Typing of Staphylococcus epidermidis using multiple-locus variable-number tandem repeat analysis
Abstract number: 1134_04_41
Johansson A., Gottfridsson P., Koskiniemi S., Wiström J., Sjöstedt A., Monsen T.
The emergence of Staphylococcus epidermidis as an important nosocomial pathogen is related to advances in modern medicine. There is a lack of methods to distinguish clinically significant S. epidermidis from contaminant strains and rapid and reproducible methods for epidemiological studies. Multiple-locus variable-number tandem repeat analysis (MLVA) has proved efficient for high-resolution typing of several bacterial species. The aim of the present study was to develop a MLVA system for molecular typing of S. epidermidis.
The genome sequence of S. epidermidis ATCC 12228 was analysed for the presence of tandem repeated sequence using software programmes. Genomic DNA of 30 clinical isolates of multi-resistant S. epidermidis was subjected to PCR amplification of multiple genomic regions that exhibited tandem repeats. Variation in repeat copy numbers among isolates was determined by sizing of PCR fragments on agarose gels and verified by sequencing. All S. epidermidis isolates were typed using SmaI digestion and pulsedfield gel electrophoresis. Diversity values (D) were calculated using the formula D = 1-SUM(allele frequency)2.
The analysis of the genome of S. epidermidis ATCC 12228 identified 137 genomic regions with tandem repeated sequences. Fourteen regions were analysed in a subset of S. epidermidis isolates. Five regions showed variable numbers of tandem repeats and were further analysed in 30 clinical S. epidermidis isolates from a University Hospital. The variable repeat motifs displayed repeat sizes ranging from 18 bp to 58 bp in length. The number of detected alleles at individual repeat loci ranged from three to ten. Calculated D-values ranged from 0.50 to 0.82. The D-value for the combination of five repeat loci (all loci treated as a single allele) was 0.87. MLVA revealed 16 genotypes among 30 isolates. Observed PFGE genotypes resulted in a D-value of 0.84. It was apparent that MLVA and PFGE resulted in very similar clustering of isolates (Figure 1). Three out of five identified polymorphic tandem repeat loci were located in genes predicted to encode surface binding proteins that may have a functional role in adherence to host fibrinogen.
MLVA is a promising typing approach for molecular epidemiology studies of S. epidermidis due to the speed of analysis, a high discriminatory power, and an ability to produce discrete character data that can be easily exchanged among laboratories.
|Session name:||XXIst ISTH Congress|
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