RESUMO
Metallo-ß-lactamases (MßLs) hydrolyze and inactivate ß-lactam antibiotics, are a pivotal mechanism conferring resistance against bacterial infections. SMB-1, a novel B3 subclass of MßLs from Serratia marcescens could deactivate almost all ß-lactam antibiotics including ampicillin (AMP), which has posed a serious threat to public health. To illuminate the mechanism of recognition and interaction between SMB-1 and AMP, various fluorescence spectroscopy techniques and molecular dynamics simulation were employed. The results of quenching spectroscopy unraveled that AMP could make SMB-1 fluorescence quenching that mechanism was the static quenching; the synchronous and three-dimensional fluorescence spectra validated that the microenvironment and conformation of SMB-1 were altered after interaction with AMP. The molecular dynamics results demonstrated that the whole AMP enters the binding pocket of SMB-1, even though with a relatively bulky R1 side chain. Loop1 and loop2 in SMB-1 undergo significant fluctuations, and α2 (71-73) and local α5 (186-188) were turned into random coils, promoting zinc ion exposure consistent with circular dichroism spectroscopy results. The binding between them was driven by a combination of enthalpy and entropy changes, which was dominated by electrostatic force in agreement with the fluorescence observations. The present study brings structural insights and solid foundations for the design of new substrates for ß-lactamases and the development of effective antibiotics that are resistant to superbugs.
Assuntos
Ampicilina , Simulação de Dinâmica Molecular , Serratia marcescens , Espectrometria de Fluorescência , beta-Lactamases , beta-Lactamases/química , beta-Lactamases/metabolismo , Ampicilina/química , Ampicilina/metabolismo , Ampicilina/farmacologia , Serratia marcescens/enzimologia , Ligação Proteica , Sítios de Ligação , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismoRESUMO
Microbial colonization and development of infections in wounds is a sign of chronicity. The prevailing approach to manage and treat these wounds involves dressings. However, these often fail in effectively addressing infections, as they struggle to both absorb exudates and maintain optimal local moisture. The system here presented was conceptualized with a three-layer design: the outer layer made of a fibrous polycaprolactone (PCL) film, to act as a barrier for preventing microorganisms and impurities from reaching the wound; the intermediate layer formed of a sodium alginate (SA) hydrogel loaded with ampicillin (Amp) for fighting infections; and the inner layer comprised of a fibrous film of PCL and polyethylene glycol (PEG) for facilitating cell recognition and preventing wound adhesion. Thermal evaluations, degradation, wettability and release behavior testing confirmed the system resistance overtime. The sandwich demonstrated the capability for absorbing exudates (≈70 %) and exhibited a controlled release of Amp for up to 24 h. Antimicrobial testing was performed against Staphylococcus aureus and Escherichia coli, as representatives of Gram-positive and Gram-negative bacteria: >99 % elimination of bacteria. Cell cytotoxicity assessments showed high cytocompatibility levels, confirming the safety of the proposed sandwich system. Adhesion assays confirmed the system ease of detaching without mechanical effort (0.37 N). Data established the efficiency of the sandwich-like system, suggesting promising applications in infected wound care.
Assuntos
Alginatos , Antibacterianos , Escherichia coli , Poliésteres , Staphylococcus aureus , Infecção dos Ferimentos , Alginatos/química , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Antibacterianos/química , Antibacterianos/administração & dosagem , Poliésteres/química , Ampicilina/farmacologia , Ampicilina/uso terapêutico , Ampicilina/química , Humanos , Hidrogéis/química , Polietilenoglicóis/química , Animais , Bandagens , Testes de Sensibilidade Microbiana , Camundongos , Cicatrização/efeitos dos fármacosRESUMO
Background: The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries. Methods: This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair. Results: Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31±0.09 mm and 12±0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10. Conclusion: The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach.
