Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: PNA probe hybridization assay for specific detection of uropathogens. A) We have designed PNA probes specific to the i) E. coli and ii) P. mirabilis species, the iii) Enterobacterales order, and the iv) eubacterial kingdom, in order to be able to detect all predominant uropathogens. We ensure that the designed probes can detect signal from target bacteria over blank urine background (N) by measuring probe fluorescence in the presence of E. coli (EC), P. mirabilis (PM), K. pneumoniae (KP), and P. aeruginosa (PA) ( p ‐values are calculated using unpaired one‐tailed t ‐tests; * p < 0.05, ** p < 0.01, *** p < 0.001, no asterisks between bars indicates no significant difference). B) Our assay works across a wide range of i) lysis temperatures and ii) hybridization temperatures (green: selected temperatures). C) Bulk‐based pheno‐molecular AST of reference E. coli ATCC 25922 and multi‐drug resistant E. coli BAA 2471 using hybridization detection of 16S rRNA is feasible, but requires >90 min of culture/antibiotic exposure to differentiate the effect of gentamicin on the susceptible and the resistant strains of E. coli . Data presented as mean +/− SD, n ≥ 3 or n ≥ 2 (bulk pheno‐molecular AST).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Hybridization, Bacteria, Fluorescence, One-tailed Test, Lysis

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Single‐cell detection of bacterial 16S rRNA from urine using microfluidic droplets. A) i) Urine samples of distinctly different color and turbidity can be discretized using flow‐focusing to generate monodisperse droplets (scale bars ≈50 µm) of ii) 4 ± 1 pL volume. B) Droplet fluorescence peak traces i) without E. coli , droplets emit baseline fluorescence signal, and have a positive droplet rate of 0.0079% (also known as the average limit of blank). ii) In the presence of 10 7 CFU mL −1 E. coli , droplets emit a higher fluorescence signal, and have a positive droplet rate of 6.67%. C) Droplet‐based quantification of E. coli in urine across four orders of magnitude within the clinically relevant dynamic range for UTIs (10 4 to 2 × 10 7 CFU mL −1 ), R 2 = 0.992 D) i) Reduction in droplet volume from 30 to 4 to 1 pL results in lower background fluorescence signals (scale bars in white ≈100 µm). ii) Compared to larger 30 pL droplets, 4 pL droplets facilitate faster generation of differentiable fluorescence signal over the reduced local background, as quickly as within 15 min. Data in (C,D(i)) presented as mean +/− SD, n ≥ 2 except for 2 × 10 7 CFU mL −1 input bacterial concentration in (C).

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Fluorescence, Concentration Assay

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: Accelerating antimicrobial susceptibility assessment via quantitative measurement of 16S rRNA from single cells. A) LIF detection of droplets containing E. coli cells suspended in MH broth i) without 30 min culture results in the expected 8% positive droplet frequency (7.02% observed), ii) following 30 min culture results in higher positive droplet intensities (indicative of higher 16S rRNA production) and 7.70% frequency, and iii) and after 30 min culture along with bactericidal gentamicin results in lower positive droplet intensities (indicative of relatively lower 16S rRNA production) and 3.12% frequency. B) Resistant E. coli can be differentiated from reference E. coli spiked into urine by comparing the positive droplet percentage from cells subject to antibiotic and no‐antibiotic conditions (“Normalized Positive Droplet Population”) for culture/drug exposure durations as low as 10 min. C) Resistant and susceptible strains of E. coli can be differentiated using our platform for three different antibiotics spanning distinct classes—gentamicin (aminoglycoside), ciprofloxacin (fluoroquinolone), and ampicillin (beta lactam). Error bars represent 1 standard deviation. The p ‐values are calculated from unpaired one‐tailed t ‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Standard Deviation, One-tailed Test

Journal: Advanced Science

Article Title: Droplet‐Based Single‐Cell Measurements of 16S rRNA Enable Integrated Bacteria Identification and Pheno‐Molecular Antimicrobial Susceptibility Testing from Clinical Samples in 30 min

doi: 10.1002/advs.202003419

Figure Lengend Snippet: DropDx clinical comparison study of 50 deidentified patient samples from Johns Hopkins Hospital. A) Each sample was simultaneously tested using clinical standard ID/AST tests as well as with 2 DropDx devices for measurements without and with ciprofloxacin. For ID, we used a combination of EC, EB, and UNI probes. B) Our 7‐outcome DropDx workflow is used to determine if there is a Gram‐negative bacterial infection present, whether the infecting pathogen is E. coli , whether the infecting pathogen is in the Enterobacterales order, or whether the infecting pathogen is a different (Gram‐negative) bacteria and to assess the susceptibility of the infecting pathogen to ciprofloxacin. λ is the proportion of droplets that contain a single cell to all droplets. C) Unbiased thresholding for each diagnostic metric was conducted in pilot studies using ROC curve analysis, and the final data groups and resulting ROC curves are plotted for i) differentiating culture‐positive from culture‐negative samples (AUC: 0.964), for ii) differentiating E. coli from non‐ E. coli samples (AUC: 1.000), for iii) differentiating Enterobacterales from non‐ Enterobacterales samples (AUC: 0.956), and for D) differentiating ciprofloxacin resistant from susceptible samples (AUC: 0.951). Importantly, DropDx's single‐cell pheno‐molecular AST results in a categorical agreement of 95.3% with no major errors. Error bars represent mean and standard error. The p ‐values are calculated from unpaired one‐tailed t‐tests.

Article Snippet: To demonstrate, we incubated EC PNA probes with either multi‐drug resistant E. coli ATCC BAA 2471 or the reference E. coli strain, each strain without and with gentamicin (at a bactericidal concentration of 8 μg mL −1 ) in 20 μL sample volume for 0, 60, 90, or 120 min at 37°C before subjecting these samples to 2 min 95 °C lysis, 30 min 60 °C hybridization, and LIF detection within 10 μm wide detection channels.

Techniques: Comparison, Infection, Bacteria, Diagnostic Assay, One-tailed Test