Di Wu

The dissemination of antibiotic resistance genes (ARGs) via conjugative plasmids is a major driver of the antimicrobial resistance crisis. The optrA gene, which confers cross-resistance to linezolid and florfenicol, is frequently detected in Enterococcus isolates and disseminated via plasmids. This study investigated the inhibitory effect of carvacrol on horizontal transfer of an optrA-carrying plasmid in porcine Enterococcus faecalis. In vitro conjugation assays showed that subinhibitory carvacrol (1/32 MIC, 16 µg/mL) significantly reduced conjugation efficiency in a dose- and time-dependent manner. In vivo (mouse model) treatment with 32 μg/mL carvacrol decreased transconjugant recovery by 62.8%. Mechanistically, carvacrol disrupted membrane integrity, depleted intracellular ATP, sup-pressed ROS production, reduced biofilm formation, and downregulated conjugation-associated genes (type VI secretion protein, conjugal transfer protein, prgB, asa1) as shown by transcriptomics and RT-qPCR. Collectively, carvacrol inhibits optrA plasmid transfer via multi-target mechanisms involving membrane damage, energy metabolism interference, and suppression of conjugation gene expression. This study highlights carvacrol as a natural agent to control the transfer of ARGs.

Bacterial subtype detection using multiplex amplification with multiple distinguishable labels is complex and costly, whereas recombinant DNA technology via fragment fusion provides a simpler, cheaper alternative. Here, we propose a label-free method for Listeria monocytogenes subtype detection based on a hybrid split G-quadruplex (G4) and fusion recombinase-aided amplification (RAA)-cascaded CRISPR/Cas13a (HGFRC) platform. Fusion RAA co-amplified species-specific and clonal complex (CC)-specific targets into 535 bp fusion products, which activated T7 transcription and Cas13a trans-cleavage activity. Activated Cas13a cleaved the RNA linker, disrupting G4 assembly and reducing fluorescence to generate a signal-off response. This design eliminated labeled probes, enabling single-signal detection. The assay achieved a sensitivity of 1.0 × 101 CFU/mL within 45 min, with good specificity and sample suitability. Overall, this study provides a label-free, sensitive, and specific method for detecting L. monocytogenes CC5, offering new insights for bacterial subtype biosensing.

Precise identification and structural elucidation of glycans have long been a challenge in analytical science. A clear understanding of their structure–function relationship is also crucial for revealing related biological activities. Although emerging nanopore technology has the potential to provide glycan fingerprints at the single-molecule level, direct detection remains significantly difficult due to limitations such as small size, insufficient charge, and efficient methods for nanopore events identification. To overcome these hurdles, we proposed a synergistic enhancement strategy that integrated charge modulation of glycans with the spatial confinement effect of the nanopore lumen. This innovation significantly strengthens the interactions between glycans and the nanopore, thereby amplifying the fingerprint signals of glycans with subtle structural differences. This method not only distinguished between glycan isomers but also enabled qualitative analysis and has been successfully validated in real samples. To further enhance the analytical objectivity, we employed a customized machine learning algorithm to automatically classify and identify translocation events, achieving an overall accuracy of over 97%. In conclusion, this study provides a novel single-molecule sensing perspective for glycan fingerprint analysis.

Argonautes (Agos) are next-generation programmable nucleases that demonstrate great potential for the detection of nucleic acid targets. However, a contemporary difficulty in the intensely active field of Ago-based biosensing is how to enhance its flexibility to meet the requirement in versatile non-nucleic acid target detection. Herein, we proposed an Ago enzymatic activation state regulation strategy to achieve diverse target detection. The normal linear guide DNA of Ago is confined in a hairpin conformation, termed the hgDNA. This unique configuration could effectively prevent Clostridium butyricum Ago (CbAgo) activation. Under the action of enzymatic hydrolysis or small-molecule-mediated attack on specific sites, the hairpin structure of hgDNA is disrupted, leading to the release of active gDNA. Therefore, the activation and inactivation states of CbAgo can be deliberately regulated. The possible activation mechanism could be ascribed to the strand displacement reaction between unstable hgDNA and the complementary strand. This tunability transforms CbAgo into a generalizable detection platform suitable for various targets such as nucleases, small molecules, and pathogens. This study advances the dynamic response of CbAgo to gDNA conformations and introduces the universal concept of Ago-based non-nucleic acid detection to broaden the next-generation biosensing applications.

Clostridium butyricum Argonaute (CbAgo) is an emerging programmable nuclease with great potential in medical diagnosis and food analysis. However, the regulation of the CbAgo enzyme activity remains challenging. Here, we found that splitting the full-length guide DNA (gDNA) into two shorter strands could prevent the activation of CbAgo. Utilizing this characteristic, an Escherichia coli isotope, an E. coli O157:H7-triggered click reaction was designed to ligate split gDNA and activate CbAgo. As a result, a complementary molecular beacon (MB) was cut off to restore the fluorescence. And the fluorescence intensity is positively related to the concentration of the target pathogen. The whole reaction process takes only 105 min, and the limit of detection (LOD) is about 10² CFU/mL. This study provided a novel strategy for the detection of E. coli O157:H7 in a fast and simple manner, which also showed a great potential application in real samples.