RESUMEN
Biological amines and amino acids play essential roles in many biochemical processes. The chemical complexity of biological samples is challenging, and the selective identification and quantification of amines and amino acid stereoisomers would be very useful for amine-focused "amino-omics" studies. Many amines and amino acids are chiral, and their stereoisomers cannot be resolved on achiral media without chiral derivatization. In prior studies, we demonstrated the use of Marfey's reagentâa chiral derivatization reagent for amines and phenolic OH groupsâfor the LC-MS/MS resolution and quantification of amines and amino acid stereoisomers. In this study, a heavy atom isotope labeled Marfey's reagent approach for the stereoselective detection and quantification of amines and amino acids was developed. Heavy (13C2) l-Marfey's (Hl-Mar) and heavy (2H3) d-Marfey's (Hd-Mar) were synthesized from 13C2-l-Ala and 2H3-d-Ala, respectively. Both light and heavy Marfey's reagents were used to derivatize standard amine mixtures, which were analyzed by LC-QToF-HRMS. Aligned peak lists were comparatively analyzed by light vs heavy Mar mass differences to identify mono-, di-, and tri-Marfey's adducts and then by the retention time difference between l- and d-Mar derivatives to identify stereoisomers. This approach was then applied to identify achiral and chiral amine and amino acid components in a methicillin-resistant Staphylococcus aureus (MRSA) extract. This approach shows high analytical selectivity and reproducibility.
Asunto(s)
Aminas , Aminoácidos , Marcaje Isotópico , Espectrometría de Masas en Tándem , Aminas/química , Estereoisomerismo , Espectrometría de Masas en Tándem/métodos , Marcaje Isotópico/métodos , Aminoácidos/química , Aminoácidos/análisis , Isótopos de Carbono/análisis , Isótopos de Carbono/química , Staphylococcus aureus Resistente a Meticilina/química , Alanina/química , Alanina/análogos & derivados , Cromatografía Liquida/métodos , DinitrobencenosRESUMEN
Invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are associated with high mortality and morbidity. The sooner the pathogen is determined, the better it is beneficial to patient. However, routine laboratory inspections are time-consuming and laborious. A thorough research was conducted in PubMed and Web of Science (until June 2021) to identify studies evaluating the accuracy of MRSA identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). STATA 15.0 software was used to analyze the pooled results of sensitivity, specificity, and 95% confidence intervals (CI). The summary receiver operating characteristic curves (SROC) and area under the curve (AUC) were utilized to show the overall performance of MALDI-TOF MS. Fifteen studies involving 2471 isolates were included in this study after the final selection in this meta-analysis. Using the random effects model forest plot to summarize the overall statistics, the sensitivity of MALDI-TOF MS for identifying MRSA was 92% (95% CI: 81%-97%), and the specificity was 97% (95% CI: 89%-99%). In the SROC curve, the AUC reached 0.99 (95% CI: 97%-99%). Deeks' test showed no significant publication bias in this meta-analysis. Compared with clinical reference methods, MALDI-TOF MS identification of MRSA shows a higher degree of sensitivity and specificity.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Humanos , Staphylococcus aureus Resistente a Meticilina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Sensibilidad y Especificidad , Programas Informáticos , Bosques AleatoriosRESUMEN
Tools for noninvasive detection of bacterial pathogens are needed but are not currently available for clinical use. We have previously shown that para-aminobenzoic acid (PABA) rapidly accumulates in a wide range of pathogenic bacteria, motivating the development of related PET radiotracers. In this study, 11C-PABA PET imaging was used to accurately detect and monitor infections due to pyogenic bacteria in multiple clinically relevant animal models. 11C-PABA PET imaging selectively detected infections in muscle, intervertebral discs, and methicillin-resistant Staphylococcus aureus-infected orthopedic implants. In what we believe to be first-in-human studies in healthy participants, 11C-PABA was safe, well-tolerated, and had a favorable biodistribution, with low background activity in the lungs, muscles, and brain. 11C-PABA has the potential for clinical translation to detect and localize a broad range of bacteria.
