Antibiotics development

Bacterial bioenergetics for better phenotype based antibiotics discovery – microcalorimetry based microbial activity assays

The challenge of developing novel antibiotics with old tools

The development of novel antibiotics faces a lot of challenges that the classic ‘Pasteur era’ technologies cannot handle. The development of effective new drugs must be able to address a number of questions hard to answer by traditional methodology:

  • How to address biofilm forming bacteria and communal resistance patterns that are different from planktonic growth resistance
  • Studying hard to reach tissues and bacterial resistance in complex matrices
  • Addressing persistent and dormant cells in biofilms, with re-growth of cells surviving initial treatment by going into quiescent states
  • Evaluation of combination treatment and studying cooperative effects of antibiotics and potentiating agents in relevant conditions
  • Accurate quantification - do you really know if a CFU is based on one or a million bacteria and errors in CFU counting due to bacterial clusters
  • Speed, sensitivity and work-load

The Symcel direct metabolic assays help solve all these challenges

The calScreener is uniquely positioned for the needs of antimicrobial research and gives a true phenotype fingerprint of the organism measured. Different bacteria and treatments create unique heat profiles that reveal significant information about the system tested. Calorimetry provides label-free, nondestructive measurement, and thus makes post-experimental analysis possible, while being independent of sample morphology. This means that assays can be performed on bacteria in solution as well as on solid media, including three-dimensional matrices such as bone biopsies and surgical and dental implant materials.

  • Observe the bacterial metabolism in real-time with continous measurement without manual intervention
  • Increased sensitivity and speed - around 1000x more sensitive than optical based assays, enabling monitoring of very small initial bacterial loads and faster answers
  • Precise determination of live bacteria - no signal from bacterial debris or metabolically inactive cells, as opposed to optical methods
  • Easy distinction between bacteriostatic and bactericidal modes of action from the continous measurement
  • MIC determination
  • Uniquely suited to monitor biofilm forming bacteria
  • Possible to monitor persister or quiescent cells as well as re-growth over time

Test concentration-dependent inhibition of bacteria

FYL25

LL-37

Heat flow curves of P. aeruginosa, testing the inhibitory effects of different concentrations of the FYL25 and LL-37 peptides.

Determine growth parameters through growth curves

FYL25

LL-37

Growth curves of P. aeruginosa, derived by plotting the accumulated energy released during the testing of the FYL25 and LL-37 concentration-dependent inhibition.

Assess dose-response relationship through total energy release

FYL25

LL-37

Through the total accumulated energy release, the inhibitory effect of each peptide concentration can be clearly seen and the dose-response assessed.

P. aeruginosa with increasing peptide concentrations.

In a new paper by Abdillahi et al, the calScreener bioactivity measurements are used to study detailed antimicrobial properties of novel collagen peptides. The direct metabolic readout is used for analysis of microbial growth kinetics and the effect on bacterial growth or inhibition properties by the peptides. The potency of bacterial killing is assessed in detail and benchmarked against the peptide LL-37.

Key results of the calScreener assay:

  • Improved determination of the characteristics of lead drug candidates and rank the desired properties
  • Direct visual results on bacteriostatic and/or bacteriocidal effects
  • Bacterial killing potency by determination of dose dependency
  • Quantifiable results on specific kinetic profile through shift in lag phase duration, maximum growth rate and maximum peak energy

The calScreener assay is demonstrated as a superior tool for determining lead candidate drugs and ranking the desired properties. This saves time and money during drug development and de-risks the development of novel antimicrobials. 

Testimonial

A picture of Dr. Suado Abdillahi who is the CSO at Colzyx

Dr. Suado Abdillahi

CSO, Colzyx

“Colzyx has developed novel antibacterial peptides derived from Collagen VI. 
We have extensively characterized the killing activity against both Gram-positive and Gram-negative bacteria in vitro and gained detailed insight in the bactericidal nature of these peptides.

The calScreener metabolic rates assay further shows the different impact on bacterial growth both on inhibition properties as well as the potency of bacterial killing in a planktonic growth system using P.aeruginosa as model organism. Calorimetric assessment of growth inhibition is a valuable tool to determine the properties of lead drug candidates and rank the desired properties.”

Scientific References

Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A–like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureusEscherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI–derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.

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