Bacterial persistence, a non-heritable high-antibiotic-tolerance phenotype, is a key driver of recurrent clinical infections and antibiotic treatment failure. The pheromone-responsive pCF10 plasmid in Enterococcus faecalis (E. faecalis) mediates antibiotic resistance gene dissemination, but its role in bacterial persister formation remains unclear. This study systematically investigated the regulatory role of pheromone cCF10 in the persister phenotype of pCF10-carrying E. faecalis and its underlying molecular mechanisms. We confirmed that cCF10 enhanced persistence against levofloxacin in OG1RF (pCF10), with the persister frequency increasing from 0.291% to 16.466% upon treatment. Transcriptomic analysis revealed that cCF10 activated the (p)ppGpp-mediated stringent response and downregulated the expression of genes associated with energy-intensive pathways, including those involved in DNA repair, protein folding, and respiration. Concurrently, cCF10 enhanced the expression of genes related to biofilm formation and cell lysis resistance and downregulated components of its own sensing and uptake systems. These findings demonstrate that cCF10 induces transcriptional reprogramming associated with increased persister formation in E. faecalis carrying the pCF10 plasmid and identify potential targets within the stringent response and associated metabolic pathways for the development of anti-persister strategies.

Introduction:Pheromone-regulated horizontal transfer serves as the core mechanism for horizontal gene transfer of antibiotic resistance genes, playing a pivotal role in driving the spread of resistance. Given the strict species-specific constraints of this regulatory system, it is imperative to determine whether novel regulatory signal peptides and cross-genus receptors responsive to these signals exist, thereby elucidating its potential for disseminating resistance across broader microbial communities. Methods:This study isolated and screened a Gram-positive coccus, Aerococcus urinae Ae1, from the gut microbiota, and confirmed that Ae1 can undergo intergeneric plasmid transfer with Enterococcus faecalis (E. faecalis), challenging the conventional understanding that the pCF10 plasmid spreads only within the same species. Results and discussion:Results showed an intergeneric plasmid transfer frequency of (3.41 ± 0.26) × 10-3 in Ae1, which increased to (7.97 ± 1.77) × 10-3 upon exogenous addition of the cCF10 signal peptide, indicating cCF10's regulatory role in this process. Furthermore, the Ae1 signal peptide SPI-WT appeared to functionally resemble the cCF10 mechanism, possibly by acting on the prgZ/prgX pathway to promote pCF10 intergeneric transfer. This study suggests that Aerococcus urinae can acquire the pCF10 plasmid via intergeneric transfer and provides preliminary evidence that its endogenous signal peptide SPI-WT may play a regulatory role via the prgZ/prgX pathway. However, direct proof of natural secretion, physical binding, intracellular uptake, and relief of transcriptional repression is lacking; these remain important questions for future investigation. Nonetheless, our findings provide new insights into the dissemination pathways of intestinal antibiotic resistance genes.

[This corrects the article DOI: 10.3389/fmicb.2026.1817926.].

The current common detection techniques are difficult to achieve in the on-site detection of low-concentration water samples. To recover pathogenic microorganisms from water, a concentration filtration system and enrichment technology was constructed with bimetallic layered hydroxides. Specifically, Mg/Al bimetallic hydroxides (Mg/Al LDH) with a Mg:Al ratio of 2.14:1 were synthesized by coprecipitation, and bimetallic oxides (CLDHs) with larger specific surface areas were obtained by high-temperature calcination. This study revealed that the adsorption kinetics of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) reached a stable equilibrium at 30 and 40 min, respectively, through static-condition experiments. The adsorption capacity of CLDHs for E. coli was about 5.321 x 109 CFU/g, while that for S. aureus was about 1.679 x 107 CFU/g. Moreover, a concentrated recovery system was designed and constructed. This study found that the system could effectively remove 99% of E. coli, S. aureus, and Candida albicans (C. albicans) from water, with a bacterial recovery rate exceeding 80%. In conclusion, the established concentration and recovery system offers an efficient means of recovering pathogenic microorganisms from water, thereby providing great convenience for subsequent microbial detection.