Assuntos
Ampicilina , Queimaduras , Colágeno , Escherichia coli , Nanofibras , Álcool de Polivinil , Povidona , Resveratrol , Staphylococcus aureus , Cicatrização , Resveratrol/farmacologia , Resveratrol/química , Resveratrol/administração & dosagem , Resveratrol/farmacocinética , Nanofibras/química , Queimaduras/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Animais , Colágeno/química , Povidona/química , Staphylococcus aureus/efeitos dos fármacos , Álcool de Polivinil/química , Humanos , Escherichia coli/efeitos dos fármacos , Ampicilina/farmacologia , Ampicilina/química , Ampicilina/farmacocinética , Ampicilina/administração & dosagem , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/administração & dosagem , Ratos , Biofilmes/efeitos dos fármacos , MasculinoRESUMO
Monitoring methods for beta-lactam (ß-lactam) antibiotics, especially for ampicillin (AMP), with simple operation and sensitivity for realtime applications are highly required. To address this need, antioxidant carbon dots (E-CDs) with excellent fluorescence properties were synthesized using citric acid and ethylenediamine as raw materials. With a quantum yield of 81.97%, E-CDs exhibited a specific and sensitive response to ËOH. The quenched fluorescence of E-CDs by the formed ËOH could be restored through a competition reaction with AMP. Leveraging the signal-quenching strategy of E-CDs, H2O2, and Fe2+, a fluorescence signal-on strategy was developed using AMP as the fluorescence recovery agent for the sensitive detection of AMP. The mechanism of the quenching of E-CDs by ËOH was attributed to the damaging effect of ËOH on E-CDs. Under optimal conditions, the detection limit of this method for AMP was determined to be 0.38 µg mL-1. This method was successful in drug quality control and the spiked detection of AMP in lake water, milk, and sea cucumber, presenting a viable option for convenient and rapid antibiotic monitoring methods.
Assuntos
Ampicilina , Carbono , Limite de Detecção , Pontos Quânticos , Espectrometria de Fluorescência , Carbono/química , Ampicilina/análise , Ampicilina/química , Pontos Quânticos/química , Espectrometria de Fluorescência/métodos , Animais , Antioxidantes/análise , Antioxidantes/química , Leite/química , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/análise , Radical Hidroxila/química , Radical Hidroxila/análise , Antibacterianos/análise , Antibacterianos/química , Corantes Fluorescentes/química , Ácido Cítrico/química , Fluorescência , EtilenodiaminasRESUMO
Periprosthetic infections caused by Staphylococcus aureus (S. aureus) pose unique challenges in orthopedic surgeries, in part due to the bacterium's capacity to invade surrounding bone tissues besides forming recalcitrant biofilms on implant surfaces. We previously developed prophylactic implant coatings for the on-demand release of vancomycin, triggered by the cleavage of an oligonucleotide (Oligo) linker by micrococcal nuclease (MN) secreted by the Gram-positive bacterium, to eradicate S. aureus surrounding the implant in vitro and in vivo. Building upon this coating platform, here we explore the feasibility of extending the on-demand release to ampicillin, a broad-spectrum aminopenicillin ß-lactam antibiotic that is more effective than vancomycin in killing Gram-negative bacteria that may accompany S. aureus infections. The amino group of ampicillin was successfully conjugated to the carboxyl end of an MN-sensitive Oligo covalently integrated in a polymethacrylate hydrogel coating applied to titanium alloy pins. The resultant Oligo-Ampicillin hydrogel coating released the ß-lactam in the presence of S. aureus and successfully cleared nearby S. aureus in vitro. When the Oligo-Ampicillin-coated pin was delivered to a rat femoral canal inoculated with 1000 cfu S. aureus, it prevented periprosthetic infection with timely on-demand drug release. The clearance of the bacteria from the pin surface as well as surrounding tissue persisted over 3 months, with no local or systemic toxicity observed with the coating. The negatively charged Oligo fragment attached to ampicillin upon cleavage from the coating did diminish the antibiotic's potency against S. aureus and Escherichia coli (E. coli) to varying degrees, likely due to electrostatic repulsion by the anionic surfaces of the bacteria. Although the on-demand release of the ß-lactam led to adequate killing of S. aureus but not E. coli in the presence of a mixture of the bacteria, strong inhibition of the colonization of the remaining E. coli on hydrogel coating was observed. These findings will inspire considerations of alternative broad-spectrum antibiotics, optimized drug conjugation, and Oligo linker engineering for more effective protection against polymicrobial periprosthetic infections.
