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Introduction: Bacterial 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. Methods: As 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 discussion: The 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.
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Pentachlorophenol (PCP) was once used as a pesticide, germicide, and preservative due to its stable properties and resistance to degradation. This study aimed to design a biosensor for the quantitative and prompt detection of capable of PCP. A cell-free fluorescence biosensor was developed while employing NalC, an allosteric Transcription Factor responsive to PCP and In Vitro Transcription. By adding a DNA template and PCP and employing Electrophoretic Mobility Shift Assay while monitoring the dynamic fluorescence changes in RNA, this study offers evidence of NalC's potential applicability in sensor systems developed for the specific detection of PCP. The biosensor showed the capability for the quantitative detection of PCP, with a Limit of Detection (LOD) of 0.21 µM. Following the addition of Nucleic Acid Sequence-Based Amplification, the fluorescence intensity of RNA revealed an excellent linear relationship with the concentration of PCP, showing a correlation coefficient (R2) of 0.9595. The final LOD was determined to be 0.002 µM. This study has successfully translated the determination of PCP into a fluorescent RNA output, thereby presenting a novel approach for detecting PCP within environmental settings.
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The composition, quantity, and function of peripheral blood mononuclear cells (PBMCs) are closely correlated with tumorigenesis. However, the mechanisms of PBMCs in lung cancer are not clear. Mitochondria are energy factories of cells, and almost all cellular functions rely on their energy metabolism level. The present study aimed to test whether the mitochondrial function of PBMCs directly determines their tumor immune monitoring function. We recruited 211 subjects, including 105 healthy controls and 106 patients with recently diagnosed with lung cancer. The model of lung carcinogenesis induced by BaP was used in animal experiment, and the Bap carcinogenic metabolite, Benzo(a)pyren-7,8-dihydrodiol-9,10-epoxide (BPDE), was used in cell experiment. We found that mitochondrial function of PBMCs decreased significantly in patients with new lung cancer, regardless of age. In vivo, BaP caused PBMC mitochondrial dysfunction in mice before the appearance of visible malignant tissue. Moreover, mitochondrial function decreased significantly in mice with lung cancers induced by BaP compared to those without lung cancer after BaP intervention. In vitro, BPDE also induced mitochondrial dysfunction and reduced the aggressiveness of PBMCs toward cancer cells. Furthermore, the changes in mitochondrial energy metabolism gene expression caused by BPDE are involved in this process. Thus, the mitochondrial function of PBMCs is a potential prognostic biomarker or therapeutic target to improve clinical outcomes in patients with lung cancer.
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Leucócitos Mononucleares , Neoplasias Pulmonares , Mitocôndrias , Humanos , Neoplasias Pulmonares/patologia , Leucócitos Mononucleares/metabolismo , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Masculino , Feminino , Camundongos , Pessoa de Meia-Idade , Carcinogênese , Benzo(a)pireno/toxicidade , Metabolismo Energético , Idoso , Camundongos Endogâmicos C57BLRESUMO
The widespread use of disinfectants during the global response to the 2019 coronavirus pandemic has increased the co-occurrence of disinfection byproducts (DBPs) and antibiotic resistance genes (ARGs). Although DBPs pose major threats to public health globally, there is limited knowledge regarding their biological effects on ARGs. This study aimed to investigate the effects of two inorganic DBPs (chlorite and bromate) on the conjugative transfer of RP4 plasmid among Escherichia coli strains at environmentally relevant concentrations. Interestingly, the frequency of conjugative transfer was initially inhibited when the exposure time to chlorite or bromate was less than 24 h. However, this inhibition transformed into promotion when the exposure time was extended to 36 h. Short exposures to chlorite or bromate were shown to impede the electron transport chain, resulting in an ATP shortage and subsequently inhibiting conjugative transfer. Consequently, this stimulates the overproduction of reactive oxygen species (ROS) and activation of the SOS response. Upon prolonged exposure, the resurgent energy supply promoted conjugative transfer. These findings offer novel and valuable insights into the effects of environmentally relevant concentrations of inorganic DBPs on the conjugative transfer of ARGs, thereby providing a theoretical basis for the management of DBPs.
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Bromatos , Cloretos , Escherichia coli , Estresse Oxidativo , Plasmídeos , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bromatos/toxicidade , Plasmídeos/genética , Cloretos/farmacologia , Desinfetantes/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Conjugação Genética/efeitos dos fármacos , Resistência Microbiana a Medicamentos/genética , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacosRESUMO
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⯵g/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10⯵g/L of CYN led to maximum fold change of â¼6.5- fold at 16â¯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.
