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      The current role of pulsed-field gel electrophoresis in methicillin-resistant Staphylococcus aureus (MRSA) typing and the retrospective identification of outbreaks.

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          Abstract

          The objective of this paper was to investigate whether retrospective pulsed-field gel electrophoresis (PFGE) of methicillin-resistant Staphylococcus aureus (MRSA) isolates at two-year intervals is suitable and sufficient to demonstrate changes in the clonal composition of MRSA isolates and to identify previously undetected local outbreaks. PFGE patterns of 400 MRSA isolates were collected between 2004 and 2008 at the University of Rostock Hospital in Germany, and were used to assess the prevalence of MRSA clones at different time points. Only minor changes were detected. The combined analysis of all isolates that were collected per year reduced the time needed to perform this laborious procedure. The retrospective identification of outbreaks may require shorter intervals. Improved infection prevention and control measures prevented further outbreaks in previously affected hospital departments. In conclusion, PGFE at two-year intervals is sufficient to detect changes in the clonal composition of local MRSA isolates. If time for identification is important during outbreak investigations, more rapid methods with a similarly high discriminatory power such as spa typing should be used.

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          Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis.

          A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.
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            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 beta-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.
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              Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing.

              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.
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                Author and article information

                Journal
                Eur J Microbiol Immunol (Bp)
                European journal of microbiology & immunology
                Akademiai Kiado Zrt.
                2062-509X
                2062-509X
                Jun 2012
                : 2
                : 2
                Article
                EuJMI_2(2012)2/5
                10.1556/EuJMI.2.2012.2.5
                3956961
                24672681
                bdb24aaf-20a2-44ea-8c74-e6ac1204616c
                History

                methicillin-resistant Staphylococcus aureus,outbreak,pulsed-field gel electrophoresis,typing,MRSA,PFGE,epidemic clone

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