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Reproducibility of Staphylococcus epidermidis plasmid profiles
A I Hartstein1, V H Morthland, A L Rashad
1Department of Hospital Infection Control, University Hospitals, Portland, Oregon.
Abstract:
The plasmid profiles of six isolates of Staphylococcus epidermidis were repetitively evaluated over an 8-month period. Each isolate was subcultured and stored at three different temperatures (-70 degrees C, -20 degrees C, and room temperature) and plasmid DNA was prepared from each subculture at 0, 1, 4, and 8 months by two different methods of plasmid extraction [using mixed alkyltrimethylammonium bromide (ATAB) or Brij 58 and deoxycholate (modified Parisi)]. Plasmid DNA bands were lost from two isolates when subcultures were kept at room temperature. This plasmid loss was confirmed by repetitive extractions and electrophoresis, as well as by restriction endonuclease analysis of the ATAB preparations. Profiles were otherwise highly related to one another, with occasional exceptions being extra or missing plasmid DNA bands of high molecular size. The latter findings were not reproducible. Plasmid DNA extracted by the modified Parisi method was not reliably digested with restriction endonuclease enzymes. We conclude that the plasmid profiles of Staphylococcus epidermidis isolates are highly reproducible as long as isolates are stored at less than or equal to -20 degrees C. Minor discrepancies in the number of plasmid DNA bands of large molecular size may occur. These are resolvable by repetitive testing or restriction endonuclease analysis of ATAB-extracted plasmid DNA preparations.
Insights
Staphylococcus epidermidis plasmid profiles are reproducible when stored below -20°C. Room temperature storage leads to plasmid DNA loss, impacting profile stability and analysis.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriology
Background:
- Plasmid profiling is crucial for bacterial identification and tracking.
- Staphylococcus epidermidis is an opportunistic pathogen with significant clinical relevance.
- Understanding factors affecting plasmid stability is essential for reliable microbiological studies.
Purpose of the Study:
- To assess the reproducibility of Staphylococcus epidermidis plasmid profiles over time.
- To determine the impact of storage temperature and extraction methods on plasmid stability.
- To identify optimal conditions for maintaining plasmid profiles for accurate analysis.
Main Methods:
- Subculturing and storage of six Staphylococcus epidermidis isolates at -70°C, -20°C, and room temperature for 8 months.
- Plasmid DNA extraction using two methods: mixed alkyltrimethylammonium bromide (ATAB) and modified Parisi (Brij 58 and deoxycholate).
- Analysis of plasmid DNA via electrophoresis and restriction endonuclease digestion.
Main Results:
- Plasmid DNA loss occurred in isolates stored at room temperature.
- Plasmid profiles were generally reproducible across different storage conditions and extraction methods, except for room temperature storage.
- Minor variations in high molecular weight plasmid DNA bands were observed but were not consistently reproducible.
- The modified Parisi extraction method showed unreliable results with restriction endonuclease digestion.
Conclusions:
- Staphylococcus epidermidis plasmid profiles are highly reproducible when isolates are stored at or below -20°C.
- Storage at room temperature compromises plasmid stability and profile accuracy.
- Minor discrepancies in large plasmid DNA bands can occur but are manageable with repetitive testing or restriction analysis of ATAB-extracted DNA.