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Alcaloides , Toxinas Bacterianas , Toxinas de Cianobactérias , Escherichia coli , Glutationa , Plasmídeos , Uracila , Plasmídeos/genética , Glutationa/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Toxinas Bacterianas/toxicidade , Uracila/análogos & derivados , Uracila/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Conjugação Genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genéticaRESUMO
In wastewater treatment plants (WWTPs), ammonia oxidation is primarily carried out by three types of ammonia oxidation microorganisms (AOMs): ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and comammox (CMX). Antibiotic resistance genes (ARGs), which pose an important public health concern, have been identified at every stage of wastewater treatment. However, few studies have focused on the impact of ARGs on ammonia removal performance. Therefore, our study sought to investigate the effect of the representative multidrug-resistant plasmid RP4 on the functional microorganisms involved in ammonia oxidation. Using an inhibitor-based method, we first evaluated the contributions of AOA, AOB, and CMX to ammonia oxidation in activated sludge, which were determined to be 13.7%, 41.1%, and 39.1%, respectively. The inhibitory effects of C2H2, C8H14, and 3,4-dimethylpyrazole phosphate (DMPP) were then validated by qPCR. After adding donor strains to the sludge, fluorescence in situ hybridization (FISH) imaging analysis demonstrated the co-localization of RP4 plasmids and all three AOMs, thus confirming the horizontal gene transfer (HGT) of the RP4 plasmid among these microorganisms. Significant inhibitory effects of the RP4 plasmid on the ammonia nitrogen consumption of AOA, AOB, and CMX were also observed, with inhibition rates of 39.7%, 36.2%, and 49.7%, respectively. Moreover, amoA expression in AOB and CMX was variably inhibited by the RP4 plasmid, whereas AOA amoA expression was not inhibited. These results demonstrate the adverse environmental effects of the RP4 plasmid and provide indirect evidence supporting plasmid-mediated conjugation transfer from bacteria to archaea.
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Archaea , Betaproteobacteria , Archaea/genética , Archaea/metabolismo , Esgotos/microbiologia , Amônia , Nitrogênio/metabolismo , Desnitrificação , Hibridização in Situ Fluorescente , Oxirredução , Bactérias/genética , Bactérias/metabolismo , Plasmídeos/genética , Betaproteobacteria/genética , Betaproteobacteria/metabolismo , Antibacterianos , Filogenia , Microbiologia do SoloRESUMO
Background: Intracorporeal esophagojejunostomy (EJ) in the context of laparoscopic total gastrectomy remains a complex and technically demanding procedure. We have previously introduced a novel method of intracorporeal circular stapled EJ utilizing a conventional purse-string suture instrument. Since May 2018, we have refined this technique, and the aim of this study was to assess its safety and efficacy. Methods: Between May 2018 and June 2022, we enrolled 92 patients who underwent laparoscopic total gastrectomy with the modified intracorporeal reconstruction method. In addition, between March 2014 and June 2022, we enrolled 121 patients who underwent the procedure with the extracorporeal reconstruction method. We retrospectively collected and compared the clinical data of these 2 patient cohorts. Results: Intracorporeal reconstruction group experienced lower postoperative pain scores (2.7 ± 1.3 versus 4.5 ± 1.4, P = .032), reduced administration of analgesics (3.1 ± 2.2 versus 5.0 ± 3.5, P = .041), and shorter postoperative hospital stays (4.9 ± 2.3 versus 6.3 ± 3.5, P = .045) compared with the extracorporeal reconstruction group. In addition, anastomotic time and postoperative pain score were not increased in the overweight patients in the intracorporeal reconstruction group. Anastomotic leakage occurred in 2 (2.2%) patients in the intracorporeal reconstruction group and 4 (3.3%) patients in the extracorporeal reconstruction group. Anastomotic stricture occurred in 1 (1.1% and 0.8%) patient in each group. There was no significant difference in the overall postoperative complication rate between the 2 groups. Conclusions: The modified intracorporeal purse-string stapling technique for EJ during laparoscopic total gastrectomy is a safe and viable option, exhibiting less invasiveness and comparable outcomes to the extracorporeal reconstruction method, especially suitable for obese patients.
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Laparoscopia , Neoplasias Gástricas , Humanos , Grampeamento Cirúrgico/métodos , Estudos Retrospectivos , Jejuno/cirurgia , Laparoscopia/métodos , Anastomose Cirúrgica/métodos , Complicações Pós-Operatórias/cirurgia , Gastrectomia/métodos , Dor Pós-Operatória/cirurgia , Neoplasias Gástricas/cirurgiaRESUMO
Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.
