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Exploiting the fitness cost of metallo-β-lactamase expression can overcome antibiotic resistance in bacterial pathogens

Nature Microbiology volume 10, pages 53–65 (2025)Cite this article

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Abstract

Carbapenems are last-resort antibiotics for treating bacterial infections. The widespread acquisition of metallo-β-lactamases, such as VIM-2, contributes to the emergence of carbapenem-resistant pathogens, and currently, no metallo-β-lactamase inhibitors are available in the clinic. Here we show that bacteria expressing VIM-2 have impaired growth in zinc-deprived environments, including human serum and murine infection models. Using transcriptomic, genomic and chemical probes, we identified molecular pathways critical for VIM-2 expression under zinc limitation. In particular, disruption of envelope stress response pathways reduced the growth of VIM-2-expressing bacteria in vitro and in vivo. Furthermore, we showed that VIM-2 expression disrupts the integrity of the outer membrane, rendering VIM-2-expressing bacteria more susceptible to azithromycin. Using a systemic murine infection model, we showed azithromycin’s therapeutic potential against VIM-2-expressing pathogens. In all, our findings provide a framework to exploit the fitness trade-offs of resistance, potentially accelerating the discovery of additional treatments for infections caused by multidrug-resistant bacteria.

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Fig. 1: Expression of VIM-2 has a fitness cost in physiologically relevant environments.
Fig. 2: VIM-2 expression induces the protein misfolding response.
Fig. 3: Targeting the σE pathway preferentially inhibits VIM-2-expressing bacteria.
Fig. 4: VIM-2 expression sensitizes bacteria to a diverse range of antibiotics.
Fig. 5: Elevated levels of VIM-2 expression sensitize P. aeruginosa clinical isolates to azithromycin.

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Data availability

RNA-seq data are available at the NCBI Sequence Read Archive under BioProject PRJNA1054171 with the accession numbers SRR27256495, SRR27256494, SRR27256493, SRR27256492, SRR27256491 and SRR27256490. The genomic data for E. coli BW25113 referenced in this study are available at the European Nucleotide Archive under the accession number CP009273. In addition, the K. pneumoniae strain ATCC 43816 genome sequence is available in GenBank under BioProject PRJNA675363 with the accession number CP064352. Genomics source data have been deposited on Code Ocean86 (for genetic suppressors—Keio collection, https://doi.org/10.24433/CO.1737239.v1; for genetic enhancers—Keio collection, https://doi.org/10.24433/CO.7249224.v1; for genetic enhancers—CRISPRi collection, https://doi.org/10.24433/CO.7144620.v1). The data presented in Fig. 2a,c–g are provided in Supplementary Tables 1, 3, 4, 5, 7 and 8, respectively. Source data are provided with this paper.

Code availability

The R script supporting the findings of this study is available on Code Ocean86 (for genetic suppressors—Keio collection, https://doi.org/10.24433/CO.1737239.v1; for genetic enhancers—Keio collection, https://doi.org/10.24433/CO.7249224.v1; for genetic enhancers—CRISPRi collection, https://doi.org/10.24433/CO.7144620.v1).

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Acknowledgements

We thank G. Wright from McMaster University for bacterial strains from the Institute for Infectious Disease Research clinical collection. We also thank the Centre for Microbial Chemical Biology at McMaster University, specifically N. Henriquez for LC–MS/MS. This research was supported by a Tier 1 Canada Research Chair award, a Foundation Grant from the Canadian Institutes of Health Research (CHIR; FRN 143215) and a grant from the Ontario Research Fund (RE09-047) to E.D.B. M.M.T. was supported by a CIHR Canada Graduate Scholarship (CGS-D). D.C. was supported by a CIHR Canada Graduate Scholarship (CGS-M) and an Ontario Graduate Scholarship. M.E.S. was supported by a CIHR Canada Graduate Scholarship (CGS-M). J.M.S. was supported by The Weston Family Foundation and the CIHR. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Authors and Affiliations

