- Rapid antimicrobial susceptibility testing (AST) using Raman spectroscopy combined with a multiwell-dielectrophoresis chip
Rapid antimicrobial susceptibility testing (AST) using Raman spectroscopy combined with a multiwell-dielectrophoresis chip
in: Infection (2019)
Introduction: Over the past yearsmulti-resistant pathogens have become an acute healthcare problem worldwide, which also critically affects sepsis patients. In order to initiate appropriate antibiotic therapy, the pathogen must be identified and its antibiotic sensitivities characterized. Due to several cultivation steps classical microbiological diagnosis takes up to several days from patient sample to result. Faster antimicrobial susceptibility testing (AST) is urgently needed to avoid both, inappropriate undertreatment resulting in increased mortality in sepsis patients and the unnecessary use of broad-spectrum antibiotics fostering further spread of multi-resistant bacteria.We develop a rapid cultivation-free procedure for isolation of the pathogen directly from patient’s body fluid, followed by spectroscopic identification and subsequent AST. The whole procedure from urine sample toASTresult takes as little as 3.5 h . Here,we present a simple and fast on-chip spectroscopic technique to identify antibiotic susceptibilities and furthermore, to determine the minimal inhibitory concentration (MIC) of bacterial isolates within 2 h. Objectives: Our aim is the development of a parallelized and automated procedure based on Raman spectroscopy in combination with a multiwell-dielectrophoresis (DEP) chip for identification and AST of sepsis pathogens within a few hours. Methods: Raman spectroscopy is a nondestructive and label-free method for analysis of chemical composition, enabling measurement of biological samples in aqueous environment. A laser is irradiated on the specimen and the inelastically scattered light yields its vibrational fingerprint, which allows identification of the pathogen and comprises information of its phenotypic behavior [1, 2]. To concentrate the bacteria from patient’s bodily fluids, e.g. urine, DEP is used. In an alternating inhomogeneous electric field, dielectric forces capture particles along the field lines. Utilizing a quadrupole electrode design, bacteria can be accumulated in the middle of the electrodes for direct spectroscopic characterization . The novel multiwell-DEP-chip allows parallelized analysis of 20 individual conditions of bacteria-drug combinations. The test panel consists of four clinically relevant antibiotics with four concentrations permitting quantitative AST results within 2 h . Results: When treated with antibiotics, spectral changes in susceptible bacteria can be observed, whereas resistant bacteria keep on growing and generate normal spectral growth signatures. The comparison of sample spectra to a database yields species identification. Principal component analysis defined spectral marker bands indicating effective antibiotic treatment. The spectroscopic approach was adapted to determine the MIC after only 90 min interaction time. The AST method using the novel multiwell-DEP-chip is demonstrated with gram-negative urosepsis pathogens, namely Klebsiella pneumoniae, Proteus mirabilis and Escherichia coli. Conclusions: We show how Raman spectroscopy can be utilized to visualize the effect of antibiotics with different mechanisms of action on sepsis pathogens with various antibiotic susceptibility patterns. Only minimal sample preparation is required and short-time cultivation of 90 min is sufficient. The combination of DEP for concentrating bacteria and Raman-spectroscopy for fast and non-destructive characterization has the potential to become a fast and reliable diagnostic tool. References:  Schro¨der et al. Anal. Chem. 2015,  Kloss et al. Anal. Chem. 2013,  Schro¨der et al. Sci. Rep. 2013,  Kirchhoff et al. Anal. Chem. 2018.