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Vírus da Hepatite A , Hepatite A , Humanos , Vírus da Hepatite A/genética , Hepatite A/diagnóstico , Vírus da Hepatite A Humana/genética , RNARESUMO
Increased disinfection of wastewater to preserve its microbiological quality during the coronavirus infectious disease-2019 (COVID-19) pandemic have inevitably led to increased production of toxic disinfection by-products (DBPs). However, there is limited information on such DBPs (i.e., trihalomethanes, haloacetic acids, nitrosamines, and haloacetonitriles). This review focused on the upsurge of chlorine-based disinfectants (such as chlorine, chloramine and chlorine dioxide) in wastewater treatment plants (WWTPs) in the global response to COVID-19. The formation and distribution of DBPs in wastewater were then analyzed to understand the impacts of these large-scale usage of disinfectants in WWTPs. In addition, potential ecological risks associated with DBPs derived from wastewater disinfection and its receiving water bodies were summarized. Finally, various approaches for mitigating DBP levels in wastewater and suggestions for further research into the environmental risks of increased wastewater disinfection were provided. Overall, this study presented a comprehensive overview of the formation, distribution, potential ecological risks, and mitigating approaches of DBPs derived from wastewater disinfection that will facilitate appropriate wastewater disinfection techniques selection, potential ecological risk assessment, and removal approaches and regulations consideration.
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COVID-19 , Desinfetantes , Poluentes Químicos da Água , Purificação da Água , Humanos , Desinfecção/métodos , Águas Residuárias , Pandemias/prevenção & controle , Cloro , Poluentes Químicos da Água/análise , COVID-19/epidemiologia , COVID-19/prevenção & controle , Purificação da Água/métodos , Halogenação , Trialometanos/análiseRESUMO
Antibiotic resistance genes (ARGs) have recently become an important public health problem and therefore several studies have characterized ARG composition and distribution. However, few studies have assessed their impact on important functional microorganisms in the environment. Therefore, our study sought to investigate the mechanisms through which multidrug-resistant plasmid RP4 affected the ammonia oxidation capacity of ammonia-oxidizing bacteria, which play a key role in the nitrogen cycle. The ammonia oxidation capacity of N. europaea ATCC25978 (RP4) was significantly inhibited, and NO and N2O were produced instead of nitrite. Our findings demonstrated that the decrease in electrons from NH2OH decreased the ammonia monooxygenase (AMO) activity, leading to a decrease in ammonia consumption. In the ammonia oxidation process, N. europaea ATCC25978 (RP4) exhibited ATP and NADH accumulation. The corresponding mechanism was the overactivation of Complex â , ATPase, and the TCA cycle by the RP4 plasmid. The genes encoding TCA cycle enzymes related to energy generation, including gltA, icd, sucD, and NE0773, were upregulated in N. europaea ATCC25978 (RP4). These results demonstrate the ecological risks of ARGs, including the inhibition of the ammonia oxidation process and an increased production of greenhouse gases such as NO and N2O.
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Nitrosomonas europaea , Nitrosomonas europaea/genética , Amônia , Transporte de Elétrons , Oxirredução , Nitritos , NitrosomonasRESUMO
Templating polymeric membranes with micro-nano-scaled solid materials is an effective method to simultaneously improve the water flux and retention ratio. However, the fabrication of a green, recyclable, and size-controlled template material remains a challenge. Here, a new green pore-forming agent, lignin particles (LP), was developed to prepare porous polysulfone (PSF) membranes via the phase inversion technique. A series of LP have uniform sizes from ~200 nm to ~1800 nm. The performances of the templated PSF membranes cast at different sizes and contents of LP were examined for their surface and crosssection morphologies. The LP-templated PSF membranes displayed a remarkable enhancement in flux, porosity, and moisture content. Particularly, the PSF membranes cast with LP from ~200 to 1800 nm broke the traditional trade-off to a certain degree, which possessed stable retentions of bovine serum albumin (> 85 %) and significantly improved water flux (174.275 to 254.775 L m-2 h-1). In addition, the LP pore-forming agent is low-cost and environmentally friendly as it was prepared from industrial by-products and can be easily recycled. Overall, this study shows that lignin particles are green pore-forming agents that can be used for the fabrication of porous polymeric membranes with improved performance for water treatment.
