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. 2018 Jul 10;9:1530.
doi: 10.3389/fmicb.2018.01530. eCollection 2018.

Rapid Phenotypic Antibiotic Susceptibility Testing of Uropathogens Using Optical Signal Analysis on the Nanowell Slide

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Free PMC article

Rapid Phenotypic Antibiotic Susceptibility Testing of Uropathogens Using Optical Signal Analysis on the Nanowell Slide

Marta Veses-Garcia et al. Front Microbiol. .
Free PMC article

Abstract

Achieving fast antimicrobial susceptibility results is a primary goal in the fight against antimicrobial resistance. Standard antibiotic susceptibility testing (AST) takes, however, at least a day from patient sample to susceptibility profile. Here, we developed and clinically validated a rapid phenotypic AST based on a miniaturized nanotiter plate, the nanowell slide, that holds 672 wells in a 500 nl format for bacterial cultivation. The multitude of nanowells allows multiplexing with a panel of six antibiotics relevant for urinary tract infections. Inclusion of seven concentrations per antibiotic plus technical replicates enabled us to determine a precise minimum inhibitory concentration for 70 clinical uropathogenic Escherichia coli isolates. By combining optical recordings of bacterial growth with an algorithm for optical signal analysis, we calculated Tlag, the point of transition from lag to exponential phase, in each nanoculture. Algorithm-assisted analysis determined antibiotic susceptibility as early as 3 h 40 min. In comparison to standard disk diffusion assays, the nanowell AST showed a total categorical agreement of 97.9% with 2.6% major errors and 0% very major errors for all isolate-antibiotic combination tested. Taking advantage of the optical compatibility of the nanowell slide, we performed microscopy to illustrate its potential in defining susceptibility profiles based on bacterial morphotyping. The excellent clinical performance of the nanowell AST, combined with a short detection time, morphotyping, and the very low consumption of reagents clearly show the advantage of this phenotypic AST as a diagnostic tool in a clinical setting.

Keywords: AST; UTI; algorithm; antibiotic resistance; diagnostics; microfabrication.

Figures

FIGURE 1
FIGURE 1
Characterization of the nwSlide as an AST platform. (A) Photograph of a nwSlide (25 mm × 75 mm) holding 672 nanowells in a 14 × 48 matrix. (B) Side view of one nanowell with dimensions and volume (V) indicated. (C) Growth measured at OD600 of a wt Escherichia coli laboratory strain (W3110, black) and a strain with mutated fnr gene (BW25113Δfnr, blue) in a nwSlide. Circa 200 nanowells were recorded for each strain, dashed lines = SD, n = 3. (D) The design of functionalized nwSlides used for nwASTs. The left side offers 24 non-functionalized nanowells each for negative (NEG., medium only) and positive (POS., inoculated medium) controls of bacterial growth. The antibiotics ampicillin (blue, AMP), ciprofloxacin (green, CIP), nitrofurantoin (red, NIT), cefadroxil (purple, CFR), mecillinam (yellow, MEC), and trimethoprim (brown, TMP) are coated and distributed in separate rows. The antibiotic concentration varies from lowest (left) to highest (right) as indicated schematically above the nwSlide. Each antibiotic is represented by seven twofold dilutions. Each concentration includes four nanowells that serve as technical replicates. Antibiotic concentrations in individual experiments are defined in Section “Materials and Methods.” (E) MIC determination of the reference strain E. coli ATCC 25922 from one nwAST functionalized as in (D). The heatmap shows OD600 recordings in each of the 216 nanocultures over 12 h at indicated conditions. A color change from yellow (low OD600) to red (high OD600) in one row indicates bacterial growth in the corresponding nanowell. Negative and positive controls include 24 wells each. For each antibiotic, growth pattern of the 28 nanocultures at 7 antibiotic concentrations is shown. The vertical gradient symbol indicates that nanocultures in the upper rows are exposed to the lowest concentration of antibiotics, whereas a gradual increase leaves the lower rows representing nanowells exposed to the highest concentration. Black dots represent the Tlag of each nanoculture.
FIGURE 2
FIGURE 2
Tlag distribution of clinical UPEC isolates. (A–F) Dot plots showing the Tlag generated by each of the 70 clinical UPEC isolates at the highest antibiotic concentration they were able to grow at. Strains are grouped as resistant strains (R), which generated a Tlag above the clinical breakpoint, and susceptible strains (S), which generated a Tlag below the clinical breakpoint of each antibiotic. Median is shown with whiskers drawn at the 25th and 75th percentile. (G) Box plots showing the overall distribution of Tlag values obtained for resistant (R) and sensitive (S) strains irrespective of the type of antibiotic. Whiskers show the minimum and maximum value of Tlag.
FIGURE 3
FIGURE 3
Imaging and morphotypic quantification of bacterial morphologies upon exposure to antibiotics. Phase contrast microscopy of the clinical UPEC isolates (A) ARD311 and (B) ARD318 in the nwAST shows the bacterial morphology after 3 h exposure to MIC (sensitive isolates) or breakpoint (resistant isolates) concentrations of each antibiotic. Scale bar = 5 μm, applies to all micrographs. Quantification of morphotypic parameters for strain (C–E) ARD311 and (F–H) ARD318 upon exposure to ampicillin (AMP), ciprofloxacin (CIP), nitrofurantoin (NIT), cefadroxil (CFR), mecillinam (MEC), and trimethoprim (TMP). Morphotypic parameters of major to minor axis, perimeter, and circularity were calculated based on image analysis from four technical replicates per antibiotic. The average and standard deviation is shown. Ctrl = no antibiotic added.

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