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.

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.

Isolates found in persons and animals in Germany and Austria show a genetic relationship.

Staphylococcus aureus is both a successful human commensal and a major pathogen. The elucidation of the molecular determinants of virulence, in particular assessment of the contributions of the genetic background versus those of mobile genetic elements (MGEs), has proved difficult in this variable species. To address this, we simultaneously determined the genetic backgrounds (spa typing) and the distributions of all 19 known superantigens and the exfoliative toxins A and D (multiplex PCR) as markers for MGEs. Methicillin-sensitive S. aureus strains from Pomerania, 107 nasal and 88 blood culture isolates, were investigated. All superantigenencoding MGEs were linked more or less tightly to the genetic background. Thus, each S. aureus clonal complex was characterized by a typical repertoire of superantigen and exfoliative toxin genes. However, within each S. aureus clonal complex and even within the same spa type, virulence gene profiles varied remarkably. Therefore, virulence genes of nasal and blood culture isolates were separately compared in each clonal complex. The results indicated a role in infection for the MGE harboring the exfoliative toxin D gene. In contrast, there was no association of superantigen genes with bloodstream invasion. In summary, we show here that the simultaneous assessment of virulence gene profiles and the genetic background increases the discriminatory power of genetic investigations into the mechanisms of S. aureus pathogenesis.

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on healthy skin. Here we show that S. epidermidis has also evolved to become a formidable nosocomial pathogen. Using genomics, we reveal that three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) have emerged in recent decades and spread globally. These lineages are resistant to rifampicin through acquisition of specific rpoB mutations that have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified dual D471E and I527M RpoB substitutions to be the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, we reveal that the D471E and I527M combination occurs almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification barriers and then performing site-specific mutagenesis, we show that these rpoB mutations not only confer rifampicin resistance, but also reduce susceptibility to the last-line glycopeptide antibiotics, vancomycin and teicoplanin. Our study has uncovered the previously unrecognized international spread of a near pan-drug-resistant opportunistic pathogen, identifiable by a rifampicin-resistant phenotype. It is possible that hospital practices, such as antibiotic monotherapy utilizing rifampicin-impregnated medical devices, have driven the evolution of this organism, once trivialized as a contaminant, towards potentially incurable infections.

The implementation of the new clustering algorithm Based Upon Repeat Pattern (BURP) into the Ridom StaphType software tool enables clustering based on spa typing data for Staphylococcus aureus. We compared clustering results obtained by spa typing/BURP to those obtained by currently well-established methods, i.e., SmaI macrorestriction analysis and multilocus sequence typing/eBURST. A total of 99 clinical S. aureus strains, including MRSA and representing major clonal lineages associated with important kinds of infections which have been prevalent in Germany and Central Europe during the last 10 years, were used for comparison. SmaI macrorestriction analysis revealed the highest discriminatory power, and clustering results for all three methods resulted in concordance values ranging from 96.8% between the two sequence-based methods to 93.4% between spa typing/BURP and SmaI macrorestriction/cluster analysis. The results of this study indicate that spa typing, together with BURP clustering, is a useful tool in S. aureus epidemiology, especially because of ease of use and the advantages of unambiguous sequence analysis as well as reproducibility and exchange of typing data.Staphylococcus aureus is one of the most frequent nosocomial pathogens. The emergence and spread of epidemic strains of methicillin-resistant S. aureus in hospitals (hMRSA) and, independent from the nosocomial setting, in the community (cMRSA) require special attention of infection control. Typing is an important prerequisite for targeted control measures. For about 30 years, phage typing has been widely used for strain typing. More recently, SmaI macrorestriction analysis (pulsedfield gel electrophoresis [PFGE]) was introduced as a typing method with high discriminatory power. PFGE is still regarded the "gold standard" of molecular typing of MRSA, despite insufficient comparability of results obtained from different laboratories (21). During the past 5 years, DNA sequencebased typing has become more popular due to progress in large-scale sequencing methodology, ease of data transfer, and excellent comparability of results (2). This first became evident by the application of multilocus sequence typing (MLST) to MRSA (4, 5). At present, however, MLST is not suitable for routine infection control due to high cost, labor intensity, and lack of broad access to high-throughput DNA sequencing.Several S. aureus typing schemes targeting polymorphic DNA repeat regions in genes for microbial surface components recognizing adhesive matrix molecules have been described previously (7,9,16,27,30). They also include typing methods based on the length polymorphism in spa amplimers (9) or, more recently, on polymorphisms in multiple fragments amplified in a multiplex PCR approach for variable-number tandem repeats (7, 27). Among sequence-based approches, spa typing was the most promising (8,12,13,15,31). The X region of the protein A gene (spa) consists of direct repeats exhibiting an extensive polymorphism based on point mutations, deletions, duplications, and i...

BackgroundStudies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans.MethodsAfter selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay.ResultsAt the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms.ConclusionThese results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.

We determined the value of spa typing in combination with BURP (based upon repeat pattern) grouping analysis as a frontline tool in the epidemiological typing of Staphylococcus aureus, based on a random collection of 1,459 clinical isolates sent to the German Reference Centre for Staphylococci within a 6-month period. The application was found to be helpful for the classification of isolates into the particular clonal lineages currently prevalent in Germany. Due to its major advantages because of the ease of interpretation and the exchangeability of the results, the use of spa typing greatly simplifies communication between laboratories on both the national and the international levels. Thus, it is an excellent tool for national and international surveillance of S. aureus as well as for analysis of the short-term local epidemiology. However, to overcome the limitations of the BURP grouping method in terms of typing accuracy and discriminatory power, the results of the default BURP grouping method must be interpreted with caution. Additional markers, like staphylococcal chromosomal cassette mec, lineage-specific genes, or alternative DNA polymorphisms, are indispensable. They should be selected by dependence on the clonal lineage indicated by spa typing and subsequent BURP analysis as well as on the basis of the particular question to be addressed.Staphylococcus aureus is well known both as a commensal organism on the human skin and as a leading cause of human disease responsible for a variety of diseases ranging from superficial skin infections to serious infections like pneumonia, bacteremia, and endocarditis (20). The occurrence and spread of methicillin-resistant S. aureus (MRSA) soon after the introduction of methicillin in clinical practice finally led to the appearance of hospital-adapted multiresistant clones, which constitute a constantly growing problem as a major cause of nosocomial infections all over the world (2, 5, 42). Additionally, the appearance of MRSA in the community (communityacquired MRSA [caMRSA]) and the potential risk of its introduction into hospitals are matters of great concern (5,16,31,34).The use of efficient and accurate epidemiological typing methods is a prerequisite for monitoring and for limiting the occurrence and spread of epidemic clones within and between hospitals. Therefore, typing systems must enable the discrimination between unrelated isolates as well as the recognition of isolates belonging to the same clonal lineage in order to determine whether epidemiologically related isolates are also genetically related (36, 38). Historically, typing of S. aureus mostly relied on phenotypic strain characteristics (for example, susceptibility to bacteriophages or antibiotics), but over the past two decades a variety of molecular technologies have been developed. Among those technologies, SmaI macrorestriction analysis became the "gold standard" for S. aureus strain typing mainly because of its excellent discriminatory power, especially for analysis of the local short-term epidemi...