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Lignina , Membranas Artificiais , Polímeros , SulfonasRESUMO
Horizontal gene transfer (HGT) mediated by conjugative plasmids greatly contributes to bacteria evolution and the transmission of antibiotic resistance genes (ARGs). In addition to the selective pressure imposed by extensive antibiotic use, environmental chemical pollutants facilitate the dissemination of antibiotic resistance, consequently posing a serious threat to the ecological environment. Presently, the majority of studies focus on the effects of environmental compounds on R plasmid-mediated conjugation transfer, and pheromone-inducible conjugation has largely been neglected. In this study, we explored the pheromone effect and potential molecular mechanisms of estradiol in promoting the conjugative transfer of pCF10 plasmid in Enterococcus faecalis. Environmentally relevant concentrations of estradiol significantly increased the conjugative transfer of pCF10 with a maximum frequency of 3.2 × 10-2, up to 3.5-fold change compared to that of control. Exposure to estradiol induced the activation of pheromone signaling cascade by increasing the expression of ccfA. Furthermore, estradiol might directly bind to the pheromone receptor PrgZ and promote pCF10 induction and finally enhance the conjugative transfer of pCF10. These findings cast valuable insights on the roles of estradiol and its homolog in increasing antibiotic resistance and the potential ecological risk.
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Antibacterianos , Feromônios , Antibacterianos/metabolismo , Feromônios/farmacologia , Feromônios/genética , Feromônios/metabolismo , Estradiol/farmacologia , Estradiol/metabolismo , Plasmídeos/genética , Resistência Microbiana a Medicamentos/genética , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Transferência Genética HorizontalRESUMO
The rapid spread of antibiotic-resistance genes (ARGs) in Enterococcus faecalis (E. faecalis) poses a great challenge to human health and ecological and environmental safety. Therefore, it is important to control the spread of ARGs. In this study, we observed that the addition of 5 µg/mL docosahexaenoic acid (DHA) reduced the conjugative transfer of pCF10 plasmid by more than 95% in E. faecalis. DHA disturbed the pheromone transport by inhibiting the mRNA levels of the prgZ gene, causing the iCF10 pheromone to accumulate in the donor bacteria and bond to the PrgX receptor to form an inhibitory phase, which resulted in the down-regulation of the expression of genes related to conjugative transfer, inhibiting biofilm formation, reducing bacterial adhesion and thus inhibiting conjugative transfer. Collectively, DHA exhibited an admirable inhibitory effect on the transfer of ARGs in E. faecalis. This study provided a technical option to control the transfer of ARGs.
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Ácidos Docosa-Hexaenoicos , Enterococcus faecalis , Humanos , Enterococcus faecalis/genética , Ácidos Docosa-Hexaenoicos/farmacologia , Antibacterianos , Feromônios , Resistência Microbiana a Medicamentos , Plasmídeos/genéticaRESUMO
BACKGROUND: Both AKT and Aurora inhibitors are a potential therapeutic agent for the treatment of malignant tumors. However, the role of combined inhibition of AKT and Aurora in colon cancer and its underlying mechanism have yet to be fully investigated. OBJECTIVE: To investigate the role of combined AKT and Aurora inhibitors in colon cancer and its underlying mechanisms. METHODS: CCK8 assay, colony formation assay, and flow cytometry were performed to analyze the proliferation and apoptosis of colon cancer cell line SW480 treated with combined AKT inhibitor MK2206 and Aurora inhibitor Alisertib, respectively. And tumor formation and growth were measured in tumor allograft model mice administered with the combined inhibitors. Western blot analysis was used to examine the expression levels of apoptosis-related proteins and signal transduction pathway components. The PI3K agonist 740Y-P and Overexpression of AKT are used to verify whether the PI3K/AKT pathway plays an anti-tumor effect when combined with inhibitory administration. RESULTS: Aurora A inhibitor Alisertib and AKT inhibitor MK2206 displayed consistent and synergistic antiproliferation and proapoptotic effects. Combined inhibition of Aurora A and AKT down-regulated the expression of Bcl-2/Bax and up-regulated the expression of cleaved-caspase-3 and cleaved-PARP. While single-drug treatment can significantly inhibit the expression of P-PI3K and P-AKT as well as increase the expression of P53 and H2A.X, the combined drugs had a more significant inhibitory effect than the single drug. Moreover, administration of PI3K agonist 740Y-P and AKT1 overexpression in experiments proved that the combined drugs exert an anticancer effect by inhibiting the PI3K/AKT pathway. Meanwhile, we showed that the combined administration had an anti-colon cancer effect on tumor allograft mice, and the underlying mechanism involved inhibition of the PI3K/AKT pathway. CONCLUSION: Combined administration of Aurora A inhibitor Alisertib and AKT inhibitor MK2206 can inhibit the proliferation of colon cancer cells and induce apoptosis, while inhibiting tumor growth in vivo. The underlying mechanism may involve the PI3K/AKT pathway and DNA damage pathway.
