Comparison of high-resolution melting curves analysis of the short-sequence repeat region of the proteinA, spa sequencing and pulsed-field gel electrophoresis for the investigation of an outbreak due to MRSA
Abstract number: P1710
Stuart J.I., Reyes R.C., Stuart A., McGavin M., Ainsworth P., John M.A., Hussain Z.
Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infection world-wide. In an outbreak situation, rapid identification and typing is an important epidemiological tool in determining clonal relatedness. Several molecular techniques are available for differentiating S. aureus but no method is superior under all conditions. Pulse field gel electrophoresis (PFGE) has been widely used and is the gold standard. More recently sequencing of the polymorphic X or short sequence repeat region of the protein a gene (spa) has been used as an alternative to current techniques. We here describe the comparison of spa sequencing, PFGE and a method using high resolution melting curve analysis (HRM) of the polymorphic X region in the investigation of an outbreak due to MRSA.
Methods: Nineteen outbreak-related MRSA strains were typed by spa sequencing, PFGE and HRM. The DNA sequences of the spa repeat region in both directions were imported as ABI files and analyzed by automated spa typing (tools.egenomics.com), using Kreiswirth nomenclature. PFGE was performed using standard techniques. MRSA types (CMRSA) were defined using Canadian MRSA PFGE typing guidelines. Amplicons of the spa region X were analyzed by HRM utilizing a difference graph format. S. aureus ATTC 43300 was included with each for standardization of curves.
Results: Strains from seventeen patients were identical by all three methods, corresponding to spa type 2 and CMRSA 2 PFGE type. One strain was closely related to the 17 strains by spa typing. It was spa-type 407. The only difference is the 6th cassette, which is G1 in spa-type 407 and D1 in spa-type 2. The D1 and G1 repeats differ by 2 nucleotides. PGFE and HRM failed to differentiate this strain from outbreak strains. One strains classified as CMRSA 9 and Spa-type 1 was unrelated to other strains, and was also deemed as unrelated by HRM analysis.
Conclusions: There was an excellent correlation clustering of strains by HRM compared to PFGE. The spa sequencing was most discriminatory, because it identified a strain, which was different though closely related to outbreak-causing strains. PFGE and HRM failed to discern this difference. HRM potentially represents a quick and inexpensive screening tool to rule out similarity of strains.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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