Assuntos
Ampicilina , Antibacterianos , Materiais Revestidos Biocompatíveis , Infecções Relacionadas à Prótese , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Staphylococcus aureus/efeitos dos fármacos , Ampicilina/química , Ampicilina/farmacologia , Ratos , Antibacterianos/química , Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/tratamento farmacológico , Infecções Relacionadas à Prótese/prevenção & controle , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/microbiologia , Ratos Sprague-Dawley , Testes de Sensibilidade Microbiana , Liberação Controlada de Fármacos , Próteses e ImplantesRESUMO
To develop a sensitive and simple ampicillin (AMP) sensor for trace antibiotic residue detection, the influencing factors of the modification effect of nanogold-functionalized nucleic acid sequences (Adenine: A, Thymine: T) were comprehensively analyzed in this study, including the modification method, base length and type. It was found that under the same base concentration, longer chains are more likely to reach saturation than shorter chains; and when the base concentration and length are both the same, A exhibits a higher saturation modification level compared to T. Based on these research findings, a highly sensitive fluorescence aptamer sensor for detecting ampicillin was constructed using the optimized functionalized sequence (ployA6-aptamer) and experimental conditions (6 hours binding time between nucleic acid aptamer and complementary strand, pH 7 working solution, 20 minutes detection time) based on the principle of fluorescence resonance energy transfer. The sensor has a detection range of 0.18 ng ml-1 to 3.11 ng ml-1 for ampicillin, with a detection limit of 0.04 ng ml-1. It exhibits significant selectivity and achieves an average recovery rate of 98.71% in tap water and 91.83% in milk. This method can be used not only for residual ampicillin detection, but also for highly sensitive detection of various antibiotics and small biological molecules by replacing the aptamer type. It provides a research basis for the design of highly sensitive fluorescence aptamer sensors and further applications of nanogold@DNA composite structures.
Assuntos
Ampicilina , Antibacterianos , Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Limite de Detecção , Leite , Aptâmeros de Nucleotídeos/química , Ampicilina/análise , Ampicilina/química , Antibacterianos/análise , Antibacterianos/química , Leite/química , Técnicas Biossensoriais/métodos , Animais , Transferência Ressonante de Energia de Fluorescência/métodos , Nanopartículas Metálicas/química , Ouro/químicaRESUMO
Early transition metal carbides (MXene) hybridized by precious metals open a door for innovative electrochemical biosensing device design. Herein, we present a facile one-pot synthesis of gold nanoparticles (AuNPs)-doped two-dimensional (2D) titanium carbide MXene nanoflakes (Ti3C2Tx/Au). Ti3C2Tx MXene exhibits high electrical conductivity and yields synergistic signal amplification in conjunction with AuNPs leading to excellent electrochemical performance. Thus Ti3C2Tx/Au hybrid nanostructure can be used as an electrode platform for the electrochemical analysis of various targets. We used screen-printed electrodes modified with the Ti3C2Tx/Au electrode and functionalized with different biorecognition elements to detect and quantify an antibiotic, ampicillin (AMP), and a mycotoxin, fumonisin B1 (FB1). The ultralow limits of detection of 2.284 pM and 1.617 pg.mL-1, which we achieved respectively for AMP and FB1 are far lower than their corresponding maximum residue limits of 2.8 nM in milk and 2 to 4 mg kg-1 in corn products for human consumption set by the United States Food and Drug Administration. Additionally, the linear range of detection and quantification of AMP and FB1 were, respectively, 10 pM to 500 nM and 10 pg mL-1 to 1 µg mL-1. The unique structure and excellent electrochemical performance of Ti3C2Tx/Au nanocomposite suggest that it is highly suitable for anchoring biorecognition entities such as antibodies and oligonucleotides for monitoring various deleterious contaminants in agri-food products.
