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. 2023 May 9;95(18):7329-7335.
doi: 10.1021/acs.analchem.3c00478. Epub 2023 Apr 21.

Bioluminogenic Probe for Rapid, Ultrasensitive Detection of β-Lactam-Resistant Bacteria

Tingting Dai  1 , Jinghang Xie  2 , Joseph A Buonomo  1   3 , Angel Moreno  4 , Niaz Banaei  4   5   6 , Carolyn R Bertozzi  1   7   3 , Jianghong Rao  1   2   3
Affiliations

Bioluminogenic Probe for Rapid, Ultrasensitive Detection of β-Lactam-Resistant Bacteria

Tingting Dai et al. Anal Chem. .

Abstract

Increasingly difficult-to-treat infections by antibiotic-resistant bacteria have become a major public health challenge. Rapid detection of common resistance mechanisms before empiric antibiotic usage is essential for optimizing therapeutic outcomes and containing further spread of resistance to antibiotics among other bacteria. Herein, we present a bioluminogenic probe, D-Bluco, for rapid detection of β-lactamase activity in viable pathogenic bacteria. D-Bluco is a pro-luciferin caged by a β-lactamase-responsive cephalosporin structure and further conjugated with a dabcyl quencher. The caging and quenching significantly decreased the initial background emission and increased the signal-to-background ratio by more than 1200-fold. D-Bluco was shown to detect a broad range of β-lactamases at the femtomolar level. An ultrasensitive RAPID bioluminescence assay using D-Bluco can detect 102 to 103 colony forming unit per milliliter (cfu/mL) of β-lactamase-producing Enterobacterales in urine samples within 30 min. The high sensitivity and rapid detection make the assay attractive for the use of point-of-care diagnostics for lactam-resistant pathogens.

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Conflict of interest statement

Notes

The authors declare the following competing financial interest(s): C.R.B. is a cofounder of Redwood Biosciences (a subsidiary of Catalent), Enable Biosciences, Palleon Pharmaceuticals, InterVenn Bio, Lycia Therapeutics, and OliLux Biosciences, and a member of the Board of Directors of Eli Lilly. All other authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Bioluminogenic substrates for the detection of β-lactamase (Bla) activity.
Figure 2.
Figure 2.
Characterization of D-Bluco with recombinant β-lactamase. (a) Longitudinal monitoring of fluorescence enhancement of D-Bluco (1 μM) with or without TEM-1 (100 nM) in PBS (pH = 7.4). (Ex-330/Em-530). (b) Fluorescence emission spectrum of D-Bluco (1 μM) incubated with TEM-1 (100 nM) for 40 min; a.u. indicates arbitrary unit. (c) Bioluminescent signal of Bluco or D-Bluco (10 μM) incubated with or without TEM-1 (20 nM) for 15 min in PBS (pH = 7.4). Experiments were conducted at 25 °C. Error bars indicate standard deviations (n = 3).
Figure 3.
Figure 3.
Verification of quenching ability with D-luciferin and Bluco analogues. Bluco, Am-Bluco, and D-Bluco (70 μM) were incubated with 4 μM of luciferase for 10 min (t0) before further luciferase addition at a 4 min interval (t1 to t4). Error bars indicate standard deviations (n = 3).
Figure 4.
Figure 4.
Sensitivity of D-Bluco toward different types of lactamase enzymes. Bioluminescent signal of D-Bluco incubated with different concentrations of enzyme for 2 h. The red dashed line indicates three times the standard deviation of the blank. RLU indicates relative light units. Experiments were conducted at 25 °C. Error bars indicate standard deviations (n = 4).
Figure 5.
Figure 5.
Bioluminescence detection of β-lactamase with clinic isolates by D-Bluco. (a) Inhibitory study with clavulanate (2 mg/mL) and avibactam (2 mg/mL) in the presence of D-Bluco (10 μM) with E. coli/TEM-1. E. coli/TEM-1 treated with D-Bluco exhibited a 260-fold increase of bioluminescent intensity over PBS, which was arbitrarily set as 1 to normalize the test samples and show percentage inhibition. (b) D-Bluco (10 μM) was incubated with different concentrations of E. coli or E. coli expressing TEM (E. coli/TEM). The BLI signal was monitored over 4 h in PBS (pH = 7.4). Statistical significance was calculated using the unpaired two-tailed Student’s t-test (*p < 0.0332, ***p < 0.0002, and ****p < 0.0001 ns: not significant). RLU indicates relative light units. Experiments were conducted at 25 °C. Error bars indicate standard deviations (n = 3).
Figure 6.
Figure 6.
Development of RAPID BLI test for clinical isolates of UTI. (a) RAPID BLI assay workflow for clinical sample detection. (b) Bioluminescent intensity of 102, 103, 104 cfu/mL of β-lactamases-expressing clinic isolates after incubation with D-Bluco (10 μM) in diluted urine. From left to right: (1) E. cloacae/IMI, (2) E. coli/TEM, (3) K. pneumoniae/KPC, (4) E. coli/NDM, (5) E. cloacae/AmpC. (c) Bioluminesecent intensity of 105 cfu/mL E. coli and 103 cfu/mL K. pneumoniae/KPC. The modified rapid BLI protocol is applied. The signal of D-Bluco in PBS was subtracted before plotting. The working concentration of D-Bluco was 10 μM. Statistical significance was calculated using the unpaired two-tailed Student’s t-test (***p < 0.0002 and ****p < 0.0001, for comparison with 105 cfu/mL E. coli) RLU indicates relative light units. Dot line represents 3SD of the negative control. Part of the image is adapted from Servier Medical Art. Error bars indicate standard deviations (n = 3).
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