Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples

Jonkers, Tim; Steenhuis, Maurice; Schalkwijk, Louis; Luirink, Joen; Bald, Dirk; Houtman, Corine J.; Kool, Jeroen; Lamoree, M.H.; Hamers, Timo

doi:10.1016/j.scitotenv.2020.139028

Cited by 14 publications

(14 citation statements)

References 47 publications

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“…To rule out that the decrease in MIC in the mutant strains is a secondary effect of increased membrane permeability in these strains, we tried to raise the permeability of the E. coli OM by expressing an open channel variant of the OMP FhuA. 33 Native FhuA is a siderophore transporter that forms a 22-stranded β-barrel, which is plugged by the N-terminal domain. Krishnamoorthy and co-workers 34 showed that deletion of the plug domain and four large external loops creates a large pore that is permeable to compounds up to approximately 2 kDa.…”

Section: Resultsmentioning

confidence: 99%

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

et al. 2021

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The development of new antibiotics is particularly problematic in Gram-negative bacteria due to the presence of the outer membrane (OM), which serves as a permeability barrier. Recently, the β-barrel assembly machine (BAM), located in the OM and responsible for β-barrel type OM protein (OMP) assembly, has been validated as a novel target for antibiotics. Here, we identified potential BAM complex inhibitors using a screening approach that reports on cell envelope σ E and Rcs stress in Escherichia coli . Screening a library consisting of 316 953 compounds yielded five compounds that induced σ E and Rcs stress responses, while not inducing the intracellular heat-shock response. Two of the five compounds (compounds 2 and 14) showed the characteristics of known BAM complex inhibitors: synergy with OMP biogenesis mutants, decrease in the abundance of various OMPs, and loss of OM integrity. Importantly, compound 2 also inhibited BAM-dependent OMP folding in an in vitro refolding assay using purified BAM complex reconstituted in proteoliposomes.

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Section: Resultsmentioning

confidence: 99%

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

et al. 2021

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“…The TTR-binding assay has been used for serum, 19 dust, 18 and water samples 21 previously, whereas the antibiotics assay is only validated for water samples. 14 …”

Section: Methodsmentioning

confidence: 99%

“…This cell-based assay monitors the bacterial growth of E. coli FhuAT, a Gram-negative strain that is susceptible to a wide range of antibiotics as it carries an open variant of an outer membrane protein channel combined with an inactivated multidrug efflux transport system. 14 Growth-inhibiting effects were determined in the logarithmic growth phase of the cells. The antibiotics assay was performed according to Jonkers et al 14 as described in Section S1 of the Supporting Information .…”

Section: Methodsmentioning

confidence: 99%

“…For this, (1) wastewater treatment plant (WWTP) effluent spiked with antimicrobial agents, (2) the dust standard reference material (SRM) 2585, and (3) fetal calf serum (FCS) spiked with thyroid hormone (TH) system disrupting compounds were extracted and analyzed using HPLC-HRMS. Furthermore, high-resolution fractionation of the samples was performed using the FractioMate and the fractions were tested for their capacity to inhibit the bacterial growth of a sensitive E. coli clone (antibiotics assay) 14 and for their capacity to compete with TH for binding to the distributor protein transthyretin (FITC-T4 TTR-binding assay). 15 Finally, a comprehensive suspect screening approach, using a reference database (i.e., CECscreen), spectral libraries (i.e., MassBank of North America and EU MassBank), and in-house standard mixtures, was applied, and the annotation quality of annotated features was assessed.…”

Section: Introductionmentioning

confidence: 99%

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High-Performance Data Processing Workflow Incorporating Effect-Directed Analysis for Feature Prioritization in Suspect and Nontarget Screening

Jonkers

Meijer

Vlaanderen

et al. 2022

Environ. Sci. Technol.

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Effect-directed analysis (EDA) aims at the detection of bioactive chemicals of emerging concern (CECs) by combining toxicity testing and high-resolution mass spectrometry (HRMS). However, consolidation of toxicological and chemical analysis techniques to identify bioactive CECs remains challenging and laborious. In this study, we incorporate state-of-the-art identification approaches in EDA and propose a robust workflow for the high-throughput screening of CECs in environmental and human samples. Three different sample types were extracted and chemically analyzed using a single high-performance liquid chromatography HRMS method. Chemical features were annotated by suspect screening with several reference databases. Annotation quality was assessed using an automated scoring system. In parallel, the extracts were fractionated into 80 micro-fractions each covering a couple of seconds from the chromatogram run and tested for bioactivity in two bioassays. The EDA workflow prioritized and identified chemical features related to bioactive fractions with varying levels of confidence. Confidence levels were improved with the in silico software tools MetFrag and the retention time indices platform. The toxicological and chemical data quality was comparable between the use of single and multiple technical replicates. The proposed workflow incorporating EDA for feature prioritization in suspect and nontarget screening paves the way for the routine identification of CECs in a high-throughput manner.

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“…17). More recently, similar high-throughput assays have been developed for screening for antibiotics in aquatic samples, 237 screening of antibacterial dental adhesives against mutants of streptococcus, 339 and monitoring and inhibiting kinase activity. 340,341 Both Fluc and Rluc have been extensively used in highthroughput screening campaigns against large compound libraries in both biochemical assays and cell-based assays.…”

Section: High-throughput Screeningmentioning

confidence: 99%

Applications of bioluminescence in biotechnology and beyond

2021

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Development of a high-throughput bioassay for screening of antibiotics in aquatic environmental samples

Cited by 14 publications

References 47 publications

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

Combining Cell Envelope Stress Reporter Assays in a Screening Approach to Identify BAM Complex Inhibitors

High-Performance Data Processing Workflow Incorporating Effect-Directed Analysis for Feature Prioritization in Suspect and Nontarget Screening

Applications of bioluminescence in biotechnology and beyond

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