Chemical reagents

Meropenem was purchased from TCI Chemicals (Shanghai). Kanamycin sulfate, Luria-Bertani (LB) Broth Powder, and LB agar were purchased from Affymetrix. All other chemicals were purchased from Sigma-Aldrich unless otherwise stated.

Bacteria

The bacteria employed for cell-based and animal studies are listed in Supplementary Table 1. All the E. coli variants were made as described below. The NDM-HK PCV was screened by 20th-generation serial passages of NDM-HK in antibiotic-free medium. The loss of bla NDM-1 gene was confirmed by both PCR and susceptibility test.

Preparation of Bi(III) compounds

CBS and RBC were kindly provided by Livzon Pharmaceutical Group. Bi(NIT) 3 (Bi(NO 3 )·5H 2 O) was purchased from Sigma-Aldrich. The chemical structures of all the ligands used are shown in Supplementary Fig. 2. Bi(EDTA) (EDTA: ethylenediaminetetraacetate) and Bi(NTA) (NTA: nitrilotriacetate) were prepared by mixing bismuth subcarbonate ((BiO) 2 CO 3 ) and appropriate amounts of ligands, followed by refluxing overnight. The solution was filtered while hot and was cooled down naturally. Crystals were collected and washed by water and ethanol the next day. Bi(NAC) 3 (NAC: N-acetyl-cysteine), Bi(GSH) 3 (GSH: glutathione)39, and Bi(TBC) 2 (TBC: tetrabromocatechol)61 were prepared by mixing Bi(NO 3 )·5H 2 O with the appropriate amounts of ligands in methanol. Any impurities were removed by filtering and the resulting solution was evaporated slowly to obtain the solid product.

Bi(TPP) (TPP: tetraphenylporphyrin) were synthesized using a modified method39. In brief, to 80 mL of reflexing pyridine containing 0.10 g (0.2 mmol) of TPP was added 1.99 g of Bi(NO 3 ) 3 ·5H 2 O (2.1 mmol), and a further 2.00 g (4.1 mmol) was then added 3 h later. After further reflexing for 5 h, a large amount of pyridine was removed via rotary evaporation and the resulting thick, green mixture was then left to dry overnight under vacuum to remove residual solvents. Green solids that were obtained were then washed with chloroform and rotary evaporated to ensure that all pyridine solvent was removed. The dark-green solids were then purified on a silica gel column. The compound was purified by washing the column with first chloroform and then chloroform:methanol in a ratio of 10:1. FAB-MS+ was used to characterize the complex with m/z of 820.9 ([Bi(L1)]+; calculated: 821.2).

Bi(CPL) 2 was prepared by mixing BiCl 3 (1 mmol) and D-captopril (3 mmol, J&K Scientific) in methanol at room temperature with constant stirring overnight until a color change from colorless to yellow had occurred. The methanol was removed under vacuum, and the resulting yellow solid product was washed successively with ethanol and water before recrystallization from methanol, yielding the product. 1H NMR (300 MHz, CD 3 OD, δ ppm): 4.73 (m, 1 H), 3.83 (m, 1 H), 3.71 (s, 3 H), 3.05 (m, 1 H), 2.26 (m, 1 H), 2.03 (m, 2 H), and 1.24 (d, 3 H, J = 7.0 Hz). ESI-MS− m/z: 639.08 (100%). The peak at m/z 639.08 corresponds to [BiL 2 ]− (calculated: 639.51).

Bi(PCM) 2 was prepared by mixing BiCl 3 (1 mmol) and D-penicillamine (3 mmol, J&K Scientific) in methanol with stirring, resulting in an immediate color change from colorless to pale yellow. The mixture was further stirred overnight at room temperature before any remaining solid was filtered off and the solvent from the filtrate was removed under vacuum. The resulting solid was recrystallized from methanol, yielding a yellow solid product. 1H NMR (300 MHz, D 2 O, δ ppm): 3.48 (s, 1 H), 0.90 (s, 3 H), 0.83 (s, 3 H). ESI-MS+ m/z: 1214.8 (80%), 860.2 (100), 505.3 (95), and 356.3 (50). The peaks at m/z 356.3, 505.3, 860.2, and 1214.8 correspond to [BiL]+ (calculated: 356.01), [BiL 2 ]+ (calculated: 505.06), [Bi 2 L 3 ]+ (calculated: 860.06), and [Bi 3 L 4 ]+ (calculated: 1214.46), respectively.