The overuse of tetracycline (TC) a novel reverse ELISA assay integrating quantum dots (QDs) with an allosteric transcription factor (TetR) was developed for rapid tetracycline (TC) detection. Specifically, biotin-modified double-stranded DNA (dsDNA) was immobilized on a streptavidin-coated 96-well plate, after which QD-TetR conjugates were added. In the presence of TC, the QD-TetR conjugate binds TC and undergoes allosteric changes that result in its dissociation from the dsDNA. The developed QD-TetR-based reverse ELISA achieved quantitative TC detection within a linear dynamic range of 0.05–100 μM in just 5 min, with a method detection limit of 0.02 μM. The recoveries ranged from 93.43

Antibiotic resistance gene (ARG) transfer affects the performance of wastewater treatment systems. However, there is a limited understanding of the transfer of ARGs in anaerobic ammonia oxidation (Anammox) system. Here, effects of ARGs transfer were investigated by constructing a conjunctive transfer model including Escherichia coli K12 (RP4) and anammox system. Results indicated that the RP4 plasmid could be successfully transferred to anaerobic ammonia oxidative bacteria (AnAOB). Furthermore, degradation efficiencies of ammonia nitrogen (NH4+-N) and nitrite nitrogen (NO2--N) were decreased by 5.14 % - 14.89 % and 4.30 % - 18.7 % respectively. Concurrently, down-regulated expressions of nirS and hzsB genes were observed. Electron transport chains and cell membrane damage were involved in anammox inhibition. Furthermore, metatranscriptomic analysis revealed the involvement of quorum sensing (QS) in anammox (RP4) metabolism. C6HSL was found to be the key QS molecule and its concentration increased gradually. Correlation analysis showed a negative correlation between C6-HSL and the specific anammox activity difference (r=-0.817). Furthermore, the addition of C6-HSL to the model increased the degradation rate of NH4+-N and NO2--N. This further confirmed that QS could alleviate the inhibition effects of RP4 plasmid transfer by up-regulating the levels of nirS and hzsB. This study will provide novel insights into the mechanisms of anammox metabolism.

Microplastics and antibiotic resistance genes are two new pollutants in water environments, and they have potential risks to human health and ecological safety. On the basis of the accumulation of pollutants and microorganisms in sediment, macrobenthic invertebrates are considered as potential practitioners of microplastic degradation and antibiotic resistance gene (ARG) transfer. However, whether microplastic degradation can affect ARG transfer in aquatic environments, especially in the gut of macrobenthic invertebrates, remains unclear. In this study, we demonstrated that microplastics including polyethylene terephthalate (PET), polyvinyl chloride(PVC), polyamide (PA), polystyrene (PS), polypropylene (PP), polyethylene (PE), and polyurethane (PU), and ARGs including tetA, sul1, sul2, and sul3 were widely distributed in sediment and benthic invertebrates in Nansi lake. The distribution of ARGs was related to the number and size of microplastic particles. In particular, it was found for the first time that the content of ARGs corresponding to individual particles was linearly and negatively correlated with the size of microplastics. The results of animal feeding experiments showed that microplastic degradation in the gut of Chironomidae larvae could promote the conjugative transfer of ARGs. The underlying molecular mechanism was SOS response. This study provides a new method for the analysis of the interaction effect of multiple pollutants in freshwater environments.

Antibiotics are extensively utilized in agriculture and livestock farming; however, their environmental residues may foster the proliferation of drug-resistant bacteria and disrupt ecosystems, underscoring the necessity for sensitive detection technologies. Allosteric transcription factors (aTFs) represent a class of proteins that can specifically recognize small molecules and regulate gene expression. Owing to their high specificity and sensitivity, aTFs have been widely adopted in the development of biosensors. This study utilized the properties of allosteric transcription factors to alter their DNA affinity upon ligand binding. By measuring the changes in affinity of TetR and MphR before and after DNA binding using SPR, the study quantitatively analyzed the concentrations of tetracycline and erythromycin. Through SPR technology, quantitative detection of these affinity changes was achieved. Under optimized conditions regarding sample incubation time and system injection time, the affinity values exhibited changes of two orders of magnitude. The detection limits for tetracycline and erythromycin using this technique were determined to be 0.8 nM and 1 nM, respectively. These results indicate that the developed SPR detection method demonstrates high sensitivity and accuracy for detecting tetracycline and erythromycin, making it a promising tool for various applications, including environmental monitoring and quality control.

