Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates

Rapid phenotypic stress-based microfluidic antibiotic susceptibility testing of Gram-negative clinical isolates



When you ask this question to a person, you are hoping that they feel better soon. When you ask the same of pathogenic bacteria, you are really wishing the opposite and that whatever treatment you use really hurts.

A team led by Dr. Alexis Sauer-Budge at Fraunhofer CMI is developing a novel rapid antibiotic susceptibility test (AST) to accelerate conventionally slow techniques for antibiotic susceptibility testing. Bacteremia, the presence of bacteria in otherwise sterile blood, is a potentially life-threatening condition for which antibiotics must be prescribed within hours of clinical diagnosis. However, identification and susceptibility profiling for both Gram-positive and Gram-negative bacterial species requires at least 48–72 h. This is because the current gold standard for bacteremia diagnosis is still based on conventional methods developed in the mid-1800s—growth in specialized media. Recent advancements in accelerated AST are related to the shift from macroscopic to microscopic observation of bacteria growth. Albeit more rapid than traditional macroscopic growth, this approach is still inherently limited by the growth rate of the bacterial population.

Our approach is fundamentally different. By applying environmental stress to bacteria in a microfluidic platform, we monitor bacterial response in the presence or absence of an antibiotic. If bacteria are resistant to antibiotic they will be able to sustain both stresses and survive, if not, they die. One of the important features of the living bacteria is the ability to sustain an outer protective layer – the plasma membrane, which allows for control of the material flow in and out of the cell. The unselective permeability of the plasma membrane is an indicator of imminent bacteria death. The Fraunhofer team uses a membrane impermeable fluorescent dye to identify the presence of dying bacteria and measure the percentage of dying bacteria of the overall population. In simple terms, the susceptible bacteria will show a high degree of fluorescence, while resistant ones will show little-to-no fluorescence.

The recent publication, in the journal of Scientific Reports, the Fraunhofer CMI team correctly assigned antibiotic susceptibility profiles of clinically relevant Gram-negative bacteria within two hours of antibiotic introduction. The sampled bacteria population consistent of Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa substantial is detected in about 40% of bacteremia cases. We also demonstrate that the platform is compatible with antibiotics with varying mechanisms of action—meropenem, gentamicin, and ceftazidime—highlighting the suitability of this platform to be included at early time points in the clinical workflow. For Full article Click HERE