DNA manipulations and construction of plasmids

All the plasmids used as templates for PCR were purified using the gel extraction kit (QIAprep Spin Miniprep Kit (250), QIAGEN). All PCR primers were synthesized from Thermo Fisher and listed in Supplementary Table 7. PCR was performed using KOD Hot Start DNA Polymerase (Novagen) based on the reaction conditions described in the protocols by the manufacturers. All restriction enzymes were used directly (New England Biolab). The amplified genes of NDM-1, VIM-2, and IMP-4 were subsequently inserted into pET-28a plasmid with an incorporated N-terminal His-Tag using T4 DNA ligase to form the vector pET-28a-NDM-1, pET-28a-VIM-2, and pET-28a-IMP-4, respectively. Expression vector for NDM-1 variant (NDM-1-C208A) was generated by PCR using the standard protocols from the Phusion Site-Directed Mutagenesis Kit (New England Labs) with pET-28a-NDM-1 as the template. The constructed plasmids were subsequently transformed into XL1-Blue competent cells for molecular cloning.

Protein purification

A single colony of E. coli BL21(DE3) transformed with the respective MBL was inoculated into LB medium supplied with 50 μg mL−1 kanamycin and grown at 37 °C. Protein overexpression was induced using 0.2 mM IPTG supplemented with 0.2 mM ZnSO 4 at OD 600 0.6. The bacterial culture was incubated at 25 °C overnight. To purify the respective protein, the cultured cells were harvested by centrifugation at 4500 × g and resuspended in a lysis buffer (20 mM HEPES, 0.5 M NaCl, and 1 mM PMSF at pH 7.0). The cells were ice-cooled and lysed by sonication and then centrifuged at 35000 × g for 30 min to remove the majority of cell debris. The supernatant was filtered using Minisart syringe filter (0.45 μm) to remove any remaining large and insoluble cell debris, and was then applied to a 5 mL Ni(II)-loaded HiTrap chelating columns (GE Healthcare) at a rate of 2 mL min−1. The column was washed using five column volumes of washing buffer (20 mM HEPES, 0.5 M NaCl, and 30 mM imidazole at pH 7.0). The protein was eluted out using four column volumes of the same buffer with gradient amounts of imidazole, and was subsequently dialyzed against the cleavage buffer (20 mM HEPES, 0.15 M NaCl at pH 7.0). The N-terminal His-tag of the fusion protein was cleaved by adding 50 NIH units of thrombin at 25 °C for 3 h with mild shaking and the cleaved His-tag was separated from the resulting proteins by passing through the Ni(II)-NTA column again using washing buffer so that >90% of the proteins were in the flow-through fraction. The enzymes were further purified using HiLoad 16/60 Superdex 200 pg gel filtration column. The samples were then concentrated using Amicon Ultra-15 Centrifugal Filter Devices (Millipore) and separated into aliquots after dialysis with storage buffer (20 mM HEPES, 0.1 M NaCl at pH 7.0 for long-term storage at −80 °C.

IC 50 enzyme inhibition assay

Freshly prepared 50 nM of Zn 2 -NDM-1, Zn 2 -VIM-2, or Zn 2 -IMP-4 in 50 mM HEPES/Na pH 7.0, 100 mM NaCl were first incubated with various concentrations of CBS for 1 h at 25 °C, then mixed with equal volume of 0.2 mM MER. The assay was performed in a 1 cm quartz cuvette using the kinetic mode on a Varian Cary50 UV-visible spectrophotometer at 25 °C. The decrease in absorbance at 300 nm due to ring-opening of MER was monitored every second for a duration of 10 min until the reaction was completed. The initial rates were extracted and calculated from each reaction curves for fitting IC 50 curves.

