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

Andrey Grushnikov,Kazuma Kikuchi,Yasushi Yagi,Yoshimi Matsumoto,Akihito Yamaguchi,Kunihiko Nishino,Hiroyuki Noji,Mohamed N Seleem,Ryota Iino,Shouichi Sakakihara

doi:10.1371/journal.pone.0148797

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.

Highlights

Bacterial antibiotic resistance is an alarming issue, affecting veterinary and human health
The key to the rapidity of the drug susceptibility testing microfluidic (DSTM) method is the introduction of a microscope for susceptibility judgment
Only Choi J. et al.[42, 61] described the susceptibility results from P. aeruginosa obtained over a 3–4 h period

Summary

Introduction

Bacterial antibiotic resistance is an alarming issue, affecting veterinary and human health. Pseudomonas aeruginosa is one of the major causative agents of hospital-acquired infections, among patients admitted to the intensive care unit. P. aeruginosa infections, those in immunocompromised patients, often result in life-threatening disease [1, 2] and are untreatable because of resistance to multiple antimicrobial agents. Multidrug-resistant P. aeruginosa (MDRP) strains resistant to major antipseudomonal agents such as carbapenems, quinolones, and aminoglycosides, have recently become prevalent [8, 9] and have caused nosocomial outbreaks in Japan.[10, 11] Few antibiotics are available for the treatment of MDRP infections, and one of the few effective drugs against this pathogen, colistin [12] was only available until very recently in Japan.

Methods

Results

Conclusion