On-site detection of antibiotics and heavy metal ions is critical due to their severe impact on the environment and human health. In this study, allosteric transcription factors (aTFs) were integrated with optical fiber sensing technology to develop a aTF-quantum dot (QD) complex fluorescent fiber biosensor. This biosensor allows for the one-step quantitative on-site detection of tetracycline and Pb2+. Within 5 min, the prepared biosensor enabled the quantitative detection of tetracycline and Pb2+ with detection limits of 16 nM and 0.14 nM, respectively. The linear detection ranges were 10 nM to 1 x 106 nM for tetracycline and 0.1 nM to 500 nM for Pb2+. The biosensors demonstrated excellent selectivity and could be reused at least five times after regeneration. In actual sample analyses, the biosensors achieved recovery rates of 96.86-112.66 % for tetracycline and 98.69-104.01 % for Pb2+. These results highlight the advantages of the prepared biosensors, including rapid detection, high sensitivity, and excellent selectivity. Furthermore, a portable water contaminant analyzer platform and corresponding software were developed, significantly enhancing the on-site rapid detection capabilities of optical fiber biosensors. This study reports a novel strategy for constructing optical fiber fluorescent biosensors, making them particularly suitable for field detection applications.

The quartz crystal microbalance (QCM) is a powerful analytical instrument that enables the quantification of substance mass by precisely measuring frequency changes. To meet the requirements of trace detection limits, traditional QCM sensors typically need to use nanomaterials to connect to target during the detection process to gradually increase the mass of the target, thereby enhancing frequency signal changes and improving the detection limit. However, this increases the processing time for the target. To address these limitations, we have developed a QCM biosensor based on allosteric transcription factors (aTFs). Through the implementation of a signal amplification synergy effect strategy-specifically, increasing the number of aTFs and employing quantum dots for mass signal amplification-the detection sensitivity for Pb2+ has been enhanced by 20-fold, achieving an ultra-low limit of detection (LOD) of 0.05 pM. Meanwhile, the dynamic range has been extended by two orders of magnitude, showing a linear response from 0.05 pM to 200 nM. It is worth noting that leveraging the rapid and specific recognition of Pb2+ by aTFs, the incubation time has been shortened to 10 min. In addition, the QCM biosensor can be regenerated up to five times. These results indicate that the QCM biosensor based on aTFs effectively overcomes the limitations of traditional QCM in the quantitative analysis of low-mass substances. It also has certain economic applicability, providing a new strategy for the development of QCM biosensors suitable for various analytes.

Background: High-altitude hypoxia is known to adversely affect bone health, leading to accelerated bone loss and metabolic alterations. Recent studies suggest that factors such as bicarbonate and gut microbiota may play key roles in bone health. Mineral water, rich in bicarbonate, may influence bone health and the gut–bone axis under such conditions. Methods: Mice were exposed to hypoxia and treated with different concentrations of drinking water. Bone-related parameters were assessed using dual-energy X-ray absorptiometry (DXA) and Micro-CT. Bone health was assessed using the measurement of serum biomarkers. Additionally, Untargeted Metabolomics was employed to analyze differential metabolites between groups, while gut microbiota composition was analyzed using 16S rRNA sequencing. Results: BMW consumption increased bone mineral density (BMD) and helped alleviate the damage to the microstructure of bones caused by hypoxia and delayed the progression of osteoporosis. Additionally, BMW was shown to enhance probiotics such as Akkermansia and Dubosiella and regulate the longevity-regulating pathway as well as the PI3K/AKT/mTOR (PAM) signaling pathway. This study also discovered changes in metabolic products due to BMW intervention, predominantly in pathways such as the amino acid, prostaglandin, and purine metabolisms, with correlation analysis further exploring the relationships between gut microbiota and these differential metabolites. Conclusions: Long-term exposure to high-altitude hypoxic conditions affects the structure of gut microbiota and bone metabolism in mice. The consumption of BMW improves the structure of gut microbiota and regulates the metabolic pathways to maintain bone health under high-altitude hypoxia.

