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Siderophores promote cooperative interspecies and intraspecies cross-protection against antibiotics in vitro

Nature Microbiology volume 9, pages 631–646 (2024)Cite this article

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Abstract

The antibiotic cefiderocol hijacks iron transporters to facilitate its uptake and resists β-lactamase degradation. While effective, resistance has been detected clinically with unknown mechanisms. Here, using experimental evolution, we identified cefiderocol resistance mutations in Pseudomonas aeruginosa. Resistance was multifactorial in host-mimicking growth media, led to multidrug resistance and paid fitness costs in cefiderocol-free environments. However, kin selection drove some resistant populations to cross-protect susceptible individuals from killing by increasing pyoverdine secretion via a two-component sensor mutation. While pyochelin sensitized P. aeruginosa to cefiderocol killing, pyoverdine and the enterobacteria siderophore enterobactin displaced iron from cefiderocol, preventing uptake by susceptible cells. Among 113 P. aeruginosa intensive care unit clinical isolates, pyoverdine production directly correlated with cefiderocol tolerance, and high pyoverdine producing isolates cross-protected susceptible P. aeruginosa and other Gram-negative bacteria. These in vitro data show that antibiotic cross-protection can occur via degradation-independent mechanisms and siderophores can serve unexpected protective cooperative roles in polymicrobial communities.

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Fig. 1: Cefiderocol resistance variants affect multiple genes and pathways.
Fig. 2: Cefiderocol resistance variants detected in evolved clones and P. aeruginosa clinical isolates.
Fig. 3: Evolved cefiderocol resistance pays fitness costs.
Fig. 4: Cross-protection evolved in cefiderocol-resistant populations.
Fig. 5: The role of pyoverdine in cross-protection.
Fig. 6: Cefiderocol resistance and cross-protection mechanisms.

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Data availability

RNA, whole-genome population and colony sequencing data are accessible in the NCBI SRA under accession number PRJNA971207. Genome sequencing data for the clinical isolates are available in the NCBI SRA under the accession number PRJNA934930. Confocal micrographs are available at https://doi.org/10.6084/m9.figshare.24898107. The reference genome sequence of P. aeruginosa PAO1 is available from GenBank, with accession number NC_002516. Source data are provided with this paper.

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Acknowledgements

This research was funded by grant numbers JORTH17F5, JORTH19P0 and MILESI21F0 from the Cystic Fibrosis Foundation and grant numbers K22AI127473, R21AI151362 and R01AI14642 from the NIH/National Institute of Allergy and Infectious Diseases and R01Hl136143 from the NIH/National Heart, Lung, and Blood Institute. We thank Applied Genomics, Computation and Translational Core at Cedars-Sinai Medical Center, for helping with whole-genome sequencing and bulk RNA sequencing. We also thank the Pulmonary Translational Research Core team at the University of Pittsburgh for the P. aeruginosa clinical isolates used in this study.

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Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Anna Clara M. Galdino, Mylene Vaillancourt, Diana Celedonio, Kara Huse & Peter Jorth

  2. Center for Innovative Antimicrobial Therapy, Division of Infectious Diseases, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, USA

    Yohei Doi

  3. Acute Lung Injury Center of Excellence, Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA

    Janet S. Lee

  4. Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO, USA

    Janet S. Lee

  5. Department of Medicine, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Peter Jorth

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Contributions

Conceptualization, A.C.M.G. and P.J.; methodology, A.C.M.G. and P.J.; formal analysis, A.C.M.G., D.C. and P.J.; investigation, A.C.M.G., M.V., D.C., K.H. and P.J.; resources, Y.D., J.S.L. and P.J.; data curation, A.C.M.G., D.C. and P.J.; writing—original draft preparation, A.C.M.G. and P.J.; writing—review and editing, A.C.M.G., M.V., D.C., Y.D., J.S.L. and P.J.; supervision, A.C.M.G. and P.J.; project administration, A.C.M.G. and P.J.; funding acquisition, A.C.M.G., J.S.L. and P.J. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Peter Jorth.

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Extended data

Extended Data Fig. 1 Time to achieve growth at high cefiderocol concentrations during experimental evolution.

Mean time to achieve growth at (a) 4 μg/ml and (b) 1,024 μg/ml cefiderocol during experimental evolution in cystic fibrosis (SCFM2 planktonic and aggregate populations) and synthetic human urine (SHU) media (Mean ± SD; p-values: one-way ANOVA with Tukey’s multiple comparison test, n = 8 parallel cultures).

Source data

Extended Data Fig. 2 Genetic diversity of populations evolved in the presence or absence of increasing concentrations of cefiderocol.

Shannon diversity indices were calculated from SNV frequencies in control populations passaged in cefiderocol-free media and in cefiderocol-evolved populations (mean; p-values, two-sided unpaired t-test, n = 8 parallel cultures).

Source data

Extended Data Fig. 3 Maximum parsimony phylogenetic trees of evolved isolates show diversity within each cefiderocol resistant population.

Maximum-likelihood phylogenetic trees were constructed based on mutations detected in cefiderocol evolved isolated colonies (20 colonies per evolved population). Phylogenetic trees were rooted on the PAO1 wild-type genome. Bootstrap values are indicated on respective branches. Trees were plotted using iTOL.

