in Mexico, which had a rather limited resistance profile. In more recent studies which involve strains collected for the period 1997 to 2003 in two Mexican hospitals, PFGE distributed the MRSA isolates into two types M (clone EMRSA-16-U.K) and C (clone New York/Japan) these two clones were distinguished by antibiogram and other molecular

The aim of this study was to identify MRSA clones circulating in a tertiary care hospital in Mexico City and their prevalence in the course of time 2002-2009. For this purpose, we used a phenotypic characterization and a combination of different molecular typing methods, including PFGE, hybridization with a Tn*554* and *mecA* probes, staphylococcal cassette

The Instituto Nacional de Cardiologia "Dr. Ignacio Chavez" (CAR) is a tertiary-care cardiology hospital located, in Mexico City with 246 beds, distributed 10 wards: surgery, adults and pediatric cardiology, neumology, nephrology, coronary unit and others. In addition the hospital has 17 external services. The microbiology laboratory receives an average of 18,000 samples annually. The hospital has 5,800 admission and 5,700 discharges

We studied a total of ninety single-patient clinical MRSA isolates, between January 2002 and December 2009. The strains were collected from several clinical sources: bronchial secretions (n=34); wound secretions (n= 25), blood (n= 16); catheter (n=3); pleural liquid (n=3); peritoneal fluid (n=1) and others (n=13). MRSA strains were collected from different wards: pediatric surgery, adult surgery, coronary unit, nephrology, surgery and cardiology. Of the 90 MRSA isolates, 24 were from children and 66 were from

Antimicrobial susceptibility testing for MRSA isolates was performed using the automated method of MicroScan® (DADE-BEHRING, Sacramento, CA) for: penicillin, oxacillin,

of collections of MRSA isolates or new MRSA clones (Enright et al., 2000).

properties (Echaniz et al., 2006; Velazquez et al., 2004).

chromosome *mec* (SCC*mec*) and MLST.

**2.3 Antimicrobial susceptibility testing** 

**2. Material and methods** 

**2.1 Hospital setting** 

**2.2 Bacterial isolates** 

per year.

adults.

The whole genomic DNA was prepared as described previously (Chung et al., 2000). After digestion with *Sma*I endonuclease, DNA was separated in a CHEF-DRII apparatus (Bio-Rad, Birmingham, U.K) (Chung et al., 2000). Strains HU25, HPV107, HDE288, BK2464, JP27 and 96/32010, representing the Brazilian, Iberian, Pediatric, New York/Japan-USA, New York/Japan-Japan and EMRSA-16-U.K clones, were included in the PFGE gels as controls. The control strains were kindly provided by Prof. Herminia de Lencastre from the Molecular Genetics laboratory Institute de Tecnología Química e Biologica da Universidad Nova de Lisboa. Criteria of Tenover were used to compare different clones (Tenover et al., 1995). Strains BK2464 and HDE288 were used as SCC*mec* controls. Hybridization of *SmaI* digests with *mecA* and Tn554 probes (de Lencastre et al., 1994), SCC*mec* typing (Oliveira & de Lencastre, 2002) and MLST (Enright et al., 2000) were performed as previously described. Briefly, MLST is based in internal fragments of seven housekeeping genes (*arcC*, *aroE*, *glpF, gmk, pta, tpi, yqiL*) for each isolates, the alleles at the seven loci defined the allelic profiles, which corresponded to a sequence type (ST). ST designations were those assigned the MLST data base (http://www.mlst.net). The SCC*mec* typing system is defined by combining the class of the *mec* gene complex with the cassette chromosome recombinase gene (*ccr*) allotypes. The polymorphism in the vicinity of the *mecA* gene detected by probe *ClaI*digested DNAs with a *mecA* probe and transposon Tn*554* insertion patterns detecting by probing *ClaI* digestion DNAs with a specific probe (de Lencastre et al., 1994, Enright et al., 2000; Oliveira & de Lencastre, 2002).