Antibiotic and heavy metal contamination in water poses a severe global public health risk, yet conventional detection methods remain constrained by reliance on complex laboratory instrumentation. Although cell-free biosensors utilizing allosteric transcription factors (aTFs) offer promising alternatives, their limited sensitivity impedes reliable detection of small-molecule contaminants. To address this limitation, we developed allosteric transcription factors (aTFs)-regulated rolling circle transcription assay (ARCTA) for ultrasensitive detection of water contaminants. The ARCTA system employs a circular DNA template comprising a double-stranded region with a T7 promoter and aTFs binding sequence, alongside a single-stranded domain transcribable into fluorescent RNA. Target-specific recognition by aTFs enables rapid, efficient transcription and amplification of fluorescent RNA, achieving an exceptionally low detection threshold. Applied to tetracycline, oxytetracycline, erythromycin, clarithromycin, Hg2+, Pb2+, Cd2+, and As3+ in water samples, ARCTA demonstrated detection limits of 1.25 nM, 0.93 nM, 0.28 nM, 0.45 nM, 0.034 nM, 0.006 nM, 0.13 nM, and 0.016 nM, respectively, and all had a broad dynamic ranges.Notably, ARCTA by designing distinct circular DNA templates to transcribe wavelength-specific fluorescent RNAs, enabling the simultaneous detection of multiple targets. In addition, the system also exhibited robust performance in real-world applications, underscoring its practicality. These results highlight ARCTA's potential as a transformative tool for ultrasensitive monitoring of diverse small-molecule contaminants.

Antibiotic resistance (AMR) in the environment has emerged as a significant threat, severely impacting public health, ecological balance, and economic stability. Concurrently, environmental chemical pollution has been verified to trigger the spread of antibiotic resistance genes (ARGs). However, studies on the impacts of environmental pollutants on pheromone-regulated plasmid-mediated conjugative transfer of ARGs remain extremely limited. In the present study, we investigated the effects and underlying mechanisms of diethylstilbestrol (DES) on the conjugative transfer of pCF10 in Enterococcus faecalis. The results showed that DES at environmental concentrations led to an increase in conjugation transfer frequency by approximately 2.4 times higher than that of control at 2 h. Through a comprehensive suite of techniques, including mass spectrometry detection, quantitative polymerase chain reaction (qPCR), gene knockout, and morphological analysis, this study revealed that DES promoted the expression of pheromone regulated genes and activated the pheromone signaling pathway. Alternatively, it entered bacterial cells and bound to pheromone molecule regulatory switch PrgX. This binding subsequently stimulated the production of iCF10, as well as the expression of downstream signaling molecules, ultimately facilitating the conjugative transfer of pCF10. This study deepens our understanding of the environmental biological effect of DES and the spread of ARGs.

As modern society places a growing emphasis on sustainable development, the demand for eco-friendly adsorbent materials to effectively remediate toxic dyes in water has become increasingly critical. This study utilizes cost-effective cellulose nanofibers as the primary raw material, along with polyethyleneimine and trimethoxysilane as modifying agents. A composite aerogel adsorbent, referred to as cellulose/polyethyleneimine (CMP), is synthesized via a combination of chemical crosslinking and directional freezing employing an ice template method. The resulting CMP composite aerogel features a three-dimensional multi-walled porous structure enriched with amino and oxygen-containing functional groups, facilitating the efficient adsorption of organic dyes from aqueous solutions. The directional porous architecture and high specific surface area of the CMP composite aerogel enhance the binding affinity of organic pollutants to its active sites, thereby accelerating ion transfer. Calculations based on the Langmuir isotherm model demonstrate that the CMP composite aerogel exhibits an impressive adsorption capacity of 513.30 mg/g for the organic dye methyl orange, offering an effective solution to dye pollution in aquatic environments. The application of directional freeze-drying technology represents a green and efficient method for the fabrication of layered microporous structures. Additionally, the resulting aerogel demonstrates a low density (0.027 g/cm3)and high hydrophobicity (water contact angle 132.3 degrees), significantly enhancing its utility. The adsorption material demonstrates considerable potential for application in the domain of water treatment, effectively removing chemical pollutants such as dyes from water.

Exposure to Pb2+ in the environment, especially in water, poses a significant threat to human health and urgently necessitates the development of highly sensitive Pb2+ detection methods. In this study, we have integrated the high sensitivity of electrochemical techniques with allosteric transcription factors (aTFs) to develop an innovative electrochemical biosensing platform. This biosensors leverage the specific binding and dissociation of DNA to the aTFs (PbrR) on electrode surfaces to detect Pb2+. Under the optimal conditions, the platform has a broad linear detection range from 1 pM to 10 nM and an exceptionally low detection threshold of 1 pM, coupled with excellent selectivity for Pb2+. Notably, the biosensor demonstrates regenerative capabilities, enabling up to five effective Pb2+ measurements. After one week of storage at 4 °C, effective lead ion detection was still possible, demonstrating the biosensor's excellent stability, this can effectively save the cost of detection. The biosensor also achieves a recovery rate of 93.3% to 106.6% in real water samples. The biosensor shows its potential as a robust tool for the ultrasensitive detection of Pb2+ in environmental monitoring. Moreover, this research provides new insights into the future applications of aTFs in electrochemical sensing.

