**1. Introduction**

178 Gel Electrophoresis – Advanced Techniques

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> Methicillin-resistant *Staphylococcus aureus* (MRSA) is an important threat to hospitalized patients worldwide and is responsible for a wide range of human diseases, including septicemia, endocarditis, pneumonia, osteomyelitis, toxic shock syndrome, and bacteremia (Tenover & Gaynes, 2000). This species nevertheless represents a serious public health burden, particularly the clones which are resistant to methicillin and other classes of antibiotics; the emergence of penicillin-methicillin-, and recently high-level vancomycinresistant strains emphasize the importance and urgency of such rational prescribing policy for the treatment of MRSA infections (Appelbaum, 2007; Goldstein 2007). Multiple studies have shown clonal spreads of epidemic MRSA strains within hospitals, between hospitals within a country (Breurec et al., 2011; Nübel et al., 2010), and also between countries and continents (Breurec et al., 2011; Deurenberg et al., 2009; Diekema et al. 2001). There are only a limited number of nosocomial MRSA clones spread worldwide (the Iberian [ST247- SCC*mec* I], the Brazilian [ST239-IIIA], the Hungarian [ST239-III], the New York/Japan [ST5- II], the Pediatric [ST5-VI], the Berlin [ST45-IV], EMRSA-15 [ST22-IV], and the EMRSA-16 [ST36-II] clones) (Enright et al., 2002; Oliveira et al., 2001).

> Molecular typing of MRSA is used to support infection control measures. Although Pulsedfield gel electrophoresis (PFGE) is well known and considered as golden standard, for establishing clonal relationships at the local level, its detection capacity seems to make it also too discriminative for global comparisons (McDougal et al.2003; Murchan et al., 2003). Recently multilocus sequence typing (MLST) has been proven to be the most adequate method both for long-term and global epidemiologic studies and for population genetic studies. Typing methods based on sequencing of more stable housekeeping genes (MLST) allow the creation of Internet-based curate databases and inter-laboratory data exchange

<sup>\*</sup> Corresponding Author

Pulsed Field Gel Electrophoresis in Molecular Typing and

**2.4 Molecular typing** 

2000; Oliveira & de Lencastre, 2002).

arithmetic average (UPGMA).

**3.1 Antimicrobial susceptibility** 

**3. Results** 

vancomycin.

**2.5 Computer-fingerprinting analysis** 

Epidemiological Detection of Methicillin Resistant *Staphylococcus aureus* (MRSA) 181

amoxicillin, cefotaxime, cephalothin, cefazolin, imipenem, trimethoprim-sulfamethoxazole, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, clarithromycin, gentamicin, rifampin, tetracycline and vancomycin, following the Clinical Laboratory Standards

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.,

The computer analysis of the banding patterns obtained by PFGE was done using the NTSYSpc software version 2.0.2.11 (Applied Biostatistics Inc.) after visual inspection. Each gel included reference strain *S. aureus* NCTC 8325 to normalize the PFGE profiles. For clusters analyses, the Dice coefficients were calculated to compute the matrix similarity and were transformed into an agglomerative cluster by the unweighted pair group method with

The 90 isolates showed resistance to penicillin (100%), oxacillin (99.3%), amoxicillin (100%) , cefotaxime (100%), cephalothin (100%), cefazolin (100%), chloramphenicol (100%), imipenem (99.3%), ciprofloxacin (87.7%); eleven strains (12.2%) showed low susceptibility for clindamycin, erythromycin, clarithromycin and were susceptible to ciprofloxacin; two strains (2.2%) showed low susceptibility for oxacillin (MIC 4µg/mL) and imipenem. All strains were susceptible to rifampin, tetracycline, gentamicin, trimethoprim-sulfamethoxazole and

Institute guidelines (Clinical Laboratory Standards Institute [CLSI], 2009).

(Enright et al., 2000) The combination of these methods allows the unambiguous assignment of collections of MRSA isolates or new MRSA clones (Enright et al., 2000).

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

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