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Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus
Published on: September 5, 2013
Staphylococcal Cassette Chromosome mec (SCCmec) Natural Excision Frequencies and Its Contributing Factors in Variant
Salman Mirza1,2, Laura Fine1,2, Jo-Ann McClure1,3
1Department of Pathology & Laboratory Medicine, University of Calgary and Alberta Health Services, Calgary, AB T2N 4N1, Canada.
Background:
Staphylococcus aureus acquires methicillin resistance genes through the SCCmec element. Although spontaneous SCCmec excision has been observed, its frequency, type-specific variation, and responsiveness to environmental conditions remain undefined. Here, we systematically quantified SCCmec excision across diverse prototypic types/subtypes and evaluated the factors that contribute to excision variability.
Methods:
Twenty five prototypic MRSA strains (SCCmec types I-VIII, XI-XIII and defined subtypes) were examined under standard growth temperature (37 °C), elevated temperature (42 °C), desiccation, prolonged continuous culture (30 days), and sub-lethal oxacillin pressure. Excision frequencies were quantified using qPCR, normalized to the gyrB housekeeping gene using the formula: 10-((Ct,orfX-Ct,gyrB)/3.32). Statistical analyses included one-way ANOVA, t-tests, and OLS regression for time-dependent trends.
Results:
At 37 °C, excision frequencies ranged from 2.40 × 10-6 to 1.32 × 10-3 and varied among representative SCCmec types/subtypes but were unrelated to SCCmec size (R2 = 0.027, p = 0.44). Type I showed no detectable excision due to a truncated ccrB gene. At 42 °C, excision increased in 14 of 24 types (median +11.2%; eight significant) and decreased in 10 (median -7.4%; four significant). Desiccation produced similar effects, with nine types increasing (median +7.1%; four significant), 14 decreasing (median -8.2%; five significant), and one unchanged. Continuous culture exhibited progressive increases in excision across multiple types (R2 = 0.3-0.94), whereas sub-lethal oxacillin uniformly maintained low detectable excision frequencies across all SCCmec types.
Conclusions:
Excision varied among representative SCCmec types and was influenced heterogeneously by distinct stress conditions. Continuous culture promoted excision, whereas oxacillin exposure maintained low detectable excision. This work quantitatively confirms spontaneous SCCmec excision and provides new insights into MRSA genome plasticity.
Insights
Spontaneous SCCmec excision in MRSA varies by type and is influenced by environmental stress. Continuous culture promotes excision, while oxacillin maintains low levels, revealing insights into MRSA genome plasticity.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Methicillin resistance in *Staphylococcus aureus* is mediated by the SCC*mec* element.
- Spontaneous excision of SCC*mec* is known but poorly characterized regarding frequency, type-specific differences, and environmental influences.
Purpose of the Study:
- To systematically quantify SCC*mec* excision across diverse prototypic types/subtypes.
- To evaluate factors contributing to excision variability under various conditions.
Main Methods:
- Examined 25 prototypic MRSA strains (SCC*mec* types I-VIII, XI-XIII) under different conditions: 37°C, 42°C, desiccation, 30-day continuous culture, and sub-lethal oxacillin.
- Quantified excision frequencies using qPCR, normalized to the *gyrB* housekeeping gene.
- Statistical analyses included ANOVA, t-tests, and OLS regression.
Main Results:
- Excision frequencies at 37°C varied significantly among SCC*mec* types and were independent of element size.
- Elevated temperature (42°C) and desiccation heterogeneously affected excision rates, with some types increasing and others decreasing.
- Continuous culture progressively increased excision across multiple types, while sub-lethal oxacillin consistently maintained low excision frequencies.
Conclusions:
- SCC*mec* excision exhibits type-specific variation and is differentially modulated by environmental stresses.
- Continuous culture enhances SCC*mec* excision, whereas oxacillin exposure suppresses it.
- This study quantitatively confirms spontaneous SCC*mec* excision and highlights its role in MRSA genome plasticity.
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