UV-vis spectroscopy

UV-vis spectra were collected on a Varian Cary 50 spectrophotometer at a rate of 360 nm min−1 using a 1-cm quartz cuvette at ambient temperature. Aliquots of 2 mM (Bi(NTA)) stock solution were stepwise titrated into apo-NDM-1 sample (50 μM) in a titration buffer (20 mM HEPES, 50 mM NaCl at pH 7.4) and UV-vis spectra were recorded in a range of 220–600 nm at least 30 min after each addition. The binding of Bi(III) to apo-NDM-1 was monitored by the increase in absorption at 340 nm due to LMCT involving the only cysteine residue (Cys208). The UV titration curve was fitted to Ryan–Weber nonlinear equation62 (1).

$$I = \frac{{I_{\max }}}{2C_{\mathrm{p}}}\left[ {\left( {K_{\mathrm{d}} + C_{\mathrm{m}} + C_{\mathrm{p}}} \right) - \sqrt {\left( {C_{\mathrm{p}} + C_{\mathrm{m}} + K_{\mathrm{d}}} \right)^2 - 4C_{\mathrm{m}}C_{\mathrm{p}}} } \right] ,$$ (1)

where I stands for UV absorbance intensity; I max for maximal UV absorbance; C p and C m refers to the total concentrations of proteins and ligands, respectively; K d is the dissociation constant. For Bi3+, the dissociation constant from NDM-1 was derived by K d ′ = K d /K a , where K d is the dissociation constant of Bi(NTA) from NDM-1 determined from Ryan–Weber nonlinear fitting35 and K a is the formation constant of Bi(NTA) with log K a = 17.55.

Zinc displacement analysis by ICP-MS

To monitor the displacement of Zn(II) by Bi(III), ICP-MS was employed to accurately quantify 209Bi and 66Zn contents in various purified NDM-1 samples. Purified Zn-bound-NDM-1 (20 μM) dissolved in trace-metal-free ICP-MS buffer containing 50 mM HEPES, pH 7.0, was incubated with various concentrations of CBS at 25 °C for 5 h with mild shaking. The samples were subsequently dialyzed in ICP-MS buffer to remove unbound-metal ions and were then acidified and subsequently analyzed using an ICP-MS spectrometer (Agilent 7500a, Agilent Technologies, CA, USA) with 115In as an internal standard for 209Bi, 66Zn, and 34S contents, which are used to quantify protein concentration.

Zinc supplementation assay

To investigate whether bismuth inhibits the enzyme reversibly, enzyme activities of Bi-NDM-1 and apo-NDM-1 were compared upon the supplementation of Zn(II). Bi-bound NDM-1 (50 nM) was prepared by pre-incubation of apo-bound NDM-1 with CBS for 2 h at 25 °C, followed by removal of unbound Bi(III) and the bound Bi was verified by ICP-MS. The above protein solutions were mixed with ZnSO 4 at concentration up to 2 molar equivalents to NDM-1 and 100 µM MER. The change in absorbance at 300 nm was monitored on a Varian Cary 50 UV-visible spectrophotometer at 25 °C for calculation of reaction rates. Reaction rate of apo-NDM-1 with addition of 2 molar equivalents of ZnSO 4 was normalized to 1. The metal content of Bi-bound NDM-1 treated with Zn(II) was measured and analyzed by ICP-MS as described in the previous section.

Limited proteolysis of NDM-1 protein

Limited proteolysis41 was performed to examine the in vitro stability of different forms of NDM-1. In brief, the aliquots (150 μg) of pure apo-bound, Zn-bound, and Bi-bound NDM-1 were treated with 2 μg of proteinase K (Fungal, Invitrogen, >20 U/mg) in 10 mM Tris, 5 mM CaCl 2 , pH 8, at 16 °C. Aliquots were taken at various time intervals. The reaction was quenched with 5 mM PMSF, and samples were then subjected to SDS-PAGE and Coomassie blue staining. A PageRuler Prestained Protein Ladder #26616 (Thermo) was used as a standard marker.