Assuntos
Ampicilina , Técnicas Eletroquímicas , Fumonisinas , Ouro , Limite de Detecção , Nanopartículas Metálicas , Titânio , Fumonisinas/análise , Ouro/química , Ampicilina/análise , Ampicilina/química , Nanopartículas Metálicas/química , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Titânio/química , Técnicas Biossensoriais/métodos , Leite/química , Antibacterianos/análise , Eletrodos , Contaminação de Alimentos/análise , AnimaisRESUMO
In this work, we present the modification of a medical-grade silicone catheter with the N-vinylimidazole monomer using the grafting-from method at room temperature and induced by gamma rays. The catheters were modified by varying the monomer concentration (20-100 vol%) and the irradiation dose (20-100 kGy). Unlike the pristine material, the grafted poly(N-vinylimidazole) chains provided the catheter with hydrophilicity and pH response. This change allowed for the functionalization of the catheters to endow it with antimicrobial features. Thus, the quaternization of amines with iodomethane and bromoethane was performed, as well as the immobilization of silver and ampicillin. The inhibitory capacity of these materials, functionalized with antimicrobial agents, was challenged against Escherichia coli and Staphylococcus aureus strains, showing variable results, where loaded ampicillin was amply better at eliminating bacteria.
Assuntos
Escherichia coli , Imidazóis , Silicones , Staphylococcus aureus , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Silicones/química , Imidazóis/química , Imidazóis/farmacologia , Catéteres/microbiologia , Testes de Sensibilidade Microbiana , Polivinil/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Ampicilina/química , Ampicilina/farmacologia , Raios gamaRESUMO
The global threat of antibiotic resistance has increased the importance of the detection of antibiotics. Conventional methods to detect antibiotics are time-consuming and require expensive specialized equipment. Here, we present a simple and rapid biosensor for detecting ampicillin, a commonly used antibiotic. Our method is based on the fluorescent properties of chitosan-coated Mn-doped ZnS micromaterials combined with the ß-lactamase enzyme. The biosensors exhibited the highest sensitivity in a linear working range of 13.1-72.2 pM with a limit of detection of 8.24 pM in deionized water. In addition, due to the biological specificity of ß-lactamase, the proposed sensors have demonstrated high selectivity over penicillin, tetracycline, and glucose through the enhancing and quenching effects at wavelengths of 510 nm and 614 nm, respectively. These proposed sensors also showed promising results when tested in various matrices, including tap water, bottled water, and milk. Our work reports for the first time the cost-effective (Mn:ZnS)Chitosan micromaterial was used for ampicillin detection. The results will facilitate the monitoring of antibiotics in clinical and environmental contexts.
Assuntos
Ampicilina , Técnicas Biossensoriais , Quitosana , Manganês , Sulfetos , Compostos de Zinco , Ampicilina/análise , Ampicilina/química , Quitosana/química , Técnicas Biossensoriais/métodos , Compostos de Zinco/química , Manganês/química , Sulfetos/química , Antibacterianos/análise , Antibacterianos/química , beta-Lactamases/análise , beta-Lactamases/metabolismo , beta-Lactamases/química , Leite/química , Limite de Detecção , Espectrometria de Fluorescência/métodos , Corantes Fluorescentes/química , AnimaisRESUMO
Timely and rapid detection of antibiotic residues in the environment is conducive to safeguarding human health and promoting an ecological virtuous cycle. A foldable paper-based photoelectrochemical (PEC) sensor was successfully developed for the detection of ampicillin (AMP) based on glutathione/zirconium dioxide hollow nanorods/aptamer (GSH@ZrO2 HS@apt) modified cellulose paper as a reactive zone with laser direct-writing lead sulfide/cadmium sulfide/graphene (PbS/CdS/LIG) as photoelectrode and cobalt hydroxide (CoOOH) as a photoresist material. Initially, AMP was introduced into the paper-based reaction zone as a biogate aptamer, which specifically recognized the target and then left the ZrO2 HS surface, releasing glutathione (GSH) encapsulated inside. Subsequently, the introduction of GSH into the reaction region and etching of CoOOH nanosheets to expose the PbS/CdS/LIG photosensitive material increased photocurrent. Under optimal conditions, the paper-based PEC biosensor showed a linear response to AMP in the range of 5.0 - 2 × 104 pM with a detection limit of 1.36 pM (S/N = 3). In addition, the constructed PEC sensing platform has excellent selectivity, high stability and favorable reproducibility, and can be used to assess AMP residue levels in various real water samples (milk, tap water, river water), indicating its promising application in environmental antibiotic detection.
