A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii

Abstract

Industry screens of large chemical libraries have traditionally relied on rich media to ensure rapid bacterial growth in high-throughput testing. We used eukaryotic, nutrient-limited growth media in a compound screen that unmasked a previously unknown hyperactivity of the old antibiotic, rifabutin (RBT), against highly resistant Acinetobacter baumannii. In nutrient-limited, but not rich, media, RBT was 200-fold more potent than rifampin. RBT was also substantially more effective in vivo. The mechanism of enhanced efficacy was a Trojan horse-like import of RBT, but not rifampin, through fhuE, only in nutrient-limited conditions. These results are of fundamental importance to efforts to discover antibacterial agents.

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Fig. 1: Summary of the ReFRAME library screen used for the identification of RBT.
Fig. 2: The role of fhuE and sensitivity to RBT.
Fig. 3: Efficacy of RBT in vivo.
Fig. 4: RBT and COL combination therapy.

Data availability

Screening data are available on ReFRAMEdb.org. Genome sequencing data are available at NCBI accession no. PRJNA629056. Source data for the figures are provided with the paper.

Change history

  • 17 June 2020

    In the version of this Article originally published, the authors’ first names were formatted as initials. This has now been corrected and the authors’ full names are displayed.

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Acknowledgements

We thank Calibr’s High Throughput Screening and Compound Management teams for assisting with the ReFRAME screen. We also thank B. Weiss at the University of North Texas for supporting the neutropenic lung infection model. This work was supported by the National Institute of Allergy and Infectious Diseases (grant nos. R01AI139052 to B.L., R.S. and B.S., and R01AI130060 and R01AI117211 to B.S.), and the FDA (BAA contract HHSF223201710199C to B.L. and B.S.). Calibr at Scripps Research Institute was supported by the Bill & Melinda Gates Foundation (OPP1107194).

Author information

Affiliations

Authors

Contributions

B.L., M.B., V.T., C.M. and B.S. designed the experiments and wrote the manuscript. M.B. and C.M. helped design the HTS assay and performed the compound screening and analysis. B.L., A.U., J.Y., T.N., P.L., J.L., J.C., W.K., H.E., N.S., R.S., C.K., S.L. and G.D. participated in performing experiments, contributed intellectually and interpreted results. B.L., A.U., P.L. and R.S. conducted the MIC testing. J.L., W.K. and H.E. conducted the LC–MS/MS experiments. J.Y., T.B.N., P.L. and B.L. conducted the in vivo experiments.

Corresponding authors

Correspondence to Brian Luna or Brad Spellberg.

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Competing interests

B.L., B.S. and T.N. own equity in ExBaq. The University of Southern California has a financial interest in ExBaq. G.E.D., V.T., C.K. and S.L. own equity in BioVersys.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1

MIC of rifabutin in RPMI+10% FCS on non-A. baumannii Gram-negative ESKAPE species.

Extended Data Fig. 2

MIC of rifabutin in RPMI + 10% FCS supplemented with increasing amount of ammonium iron(III) citrate.

Extended Data Fig. 3 Rifabutin MICs against A. baumannii AB5075 transposon disruption mutants.

Mutants were deficient in amino acid transport genes. MIC assay was done in both MHII and RPMI media. AB5075-UW is the parent strain for the transposon mutants.

Extended Data Fig. 4 Rifabutin MICs against A. baumannii AB5075 transposon disruption mutants.

MIC assay was done by culturing the bacteria in either MHII or RPMI media.

Extended Data Fig. 5

MIC of rifabutin and rifampicin on HUMC1 and the serial passaged mutants, and WGS of mutants with increased rifabutin MIC.

Extended Data Fig. 6

MIC of rifabutin and rifampicin on the fhuE deleted mutants and their parental strains.

Extended Data Fig. 7 Clustering of fhuE by amino acid sequence.

MICs were done for 43 A. baumannii clinical isolates to determine if the isolates were hypersensitive to RBT in RPMI medium. 28 of the 33 isolates in the outer rings exhibit the hypersensitive phenotype (open circles) in RPMI (RBT MIC = 0.05 µg/mL). 9 of 10 isolates in the inner ring are not hypersensitive (filled-in shapes). Of the 41 carbapenem-resistant isolates tested, 26 of those are hypersensitive to RBT. Source data

Extended Data Fig. 8

MIC of rifabutin on the plasmid mediated fhuE expressing ATCC-17978 strains.

Extended Data Fig. 9 RPMI MIC predicts in vivo response to treatment.

Galleria mellonella larvae (10 per group) were infected with A. baumannii. a, Larvae were infected with strain HUMC1 at 1.6 ×104 cfu/larvae and treated with rifabutin (plain lines) or rifampicin (dashed lines) at 0.1 mg/kg (blue lines), 1 mg/kg (red lines) and 10 mg/kg (green lines) and survival was measured over 72 hours. b, G. mellonella larvae were infected with A. baumannii LAC-4 and treated with RBT or c, RIF. Consistent with the RPMI MIC data, there was no difference in outcomes based on treatment. Source data

Extended Data Fig. 10 Drug -drug interaction between rifabutin or rifampin and colistin in MHII The drug-drug interaction were evaluated by calculating the fractional inhibitory concentration index (FICI).

FICI = FICA + FICB = (CA/MICA) + (CB/MICB), in which CA and CB are drug concentration of drug A and drugB in combination and MICA and MICB are the MIC of drug A and drug B alone. Synergy was defined as FICI ≤ 0.5, no interaction was defined as FICI > 0.5–4.0 and antagonism was defined as FICI > 4.0.

Supplementary information

Reporting summary

Supplementary Table 1

ReFRAME screen results.

Supplementary Table 2

WGS sequencing data summary.

Supplementary Table 3

List of bacteria strains used.

Source data

Source Data Fig. 1

Numerical data.

Source Data Fig. 2

Numerical data.

Source Data Fig. 3

Numerical data..

Source Data Fig. 4

Numerical data

Source Data Extended Data Fig. 7

Numerical data.

Source Data Extended Data Fig. 9

Numerical data.

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Luna, B., Trebosc, V., Lee, B. et al. A nutrient-limited screen unmasks rifabutin hyperactivity for extensively drug-resistant Acinetobacter baumannii. Nat Microbiol (2020). https://doi.org/10.1038/s41564-020-0737-6

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