Michaelis–Menten kinetics

NDM-1 (50 nM) was incubated with Bi(NIT) 3 (0.5, 1.0, and 1.5 µM) for 1 h at 25 °C with gentle shaking. The assay was performed in a 96-well microplate reader at 298 K. The final assay buffer contains 50 mM HEPES at pH 7.0, 100 mM NaCl, with MER as the substrate ranging from 10 to 150 µM. Control experiment was also performed in the absence of inhibitors under the same conditions. The K m and V max for both the uninhibited and inhibited reactions were obtained by fitting the data into the double reciprocal Lineweaver–Burk plots.

Detection of NDM-1 expression level by western blot

NDM-1 protein level was determined by SDS-PAGE followed by western blot over the whole-cell lysates of different E. coli stains. Typically, each logarithmic phase culture was lysed by sonication in sonication buffer (50 mM HEPES, pH 7.3, 100 mM NaCl). Bacterial lysates were harvested by centrifugation and normalized according to total protein concentrations, as quantified by bicinchoninic acid assay (Pierce BCA Protein Assay Kit, Thermo Scientific). All the samples were resolved on a 13% SDS-PAGE gel and electrotransferred to a PVDF membrane (Hybond-P, GE Healthcare). A PageRuler Prestained Protein Ladder #26616 (Thermo) was used as a standard marker. Diluted NDM-1 monoclonal antibody (NOVUS Biologicals) and the secondary antibody goat anti-mouse immunoglobulin G (IgG)/alkaline phosphatase (AP) conjugate were applied after performing the standard blotting procedures. The NDM-1 bands were calorimetrically developed with specified ratio of substrates comprising nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) for 15 min. The software ImageJ63 was used to quantify the signal of each of the bands for analysis. The software GraphPad Prism (version 6.2 for Mac, GraphPad Software, La Jolla CA, USA, www.graphpad.com) was used to analyze the resulting plots.

Cellular thermal shift assay

The cellular thermal shift assay was performed according to a standard method38. Clinical isolate of NDM-1-positive E. coli (NDM-HK) was cultured overnight in the absence or presence of 100 µM Bi(III) compounds, i.e., CBS, Bi(NAC) 3 , Bi(NIT) 3 . The bacterial pellets were harvested and washed with PBS for 4 times. The cell suspensions were aliquoted into PCR tubes and heat treatment was performed at the designated temperature ranging from 40 °C to 70 °C for 3 min in a 96-well thermal cycler. The tubes were cooled immediately at room temperature for another 3 min after heating and the heating procedures were repeated for three cycles. For the cell lysis, the samples were frozen–thawed for two cycles in liquid nitrogen and thermal cycler set at 25 °C. The samples were vortexed gently after each cycle and were centrifuged at 20,000 × g to obtain the supernatant when the second cycle was finished. All the samples were subjected to western blot analysis for detection and quantification of NDM-1 content as described above.

Primary screening upon different metal compounds

Metal salts used for the screening involved bismuth nitrate (Bi(III)), gallium nitrate (Ga(III)), sodium stibogluconate (Sb(V)), chromium chloride (Cr(III)), cobalt chloride (Co(II)), nickel chloride (Ni(II)), ruthenium chloride (Ru(II)), and copper sulfate (Cu(II)). Briefly, different concentrations (10, 50, and 200 μM) of metal compounds were added to LB medium containing 8 μg mL−1 of MER in 96-well plates. About 2 × 106 CFU mL−1 logarithmic cultures of NDM-HK were added to each well of the plates and co-incubated for overnight. The growth inhibition of bacteria was monitored by OD reading at 600 nm and serial dilution in LB agar plate. Wells with no antibiotics or metal compounds served as growth controls, and wells with no bacteria added served as background controls. Each test was performed in triplicate. The inhibition was calculated as [1−(OD sample −OD background )/(OD control −OD background )] ×100%. CFU was counting by 10-fold serial dilution in PBS and 10 μL was spotted in LB agar plate.