Assuntos
Ampicilina , Técnicas Biossensoriais , Compostos de Cádmio , Cobalto , Técnicas Eletroquímicas , Grafite , Chumbo , Papel , Sulfetos , Grafite/química , Sulfetos/química , Técnicas Biossensoriais/métodos , Cobalto/química , Técnicas Eletroquímicas/métodos , Compostos de Cádmio/química , Ampicilina/análise , Ampicilina/química , Chumbo/análise , Chumbo/química , Lasers , Hidróxidos/química , Antibacterianos/análise , Antibacterianos/química , Óxidos/química , Zircônio/química , Processos Fotoquímicos , Limite de Detecção , Aptâmeros de Nucleotídeos/química , Glutationa/química , Glutationa/análise , Animais , Nanoestruturas/químicaRESUMO
In this study, we purified a lectin isolated from the seeds of Dioclea bicolor (DBL) via affinity purification. Electrophoresis analysis revealed that DBL had three bands, α, ß, and γ chains, with molecular masses of approximately 29, 14, and 12 kDa, respectively. Gel filtration chromatography revealed that the native form of DBL had a molecular mass of approximately 100 kDa, indicating that it is a tetramer. Interestingly, DBL-induced hemagglutination was inhibited by several glucosides, mannosides, ampicillin, and tetracycline with minimum inhibitory concentration (MIC) values of 1.56-50 mM. Analysis of the complete amino acid sequence of DBL revealed the presence of 237 amino acids with high similarity to other Diocleinae lectins. Circular dichroism showed the prominent ß-sheet secondary structure of DBL. Furthermore, DBL structure prediction revealed a Discrete Optimized Protein Energy (DOPE) score of -26,642.69141/Normalized DOPE score of -1.84041. The DBL monomer was found to consist a ß-sandwich based on its 3D structure. Molecular docking showed the interactions between DBL and α-D-glucose, N-acetyl-D-glucosamine, α-D-mannose, α-methyl-D-mannoside, ampicillin, and tetracycline. In addition, DBL showed antimicrobial activity with an MIC of 125 µg/mL and exerted synergistic effects in combination with ampicillin and tetracycline (fractional inhibitory concentration index ≤ 0.5). Additionally, DBL significantly inhibited biofilm formation and showed no toxicity in murine fibroblasts (p < 0.05). These results suggest that DBL exhibits antimicrobial activity and works synergistically with antibiotics.
Assuntos
Antibacterianos , Dioclea , Lectinas de Plantas , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Camundongos , Animais , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Lectinas de Plantas/isolamento & purificação , Dioclea/química , Simulação de Acoplamento Molecular , Testes de Sensibilidade Microbiana , Ampicilina/farmacologia , Ampicilina/químicaRESUMO
A new iridium(III) complex was synthesized and characterized. Its photophysical properties and aggregation-induced emission and electrochemiluminescence in the near-infrared range were studied. The large conjugated cyclometallic ligand 1,2-phenylbenzoquinoline (pbq) was selected to form the Ir-C bond with the metal iridium(III) center and provide near-infrared emission of the complex. The auxiliary ligand 4,4'-diamino-2,2'-bipyridine (dabpy) can form hydrogen bonds, which was beneficial for the generation of aggregation-induced emission. The complex was aggregated into small spherical nanoparticles in 80% water and fascinating nanorings in 90% water. The sensing of ampicillin sodium (AMP) antibiotic by the iridium(III) complex were also investigated by photoluminescent and electrochemiluminescent methods. The complex showed a good selectivity toward AMP antibiotic compared to sodium phenylacetate and other eight antibiotics. The detection limits for AMP antibiotic was 0.76 µg/mL. This work provided a new strategy for the design of iridium(III) complex-based aggregation-induced emission and electrochemiluminescence probes for the sensing application.