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Aurora Quinase A , Neoplasias , Proteínas Proto-Oncogênicas c-akt , Animais , Camundongos , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais , Aurora Quinase A/antagonistas & inibidoresRESUMO
The enrichment and spread of antibiotic resistance genes (ARGs) induced by environmental chemical pollution further exacerbated the threat to human health and ecological safety. Several compounds are known to induce R plasmid-mediated conjugation through inducing reactive oxygen species (ROS), increasing cell membrane permeability, enhancing regulatory genes expression, and so forth. Up to now, there has been no substantial breakthrough in the studies of models and related mechanisms. Here, we established a new conjugation model using pheromone-responsive plasmid pCF10 and confirmed that five kinds of bisphenols (BPs) at environmentally relevant concentrations could significantly promote the conjugation of ARGs mediated by plasmid pCF10 in E. faecalis by up to 4.5-fold compared with untreated cells. Using qPCR, gene knockout and UHPLC, we explored the mechanisms behind this phenomenon using bisphenol A (BPA) as a model of BPs and demonstrated that BPA could upregulate the expression of pheromone, promote bacterial aggregation, and even directly activate conjugation as a pheromone instead of producing ROS and enhancing cell membrane permeability. Interestingly, the result of mathematical analysis showed that the pheromone effect of most BPs is more potent than that of synthetic pheromone cCF10. These findings provide new insight into the environmental behavior and biological effect of BPs and provided new method and theory to study on enrichment and spread of ARGs induced by environmental chemical pollution.
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Antibacterianos , Compostos Benzidrílicos , Enterococcus faecalis , Fenóis , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Conjugação Genética , Resistência Microbiana a Medicamentos , Enterococcus faecalis/genética , Feromônios/genética , Feromônios/metabolismo , Plasmídeos , Espécies Reativas de Oxigênio/metabolismo , Compostos Benzidrílicos/farmacologia , Fenóis/farmacologiaRESUMO
In recent years, the issue of food safety has received a lot of attention. The Food and Drug Administration (FDA) prescribes the antibiotic's maximum residue limit (MRL) in food production. The standard detection methods of antibiotics are liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC), with complex operations and precision instruments. In this study, allosteric transcription factor (aTF)-based in vitro transcription (IVT) cell-free biosensors were developed for tetracyclines and macrolides with nucleic acid sequence-based amplification (NASBA). Characterization of binding and dissociation processes between aTF and DNA was carried out by BIAcore assay and electrophoretic mobility shift assay (EMSA). BIAcore was innovatively used to directly observe the real-time process of binding and dissociation of aTF with DNA. The biosensors produce more fluorescence RNA when target antibiotics are added to the three-way junction dimeric Broccoli (3WJdB). Four tetracyclines and two macrolides were quantified in the 0.5-15 µM range, while erythromycin and clarithromycin were detected over a range of 0.1-15 µM. NASBA, commonly used for viral detection, was used to amplify 3WJdB RNA generated by IVT, which greatly increased the LOD for tetracyclines and macrolides to 0.01 µM. The use of biosensors in milk samples demonstrated their on-site detection performance. Overall, our proposed biosensors are simple, rapid, selective, and sensitive, with the potential for field application.
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Técnicas Biossensoriais , Macrolídeos , Estados Unidos , Animais , Macrolídeos/análise , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Fatores de Transcrição/análise , Leite/química , Tetraciclinas/análise , Antibacterianos/análise , Antibacterianos/química , Tetraciclina/análise , DNA/análise , RNA/análiseRESUMO
The rapid quantitative detection of Escherichia coli (E. coli) is of great significance for evaluating water and food safety. At present, the conventional bacteria detection methods cannot meet the requirements of rapid detection in water environments. Herein, we report a method based on chronoamperometry to rapidly and quantitatively detect live E. coli. In this study, the current indicator i0 and the electricity indicator A were used to record the cumulative effect of bacteria on an unmodified glassy carbon electrode (GCE) surface during chronoamperometric detection. Through the analysis of influencing factors and morphological characterization, it was proved that the changes of the two set electrochemical indicator signals had a good correlation with the concentration of E. coli; detection time was less than 5 min, the detection range of E. coli was 104−108 CFU/mL, and the error range was <30%. The results of parallel experiments and spiking experiments showed that this method had good repeatability, stability, and sensitivity. Humic acid and dead cells did not affect the detection results. This study not only developed a rapid quantitative detection method for E. coli in the laboratory, but also realized a bacterial detection scheme based on the theory of bacterial dissolution and adsorption for the first time, providing a new direction and theoretical basis for the development of electrochemical biosensors in the future.