Asunto(s)
Ácido 4-Aminobenzoico/análisis , Radioisótopos de Carbono/análisis , Staphylococcus aureus Resistente a Meticilina , Tomografía de Emisión de Positrones/métodos , Infecciones Estafilocócicas , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Ácido 4-Aminobenzoico/farmacocinética , Adulto , Animales , Radioisótopos de Carbono/química , Radioisótopos de Carbono/metabolismo , Radioisótopos de Carbono/farmacocinética , Medios de Contraste/análisis , Medios de Contraste/química , Medios de Contraste/metabolismo , Medios de Contraste/farmacocinética , Femenino , Humanos , Masculino , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/metabolismo , Conejos , Ratas , Infecciones Estafilocócicas/diagnóstico por imagen , Infecciones Estafilocócicas/microbiología , Distribución Tisular , Adulto JovenRESUMEN
Methicillin resistant Staphylococcus aureus is considered multidrug resistant bacterium due to developing biofilm formation associated with antimicrobial resistance mechanisms. Therefore, inhibition of biofilm formation is an alternative therapeutic action to control MRSA infections. The present study revealed the non-antibacterial biofilm inhibitory potential of hesperidin against ATCC strain and clinical isolates of S. aureus. Hesperidin is a flavanone glycoside commonly found in citrus fruit. Hesperidin has been shown to exhibits numerous pharmacological activities. The present study aimed to evaluate the antibiofilm and antivirulence potential of hesperidin against MRSA. Results showed that hesperidin treatment significantly impedes lipase, hemolysin, autolysin, autoaggregation and staphyloxanthin production. Reductions of staphyloxanthin production possibly increase the MRSA susceptibility rate to H2O2 oxidative stress condition. In gene expression study revealed that hesperidin treatment downregulated the biofilm-associated gene (sarA), polysaccharide intracellular adhesion gene (icaA and icaD), autolysin (altA), fibronectin-binding protein (fnbA and fnbB) and staphyloxanthin production (crtM). Molecular docking analysis predicted the ability of hesperidin to interact with SarA and CrtM proteins involved in biofilm formation and staphyloxanthin production in MRSA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Farnesil Difosfato Farnesil Transferasa/metabolismo , Hesperidina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/fisiología , Transactivadores/metabolismo , Xantófilas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/química , Farnesil Difosfato Farnesil Transferasa/química , Regulación Bacteriana de la Expresión Génica , Hesperidina/química , Humanos , Staphylococcus aureus Resistente a Meticilina/química , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Transactivadores/química , Virulencia/efectos de los fármacos , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: This study aimed to determine the frequency of methicillin-resistant Staphylococcus aureus (MRSA), antibiotic resistance patterns, superantigenic toxins profile, and clonality of this pathogen in patients with cancer. RESULTS: In total, 79 (25.7%) isolates were confirmed as Staphylococcus species, from which 38 (48.1%) isolates were S. aureus, and 29 (76.3%) isolates were confirmed as MRSA. The highest resistance in MRSA strains was seen against ciprofloxacin (86.2%) and erythromycin (82.8%). Teicoplanin, and linezolid were the most effective antibiotics. From all MRSA isolates, 3 strains (10.3%) were resistant to vancomycin with minimum inhibitory concentration values of 128 µg/ml. The prevalence of superantigenic toxins genes was as follows: pvl (10.5%), tsst-1 (36.8%), etA (23.7%), and etB (23.7%). The t14870 spa type with frequency of 39.5% was the most prevalent clone type circulating in the cancer patients. CONCLUSIONS: This study showed the circulating of spa t14870 as the most predominant MRSA clone in cancer patients of southwest Iran. Also, a diverse antibiotic resistance pattern and toxin profiles were seen among MRSA isolates.