Assuntos
Medições Luminescentes , Espectroscopia de Luz Próxima ao Infravermelho , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ampicilina/química , Antibacterianos/química , Irídio/química , Medições Luminescentes/métodosRESUMO
Understanding evolution of antibiotic resistance is vital for containing its global spread. Yet our ability to in situ track highly heterogeneous and dynamic evolution is very limited. Here, we present a new single-cell approach integrating D2 O-labeled Raman spectroscopy, advanced multivariate analysis, and genotypic profiling to in situ track physiological evolution trajectory toward resistance. Physiological diversification of individual cells from isogenic population with cyclic ampicillin treatment is captured. Advanced multivariate analysis of spectral changes classifies all individual cells into four subsets of sensitive, intrinsic tolerant, evolved tolerant and resistant. Remarkably, their dynamic shifts with evolution are depicted and spectral markers of each state are identified. Genotypic analysis validates the phenotypic shift and provides insights into the underlying genetic basis. The new platform advances rapid phenotyping resistance evolution and guides evolution control.
Assuntos
Bactérias , Análise Espectral Raman , Análise Espectral Raman/métodos , Ampicilina/farmacologia , Ampicilina/química , Resistência Microbiana a Medicamentos , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
The presence of antibiotics and their metabolites in milk and dairy products is a serious concern because of their harmful effects on human health. In the current study, a novel synergistic bimetallic nanocluster with gold and silver as an emission fluorescence probe was investigated for the simultaneous determination of tetracycline (TC), ampicillin (AMP), and sulfacetamide (SAC) antibiotics in the milk samples using excitation-emission matrix fluorescence (EEMF) spectroscopy. The multivariate curve resolution-alternating least squares (MCR-ALS) method was implemented to analyze augmented EEMF data sets to quantify the multicomponent systems in the presence of interferences with considerable spectral overlap. A pseudo-univariate calibration curve of the resolved emission spectra intensity against the concentration of the mentioned antibiotics was linear in the range of 5-5000 ng mL-1 for AMP and 50-5000 ng mL-1 for TC and SAC. The calculated values of the limit of detection ranged between 1.4 and 14.6 ng mL-1 with a relative standard deviation (RSD) of less than 4.9%. The obtained results show that the EEMF/MCR-ALS methodology using an emission fluorescence probe is a powerful tool for the simultaneous quantification of TC, AMP, and SAC in complex matrices with highly overlapped spectra.
Assuntos
Antibacterianos , Leite , Animais , Humanos , Ampicilina/análise , Ampicilina/química , Corantes Fluorescentes , Análise dos Mínimos Quadrados , Análise Multivariada , Tetraciclina/análise , Tetraciclina/químicaRESUMO
The aim of this study was to investigate the transfer of cephalexin, penicillin-G, and ampicillin & cloxacillin from cow's milk to cheese and whey. For this purpose, raw milk was artificially contaminated to different antibiotic levels and then heat-treated to prepare fresh cheese from it. Antibiotic levels of the milk, whey and cheese were measured with LC-MS/MS. The extent of heat degradation was not sufficient to remove the antibiotic residues from milk. Antibiotic concentrations in whey and fresh cheese were in good accordance with the concentration of the same compound in milk suggesting that contamination of the milk will result in contamination of the product. The investigated antibiotics were transferred less into the cheese curd (1.6-12.5% of the original amount), than into the whey (33.2-74.1%). For penicillin-G even 100% (complete removal) was experienced.
