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Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in two metropolitan areas of São Paulo State, southeast Brazil

Abstract

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

MRSA; nosocomial; molecular characterization; PCR


Molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in two metropolitan areas of São Paulo State, southeast Brazil

Gisele Gentile CuryI; Cristiane MobilonII; Eliana Guedes StehlingII; Marcelo LancellottiII; Marcelo de Carvalho RamosI; Roberto MartinezIII; Marcelo BrocchiII; Wanderley Dias da SilveiraII

IDepartment of Internal Medicine, School of Medicine, Unicamp

IIDepartment of Microbiology and Immunology, Biology Institute, Campinas State University, Campinas (Unicamp), SP

IIIDepartment of Internal Medicine; School of Medicine; São Paulo State University at Ribeirão Preto-USP, Ribeirão Preto, SP; Brazil

Address for correspondence

ABSTRACT

One hundred and fifty-one methicillin-resistant z (MRSA) strains have been isolated from patients admitted in tertiary care hospitals in two metropolitan areas (Campinas City and Ribeirão Preto City) in the southeast region of Brazil and analyzed through PCR-based techniques [(PCR amplification of spa, coa, and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] and further restriction fragment typing of coa and of housekeeping genes. The heterogeneity of spa gene was determined directly by agarose gel electrophoresis migration. The results obtained indicate the existence of three (A, B, C) main clusters. Since the strain distribution in these three clusters is much characteristic, it denotes the existence of three main clones. All strains isolated in Campinas were grouped in clusters A and B, while most of the strains isolated in Ribeirão Preto were grouped in cluster C. This distribution denotes the existence of different founder strains that undergo independent genetic variability. The strains considered representative of the Brazilian Epidemic Clone (BEC) were categorized as cluster A. These results indicate a possible higher variability among Brazilian MRSA strains than currently described and indicate that the techniques herein used can be used as an alternative to Pulsed Field Gel Electrophoresis (PFGE).

Key-Words: MRSA, nosocomial, molecular characterization, PCR.

Methicillin-resistant strains of Staphylococcus aureus (MRSA) have become one of the most significant nosocomial pathogens worldwide, being capable of causing an wide range of hospital infections and clinical syndromes associated with severe diseases, including bacteremia, pneumonia, endocarditis, septic arthritis, osteomyelitis, and deep abscess formation [1]. As MRSA strains can disseminate very rapidly, it is necessary to implement effective monitoring programs for the identification and control of epidemic strains [2].

Studies have revealed a highly clonal population, with low variability, between MRSA strains, consistent with the view that S. aureus, unlike some of the freely recombining pathogenic species such as Streptococcus pneumoniae [3], Neisseria meningitidis [4], and Helicobacter pylori [5] is not naturally transformable [6] presenting, then, a low number of clones.

One of these MRSA described clones, the Iberian MRSA, first identified as the dominant clone in a major MRSA disease outbreak in a hospital in Spain in 1989 [7] was subsequently detected in at least eight Portuguese hospitals [8] as well as in hospitals in Western Scotland, United Kingdom, Italy, Belgium, and Germany [9], and in one hospital in the USA [10]. A second multiresistant clone (Brazilian Epidemic clone - BEC) was shown to be widely spread in Brazilian hospitals several thousands of kilometers apart [11] and later it was also associated with infections in Portugal, Argentina, Uruguay, Czech Republic, and Canada [12-15]. Several other studies [16-18] in Brazil, using pulsed-field gel electrophoresis (PFGE) technique, to analyze Brazilian MRSA strains, also indicated a low variability between strains.

In this work, to assess the existing genomic variability of 151 methicillin-resistant Staphylococcus aureus (MRSA) strains recovered from patients attended in two tertiary care hospitals, at the Medical School Hospital of Campinas State University (UNICAMP) in Campinas City and at the Medical School Hospital of São Paulo University in Ribeirão Preto City (FCMRP-USP), both cities located 155.34 miles apart in the Southeast region of Brazil, was used a combination of PCR-based techniques [(PCR amplification of spa and coa genes and housekeeping genes (arcC, aroE, gmk, pta, tpi, yqiL)] with further restriction and fragment typing of the coa gene and housekeeping genes (RFLP), plus the direct determination of the heterogeneity of the spa gene by agarose gel electrophoresis migration [3,6,19]. This approach would be able to show a possible clonal variability in these MRSA strains that was not demonstred in previously published works [16-18].

Material and Methods

Strains

One-hundred and forty-five MRSA strains investigated in this work were recovered from patients in two Medical School Hospitals located in metropolitan areas of São Paulo State, Southeast Brazil between 2002 and 2005. Four strains (257A, 20COA, 256B, and 24COA) also obtained in the Medical School Hospital of Campinas State University were considered in this work as representative strains of a previously described work [20]. As control strains and for comparison sake in the clustering analysis, another 2 strains (BEC 9393; HC 562) [17] were used.

Genomic DNA Extraction

MRSA strains were grown overnight in LB medium [21] at 37ºC and total DNA was extracted as described by Ausubel et al. [22].

PCR Amplification and Analysis of the Polymorphism of spa Gene

The amplification (Perkin Elmer® DNA Thermal Cycler 480 Waltham, Massachusetts, USA) of DNA fragments of the spa gene and the polymorphisms of the fragments were analyzed by agarose gel electrophoresis as described by Koreen et al. [23] (Figure 1A).


PCR Amplification and Analysis of the Polymorphism of the coa Gene

The amplification of DNA fragments, by PCR, of the coa gene and further restriction with endonucleases and agarose gel electrophoresis followed the protocol described by Montesinos et al. [24] using a Perkin Elmer® DNA apparatus (Thermal Cycler 480 Waltham, Massachusetts, USA) (Figure 1B).

PCR Amplification of the Housekeeping Genes and Polymorphism Analysis

Amplification of the housekeeping genes (Perkin Elmer® DNA Thermal Cycler 480 Waltham, Massachusetts, USA) and restriction-generated fragments were performed as described by Diep et al. [25]. Fragments polymorphisms were analyzed by agarose gel electrophoresis as described by Sambrook et al. [21] (Figure 2).


Genetic Similarity and Statistical Analysis

The genomic similarity between strains was assessed throughout the construction of a similarity dendrogram generated by the NTSys software (UPGMA algorithm) using the combination of all generated fragments obtained by all PCR-based techniques [26] (Figure 3).


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Publication Dates

  • Publication in this collection
    10 Feb 2010
  • Date of issue
    June 2009

History

  • Accepted
    29 May 2009
  • Received
    20 Feb 2009
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