Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing

Abstract

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

Keywords: Antibiotic resistance; Bacteria; Infection; Microfluidic; Urinary tract infection.

Fig 1.. Nanotube assisted microwave electroporation (NAME) for single cell pathogen identification.

(A) Schematic of microwave electroporation enhanced by multiwall carbon nanotubes for delivering molecular probes into viable bacteria. (B) Multicolor double-stranded probes for multiplex 16S rRNA detection. Hybridization of the target 16S rRNA with the donor probe displaces the quencher probe allowing the fluorophore to fluoresce. Fluorescence is detected only when a specific probe is transformed into the bacteria for pathogen identification. (C) Intracellular detection of bacterial 16S rRNA in viable cells enables pathogen identification and subsequent antimicrobial susceptibility testing at the single cell level. (D) Multiplex detection of E. coli and P. aeruginosa by NAME. Multicolor double-stranded probes targeting E. coli (EC probe, red) and P. aeruginosa (PA probe, green) were transformed into samples with E. coli, P. aeruginosa, or a mixture of both bacteria at 1:1 ratio. Fluorescence images with merged red and green channels (top) and bright-field images (bottom) demonstrate pathogen identification at the single cell level. Images are representative of three experiments. Scale bars, 25 μm.

Fig 2.. Transformation of double-stranded probes into viable bacteria.

(A) Overlay images of E. coli clinical isolates (EC137) transformed with or without multiwall carbon nanotubes. Fluorescence was observed only in samples with nanotubes. E. coli treated with the same microwave duration (i.e., no probe) was applied as control. Scale bars, 50 μm. (B) Overlay images illustrating the effect of the microwave time on the transformation efficiency. Scale bars, 50 μm. Images are representative of three experiments. (C) Effects of the transformation solution on the transformation efficiency and ability of the bacteria to grow. (D) The effect of the incubation time on the transformation efficiency (n=3).

Fig 3.. Single cell pathogen identification of clinical specimens.

(A) Overlay images demonstrating detection of E. coli, P. aeruginosa and K. pneumoniae clinical isolates from patient urine and blood samples. Scale bars, 25 μm. (B) Culture-free pathogen identification of patient urine samples. Scale bar, 25 μm. (C) Multiplex detection of E. coli and P. aeruginosa with EC, PA, and UNI probes (n=3).

Figure 4.. Microfluidic single cell pathogen identification and antimicrobial susceptibility testing (AST).

(A) Schematic of the microfluidic single cell AST device. Bacteria are loaded into the channels by capillary force. Physical trapping of the bacteria allows rapid phenotypic AST by monitoring the bacterial growth as the single cell level. (B) Overlay images showing pathogen identification and growth monitoring of a single bacterium (EC 137). Scale bars, 5 μm. (C) For single cell pathogen identification and AST, fluorescence detection was first performed at the beginning of the experiment. Time-lapse bright-field microscopy is performed to monitoring the growth kinetics of the bacteria in the microfluidic channel. Time-lapse images illustrate the growth of the two uropathogenic clinical isolates EC137 (ciprofloxacin susceptible) and EC132 (ciprofloxacin resistant). Yellow boxes indicate bacteria trapped in the channel. Scale bars, 5 μm. (D-E) Representative growth curves of bacteria with and without antibiotics. Each curve represents the growth of a single bacterium.