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Técnicas Biossensoriais , Infecções por Escherichia coli , Humanos , Escherichia coli , Técnicas Eletroquímicas/métodos , Substâncias Húmicas , Técnicas Biossensoriais/métodos , Água , CarbonoRESUMO
Water quality monitoring requires a reliable and practical on-site detection method for heavy metal ions. Combining an in vitro transcription (IVT) technology with allosteric transcription factors (aTFs), we developed a cell-free paper-based biosensor for on-site detection of Hg2+ and Pb2+ in water. Suitable aTFs screened using surface plasmon resonance (SPR) were employed for building biosensors. ATFs could disassociate from DNA due to their specific affinity to metal ions, and fluorescent RNA was transcribed as a signal. The developed biosensor could quantitatively detect Hg2+ in a linear dynamic range of 0.5-500 nM and Pb2+ in a 1-250 nM range in a 1 h period. The LOD of the biosensor was 0.5 nM for Hg2+ and 0.1 nM for Pb2+. The recoveries ranged from 91.09% to 123.24% for actual water samples detection. Furthermore, freeze-drying was used to create a paper-based biosensor that could detect Hg2+ and Pb2+ simultaneously on-site. This research presents a useful technique for various heavy metal ion detections.
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Técnicas Biossensoriais , Mercúrio , Metais Pesados , Técnicas Biossensoriais/métodos , Íons , Chumbo , Fatores de Transcrição/genéticaRESUMO
The widespread contamination of antibiotic resistance genes (ARGs) in freshwater environment are becoming a serious challenge to human health and ecological safety. Rapid and efficient monitoring of ARGs pollution is of great significance to ARGs control. Water, bottom mud, and fish have all been used to indicate ARG contamination in aquatic environments. However, it is unclear whether macrobenthic invertebrates in the food chain of aquatic environments can be indicators of ARG contamination. In this study, we demonstrated that ARGs including tetA gene, sul2 gene, and km gene were distributed in Chironomidae larvae in Weishan Lake. The ARG distribution was related to animal species, body parts, sampling sites, time, urban environment, animal farming, south-to-north water diversion, food chain, antibiotics, and water storage. Mathematical model predictions of ARG contamination in Weishan Lake were constructed based on the structural equation model (SEM) and the distribution of ARG sul2 in Chironomidae larvae. Influencing factors such as water storage, metal elements, antibiotic, and temperature were found to be closely related to the prediction of ARG contamination. This study provided a new indicator for ARG contamination in freshwater environments and a method to predict ARGs contamination.
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Chironomidae , Lagos , Animais , Antibacterianos/análise , Antibacterianos/farmacologia , China , Chironomidae/genética , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Humanos , Lagos/química , Larva , ÁguaRESUMO
Nanomaterials with bactericidal effects might provide novel strategies against bacteria. However, some bacteria can survive despite the exposure to nanomaterials, which challenges the safety of antibacterial nanomaterials. Here, we used a high dose of antibiotics to kill the E. coli. that survived under different concentrations of nanoalumina treatment to screen persisters, and found that nanoalumina could significantly trigger persisters formation. Treatment with 50 mg/L nanoalumina for 4 h resulted in the formation of (0.084 ± 0.005) % persisters. Both reactive oxygen species (ROS) and toxin-antitoxin (TA) system were involved in persisters formation. Interestingly, RT-PCR analysis and knockout of the five genes related to ROS and TA confirmed that only hipB was associated with the formation of persisters, suggesting the involvement of other mechanisms. We further identified 73 differentially expressed genes by transcriptome sequencing and analyzed them with bioinformatics tools. We selected six candidate genes and verified that five of them closely related to quorum sensing (QS) that were involved in persisters formation, and further validated that the coexpression of QS factors lrsF and qseB was a novel pathway for persisters. Our findings provided a better understanding on the emergence of bacterial persistence and the microbial behavior under nanomaterials exposure.