Loading...
Please wait, while we are loading the content...
Similar Documents
Unsuitable Distinction between Viable from Dead Staphylococcus Aureus and Staphylococcus Epidermidis by Ethidium Bromide Monoazide in Combination with Real-time qPCR
| Content Provider | Semantic Scholar |
|---|---|
| Copyright Year | 2009 |
| Abstract | by Ethidium Bromide Monoazide in Combination with Real-time qPCR Kobayashi, H; Oethinger, M; Tuohy, M J; Procop, G W; Kawamoto, T; Hall G S; +Bauer T W +The Cleveland Clinic Foundation, Cleveland OH osteoclast@aol.com Introduction The DNA-intercalating dye ethidium bromide monoazide (EMA) has recently been used as a DNA binding agent to differentiate viable and dead bacterial cells. EMA has the ability to reduce the real-time PCR signal from dead bacteria by selectively penetrating through the damaged membrane of dead cells and blocking the DNA for PCR amplification via photoactivation. Many investigators have tested various concentrations of EMA and various light exposure time, but little is known about the effectiveness of this assay for detecting staphylococci (mainly, Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epi)), which cause most cases of periprosthetic infections. The purpose of this study was to optimize and test the assay in vitro using S. aureus and S. epi cultures that were killed with heat inactivation. Materials and Methods Bacteria and cultures: S. aureus (ATCC 12600) and S. epi (289F22) were used in this in vitro study. 1.5 x 10 colony-forming units (CFU) /ml of each bacteria were grown to mid-exponential phase in Brain Heart Infusion broth for 8 hours at 37°C and split into two cultures. One culture was heated at 100 °C for 10 min, the other was left untreated. 500μl of each suspension was harvested for DNA extraction followed by EMA treatment or not (control). At the same time, cell suspensions were plated for colony counts in duplicate. The heat-treated tubes were cooled to room temperature and the absence of viable CFU was determined. DNA extraction was performed in triplicate. EMA condition: EMA was added to bacterial suspension for 5 min in the dark, and were light exposed using a 650-W halogen light source. The sample tubes were placed at 20 cm from the light source and were laid horizontally on ice. After photo-induced cross-linking, bacterial suspensions were centrifuged at 7600 rpm for 10 min for DNA extraction. Four different EMA concentrations (0, 2.5, 10, 50, and 100μg/ml) and three different light exposure time points (0, 1, 5, and 15 min) were tested for optimization. For S. epi, three different bacterial concentrations were treated with EMA. DNA extraction and Real-time qPCR: DNA extraction was performed from 500μl of each suspension using DNeasy Blood & Tissue kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. One hundred microliter of DNA extract was obtained for each sample. Quantitative PCR assays targeted tuf using a Rotorgene 3000® (Corbett Research, Sydney, Australia). The threshold cycle (Ct) was defined as the cycle number at which fluorescence passed a fixed threshold and corresponded to the number of cycles of target amplification necessary for detection. Samples with higher concentrations of a target require fewer cycles of amplification for detection. Conversely, a larger Ct number indicates a lower concentration. Results Effect of EMA concentration: DNA amplification of S. aureus was inhibited in a concentration dependent manner, irrespective of the viability (Fig.1). The Ct difference of dead S. aureus with and without EMA treatment was large (ዊ� Ct=14.73) at a concentration of 50μg/ml, but the Ct difference of viable S. aureus was also high (ዊ�Ct=6.05), indicating that EMA influences not only dead but also viable bacteria (Fig.1). The effect of EMA was almost identical between viable and dead S. epi. Surprisingly, viable S. epi were influenced more effectively than dead bacteria at a concentration of 100μg/ml (Fig.2). Effect of light exposure time: There was no difference of Ct values based on light exposure time (1, 5, 15 min) in both strains, irrespective of the viability. Effect of bacterial number: When 3.4 x 10 CFU/ml of S. epi was treated with EMA, there was no difference of Ct values between viable and dead bacteria (Fig.3). When a higher number (7.0 x 10 CFU/ml and 7.8 x 10 CFU/ml) of S. epi were tested with EMA, viable S. epi were inhibited more effectively than dead bacteria (Fig.3), indicating that the ability of EMA to distinguish viable from necrotic bacteria is poor. Discussion: Our in vitro assay indicated that EMA was most effective detecting S. aureus at a concentration of 50μg/ml, but inhibited viable S. aureus amplification to a considerable extent. When higher concentrations of S. epi were treated with EMA, viable S. epi was inhibited more effectively than dead bacteria. One other study has also recently suggested that EMA may be unsuitable for live-dead differentiation of some bacterial strains. Our results indicate that EMA can penetrate viable bacteria as well as dead bacteria, and the effect of EMA depends on the EMA concentration and bacterial number. Therefore, EMA is not a suitable indicator of bacterial viability, at least with respect to bacteria strains commonly involved in orthopaedic infections. References: 1. Nocker A, Cheung C, Camper AK. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods 2006; 67: 310-20. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.ors.org/Transactions/55/1549.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
