Ho-Wah Au, Man-Wah Tsang, Yu Wai Chen, Pui-Kin So, Kwok-Yin Wong, Yun-Chung Leung
Abstract
β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for β-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP β-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to β-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding.
Introduction
β-Lactam antibiotics have been important therapeutics in human and animal medications for decades [1]. Owing to their high cost-effectiveness in treating bacterial infections, they have been nowadays the most frequently used antibiotics worldwide [2, 3]. With the extensive application of these drugs, concerns have been raised on the β-lactam antibiotic contamination in food and environment which leads to a health risk on hypersensitive individuals, a spread of antibiotic resistance and technical difficulties in fermentation [4–9]. In addition, the effectiveness of the once-dependable β-lactam antibiotics has been greatly challenged by a rapid emergence of the bacterial resistance associated with the abusive and indiscriminate antibiotic uses [6–8]. Thus, there has been a rising number of untreatable infectious diseases in recent years, posing a global health crisis [6–8]. To tackle these problems, β-lactam antibiotic level in food and biological samples (e.g. plasma) must be stringently controlled and its monitoring has been an essential task in food industry and clinical practice.
Materials and methods
Materials
BADAN was purchased from Molecular Probes Inc. (Eugene, OR, USA). Cefotaxime, moxalactam, penicillin G, penicillin V, kanamycin and lysozyme were purchased from Sigma (St. Louis, MO, USA). Tryptone and yeast extract used for preparing 2×TY medium were obtained from Oxoid (Nepean, Ontario, Canada). The chemical structures of BADAN and β-lactam antibiotics involved in this study were illustrated in S1 Fig.
Conclusion
In conclusion, the BADAN-labeled β-lactamases, E166Cb and E166Cb/N170Q, showed the potential as efficient systems for β-lactam detection. Not only demonstrating a rapid detection, they also illustrated an improved sensitivity than their fluorescein-modified counterparts. Further work will be focus on the application of the BADAN-labeled β-lactamases as high throughput screening tools for food and clinical specimens.
Citation: Au H-W, Tsang M-W, Chen YW, So P-K, Wong K-Y, Leung Y-C (2020) BADAN-conjugated β-lactamases as biosensors for β-lactam antibiotic detection. PLoS ONE 15(10): e0241594. https://doi.org/10.1371/journal.pone.0241594
Editor: Sabato D’Auria, Consiglio Nazionale delle Ricerche, ITALY
Received: August 18, 2020; Accepted: October 16, 2020; Published: October 30, 2020.
Copyright: © 2020 Au et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: This work was supported by the RGC GRF grant (151019/14M and 151069/17M) and RGC grants (C5031-14E, G-YBB3, G-YB RX, BBX4 and BBX8) from Research Grants Council, University Grants Committee (Hong Kong). For the following, the recipients are Kwok-Yin Wong and Yun-Chung Leung: 151019/14M, 151069/17M, BBX4, BBX8 For the following, the recipient is Yun-Chung Leung: C5031-14E, G-YBB3, G-YB RX.
Competing interests: The authors have declared that no competing interests exist.