Asunto(s)
Toxinas Bacterianas/genética , Resistencia a la Meticilina , Neoplasias/complicaciones , Infecciones Estafilocócicas/complicaciones , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Humanos , Irán/epidemiología , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Prevalencia , Infecciones Estafilocócicas/epidemiología , Superantígenos/genéticaRESUMEN
Staphylococcus aureus is a leading pathogen responsible for mild to severe invasive infections in humans. Especially, methicillin resistant Staphylococcus aureus (MRSA) is prevalent in hospital and community associated infections. Staphyloxanthin is a golden yellow color eponymous pigment produced by S. aureus and provides resistance to reactive oxygen species (ROS) and host neutrophil-based killing. In addition, this membrane pigment contributes to membrane rigidity and helps MRSA to survive under stress conditions. Targeting virulence of pathogen without exerting selection pressure is the recent approach to fight bacterial infections without developing drug resistance. The present study for the first time evaluated the staphyloxanthin inhibitory potential of thymol against MRSA. Qualitative and quantitative analyses demonstrated 90% of staphyloxanthin inhibition at 100 µg/mL concentration of thymol without alteration in growth. Molecular docking analysis and in vitro measurement of metabolic intermediates of staphyloxanthin revealed that thymol could possibly interact with CrtM to inhibit staphyloxanthin. Absorbance and infra red spectra further validated the inhibition of staphyloxanthin by thymol. In addition, thymol treatment significantly reduced the resistance of MRSA to ROS and neutrophil-based killing as exhibited by oxidant susceptibility assays and ex vivo innate immune clearance assay using human whole blood and neutrophils. Further, reduction in staphyloxanthin by thymol treatment increased the membrane fluidity and made MRSA cells more susceptible to membrane targeting antibiotic polymyxin B. Especially, thymol was found to be non-cytotoxic to human peripheral blood mononuclear cells. Our study validated the antivirulence potential of thymol against MRSA by inhibiting staphyloxanthin and suggests the prospective therapeutic role of thymol to combat MRSA infections.
Asunto(s)
Antioxidantes/farmacología , Fluidez de la Membrana/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Neutrófilos/metabolismo , Timol/farmacología , Xantófilas/farmacología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Fluidez de la Membrana/fisiología , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Simulación del Acoplamiento Molecular/métodos , Neutrófilos/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
Staphylococcus aureus is a serious human and animal pathogen threat exhibiting extraordinary capacity for acquiring new antibiotic resistance traits in the pathogen population worldwide. The development of fast, affordable and effective diagnostic solutions capable of discriminating between antibiotic-resistant and susceptible S. aureus strains would be of huge benefit for effective disease detection and treatment. Here we develop a diagnostics solution that uses Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry (MALDI-TOF) and machine learning, to identify signature profiles of antibiotic resistance to either multidrug or benzylpenicillin in S. aureus isolates. Using ten different supervised learning techniques, we have analysed a set of 82 S. aureus isolates collected from 67 cows diagnosed with bovine mastitis across 24 farms. For the multidrug phenotyping analysis, LDA, linear SVM, RBF SVM, logistic regression, naïve Bayes, MLP neural network and QDA had Cohen's kappa values over 85.00%. For the benzylpenicillin phenotyping analysis, RBF SVM, MLP neural network, naïve Bayes, logistic regression, linear SVM, QDA, LDA, and random forests had Cohen's kappa values over 85.00%. For the benzylpenicillin the diagnostic systems achieved up to (mean result ± standard deviation over 30 runs on the test set): accuracy = 97.54% ± 1.91%, sensitivity = 99.93% ± 0.25%, specificity = 95.04% ± 3.83%, and Cohen's kappa = 95.04% ± 3.83%. Moreover, the diagnostic platform complemented by a protein-protein network and 3D structural protein information framework allowed the identification of five molecular determinants underlying the susceptible and resistant profiles. Four proteins were able to classify multidrug-resistant and susceptible strains with 96.81% ± 0.43% accuracy. Five proteins, including the previous four, were able to classify benzylpenicillin resistant and susceptible strains with 97.54% ± 1.91% accuracy. Our approach may open up new avenues for the development of a fast, affordable and effective day-to-day diagnostic solution, which would offer new opportunities for targeting resistant bacteria.
Asunto(s)
Diagnóstico por Computador/veterinaria , Mastitis Bovina/diagnóstico , Penicilina G/farmacología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus , Animales , Proteínas Bacterianas/química , Bovinos , Biología Computacional , Diagnóstico por Computador/métodos , Diagnóstico por Computador/estadística & datos numéricos , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Mastitis Bovina/tratamiento farmacológico , Mastitis Bovina/microbiología , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mapas de Interacción de Proteínas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/aislamiento & purificación , Aprendizaje Automático Supervisado , Reino UnidoRESUMEN
In S. aureus biofilms, bacteria are embedded in a matrix of extracellular polymeric substances (EPS) and are highly tolerant to antimicrobial drugs. We thus sought to identify non-antibiotic substances with broad-spectrum activity able to destroy the EPS matrix and enhance the effect of antibiotics on embedded biofilm bacteria. Among eight substances tested, subtilisin A (0.01 U/mL) and calcium gluconate (CaG, Ca2+ 1.25 mmol/L) significantly reduced the biomass of biofilms formed by at least 21/24 S. aureus isolates. Confocal laser scanning microscopy confirmed that they both eliminated nearly all the proteins and PNAG from the matrix. By contrast, antibiotics alone had nearly no effect on biofilm biomass and the selected one (oxytetracycline-OTC) could only slightly reduce biofilm bacteria. The combination of OTC with CaG or subtilisin A led to an additive reduction (average of 2 log10 CFU/mL) of embedded biofilm bacteria on the isolates susceptible to OTC (MBC < 10 µg/mL, 11/24). Moreover, these two combinations led to a reduction of the embedded biofilm bacteria higher than 3 log10 CFU/mL for 20-25% of the isolates. Further studies are now required to better understand the factors that cause the biofilm produced by specific isolates (20-25%) to be susceptible to the combinations.
