A bright spot for microbiological diagnostics

Electron microscopic image of Staphylococcus aureus, one of the pathogenic bacterial species that can be detected with the new molecular probes.

© HZI/Manfred Rohde

The detection of bacterial pathogens in clinical samples is an important prerequisite for successful antimicrobial treatment. A team from the German Center for Infection Research (DZIF), the Helmholtz Centre for Infection Research (HZI) and Tel Aviv University, Israel, has now developed molecular probes that can detect different bacterial species with high sensitivity. Bacterial cells can activate the probes and make them glow. This signal enables pathogen detection directly in clinical samples. The results were published in the international edition of the journal Angewandte Chemie.

In view of the increasing threat of antibiotic resistance, diseases should not be treated with antibiotics on suspicion. Therefore, antibiotic stewardship requires prior verification that a bacterial infection is indeed present. However, microbiological methods in which pathogens are cultivated on growth media take time, and are often not successful, for instance, if patients have already been treated with antibiotics. Improved diagnostics therefore constitutes an important component in the fight against antibiotic resistance.

A research team led by Prof Mark Brönstrup from the German Center for Infection Research (DZIF) and the Helmholtz Centre for Infection Research (HZI) now contributes to the fight with the development of novel probes for bacterial pathogens. The researchers make use of a class of molecules that bacteria employ to sequester iron-containing compounds—the siderophores.

“Iron is a scarce resource in the body. Therefore, bacteria also sequester siderophores that are not chemically identical to those they produce themselves,” says Carsten Peukert, PhD student in the Department “Chemical Biology” at HZI and first author of the study. “This predestines siderophores for use as molecular Trojan horses.”

The researchers coupled a dioxetane molecule to natural or synthetic siderophores. Dioxetane-related molecules are also responsible for the characteristic glow in fireflies and don’t require an external light source. However, to ensure that the probes only light up in presence of bacteria, the researchers integrated a molecular light switch. This is activated when bacteria enzymatically cleave the probes in their cells.

Using the probes, the research team was able to detect all bacterial pathogens known under the acronym ESKAPE. The term covers the pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. These pathogens are considered typical representatives of hospital germs and are frequently resistant to antibiotics.

“Especially Gram-negative bacteria such as Pseudomonas or Klebsiella, which have two cell membranes, could not be reliably labelled with the precursors of our probes. We have now further developed the system so that this group of bacteria can also be detected,” says corresponding author Prof Mark Brönstrup, a scientist in the DZIF research area "Novel Antibiotics" and head of the Department “Chemical Biology” at HZI.

The probes can also detect bacteria that multiply inside host cells. The technical requirements for the detection of the probes in clinical material such as blood plasma are comparatively low, allowing the system to be used outside specialised laboratories in the future.

Alternative molecular diagnostic methods such as mass spectrometry or the polymerase chain reaction do not distinguish between dead and living bacterial cells. “Our method offers the advantage that only living bacteria are detected. Hence, lighting up of the probes indicates an active infection,” says Prof Brönstrup.

In addition to the probes’ use in clinical samples, application in food safety is also conceivable, adds Prof Brönstrup. Moreover, the siderophore-based system can be adapted to deliver antimicrobial agents instead of the luminescent probes. The bright spot for diagnostics is thus only the beginning of applications for the molecular Trojan horses, the researchers say.

The study was funded by the German Research Foundation, the Joint Programming Initiative on Antimicrobial Resistance and the Fonds der Chemischen Industrie.

Source: Press release of the Helmholtz-Centre for Infection Research (HZI).

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