Assuntos
Antibacterianos/análise , Queijo/análise , Contaminação de Alimentos/análise , Leite/química , Soro do Leite/química , beta-Lactamas/análise , Ampicilina/química , Animais , Bovinos , Cefalexina/química , Cromatografia Líquida de Alta Pressão , Cloxacilina/química , Feminino , Humanos , Penicilinas/química , Espectrometria de Massas em TandemRESUMO
Herein, we propose an element probe based CRISPR/Cas14 detection platform and apply it to the detection of non-nucleic-acid targets. Combining metal isotope detection and CRISPR/Cas14 biosensing, the sensitive detection of non-nucleic-acid targets could be realized. We designed and optimized the element probe, which proved that Cas14 has a preference for longer lengths in element probe cleavage. Using this method, the quantitative detection of trace aqueous ampicillin can be achieved within 45 minutes at room temperature (25 °C). A detection limit as low as 2.06 nM is obtained with excellent performance in anti-interference tests and complex matrix detection.
Assuntos
Ampicilina/análise , Antibacterianos/análise , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/química , Ampicilina/química , Antibacterianos/química , Aptâmeros de Nucleotídeos/química , Proteínas Associadas a CRISPR/química , Endodesoxirribonucleases/química , Limite de Detecção , Rios/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/químicaRESUMO
Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Tioléster Hidrolases/química , beta-Lactamases/química , Ampicilina/química , Ampicilina/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Glutationa/análogos & derivados , Glutationa/química , Glutationa/metabolismo , Simulação de Acoplamento Molecular , Filogenia , Conformação Proteica , Stenotrophomonas maltophilia/enzimologiaRESUMO
As an important zoonotic pathogen, Streptococcus suis (S. suis) can cause a variety of diseases both in human and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which commonly appears in severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of host death. Therefore, it is urgent to find a new strategy to relieve the damage caused by STSLS. In this study, we found, for the first time, that apigenin, as a flavonoid compound, could combine with ampicillin to treat severe S. suis infection. Studies found that apigenin did not affect the growth of S. suis and the secretion of suilysin (SLY), but it could significantly inhibit the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. In cell assays, apigenin was found to have no significant toxic effects on effective concentrations, and have a good protective effect on S. suis-infected cells. More importantly, compared with the survival rate of S. suis-infected mice treated with only ampicillin, the survival rate of apigenin combined with an ampicillin-treated group significantly increased to 80%. In conclusion, all results indicate that apigenin in combination with conventional antibiotics can be a potential strategy for treating severe S. suis infection.
Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Apigenina/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Ampicilina/química , Ampicilina/uso terapêutico , Animais , Antibacterianos/química , Apigenina/química , Apigenina/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/química , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/metabolismo , Relação Estrutura-Atividade , Resultado do TratamentoRESUMO
Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria.
Assuntos
Proteínas de Bactérias/química , Neisseria meningitidis/metabolismo , Porinas/química , Ampicilina/química , Ampicilina/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Farmacorresistência Bacteriana/efeitos dos fármacos , Lipossomos/química , Lipossomos/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Permeabilidade/efeitos dos fármacos , Porinas/genética , Porinas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
In this work, we treated chitin with 2-(azidomethyl)oxirane and successfully involved the resultant azido chitin derivatives in the ultrasound-assisted Cu(I)-catalyzed azido-alkyne click (CuAAC) reaction with propargylic ester of N,N,N-trimethyl glycine. Thus, we obtained novel water-soluble triazole chitin derivatives. The triazole chitin derivatives and their nanoparticles are characterized by a high in vitro antibacterial activity, which is the same or even higher than that of commercial antibiotics ampicillin and gentamicin. The obtained derivatives are non-toxic. Moreover, the obtained water-soluble polymers are highly efficient green catalysts for the aldol reaction in green solvent water. The catalysts can be easily extracted from the reaction mixture by its precipitation with green solvent ethanol followed by centrifugation and they can be reused at least 10 times.