A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa

Abstract

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller–Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

Multiparameter antibiotic resistance detection based on hydrodynamic trapping of individual E. coli

“…Firstly, we demonstrate that the combined assessment of motility and morphology at the single-cell level could guide the design of novel susceptibility tests and, more generally, to study bacterial behaviour upon antibiotics administration. While the two parameters have been used separately as indicators of susceptibility, 4,26,34,35,39 their combination at the single cell level is presented here for the first time. In particular, we have shown the possibility to correctly classify susceptible and resistant strains in less than two hours (from spiking the culture) for three drugs of a very different nature, i.e.…”

“…To this purpose, we also exploit bacterial morphology as a second indicator of response to antibiotics, which has already been shown to be a suitable parameter to detect susceptibility. 4,39 However, to the best of our knowledge, the combined analysis has never been performed at the single-cell level before.…”

Microfluidics for Antibiotic Susceptibility and Toxicity Testing

“…In contrast to determining antibiotic resistance in terms of bacterial growth, monitoring cell death that is facilitated by applying mechanical and enzymatic stress on a microfluidic chip can achieve rapid identification of antibiotic resistant strains within 60 min [ 23 ]. In addition to rapid AST, the MIC determined by microfluidic AST has been shown to be in good agreement with those by conventional methods for standard Clinical Laboratory Standard Institute (CLSI) strains [ 18 , 22 , 24 , 25 ]. Although there are a number of papers dealing with microfluidic rapid AST, few commercial systems have been developed for clinical use.…”

Microfluidic advances in phenotypic antibiotic susceptibility testing

“…recently reported on a microfluidic platform in which bacteria are loaded into channels containing pre-loaded, dried antibiotic (Matsumoto et al 2016). Three concentrations of five antibiotics (plus controls) can be tested on a single chip, giving a susceptible or resistant result.…”