Asunto(s)
Antibacterianos/farmacología , Gluconato de Calcio/farmacología , Matriz Extracelular de Sustancias Poliméricas/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Polisacáridos Bacterianos/antagonistas & inhibidores , Subtilisinas/farmacología , Aminoglicósidos/farmacología , Combinación de Medicamentos , Sinergismo Farmacológico , Matriz Extracelular de Sustancias Poliméricas/química , Fluoroquinolonas/farmacología , Glicopéptidos/farmacología , Humanos , Macrólidos/farmacología , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Polisacáridos Bacterianos/química , Infecciones Estafilocócicas/microbiología , beta-Lactamas/farmacologíaRESUMEN
The detection of Staphylococcus aureus specific gene in combination with the mecA gene is vitally important for accurate identification of methicillin-resistant Staphylococcus aureus (MRSA). A homogeneous electrochemical DNA sensor was fabricated for simultaneous detection of mecA and nuc gene in MRSA. Metal-organic framework (type UiO-66-NH2) was applied as nanocarrier. Two electroactive dyes, methylene blue (MB) and epirubicin (EP), were encapsulated in UiO-66-NH2, respectively, and were locked by the hybrid double-stranded DNA. Based on the target-response electroactive dye release strategy, once target DNA exists, it completely hybridizes with displacement DNA (DEP and DMB). So DEP and DMB is displaced from the MOF surface, causing the release of electroactive dyes. Co-Zn bimetallic zeolitic imidazolate framework-derived N-doped porous carbon serves for electrode modification to improve electrocatalytic performance and sensitivity. The differential pulse voltammetry peak currents of MB and EP were accurately detected at - 0.14 V and - 0.53 V versus the Ag/AgCl reference electrode, respectively. Under the optimal conditions, the detection limits of mecA gene and nuc gene were 3.7 fM and 1.6 fM, respectively. Combining the effective application of MOFs and the homogeneous detection strategy, the sensor exhibited satisfactory performance for MRSA identification in real samples. The recovery was 92.6-103%, and the relative standard deviation was less than 5%. Besides, MRSA and SA can also be distinguished. This sensor has great potential in practical applications.
Asunto(s)
Carbono/química , ADN Bacteriano/análisis , Técnicas Electroquímicas/métodos , Ácidos Nucleicos Inmovilizados/química , Estructuras Metalorgánicas/química , Staphylococcus aureus Resistente a Meticilina/química , Animales , Proteínas Bacterianas/genética , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Colorantes/química , ADN Bacteriano/química , ADN Bacteriano/genética , Agua Potable/análisis , Agua Potable/microbiología , Técnicas Electroquímicas/instrumentación , Electrodos , Epirrubicina/química , Ácidos Nucleicos Inmovilizados/genética , Límite de Detección , Azul de Metileno/química , Nucleasa Microcócica/genética , Leche/microbiología , Hibridación de Ácido Nucleico , Compuestos Organometálicos/química , Proteínas de Unión a las Penicilinas/genética , Ácidos Ftálicos/química , Reproducibilidad de los ResultadosRESUMEN
RATIONALE: Methicillin-resistant Staphylococcus aureus (MRSA) is one of major clinical pathogens responsible for both hospital- and community-acquired infections worldwide. A delay in targeted antibiotic treatment contributes to longer hospitalization stay, higher costs, and increasing in-hospital mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been integrated into the routine workflow for microbial identification over the past decade, and it has also shown promising functions in the detection of bacterial resistance. Therefore, we describe a rapid MALDI-TOF MS-based methodology for MRSA screening with machine-learning algorithms. METHODS: A total of 452 clinical S. aureus isolates were included in this study, of which 194 were MRSA and 258 were methicillin-sensitive S. aureus (MSSA). The mass-to-charge ratio (m/z) features from MRSA and MSSA strains were binned and selected through Lasso regression. These features were then used to train a non-linear support vector machine (SVM) with radial basis function (RBF) kernels to evaluate the discrimination performance. The classifiers' accuracy, sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC) were evaluated and compared with those from the random forest (RF) model. RESULTS: A total