Surface-Bound Antibiotic for the Detection of β-Lactamases

Antimicrobial resistance (AMR) has been identified as a major threat to public health worldwide. To ensure appropriate use of existing antibiotics, rapid and reliable tests of AMR are necessary. One of the most common and clinically important forms of bacterial resistance is to β-lactam antibiotics (e.g., penicillin). This resistance is often caused by β-lactamases, which hydrolyze β-lactam drugs, rendering them ineffective. Current methods for detecting these enzymes require either time-consuming growth assays or antibiotic mimics such as nitrocefin. Here, we report the development of a surface-bound, clinically relevant β-lactam drug that can be used to detect β-lactamases and that is compatible with a range of high-sensitivity, low-cost, and label-free analytical techniques currently being developed for point-of-care-diagnostics. Furthermore, we demonstrate the use of these functionalized surfaces to selectively detect β-lactamases in complex biological media, such as urine.


S1.1. Tether Design
Co-crystal structure PDB2a49 was used as a representative b-lactamase in the design of the initial tether length, compound 2. 1 Figure S1 shows the active site of this exemplar b-lactamase (SHV-1) with a b-lactamase inhibitor (linearised, decarboxylated clavulanic acid) covalently bound to Ser70. It was noted that the active site is readily accessible; it was hypothesised that the tether of compound 2 would extend beyond the protein when bound, thus allowing for successful hydrolysis of the surface bound drugs by b-lactamases. Ser70 -compound 2 -surface 1 H and 13 C NMR spectra were recorded on a Jeol ECS 400 (400 MHz for 1 H, 101 MHz for 13 C) at ambient temperature. Chemical shifts are reported relative to residual solvent peaks and coupling constants (J) are given in Hertz. High-resolution ESI mass spectra were recorded on a Bruker microTOF electrospray mass spectrometer. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum Two (ATIR). Analytical HPLC measurements were performed on a Shimadzu HPLC system (Prominence) equipped with a LC-20AD pump, SIL-20A autosampler, DGU-20AS degasser, CTO-20AC column oven, CBM-20A communication bus module and SPD-M20A diode array detector using a SunFire C18 column (Waters, 4.6 x 150 mm, 5 µm). Eluent gradient: 5-95% MeCN/H2O with a 0.1% formic acid modifier, over 15 minutes.

S1.7. Control Thiol Addition
To a solution of compound S10 (50 mg, 0.093 mmol, 1 equiv.) in 3:1, D2O:DMSO-d6 (1 mL) was added cysteine (11 mg, 0.093 mmol, 1 equiv.). The resulting solution was stirred at RT for 3 h after which the reaction was concentrated under reduced pressure. The residue was diluted to 600 uL in DMSO-d6 and analysed by 1 H NMR. Analysis confirmed the b-lactam was still intact, whereas the peak assigned to the maleimide alkene (7.00 ppm) was significantly reduced; thus indicating that the thiol addition occurred preferentially at the maleimide.

S1.8. Stability of Cephalexin in Urine
Control experiments were carried out using the urine sample to confirm the absence of b-lactamases and the stability of cephalexin in this biological media.
Cephalexin monohydrate (25 mg, 0.68 mmol) was dissolved in 1.5 mL deionised water to produce a 46 mM stock solution. 100 uL of the cephalexin stock solution was then added to 900 uL urine to give a final concentration of 4.6 mM. The resultant solution was then incubated at 37 °C for 24 h. Aliquots were taken for analysis by HPLC after 24 h. Concentration of remaining cephalexin was calculated using the predetermined calibration graph and reported as %remaining, Table S1. The urine tests were carried out in duplicate with a buffer sample as a control.

S2. PBP Thermal Shift Assay
To test the binding activity of the purified E. coli PBP3, a thermal shift assay was carried out using the Protein Thermal Shift™ assay kit (Applied Biosystems). 3 µM of purified E. coli PBP3 was mixed with a selection of four b-lactam antibiotics (cephalexin, carbenicillin, amoxicillin, and ampicillin) at 300 µM in a mixture containing the Protein Thermal Shift™ Dye. The samples were then heated in a StepOnePlus™ Real-Time PCR System from 25 to 95 °C at a rate of 1 °C/min. Tests were carried out in triplicate and the averages plotted as the negative first derivative vs. temperature, Figure S2. Reference wells, i.e. solutions consisting only of only PBP3 with dye, PBP3 only and dye only, were used as controls. Melting temperature (Tm) values were determined with and without carbenicillin, and the change in melting temperature (DTm) was obtained, Table S2. b-lactamase exposed surface of gold, no functionalisation. Experimental conditions: Once cleaned, sample was immersed in a solution of b-lactamase in 50 mM KPi for 2 h at 37 °C, then gently cleaned with ultrapure water and dried with N2.
Spectrum: IR02 Surface Functionalisation: Cephalexin-PEG-maleimide (2) Description: Spectra of surface bound cephalexin-PEG. Experimental conditions: Surface post functionalisation, no further reaction. Spectrum: IR04 Surface Functionalisation: Cephalexin-PEG-maleimide (2) Description: Spectra of surface bound cephalexin-PEG before urine. Experimental conditions: Sample functionalised, analysed prior to urine test. Spectra of surface bound cephalexin-PEG after exposure to urine spiked with b-lactamase. Experimental conditions: Once functionalised, sample was immersed in a urine solution spiked with b-lactamase for 24 h at 37 °C, then gently cleaned with 2% SDS, followed by ultrapure water and then dried with N2.

Spectrum:
IR12 Surface Functionalisation: Cephalexin-C6-maleimide (S10) Description: Spectra of surface functionalised with cephalexin-C6 post enzyme hydrolysis. Experimental conditions: Once functionalised, sample was immersed in a solution of b-lactamase in 50 mM KPi for 2 h at 37 °C, then gently cleaned with ultrapure water and dried with N2.

S3.2. QCM-D Analysis -Sauerbrey Equation
Surface concentrations from QCM-D data were calculated using the Sauerbrey equation. We note, this model assumes a thin, rigid, and uniform layer, and is therefore only an estimated value.

S3.3. QCM-D Experimental Procedures and Spectra
QCM-D spectra show both the frequency and dissipation shifts for each experiment, with noteworthy shifts in frequency being indicated by shaded horizontal bars. The introduction of each new solution has been marked with a vertical dotted line as well as a label describing the solution. Horizontal dashed lines between data points indicate where data has been shifted to enable comparison between experiments in which samples were injected at different times.
Data: QCM01 Surface Functionalisation: 1, 3-Propanedithiol and MPTES on Au and SiO2 sensors, respectively Description: Spectra comparing the binding of cephalexin-PEG to both Au and SiO2.