Faster differentiation of Staphylococcus aureus versus coagulase-negative Staphylococci from blood culture material: a comparison of different bacterial DNA isolation methods

Introduction

Frequent usage of medical devices, such as intravenous lines, often results in sepsis, which is characterized by high morbidity and mortality. Rapid and reliable detection and differentiation between Staphylococcus aureus and coagulase-negative Staphylococci (CNS) is therefore clinically relevant to be able to provide adequate early treatment. Blood culture is still the gold standard method in identifying these pathogens but is time consuming. Molecular diagnostics might be a promising alternative to reduce this time-to-result delay.

Objective

This study aims to compare different DNA extraction methods from two commonly used blood culture materials, BACTEC (BD) and Bact/ALERT (Biomerieux), to accelerate differentiation between S. aureus and CNS.

Methods

Two fast real-time PCR duplex test assays, targeting the Tuf gene, to differentiate S. aureus from CNS, were developed in order to select the most sensitive one. This Tuf RT-PCR was used to compare three different DNA isolation methods on two different blood culture systems. Negative blood culture material was spiked with S. aureus; bacterial DNA was isolated with: automated extractor EasyMAG (Biomerieux), automated extractor MagNA Pure (Roche), and a manual kit MolYsis Plus (MolZyme).

Results

The best Tuf RT-PCR method appeared to have a sensitivity of 100 CFU/ml. Approximately 50 positive blood cultures containing Gram-positive cocci in clusters were tested in the Tuf RT-PCR and all were identified correctly. Bacterial DNA isolation, from spiked blood culture material, with the EasyMAG showed the highest analytical performance with a detection limit of 10³ CFU/ml in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 10⁴ CFU/ml. Hand-on time, for 26 samples, was lowest for the EasyMAG (10 minutes) and highest for the manual kit of MolZyme (2 hours). Total handling time was highest for the MolYsis Plus kit (3.5 hours) and lowest for the automated extractor EasyMAG (50 minutes).

Conclusion

A sensitive RT-PCR was developed for detection and differentiation of S. aureus versus CNS. Bacterial DNA isolation from Bact/ALERT blood culture material seems to show better reproducibility compared with isolation from BACTEC blood culture material. In this preliminary study the EasyMAG performed better when compared with MolYsis Plus and the MagNA Pure system. In future work this method will be further evaluated with reduced culture times.