Programmpunkt

14:15

Titel: Using phage-coated magnetic beads to capture low amounts of viable pathogenic bacteria
ID: 15/DKMV
Art: Abstractautor
Session: Workshop 03
Diagnostic Microbiology (StAG DV)

Referent: Mathias Jansen (Aachen)


Abstract - Text

Background:

Infections caused by multi-drug resistant bacteria are one of the greatest threats for human health. However, the time gap between the initial signs of an infection and targeted antibiotic therapy depends on the availability of pure bacterial cultures for antimicrobial susceptibility testing. Therefore, innovative approaches regarding the rapid separation of bacterial cells from patient samples are urgently needed.

Objective:

The objective of this study was to test the capacity of phages coupled with magnetic beads for specifically capturing viable multi-drug resistant bacterial cells.

Methods:

Newly isolated and characterized phages were treated with UV-radiation in order to obtain non-infective DNA-free phage ghosts. Inactive phages were covalently linked with different magnetic beads. Capture experiments were performed with bacteria spiked in LB medium or in blood samples at defined concentrations. The capture performance and verification of sustained growth capacity was analyzed by counting CFU and via liquid growth assays.

Results:

With increasing UV-doses the number of resulting phage ghosts gradually increased under maintenance of phage integrity and binding capacity. Phages could equally well be bound to tosylactivated Dynabeads and Dynabeads coated in Epoxy groups, each with a size of 4.5 µm, or to MACS MicroBeads with around 50 nm in size. Using phage-coated Dynabeads, capture efficiencies of up to 27% could be achieved when bacteria were spiked at concentrations between 105 and 101 CFU/mL. However, the capture efficiency could be increased up to 50% by using MicroBeads, even when the number of bacteria was only 101 CFU/ml. Further tests showed a reduced but still reasonable effectiveness under more challenging conditions, as about 17% of 2*102 CFU/mL Acinetobacter baumannii cells could be purified from spiked blood samples in viable form using phage KARL-1. Moreover, the functionality of this approach could also be confirmed with different phages using Staphylococcus aureus or Pseudomonas aeruginosa as host.

Conclusion:

Even low numbers of bacterial cells, as they typically occur in clinical samples, such as from sepsis patients, can be extracted in viable form within a very short time using magnetized phages. Those bacteria are then readily available for antimicrobial susceptibility testing. This system may have the potential to significantly contribute to an accelerated diagnosis of life-threatening infectious diseases.