  1. Institute of Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

    Megan M. Tu, Lindsey A. Carfrae, Kenneth Rachwalski, Shawn French, Denise Catacutan, Rodion Gordzevich, Craig R. MacNair, Melissa E. Speagle, Firas Werah, Jonathan M. Stokes & Eric D. Brown

  2. Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Megan M. Tu, Lindsey A. Carfrae, Kenneth Rachwalski, Shawn French, Denise Catacutan, Rodion Gordzevich, Craig R. MacNair, Melissa E. Speagle, Firas Werah, Jonathan M. Stokes & Eric D. Brown

  3. David Braley Centre for Antibiotic Discovery, McMaster University, Hamilton, Ontario, Canada

    Megan M. Tu, Lindsey A. Carfrae, Kenneth Rachwalski, Shawn French, Denise Catacutan, Rodion Gordzevich, Craig R. MacNair, Melissa E. Speagle, Firas Werah, Jonathan M. Stokes & Eric D. Brown

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Contributions

M.M.T. conceived the research, designed and carried out experiments and data analysis, and wrote the manuscript. L.A.C. and R.G. assisted in the animal experiments. K.R. assisted with the design of the mobile plasmids and the execution of the genomic studies. S.F. performed the microscopy analysis. D.C. aided in the analysis of the RNA-seq dataset. C.R.M. assisted with manuscript editing. M.E.S. and F.W. acquired MIC data. J.M.S. assisted with data interpretation and manuscript editing. E.D.B. conceived the research and assisted with data interpretation and manuscript editing.

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Correspondence to Eric D. Brown.

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Extended data

Extended Data Fig. 1 VIM-2 expression reduces K. pneumoniae growth in zinc-limited human serum.

(a, b) CFU/mL of viable cell counts of K. pneumoniae expressing VIM-2 (blue) and the empty vector (dark red) in 50% human serum (a, n = 5 for all time points except for 12 hours (n = 3)) and 50% human serum supplemented with 10 µM ZnSO4 (b, n = 6 for all time points except for 12 hours (n = 3) and 24 hours (n = 5)) over 24 hours. Data are mean ± s.e.m.

Source data

Extended Data Fig. 2 Overexpression of degP is associated with improved growth of carbapenem-resistant bacteria.

(a) mRNA levels of degP compared in wildtype and mutant Keio strains, transcript levels were normalized to the level of rrsE. The dotted line delineates degP levels in wildtype (WT), at a fold change of 1. Data are mean ± s.e.m. (WT, n = 7; ∆waaP, n = 4; ∆gmhA, n = 7; ∆atpD, n = 6; ∆hldE, n = 3; ∆waaF; n = 6; ∆yiiS, n = 2; n is defined as biological replicates and each biological replicate consists of at least two technical replicates). Statistical significance was determined using a Kruskal-Wallis test with Dunn’s test for multiple comparisons, relative to the WT (∆waaP, *P = 0.039; ∆gmhA, ****P < 0.0001; ∆atpD, *P = 0.013; ∆hldE, ****P < 0.0001; ∆waaF, ****P < 0.0001; ∆yiiS, ***P = 0.0005). (b) Protein levels of DegP from whole cell lysates in wildtype and mutant Keio strains. RNA Polymerase subunit α served as a loading control. Data is a representative blot of three biological replicates. (c) mRNA levels of degP compared in K. pneumoniae carrying the empty vector (Empty; n = 3) and expressing VIM-2 (VIM-2; n = 3) following growth in vivo. Samples were collected 7 hours post-infection from the blood. Transcript levels were normalized to the level of recA. Data are mean ± s.e.m, and each point represents an individual mouse. Statistical analysis was performed using a two-tailed unpaired t-test, **P < 0.01 (t = 6.727, df=4).

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Extended Data Fig. 3 Inhibiting DegS is a viable strategy to treat VIM-2-expressing K. pneumoniae in vivo.

