Typing of Methicillin-Resistant Staphylococcus aureus in a University Hospital Setting by Using Novel Software for spa Repeat Determination and Database Management

Abstract

The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA). We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant spa types was replaced by a clonally related variant in the second study period. Ten unique spa types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting. spa typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA.Staphylococcus aureus is a major human pathogen causing skin and tissue infections, pneumonia, septicemia, and deviceassociated infections. The emergence of strains resistant to methicillin and other antibacterial agents has become a major concern especially in the hospital environment, because of the higher mortality due to systemic methicillin-resistant Staphylococcus aureus (MRSA) infections (2). Typing of MRSA is used to support infection control measures. While pulsed-field gel electrophoresis (PFGE) is a "gold standard" for strain typing of MRSA (20), DNA sequence-based approaches are becoming more frequently used because sequence data can easily be transferred between laboratories via the Internet. Multilocus sequence typing (MLST), which was developed by using Neisseria meningitidis as the model species (9, 18), has been successfully adapted to S. aureus (7,8). However, MLST is not suitable for routine surveillance of MRSA because of the high cost and the necessity of access to a high-throughput DNA sequencing facility.Although there is evidence for recombination in S. aureus (10), it has been shown that point mutations by far exceed recombination events, in contrast to N. meningitidis or Streptococcus pneumoniae (11). Furthermore, there is only a small number of clonal groupings of MRSA circulating worldwide (7). Therefore, single-locus DNA sequencing of repeat regions of the coa (coagulase) gene and the spa gene (protein A), respectively, could be used for reliable and accurate typing of MRSA (12,13,(26)(27)(28)(29). spa typing is especially interesting for rapid typing of MRSA in a hospital setting since it offers higher resolution than coa typing (27). The repeat region of the spa gene is subject to spontaneous mutations, as well as loss and gain of repeats. ...

Biofilm formation in invasive Staphylococcus aureus isolates is associated with the clonal lineage

“…The clones were classified based on the number of isolates and unique PFGE types as major (> 10 isolates per PFGE type), intermediate (4-9 isolates per PFGE type) or minor (2-3 isolates per PFGE types). In addition, spa typing and SCCmec typing were also performed (Harmsen et al, 2003;Milheirico et al, 2007). MLST was performed on a representative set of isolates from each major PFGE cluster (Enright et al, 2000).…”

Characterisation of the Virulence Factors and Genetic Types of Methicillin Susceptible Staphylococcus aureus from Patients and Healthy Individuals

“…spa typing was performed as described by Harmsen et al [22]. The amplicons of spa were purified using Megaquick-DNA purification kit (Intron Biotechnology, Korea) and sequenced.…”

Characterization of Dutch Staphylococcus aureus from bovine mastitis using a Multiple Locus Variable Number Tandem Repeat Analysis

“…Amplification and sequencing of the repeat region of the S. aureus Protein A gene (spa) was performed by using a specific primer set as described (Harmsen et al, 2003). The amplicon was sequenced using BigDye terminator version 3.1 on ABI 3100 sequencer (Applied Biosystem).…”

“…Many different typing methods have been used. From a ''gold standard'' Pulsed-Field Gel Electrophoresis (PFGE) to Multi Locus Sequence Typing (MLST) and S. aureus-specific staphylococcal Protein A typing known as spa-typing (Enright et al, 2000;Harmsen et al, 2003;Struelens et al, 1992). However, not all methods can be used in all centers because some methods are still limited to well-equipped laboratories.…”