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Real-time monitoring β-lactam/β-lactamase inhibitor (BL/BLI) mixture towards the bacteria porin pathway at single molecule level

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Abstract

Multidrug-resistant bacteria are a great concern and a problem that must be addressed. Extended-spectrum β-lactamases are a common defence mechanism of bacteria to make β-lactam (BL) antibiotics ineffective. β-Lactamase inhibitors (BLIs) are consequently designed and are often clinically prescribed with a BL antibiotic to hinder degradation. Current studies focusing on how BL antibiotics or BLIs interact solely with the bacterial outer membrane nanopores (porins) on reaching the periplasmic side using a nanopore-based sensing technique. In electrochemical studies, the bias voltage allows real-time monitoring of BL antibiotics, BLIs and their mixture through the porin pathway at the single-molecule level. Here we consider the most abundant membrane protein from Escherichia coli (i.e. OmpF), purify and reconstitute the membrane protein in an artificial lipid bilayer and then study its ex vivo electrochemical behaviour. We show the piperacillin/tazobactam mixture interacts with OmpF, whereas the substrate interacts under the maximum bandwidth. The power spectrum analysis of the ionic current trace demonstrates the ampicillin/sulbactram mixture requires more energy than ampicillin alone to pass through the porin pathway. Our results demonstrate that clinically relevant combinations (e.g. piperacillin/tazobactam and ampicillin/sulbactam) interact more strongly with OmpF than either the BL antibiotic or the BLI alone. We suggest a quick and relatively cheap screening method to test the ability of BL antibiotics/BLIs to cross the bacterial cellular membrane.

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Acknowledgements

The authors thank Prof. Yi-Tao Long for providing kind advice on nanopore experiments and data analysis. JW thanks Prof. Mathias Winterhalter's group for kindly providing purified proteins and fruitful discussions and Nanion Technologies GmbH for providing technical support for use of the Orbit 16 system during the experimental work. Y-LY is sponsored by the Chen Guang project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (17CG27).

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Authors and Affiliations

  1. School of Chemistry & Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Jiajun Wang & Yi-Lun Ying

  2. Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759, Bremen, Germany

    Jiajun Wang

  3. Nanion Technologies GmbH, 80339, Munich, Germany

    Niels Fertig

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  1. Jiajun Wang
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Correspondence to Jiajun Wang or Yi-Lun Ying.

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Published in the topical collection Young Investigators in (Bio-)Analytical Chemistry with guest editors Erin Baker, Kerstin Leopold, Francesco Ricci, and Wei Wang.

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Wang, J., Fertig, N. & Ying, YL. Real-time monitoring β-lactam/β-lactamase inhibitor (BL/BLI) mixture towards the bacteria porin pathway at single molecule level. Anal Bioanal Chem 411, 4831–4837 (2019). https://doi.org/10.1007/s00216-019-01650-3

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