Extended Data Fig. 4 Increased efflux pump gene expression in cpxS variants and decreased antibiotic cross-resistance after drug-free selection.

a, Expression of mexA and muxA genes known to be under the control of the CPX two-component system, by RT-qPCR in PAO1 wild-type and cpxSSNV variants (T163P, S227G, and S235A) (mean ± SEM, ANOVA Dunnett′s multiple comparisons test, n = 6 independent experiments). b, Heatmap of antimicrobial susceptibilities of populations evolved in the absence of cefiderocol for 15 d. Heatmap indicates the mean log2 MIC fold change of drug-free-passaged populations compared to cefiderocol resistant populations (n = 8 populations per growth condition). CFDC, cefiderocol; CAZ, ceftazidime; CEP, cefepime, ATM, aztreonam, TOB, tobramycin, COL, colistin, PMB, polymyxin B, CIP ciprofloxacin.

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Extended Data Fig. 5 In vitro competitions between ancestral and cefiderocol-resistant evolved populations.

a, Planktonic competitions between ancestral and cefiderocol-resistant evolved populations in SCFM2 and SHU. b, Biofilm competitions between ancestral and cefiderocol-resistant evolved populations in SCFM2 with (right) or without (left) cefiderocol. Evolved and ancestral populations were tagged with eYFP and mApple fluorescent proteins, respectively. The fluorescent populations were competed (1:1 ratios) in the presence or absence of cefiderocol (64 μg/ml). Planktonic populations growth was determined by monitoring fluorescence over time (h). Experiments were performed in triplicate, in three independent experimental sets (mean ± SD n = 3 independent experiments). Biofilm competitions were visualized by confocal microscopy.

Source data

Extended Data Fig. 6 Ancestral populations are unable to grow in the presence of cefiderocol.

Growth in the presence and absence of 64 μg/ml cefiderocol of pre-adapted populations was determined by monitoring fluorescence over time (h). Experiments were performed in triplicate, in three independent experimental sets (mean ± SD n = 3 independent experiments).

Source data

Extended Data Fig. 7 Production of pyoverdine and pyochelin and cefiderocol susceptibilities of evolved isolated colonies.

Pyoverdine and pyochelin production by evolved isolated colonies in relation to cefiderocol susceptibility (Two-sided Spearman correlation, n = 160).

Source data

Extended Data Fig. 8 Bacterial siderophores confer cefiderocol cross-protection.

a, Ferric iron chelating activity of cefiderocol and bacterial siderophores. The chelating activity was detected by the colorimetric changes of chrome azurol B (OD630nm) at different chelator concentrations (0, 1, 2.5, 5, 10, 25, 50, 100 and 250 µM) (mean ± SEM, ANOVA, n = 2 independent experiments). b, Enterobactin protects K. pneumoniae (Kp), E. coli (Ec), B. cenocepacia (Bc), and B. multivorans (Bm) from cefiderocol killing in a dose-dependent manner. The combinatorial effect of enterobactin with cefiderocol is expressed by the log2 cefiderocol fractional inhibitory concentration (FIC; n = 3 independent experiments, mean ± SD). c, Planktonic competitions between P. aeruginosa (PAO1::mApple or cpxSS227G::mApple) and K. pneumoniae (ATCC 13883::eYFP – left) or E. coli (ATCC 25922::eYFP – right) in the presence of inhibitory concentrations with or without additional pyoverdine (8 µg/ml). The growth of K. pneumoniae and E. coli was measured by eYFP fluorescence area under the curve (AUC) (mean ± SEM, ANOVA, Dunnett′s multiple comparisons test, n = 5 independent experiments). d, Pyoverdine production by P. aeruginosa lab strains and clinical isolates in SCFM2 (mean ± SEM, ANOVA, Tukey′s multiple comparisons test, n = 3 independent experiments).

Source data

Supplementary information

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Supplementary Table 1

Material list. Supplementary Table 2 SCFM2 and SHU chemical composition. Supplementary Table 3 Primer list. Supplementary Table 4 Complete list of mutated genes detected in PAO1 after 10 days of pre-adaptation to SCFM2 and SHU. Supplementary Table 5 Complete list of mutated genes in the evolved populations with increasing concentrations of cefiderocol. Mutations detected in ancestral and control populations were filtered out. Supplementary Table 6 Complete list of mutated genes detected in isolated colonies from cefiderocol evolved SCFM2-planktonic populations. Supplementary Table 7 Complete list of mutated genes in clinical isolates sequentially recovered from ICU-admitted patients infected with P. aeruginosa. Supplementary Table 8 Complete list of mutated genes evolving in cefiderocol-resistant populations propagated for 14 days in the absence of cefiderocol. Supplementary Table 9 Differentially expressed genes in protective interactions compared with non-protective interactions. Supplementary Table 10 Summary of statistical analyses.

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Galdino, A.C.M., Vaillancourt, M., Celedonio, D. et al. Siderophores promote cooperative interspecies and intraspecies cross-protection against antibiotics in vitro. Nat Microbiol 9, 631–646 (2024). https://doi.org/10.1038/s41564-024-01601-4

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