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, 52 (9), 3310-7

Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing

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Bacterial Nanoscale Cultures for Phenotypic Multiplexed Antibiotic Susceptibility Testing

Emilie Weibull et al. J Clin Microbiol.

Abstract

An optimal antimicrobial drug regimen is the key to successful clinical outcomes of bacterial infections. To direct the choice of antibiotic, access to fast and precise antibiotic susceptibility profiling of the infecting bacteria is critical. We have developed a high-throughput nanowell antibiotic susceptibility testing (AST) device for direct, multiplexed analysis. By processing in real time the optical recordings of nanoscale cultures of reference and clinical uropathogenic Escherichia coli strains with a mathematical algorithm, the time point when growth shifts from lag phase to early logarithmic phase (Tlag) was identified for each of the several hundreds of cultures tested. Based on Tlag, the MIC could be defined within 4 h. Heatmap presentation of data from this high-throughput analysis allowed multiple resistance patterns to be differentiated at a glance. With a possibility to enhance multiplexing capacity, this device serves as a high-throughput diagnostic tool that rapidly aids clinicians in prescribing the optimal antibiotic therapy.

Figures

FIG 1
FIG 1
Nanowell AST device. (A) Illustration of the nanowell slide with 672 wells (500 nl/well). (B) Illustration of the tilted walls of the wells providing large surface-to-volume ratio, fast diffusion, and gas exchange. Each well is labeled with a unique x-y position indicator. (C) Side view illustrating a well precoated with an antibiotic (orange). (D) Top-view illustration of a slide with defined areas for bacterial growth in the absence or presence of an antibiotic concentration. Each number represents a different antibiotic concentration in μg/ml. (E) Custom-designed adaptor for placing the nanowell AST device in a microplate reader. W, water bath.
FIG 2
FIG 2
Heatmap representation and Tlag determinations for MIC analysis. (A) Heatmap representation of OD600 recordings from ATCC 25922 in M-H II broth with 0 to 32 μg/ml of ampicillin (Amp) at different inoculum sizes (5, 50, and 500 CFU/well). The vertical axis indicates experimental conditions and inoculum sizes (5, 50, 500 CFU/well). “Exp no.” refers to the 3 experiments for each inoculum, each including 4 rows corresponding to OD600s recorded from wells 1 to 4 every 10 min over 10 h. Increased absorbance is depicted as a change from yellow to red along the horizontal axis. Black dots indicate Tlag in each culture positive for growth. (B) Dot plots showing Tlag calculated from the 4 wells per experiment at 0 to 32 μg/ml of Amp for 5 CFU/well (left), 50 CFU/well (middle), and 500 CFU/well (right). Shapes and colors of dots represent data collected from wells derived from the same experiment. The grand mean for each condition is depicted as a black horizontal line.
FIG 3
FIG 3
Multiplex nanowell AST for MIC determination. Shown is a heatmap representation of growth curves (ATCC 25922) in the multiplex nanowell AST. The vertical axis indicates the 11 conditions tested. Positive (ATCC 25922 in M-H II broth) and negative (MH-II broth and M-H II broth plus antibiotics: 16 μg/ml of ampicillin [Amp], 0.03 μg/ml of ciprofloxacin [Cipro], and 0.03 μg/ml of cefotaxime [Cefo]) controls were included. “Exp no.” refers to the 3 independent experiments, each including 4 rows corresponding to OD600s from wells 1 to 4 recorded every 5 min over 12 h. Increased absorbance is depicted as a change from yellow to red along the horizontal axis.
FIG 4
FIG 4
Clinical strains analyzed in the multiplexed nanowell AST. Shown is a heatmap representation of antibiotic susceptibility of clinical strains ARD144 (A), ARD145 (B), and ARD146 (C) against ciprofloxacin (Cipro) and cefotaxime (Cefo) at the indicated concentrations (μg/ml). Positive (strains in M-H II broth) and negative (M-H II broth and M-H II broth plus antibiotics [4 μg/ml of Cipro or 64 μg/ml of Cefo]) controls were included. “Exp no.” refers to the 2 independent experiments, each including 4 rows corresponding to OD600s from wells 1 to 4 recorded every 5 min over 12 h. Increased absorbance is depicted as a change from yellow to red along the horizontal axis. Classification of strains at each condition is indicated as susceptible (S) and resistant (R) based on CLSI guidelines.

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