Microdilution MIC and MBC assay

MIC values were determined by standard broth micro-dilution method (Clinical and Laboratory Standards Institute (CLSI) M100-S20, 2010)64. Briefly, bacterial cells were cultured in LB broth overnight at 37 °C at 250 rpm and the OD was measured at 600 nm (OD 600 ). The bacterial density was adjusted to about 1 × 106 CFU mL−1 and checked by CFU counting on agar plates afterwards. Tested antibiotics or Bi(III) compounds were added triplicately into individual wells of flat-bottomed 96-well plates and performed 2-fold serial dilution, followed by addition of prepared bacterial inocula. The plate was then incubated at 37 °C for overnight. Wells with no antibiotics or Bi(III) compounds served as growth controls and wells with no bacteria added served as background controls. The MIC was determined as the lowest concentration of a drug that could inhibit the growth of microorganism by both visual reading and OD reading at 600 nm using a microtiter plate reader.

For the test of NDM-Rosetta OX, the growth condition briefly goes as follow. Overnight culture of NDM-Rosetta was 1000-fold diluted into fresh LB medium and regrew to OD ~0.6. The overexpression of NDM-1 was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 h at 37 °C. The following susceptibility test was performed as described in the Microdilution MIC and MBC assay section in the methodology by using the resulting bacterial pellets with the supplementation of 200 µM IPTG.

For the test of C208A-Rosetta, the growth condition briefly goes as follow. Overnight culture of C208A-Rosetta was diluted into fresh LB medium (1000-fold) and regrew to OD ~0.6. The overexpression of NDM-1-C208A was induced by addition of 200 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 25 °C. The following susceptibility test was performed as described in the Microdilution MIC and MBC assay section in the methodology by using the resulting bacterial pellets with the supplementation of 200 μM IPTG.

At the end of MIC assay, 50 μL of aliquots of each well (containing a specified antimicrobial concentration) for each isolate tested were applied to a LB agar plate and incubated at 37 °C overnight. Resulting growth (or lack of growth) was examined after overnight culturing and the lowest concentration that inhibits 99.9% of the original culture was taken as MBC.

For drug combination test, antibiotics and Bi(III) compounds were co-added at concentrations up to eight times higher than the MIC of the drugs tested alone. Other procedures were kept strictly the same. The FICI was determined according to the following equation: FICI = FIC A + FIC B = C A /MIC A + C B /MIC B , where MIC A and MIC B are the MIC values of compounds A and B, respectively, when functioning alone, and C A and C B are the concentrations of compounds A and B at the effective combinations. Synergism was defined when FICI ≤ 0.5, indifference was defined when FICI > 0.5 and < 4, and antagonism was defined when FICI≥465. A volume of 256µg mL−1 was set as the MIC of Bi(III) (except for Bi(NAC) 3 ) for the determination of FIC values. All of the determinations were performed at least in triplicate on different days.

Time kill assay

Time kill assay was used to further explore the synergy between MER and Bi(III) compounds. In a typical assay, NDM-HK was cultured overnight and diluted 1:250 into LB broth at 37 °C for 3 h to reach log phase. The initial bacterial density was adjusted to ~107 CFU mL−1 and then exposed to MER, Bi(III) compounds either alone or in combination. LB broth with no drugs served as a control. Aliquots of bacterial suspension were withdrawn at different time intervals (0, 1, 2, 4, 6, 8, and 24 h) for inspection of bacterial viability by agar plating. The concentrations of the drugs used in the test are 24 µg mL−1 for MER, 64 µg mL−1 for CBS, and 32 µg mL−1 for Bi(NAC) 3 . Data from three independent experiments were averaged and plotted as log 10 CFU mL−1 vs. time (h) for each time point over 24 h. All assays were triplicated and performed three times on different days.

Measurement of bismuth uptake by ICP-MS

Five colonies of NDM-HK were grown in LB broth to ~OD 600 0.1. Bi(III) compounds at various concentrations (0, 1, 2, 5, 10, 20, 30, 50, 100, 200, and 500 μM) were added to the respective wells in 24-well plate in triplicate. Bacterial cell pellets were collected after 24 h incubation at 37 °C, followed by washing with PBS for six times. The harvested bacterial pellets were dissolved by 60 μL of 68% HNO 3 at 60 °C overnight using a Thermolyne DriBath. The dissolved samples were diluted to appropriate concentration for quantification of metals by ICP-MS (Agilent 7500a, Agilent Technologies, CA, USA) with 115In as an internal standard. Metal quantifications were triplicated and average values were used.

