Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods

• Published in: European journal of clinical microbiology & infectious diseases Vol. 30; no. 3; pp. 337 - 342
• Main Authors: Loonen, A. J. M, Jansz, A. R, Kreeftenberg, H, Bruggeman, C. A, Wolffs, P. F. G, van den Brule, A. J. C
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01-03-2011; Springer-Verlag; Springer; Springer Nature B.V
• Subjects: Automation; Bacterial diseases; Bacteriological Techniques; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Blood; Blood - microbiology; Coagulase - metabolism; Deoxyribonucleic acid; DNA; DNA, Bacterial - isolation & purification; Hospitals; Human bacterial diseases; Humans; Infectious diseases; Intensive care; Internal Medicine; Laboratories; Medical Microbiology; Medical sciences; Methods; Microbiology; Mortality; Polymerase Chain Reaction; Sepsis; Staphylococcal Infections - diagnosis; Staphylococcal Infections - microbiology; Staphylococcal infections, streptococcal infections, pneumococcal infections; Staphylococcus - classification; Staphylococcus - genetics; Staphylococcus - isolation & purification; Staphylococcus aureus; Staphylococcus aureus - classification; Staphylococcus aureus - genetics; Staphylococcus aureus - isolation & purification; Staphylococcus infections; Netherlands; Blood Culture Bottle; Reduce Incubation Time; Blood Culture System; Blood Culture; Accelerate Detection; Microbiology; Identification; Method; Infection; Microbiological investigation; DNA; Bacteriosis; Bacteria; Micrococcales; Micrococcaceae; Blood culture; Staphylococcal infection; Comparative study; Staphylococcus aureus
• ISSN: 0934-9723; 1435-4373
• DOI: 10.1007/s10096-010-1090-0

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.