A high-throughput fluidic chip for rapid phenotypic antibiotic susceptibility testing

Abstract

Many patients with severe infections receive inappropriate empirical treatment and rapid detection of bacterial antibiotic susceptibility can in this context improve clinical outcome and reduce mortality. We have to this end developed a high-throughput fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime and meropenem (Gramnegative bacteria) or gentamicin, ofloxacin and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in 8 separate growth chambers in the fluidic chip. The chip was thereafter connected to a reservoir lid containing different antibiotics and a pump used to draw growth media with or without antibiotics into the chip for generation of diffusion-limited antibiotic gradients in the growth chambers. Bacterial microcolony growth was monitored using darkfield time-lapse microscopy and quantified using a cluster image analysis algorithm. Minimum inhibitory concentration (MIC) values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2-4 hours and the results showed categorical agreement to reference MIC values as determined with broth microdilution in 86% of the cases.\n\nImportancePrompt and effective antimicrobial therapy is crucial for the management of patients with severe bacterial infections but is becoming increasingly difficult to provide due to emerging antibiotic resistance. The traditional methods for antibiotic susceptibility testing (AST) used in most clinical laboratories are reliable but slow with turnaround times of 2-3 days, which necessitates the use of empirical therapy with broad-spectrum antibiotics. There is a great need for fast and reliable AST methods that enable start of targeted treatment within a few hours to improve patient outcome and reduce overuse of broad-spectrum antibiotics. The high-throughput fluidic chip for phenotypic AST described in the present study enables data on antimicrobial resistance within 2-4 hours allowing for an early initiation of appropriate antibiotic therapy.