X-ray crystallography

Crystals of native NDM-1 were grown using hanging drop vapor diffusion method. The crystals were grown using precipitant containing 0.1 M Bis-Tris at pH 5.5, 15% PEG 3350 (w/v), and 20 mM L-proline. One microliter (µl) of protein solution at concentration of 50 ~ 100 mg ml−1 was mixed with 1 µl of precipitant, sealed, and incubated at 20 °C. Diamond-like or rectangular crystals appeared within a day after seeding and grew up to full size within a week. They generally diffracted to resolutions of 0.93–1.0 Å.

NDM-1 crystals were cross-linked with 25% (v/v) glutaraldehyde at 25 °C for 30 min and then soaked in a chelating solution (0.1 M sodium acetate, pH 4.6, 25% PEG 3350, 20% glycerol, 10 mM EDTA) overnight. The crystals were washed three times in cryo-protectant solution (0.1 M Bis-Tris, pH 5.5, 25% PEG 3350, 20% glycerol) and then soaked with bismuth compounds. Soaking was done for 17 h by adding 5 mM TCEP and 1 mM bismuth compounds (bismuth nitrate or CBS). The crystals were further washed with the cryo-protectant solution for four times and flash-frozen into liquid nitrogen.

Two data sets were collected at BL17U1 at the Shanghai Synchrotron Radiation Facility (SSRF) at two specific wavelengths of 0.92 and 0.93 Å, which crossed the L 3 absorption edge of elemental Bi. Excitation scan was performed to further confirm the absence of zinc ions after soaking. The diffraction data were processed with HKL2000 at SSRF. Molecular replacement was performed using the program Phaser66 from the CCP4 suite and the ampicillin-bound NDM-1 (PDB code: 3Q6X) as a searching model. Cycles of refinement with the anomalous data were done using Refmac67 and with careful manual rebuilding in Coot68. The anomalous signal strength was compared between the two data sets collected at wavelength of 0.92 Å and 0.93 Å. The Bi(III) occupancy was refined based on the Bi anomalous signal at early refinement stage and was assessed by atomic B-factor in later stages. TLS refinement was incorporated into later refinement processes. Solvents were added automatically in Coot and then manually inspected and modified. The final models were analyzed with MolProbity69. Data collection and model refinement statistics are summarized in Supplementary Table 4.

Resistance study

To measure MPC70, NDM-HK at 1–2 × 1010 CFU was plated onto LB agar containing MER and CBS at different concentrations and incubated at 37 °C. After 48 h incubation, to any plates with observable colonies, up to four colonies were picked and re-cultured, followed by the measurement of their MIC values. Any MICs of MER that were greater than the original value were determined as higher-level resistant mutant colony. The concentration that restricted the growth of mutant colonies was determined as MPC. In an identical experiment, the higher-level resistant mutant colonies were enumerated. The relative mutation frequency at each MIC for each strain/antibiotic pair was calculated as the proportion of resistant colonies per inoculum.

For the serial passage assay71, an overnight culture of NDM-HK was diluted to ~107 CFU mL−1 in LB broth. The as-diluted bacterial suspension was added to each well of 96-well plate supplemented with the drug at 0.5-fold, 0.75-fold, 1-fold, 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, and 4-fold MIC, respectively. The drug concentrations for in vitro selection were increased to 2-fold, 4-fold, 6-fold, 8-fold, 16-fold, 24-fold, and 32-fold of MIC, respectively after 12 bacterial passages. All the plates were incubated at 37 °C and the growth of cultures was checked at 24 h intervals. Cultures from the second highest concentrations that allowed growth were performed 1:1000 dilution into fresh medium supplemented with the same concentrations of drugs. For MER, 1-fold of MIC was set as 16 μg mL−1. For the combination of MER and CBS, 1-fold of MIC was set as 2 μg mL−1 MER + 32 μg mL−1 CBS. This in vitro passage was repeated for 20 days. MIC of MER was determined every four passages.