Vibrio parahaemolyticus (V. parahaemolyticus) is a significant concern, as it can cause severe infections and hemolytic trauma. Given its prevalence in seawater and coastal seafood, it poses a substantial risk as a foodborne pathogen. Biosensor-based detection technology has been continuously evolving, and toehold switches have emerged as a promising area within it, especially in the detection of RNA viruses. Here, we have developed a cell-free toehold switch sensor for V. parahaemolyticus detection. Traditional toehold switch detection methods usually use green fluorescent protein (GFP) or enzyme LacZ as the output signal, with an incubation time as long as 2 h, and are also mainly applied to the detection of RNA viruses. In this study, we introduced a novel, artificially designed luciferase (LuxSit-i) as an output signal and constructed toehold switches with two different output signals (sfGFP, LuxSit-i), aimed at reducing the incubation time of toehold switches. Moreover, to further improve the detection process, we separately utilize recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA) to amplify dead and live bacterial suspensions for detection and attempt to distinguish between dead and live bacteria. This study provided a convenient, rapid, and accurate method for the on-site detection of V. parahaemolyticus, especially beneficial for resource-limited settings. By eliminating the requirement for specialized facilities and personnel, this system has the potential to be a valuable tool in improving public health responses, especially in developing regions.

IntroductionBacterial resistance presents a major challenge to both the ecological environment and human well-being, with persistence playing a key role. Multiple studies were recently undertaken to examine the factors influencing the formation of persisters and the underlying process, with a primary focus on Gram-negative bacteria and Staphylococcus aureus (Gram-positive bacteria). Enterococcus faecalis (E. faecalis) is capable of causing a variety of infectious diseases, but there have been few studies of E. faecalis persisters. Previous studies have shown that the sex pheromone cCF10 secreted by E. faecalis induces conjugative plasmid transfer. However, whether the pheromone cCF10 regulates the persistence of E. faecalis has not been investigated.MethodsAs a result, we investigated the effect and potential molecular mechanism of pheromone cCF10 in regulating the formation of persisters in E. faecalis OG1RF using a persistent bacteria model.Results and discussionThe metabolically active E. faecalis OG1RF reached a persistence state and temporarily tolerated lethal antibiotic concentrations after 8 h of levofloxacin hydrochloride (20 mg/mL) exposure, exhibiting a persistence rate of 0.109 %. During the growth of E. faecalis OG1RF, biofilm formation was a critical factor contributing to antibiotic persistence, whereas 10 ng/mL cCF10 blocked persister cell formation. Notably, cCF10 mediated the antibiotic persistence of E. faecalis OG1RF via regulating metabolic activity rather than suppressing biofilm formation. The addition of cCF10 stimulated the Opp system and entered bacterial cells, inhibiting (p)ppGpp accumulation, thus maintaining the metabolically active state of bacteria and reducing persister cell generation. These findings offer valuable insights into the formation, as well as the control mechanism of E. faecalis persisters.

The massive use of acetaminophen (APAP), an antipyretic and analgesic drug, especially during the COVID-19 pandemic, has caused the environmental residue and pollution of acetaminophen and its metabolites. However, the environmental ecological and health issues they may cause are still unclear. In this study, we found that environmental concentrations of APAP and its metabolites could promote the conjugative transfer of antibiotic resistance genes mediated by plasmid pCF10 through pheromone -like effect rather than the commonly mechanism found in current studies. Conjugative transfer studies showed that APAP and its metabolites increased the frequency of conjugative transfer of ARGs by up to 3.3 times, reaching 3.2 x 10 -2 . We explored the underling mechanisms using qPCR, western blot, and SEM images. This study demonstrates that APAP stimulates pheromone secretion, subsequently triggering conjugation and accelerating downstream gene expression. Furthermore, APAP functions as a pheromone, initially facilitating plasmid cleavage and peptidoglycan hydrolysis, followed by the promotion of adhesion protein PrgB expression and T4SS scaffold construction, ultimately enhancing DNA strand transfer via T4CP. This study provides a theoretical basis for understanding the environmental hazards of APAP and the environmental sustainability.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1–100 μg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 μg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.