Comparison of DNA Sequencing of the Protein A Gene Polymorphic Region with Other Molecular Typing Techniques for Typing Two Epidemiologically Diverse Collections of Methicillin-Resistant Staphylococcus aureus

Abstract

The aim of this study was to compare the recently developed typing approach for methicillin-resistant Staphylococcus aureus (MRSA) based on the DNA sequencing of the protein A gene polymorphic region (spaA typing) with a combination of three well-established molecular typing techniques: ClaI-mecA vicinity polymorphisms, ClaI-Tn554 insertion patterns, and SmaI pulsed-field gel electrophoresis (PFGE) profiles. In order to evaluate the applicability of this typing technique in different types of studies, two groups of MRSA clinical isolates were analyzed: a collection of 185 MRSA isolates circulating in Hungary recovered from 17 hospitals in seven cities during a 3-year period (1994 through 1996), and a selection of 53 MRSA strains isolated in a single hospital in Hungary between 1997 and 1998. The 238 MRSA clinical strains from Hungary were first classified in clonal types (defined as ClaI-mecA::ClaI-Tn554::SmaI-PFGE patterns), and 65 of the 238 strains, representing major MRSA clones and some sporadic clones, were further analyzed by spaA typing. Our results showed that the lineages most recently introduced in the hospital setting showed little variability in spaA types, whereas the MRSA clones circulating for a longer period of time and spread among several hospitals showed a higher degree of variability. The implementation of the spaA typing method was straightforward, and the results obtained were reproducible, unambiguous, and easily interpreted. This method seems to be adequate for outbreak investigations but should be complemented with other techniques in long-term surveillance or in studies comparing distant clonal lineages.Three molecular typing techniques (5) have been largely used for the characterization of clones of methicillin-resistant Staphylococcus aureus (MRSA) and enabled the detection of widely spread MRSA lineages, such as the Iberian, Brazilian, New York/Tokyo, and pediatric MRSA clones (1,7,11,17,20,21,24). The combined methods consist of (5, 10) Southern blot analysis of chromosomal ClaI digests with a mecA DNA probe (ClaI-mecA polymorphisms) and with a Tn554 transposon probe (ClaI-Tn554 insertion patterns) and restriction fragment length polymorphism analysis of chromosomal DNA generated after cleavage with SmaI and pulsed-field gel electrophoresis (PFGE) (SmaI-PFGE). ClaI-mecA polymorphisms are a consequence of the variability in the vicinity of the mecA gene, the central element of methicillin resistance, and ClaITn554 patterns reflect the location and copy number of the transposon Tn554, present in most MRSA clinical isolates (10). PFGE provides fine fingerprinting of the chromosomal background with high discriminatory power and has been suggested as the gold standard for the molecular typing of MRSA (25,26).DNA sequencing-based typing techniques are being developed with obvious advantages in speed, unambiguous data interpretation, simplicity of large-scale database creation, and standardization among laboratories (8). Recently, DNA sequencing of the spaA gene (protein A determinant) po...

Update on the Major Clonal Types of Methicillin-Resistant Staphylococcus aureus in the Czech Republic

“…(6). The Hungarian (9) and Taiwan clones, which belong to ST239 and ST241 (single locus variant of ST239) (1,23,26,27) and ribotypes H1 and H2, respectively, may also be progenitors of the Czech clone. Representatives of ST239 were also reported from other countries all over the world, including the countries neighboring the Czech Republic (Hungary, Poland, and Germany) (13,23).…”

Two International Methicillin-Resistant Staphylococcus aureus Clones Endemic in a University Hospital in Patras, Greece

“…When the PFGE results were interpreted, one to six band differences defined a PFGE subtype and seven or more band differences defined a distinct PFGE type (38). The primers used for spaA typing were as follows: primer spaAF1 (GAC GAT CCT TCG GTG AGC; nucleotides 1096 to 1113) and primer spaAR1 (CAG CAG TAG TGC CGT TTG C; nucleotides 1534 to 1516) (GenBank accession number J01786) (23). Previously described primers were adopted for MLST (10); the exception was primer arcCF2 (CCT TTA TTT GAT TCA CCA GCG; the forward primer for the arcC gene) (5).…”

“…During the last decade, five major internationally spread MRSA clones, the Iberian (8,34), Brazilian (37), Hungarian (7,23), New York-Japan (1,31), and pediatric (32) clones, were identified by using the combination of ClaI-mecA polymorphisms, Tn554 insertion patterns, and PFGE. Multilocus sequence typing (MLST) has recently been proven to be the most adequate method both for long-term and global epidemiologic studies and for population genetic studies (10,27), whereas for short-term or local epidemiologic studies of S. aureus, PFGE continues to be the method of choice (27).…”

Evolution of Sporadic Isolates of Methicillin-Resistant Staphylococcus aureus (MRSA) in Hospitals and Their Similarities to Isolates of Community-Acquired MRSA

“…The primers used were as follows: spaAF1, 5Ј-GAC GAT CCT TCG GTG AGC-3Ј, nucleotides 1096 to 1113; and spaAR1, 5Ј-CAG CAG TAG TGC CGT TTG C-3Ј, nucleotides 1534 to 1516 (accession no. J01786) (27).…”