In vitro cell infection assay

MDCK cell was cultured in minimum essential Media (MEM) supplemented with fetal bovine serum (FBS, 10%) and grown at 37 °C in 5% CO 2 -humidified atmosphere for 3 days. About 1.0 × 105 MDCK cells were seeded per well in 24-well plates and incubated as described above for 48 h to ensure confluency. Logarithmic cultures of NDM-HK were washed with PBS for three times and re-suspended in MEM/10% FBS resulting in the initial bacterial density of about 2.0 × 107 CFU mL−1. Then, 500 μL of bacterial suspension were added to each well and substituted for the previous MDCK culture medium. The plates were centrifuged at 800 × g for 10 min and then incubated for another 6 h, executing the bacterial infection at multiplicity of infection (MOI) of 200. We then used two infection models for examination, viz, the cell-associated bacterial infection and cell-invaded bacterial infection. The cell-associated bacteria are herein defined as bacteria that attach to, penetrate, or transcytose in MDCK cells, while the cell-invaded bacteria are defined as bacteria that penetrate or transcytose MDCK cells. For the cell-invaded infection, the infected cells were incubated in culture medium supplemented with ciprofloxacin (100 µg mL−1) for 1 h to remove extracellular bacteria. Then, the treated cells were washed vigorously with PBS for six times and replenished with culture medium. For the cell-associated infection, the infected cells were only washed vigorously with PBS for six times to remove unbound bacteria. The infected MDCK cells were then exposed to either MER or CBS, or their combination for overnight under identical cell culture condition. Cells in the absence of drugs served as a control. The bacterial loads were examined by lysing MDCK cells with 1% Triton X-100 in PBS and serially diluting the resulting lysates to enumerate bacterial colonies by agar plating. The assay was performed in triplicate, repeated three times, and results were expressed as average ± SD.

Murine peritonitis infection

All experiments were approved by, and performed in accordance with the guidelines approved by Committee on the Use of Live Animals in Teaching and Research (CULATR), the University of Hong Kong. A total of 6–8-week-old, female BALB/c mice (18–22 g) were purchased from Charles River Laboratories, Inc. and were used in all mouse studies. The animals were randomized to cages for each experiment.

In mucin-assisted model, for infection with NDM-1-positive bacteria, an overnight culture of NDM-HK was performed 1:250 dilution in 100 mL LB broth and re-grew to about OD 600 0.3 in a 500-mL flask after 2.5 h shaking at 37 °C, 250 rpm. Bacterial pellets were collected and washed by PBS buffer three times for further use. Mice were infected intraperitoneally (i.p.) with a dose of 1 × 105 CFU of bacteria in PBS supplemented with 2% mucin. Four groups of mice were i.p. administered 4 h post infection with a 100-μL aliquot of vehicle control, monotherapy of MER (10 mg kg−1) or CBS (10 mg kg−1), or combination therapy (n = 8, 8, 8, and 12, respectively). Twice-daily treatment via i.p. injection was continued throughout the whole experimental course. For the infection with NDM-1-negative bacteria, all the operations of infection were similar to those used for NDM-HK, except that NDM-HK PCV was used as infection-causing bacteria. Two groups of mice were i.p. administered 4 h post-infection with a 100-μL aliquot of vehicle control and monotherapy of MER (10 mg kg−1) (n = 5 for each group). Twice-daily treatment via i.p. injection was continued throughout the whole experimental course. Body weights and mice survival were monitored till endpoint of the experiment.

In mucin-free survival experiment, the mice were infected with a dose of 5 × 107 CFU of NDM-HK in PBS. The mice received monotherapy of MER (50 mg kg−1) or CBS (50 mg kg−1), or combination therapy 0.5 h post infection (n = 5 for each group). Other experimental operations and conditions were kept the same as that in mucin-assisted model. Body weights and mice survival were monitored for endpoint till endpoint of the experiment.

Data availability

The coordinates and structure factors for both zinc-bound native NDM-1 and Bi(III)-bound NDM-1 were deposited at Protein Data Bank with accessing code 5XP6 and 5XP9, respectively. Other data are available from the corresponding author upon reasonable request.