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. ...

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The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drugresistant bacteria that are adapted to thrive in hospitalized patients. Of particular concern are globally disseminated methicillin-resistant Staphylococcus aureus (MRSA) clones that cause outbreaks and epidemics associated with health care. The most rapidly spreading and tenacious health-care-associated clone in Europe currently is EMRSA-15, which was first detected in the UK in the early 1990s and subsequently spread throughout Europe and beyond. Using phylogenomic methods to analyze the genome sequences for 193 S. aureus isolates, we were able to show that the current pandemic population of EMRSA-15 descends from a health-care-associated MRSA epidemic that spread throughout England in the 1980s, which had itself previously emerged from a primarily community-associated methicillin-sensitive population. The emergence of fluoroquinolone resistance in this EMRSA-15 subclone in the English Midlands during the mid-1980s appears to have played a key role in triggering pandemic spread, and occurred shortly after the first clinical trials of this drug. Genome-based coalescence analysis estimated that the population of this subclone over the last 20 yr has grown four times faster than its progenitor. Using comparative genomic analysis we identified the molecular genetic basis of 99.8% of the antimicrobial resistance phenotypes of the isolates, highlighting the potential of pathogen genome sequencing as a diagnostic tool. We document the genetic changes associated with adaptation to the hospital environment and with increasing drug resistance over time, and how MRSA evolution likely has been influenced by country-specific drug use regimens.

Pulsed-field gel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium,

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-and community-associated infections worldwide. Within the healthcare setting alone, MRSA infections are estimated to affect more than 150,000 patients annually in the European Union (EU), resulting in attributable extra in-hospital costs of EUR 380 million for EU healthcare systems. Pan-European surveillance data on bloodstream infections show marked variability among EU Member States in the proportion of S. aureus that are methicillin-resistant, ranging from less than 1% to more than 50%. In the past five years, the MRSA bacteraemia rates have decreased significantly in 10 EU countries with higher endemic rates of MRSA infections. In addition to healthcare-associated infections, new MRSA strains have recently emerged as communityand livestock-associated human pathogens in most EU Member States. The prevention and control of MRSA have therefore been identified as public health priorities in the EU. In this review, we describe the current burden of MRSA infections in healthcare and community settings across Europe and outline the main threats caused by recent changes in the epidemiology of MRSA. Thereby, we aim at identifying unmet needs of surveillance, prevention and control of MRSA in Europe.

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In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.

European Antimicrobial Resistance Surveillance System shows large variations in methicillin-resistant S. aureus .