Viable cell counts of K. pneumoniae carrying the empty vector (Empty) or expressing VIM-2 (VIM-2) in the (a) blood, (b) kidney, and (c) liver. Each strain was treated with a vehicle (grey; blood n = 5,5; kidney n = 7,6, liver n = 7,7) or 50 mg/kg of the DegS inhibitor (orange; blood n = 4,8, kidney n = 6,8, liver n = 6,10) one hour after infection. Each point represents an individual mouse. The centre line delineates the median, the box limits mark the upper and lower quartiles, and the whiskers depict the range. CFU were enumerated at 6 hours post-infection. Statistical analysis was performed using a two-sided Mann-Whitney test with Benjamini, Krieger and Yekutieli’s multiple comparisons test, *P < 0.05, **P < 0.01. The mouse infection model was repeated on three separate days.

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Extended Data Fig. 4 VIM-2 expression impairs cell septation under zinc-limited conditions.

(a, b) Fluorescent microscopy of wildtype (WT) E. coli, E. coli carrying the empty vector, and E. coli expressing VIM-2 in (a) M9 minimal media and (b) M9 minimal media supplemented with 10 µM ZnSO4. Cells are stained with FM 4-64. Scale bar = 10 µm. (c) Fluorescent microscopy of wildtype (WT) K. pneumoniae, K. pneumoniae carrying the empty vector, and K. pneumoniae expressing VIM-2 in M9 minimal media. Cells are stained with FM 4-64. Scale bar = 10 µm. Microscopy was repeated in duplicate to ensure consistent phenotypes.

Extended Data Fig. 5 A22 selectively targets VIM-2 expressing K. pneumoniae in human serum.

Competitive index (CI) for the co-inoculation of K. pneumoniae expressing VIM-2 and the empty vector in 50% human serum at 24 hours in the presence of two-fold dilutions of A22 (0 µg/mL, n = 7; 2 µg/mL, n = 2; 4 µg/mL, n = 4; 8 µg/mL, n = 5; 16 µg/mL n = 3; 32 µg/mL, n = 4). Data are mean ± s.e.m. The dotted line represents a CI of 1.

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Extended Data Fig. 6 VIM-2 expression disrupts the integrity of the outer membrane.

N-Phenly-1-naphthylamine (NPN) uptake of E. coli expressing VIM-2 (light blue) relative to the empty vector control (red) in M9 minimal media (n = 6,6), M9 minimal media supplemented with 10 µM ZnSO4 (n = 5,6), and M9 minimal media supplemented with 10 µM MgSO4 (n = 4,6). Data are mean ± s.e.m. Statistical significance was assessed using a two-sided unpaired t-test with false discovery rate correction, applying the two-stage linear step-up method by Benjamini, Krieger, and Yekutieli, ***P < 0.001 (M9: t = 11.35, df=10; M9 + Zn2+: t = 1.333, df = 9; M9 + Mg2+: t = 26.20, df=8).

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Extended Data Fig. 7 Azithromycin is a viable treatment option for VIM-2-expressing K. pneumoniae in vivo.

Viable cell counts of K. pneumoniae carrying the empty vector (Empty) or expressing VIM-2 (VIM-2) in the (a) spleen, (b) kidney, and (c) liver. Each strain was treated with a vehicle (grey; spleen n = 11,12; kidney n = 7,11, liver n = 11,12) or 10 mg/kg of azithromycin (purple; blood n = 6,6, kidney n = 4,5, liver n = 6,6) one hour after infection and 3.5 hours post-infection. Each point represents an individual mouse. The centre line delineates the median, the box limits mark the upper and lower quartiles, and the whiskers depict the range. CFU were enumerated at 6 hours post-infection. Statistical analysis was performed using a two-sided Mann-Whitney test with Benjamini, Krieger and Yekutieli’s multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 8 Clinical isolate of P. aeruginosa naturally expressing VIM-2 displays a fitness cost in zinc-limited environments.

