Wolfgang Witte

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.

Methicillin-resistant Staphylococcus aureus in Europe, 1999–2002

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

Multiplex PCR Assay for Simultaneous Detection of Nine Clinically Relevant Antibiotic Resistance Genes in Staphylococcus aureus

ABSTRACT 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.

Methicillin-resistant Staphylococcus aureus ST398 in Humans and Animals, Central Europe

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

Effect of Subinhibitory Antibiotic Concentrations on Polysaccharide Intercellular Adhesin Expression in Biofilm-Forming Staphylococcus epidermidis

ABSTRACT Biofilm production is an important step in the pathogenesis ofStaphylococcus epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin. A chromosomally encoded reporter gene fusion between the icapromoter and the beta-galactosidase gene lacZ fromEscherichia coli was constructed and used to investigate the influence of both environmental factors and subinhibitory concentrations of different antibiotics on ica expression in S. epidermidis. It was shown that S. epidermidis biofilm formation is induced by external stress (i.e., high temperature and osmolarity). Subinhibitory concentrations of tetracycline and the semisynthetic streptogramin antibiotic quinupristin-dalfopristin were found to enhance icaexpression 9- to 11-fold, whereas penicillin, oxacillin, chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin, and teicoplanin had no effect on ica expression. A weak (i.e., 2.5-fold) induction of ica expression was observed for subinhibitory concentrations of erythromycin. The results were confirmed by Northern blot analyses of ica transcription and quantitative analyses of biofilm formation in a colorimetric assay.

Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens

During the past 15 years, emergence and dissemination of beta-lactam resistance in nosocomial Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, became a serious problem worldwide. Especially the increasing resistance to 3rd and 4th generation cephalosporins and carbapenems is of particular concern. Gram-negative bacteria pursue various molecular strategies for development of resistance to these antibiotics: (a) generation of extended-spectrum beta-lactamases (ESBL) according to the original definition due to extension of the spectrum of already widely disseminated plasmid-encoded beta-lactamases by amino acid substitution; (b) acquisition of genes encoding ESBL from environmental bacteria as, for instance the CTX-M-type beta-lactamases from Kluyvera spp.; (c) high-level expression of chromosome-encoded beta-lactamase (bla) genes as bla(OXA) or bla(ampC) genes due to modifications in regulatory genes, mutations of the beta-lactamase promoter sequence as well as integration of insertion sequences containing an efficient promoter for intrinsic bla genes; (d) mobilization of bla genes by incorporation in integrons and horizontal transfer into other Gram-negative species such as the transfer of the ampC gene from Citrobacter freundii to Klebsiella spp.; (e) dissemination of plasmid-mediated carbapenemases as KPC and metallo-beta-lactamases, e.g. VIM and IMP; (f) non-expression of porin genes and/or efflux pump-based antibiotic resistance. This mini-review summarizes the historical emergence of beta-lactam resistance and beta-lactamases as major resistance mechanism in enteric bacteria, and also highlights recent developments such as multidrug- and carbapenem resistance.

Assignment of Staphylococcus Isolates to Groups by spa Typing, SmaI Macrorestriction Analysis, and Multilocus Sequence Typing

ABSTRACT 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.

A genomic portrait of the emergence, evolution and global spread of a methicillin resistant Staphylococcus aureus pandemic

The widespread use of antibiotics in association with high-density clinical care has driven the emergence of drug-resistant 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.

Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus

A small number of clonal lineages dominates the global population structure of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the concept that MRSA has emerged on a few occasions after penicillinase-stable β-lactam antibiotics were introduced to clinical practice, followed by intercontinental spread of individual clones. We investigated the evolutionary history of an MRSA clone (ST5) by mutation discovery at 108 loci (46 kb) within a global collection of 135 isolates. The SNPs that were ascertained define a radial phylogenetic structure within ST5 consisting of at least 5 chains of mutational steps that define geographically associated clades. These clades are not concordant with previously described groupings based on staphylococcal protein A gene (spa) typing. By mapping the number of independent imports of the staphylococcal cassette chromosome methicillin-resistance island, we also show that import has occurred on at least 23 occasions within this single sequence type and that the progeny of such recombinant strains usually are distributed locally rather than globally. These results provide strong evidence that geographical spread of MRSA over long distances and across cultural borders is a rare event compared with the frequency with which the staphylococcal cassette chromosome island has been imported.

Selective pressure by antibiotic use in livestock.

Selective pressure exerted by the use of antibiotics as growth promoters in food animals appears to have created large reservoirs of transferable antibiotic resistance in these ecosystems. This first became evident for oxytetracycline and later for the streptothricin antibiotic nurseothricin, for which a transfer of relevant resistance determinants (sat genes) to bacterial pathogens of humans was demonstrated. With the emergence of glycopeptide resistance in Enterococcus faecium outside hospitals, a large reservoir of transferable resistance (vanA gene cluster) was identified in animal husbandry due to the use of avoparcin as feed additive. The spread of resistance, which reaches the human enterococcal flora via meat products, is probably due to the dissemination of the vanA gene cluster integrated into different conjugative plasmids among a variety of different strains. Streptogramin resistance associated with the resistance genes vatA and vatG has been found in E. faecium of animal and of clinical origin. Because virginiamycin has been used as growth promoter in animals but streptogramins have been used infrequently in human medicine, this again suggests an animal origin of resistance. Since the use of avoparcin ended, a decline in the rates of glycopeptide-resistant E. faecium (GREF) from animals and humans in the community has been recorded. This supports the ban of antibacterial growth promoters that might interfere with human chemotherapy that has been introduced in European Union countries.