Licochalcone A inhibits the assembly function of ß-barrel assembly machinery in Escherichia coli.

Antimicrobial resistance (AMR) crisis urges the development of new antibiotics. In the present work, we for the first time used bio-affinity ultrafiltration combined with HPLC-MS (UF-HPLC-MS) to examine the interaction between the outer membrane ß-barrel proteins and natural products. Our results showed that natural product licochalcone A from licorice interacts with BamA and BamD with the enrichment factor of 6.38 ± 1.46 and 4.80 ± 1.23, respectively. The interaction was further confirmed by use of biacore analysis, which demonstrated that the Kd value between BamA/D and licochalcone was 6.63/28.27 µM, suggesting a good affinity. To examine the effect of licochalcone A on BamA/D function, the developed versatile in vitro reconstitution assay was used and the results showed that 128 µg/mL licochalcone A could reduce the outer membrane protein A integration efficiency to 20%. Although licochalcone A alone can not inhibit the growth of E. coli, but it can affect the membrane permeability, suggesting that licochalcone A holds the potential to be used as a sensitizer to combat AMR.

Targeting Bacterial Membrane Proteins to Explore the Beneficial Effects of Natural Products: New Antibiotics against Drug Resistance.

Antibiotic resistance is currently a world health crisis that urges the development of new antibacterial substances. To this end, natural products, including flavonoids, alkaloids, terpenoids, steroids, peptides and organic acids play a vital role in the development of medicines and thus constitute a rich source in clinical practices, providing an important source of drugs directly or for the screen of lead compounds for new antibiotic development. Because membrane proteins, which comprise more than 60% of the current clinical drug targets, play crucial roles in signal transduction, transport, bacterial pathogenicity and drug resistance, as well as immunogenicity, it is our aim to summarize those natural products with different structures that target bacterial membrane proteins, such as efflux pumps and enzymes, to provide an overview for the development of new antibiotics to deal with antibiotic resistance.

Rapid Nanopore Assay for Carbapenem-Resistant Klebsiella pneumoniae.

The prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is rapidly increasing worldwide in recent decades and poses a challenge for today's clinical practice. Rapid detection of CRKP can avoid inappropriate antimicrobial therapy and save lives. Traditional detection methods for CRKP are extremely time-consuming; PCR and other sequencing methods are too expensive and technologically demanding, making it hard to meet the clinical demands. Nanopore assay has been used for screening biomarkers of diseases recently because of its high sensitivity, real-time detection, and low cost. In this study, we distinguished CRKP from carbapenem-sensitive K. pneumoniae (CSKP) by the detection of increasing amount of extracted 16S ribosomal RNA (16S rRNA) from bacterial culture with antibiotics imipenem, indicating the uninhibited growth of CRKP by the imipenem. Specific signals from single channel recording of 16S rRNA bound with probes by MspA nanopore allowed the ultra-sensitive and fast quantitative detection of 16S rRNA. We proved that only 4 h of CRKP culture time was needed for nanopore assay to distinguish the CRKP and CSKP. The time-cost of the assay is only about 5% of disk diffusion method while reaching the similar accuracy. This new method has the potential application in the fast screening of drug resistance in clinical microorganism samples.

Multimode MicroRNA Sensing via Multiple Enzyme-Free Signal Amplification and Cation-Exchange Reaction.

The detection of biomarkers requires not only high sensitivity but also different signal reading methods depending on the actual situation. Herein, the luminescent properties of CdTe quantum dots (QDs) were exploited, where CdTe QDs were used as shared signal molecules. Combining multiple types of nucleic acid and chemical signal amplification techniques, and various signal detection techniques, a magnetic nanoparticle (NP) and filter-assisted separation multimode sensing strategy has been developed. In this work, miRNA-141 was selected as a representative target, which can trigger the catalyzed hairpin assembly and hybrid chain reaction enzyme-free nucleic acid signal amplification that generates long double-stranded DNA. Subsequently, the chemical amplification of silver NPs (Ag NPs) that release a large amount of Ag+ was introduced into the system. Finally, the cation-exchange reaction between CdTe QDs and Ag+ was utilized to quench the fluorescence (FL) of the CdTe QDs, releasing free Cd2+. The visual/FL/chemical vapor generation-atomic fluorescence spectrometry (CVG-AFS)/inductively coupled plasma mass spectrometry (ICP-MS) method could then be performed for the analysis of miRNA. After investigating its experimental performance, it has been found that 10 fM can be differentiated from the blank solution with the naked eye. In addition, FL/CVG-AFS/ICP-MS methods all displayed good analytical capability for target detection, and the limits of detection (LODs) are as low as fM, which show high target sequence selectivity. This platform was applied to investigate miRNA-141 expression in various cancer cells, which can accurately detect in the range of 100-100 000 MDA-MB-231 cells (breast cancer cell lines), with an LOD of 15 cells. Therefore, the multimode sensing strategy based on a single signal molecule and multiple signal amplification strategies is an applicable and versatile detection method of biomarkers; it can even achieve point-of-care testing, improving the accuracy and efficiency of medical diagnosis.