(a) Growth kinetics of a clinical P. aeruginosa isolate that naturally expresses VIM-2 (GDW923) and (b) PAO1, a control strain, in M9 minimal media (n = 5,2; light blue) and M9 minimal media supplemented with 10 µM of ZnSO4 (n = 6,2; dark blue). Data are mean ± s.e.m. Data is normalized to OD600 of each strain at 48 hours and fit to Gompertz growth. (c) CFU/mL of viable cell counts of P. aeruginosa expressing VIM-2 and PAO1, a VIM-2 negative strain, in 50% human serum (n = 3,4; light blue) and 50% human serum supplemented with 10 µM ZnSO4 (n = 3,4; dark blue) at 24 hours. Data are mean ± s.e.m. Statistical significance was assessed using a two-sided unpaired t-test with false discovery rate correction, applying the two-stage linear step-up method by Benjamini, Krieger, and Yekutieli, **P < 0.01 (GDW923: t = 5.050, df=4; PAO1: t = 0.4850, df=6). (d) Fluorescent microscopy of a clinical P. aeruginosa isolate that naturally expresses VIM-2 (GDW923) and PAO1, a VIM-2 negative strain, in M9 minimal media and M9 minimal media supplemented with 10 µM ZnSO4. Cells are stained with FM 4-64. Scale bar = 10 µm. (e) NPN uptake of P. aeruginosa expressing VIM-2 (GDW923) in M9 minimal media (n = 6,6) and M9 minimal media supplemented with 10 µM ZnSO4 (n = 6,6). A carbapenem-susceptible P. aeruginosa strain (PA01) is used as a control. Data are mean ± s.e.m. Statistical significance was assessed using a two-sided unpaired t-test with false discovery rate correction, applying the two-stage linear step-up method by Benjamini, Krieger, and Yekutieli, ****P < 0.0001 (GDW923: t = 9.809, df=10; PAO1: t = 3.137, df=10).

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Extended Data Fig. 9 Subinhibitory concentrations of meropenem sensitize clinical isolates to azithromycin and elevate VIM-2 expression.

(a) MIC of azithromycin against a panel of P. aeruginosa clinical isolates harbouring VIM-2 and PAO1, a VIM-2 negative strain in M9 minimal media and M9 minimal media supplemented with ½ MIC of meropenem. (b) mRNA levels of vim-2 compared in clinical isolates in M9 minimal media (CDC 230, n = 3; CDC 242, n = 3; CDC 254, n = 3; CDC 255, n = 6) and M9 minimal media supplemented with ½ MIC of meropenem (CDC 230, n = 4; CDC 242, n = 3; CDC 254, n = 5; CDC 255, n = 5). Transcript levels were normalized to the level of 16S rRNA. The dotted line delineates vim-2 levels in CDC 230, at a fold change of 1. Data are mean ± s.e.m. Statistical significance was assessed using a two-sided unpaired t-test with false discovery rate correction, applying the two-stage linear step-up method by Benjamini, Krieger, and Yekutieli, *P < 0.05, ****P < 0.0011 (CDC 230: t = 3.682, df=5; CDC 242: t = 2.809, df=4; CDC 254: t = 1.380, df=6; CDC 255: t = 9.033,df=9).

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Extended Data Fig. 10 Azithromycin reduces the bacterial load of a clinical P. aeruginosa isolate naturally expressing VIM-2 in vivo.

Log10 reduction of viable cell counts P. aeruginosa CDC 243 in the spleen, kidney, and liver treated with 50 mg/kg of azithromycin one-hour post-infection relative to vehicle-treated mice (n = 4). Mice also received a treatment of 100 mg/kg of ZnSO4 every four hours or a vehicle (n = 4). Each point represents an individual mouse. The centre line delineates the median, the box limits mark the upper and lower quartiles, and the whiskers depict the range. CFUs were enumerated at 18 hours post-infection. Statistical analysis was performed using a two-tailed unpaired t-test, *P < 0.05, **P < 0.01 (Spleen: t = 4.982, df=6; Kidney: t = 4.745, df=6; Liver: t = 3.003, df=6).

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Tu, M.M., Carfrae, L.A., Rachwalski, K. et al. Exploiting the fitness cost of metallo-β-lactamase expression can overcome antibiotic resistance in bacterial pathogens. Nat Microbiol 10, 53–65 (2025). https://doi.org/10.1038/s41564-024-01883-8

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