of 2601 unique spectral peaks of all isolates were identified and 38 m/z features were selected for the classifying model. The AUCs of the non-linear RBF-SVM model and the RF model were 0.89 and 0.87, respectively, and the accuracy ranged between 0.86 (RBF-SVM) and 0.82 (RF). CONCLUSIONS: Our study demonstrates that MALDI-TOF MS coupled with machine-learning algorithms could be used to develop a rapid and easy-to-use method to discriminate MRSA from MSSA. Considering that this method is easy to implement in routine microbiology laboratories, it suggests a cost-effective and time-efficient alternative to conventional resistance detection in the future to improve clinical treatment.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Tipificación Molecular/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Infecciones Estafilocócicas/microbiología , Algoritmos , Humanos , Aprendizaje Automático , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/clasificación , Sensibilidad y Especificidad , Staphylococcus aureus/química , Staphylococcus aureus/clasificaciónRESUMEN
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) threatens human health. A local CA-MRSA with ST8/SCCmecIVl (CA-MRSA/J) has emerged in Japan, being associated with progression from bullous impetigo to potentially fatal invasive infection. We found that CA-MRSA/J has unique bacterial surface structures, spikes, spikes with a cap, and long spikes, reflecting clinical origins.
Asunto(s)
Infecciones Comunitarias Adquiridas/microbiología , Staphylococcus aureus Resistente a Meticilina/química , Humanos , Japón , Leucocidinas/genética , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/ultraestructura , Microscopía Electrónica , Infecciones Estafilocócicas/microbiologíaRESUMEN
The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.
Asunto(s)
Biopelículas , Enterococcus faecium/química , Listeria monocytogenes/química , Staphylococcus aureus Resistente a Meticilina/química , Salmonella enterica/química , Enterococcus faecium/fisiología , Listeria monocytogenes/fisiología , Staphylococcus aureus Resistente a Meticilina/fisiología , Microscopía Confocal , Salmonella enterica/fisiologíaRESUMEN
Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.
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Teléfono Celular , ADN Bacteriano/análisis , Fluorescencia , Staphylococcus aureus Resistente a Meticilina/química , Técnicas de Amplificación de Ácido Nucleico , Imagen Óptica , Tamaño de la Partícula , Porosidad , Propiedades de Superficie , Factores de TiempoRESUMEN
A series of homoleptic and heteroleptic bismuth(III) flavonolate complexes derived from six flavonols of varying substitution have been synthesised and structurally characterised. The complexes were evaluated for antibacterial activity towards several problematic Gram-positive (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE)) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. The cell viability of COS-7 (monkey kidney) cells treated with the bismuth flavonolates was also studied to determine the effect of the complexes on mammalian cells. The heteroleptic complexes [BiPh(L)2 ] (in which L=flavonolate) showed good antibacterial activity towards all of the bacteria but reduced COS-7 cell viability in a concentration-dependent manner. The homoleptic complexes [Bi(L)3 ] exhibited activity towards the Gram-positive bacteria and showed low toxicity towards the mammalian cell line. Bismuth uptake studies in VRE and COS-7 cells treated with the bismuth flavonolate complexes indicated that Bi accumulation is influenced by both the substitution of the flavonolate ligands and the degree of substitution at the bismuth centre.
Asunto(s)
Antibacterianos/farmacología , Bismuto/química , Complejos de Coordinación/química , Escherichia coli/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/química , Escherichia coli/química , Bacterias Grampositivas/química , Humanos , Staphylococcus aureus Resistente a Meticilina/química , Pseudomonas aeruginosa/química , Staphylococcus aureus/químicaRESUMEN
The wall teichoic acid (WTA) is a major cell wall component of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), a common cause of fatal clinical infections in humans. Thus, the indispensable ABC transporter TarGH, which flips WTA from cytoplasm to extracellular space, becomes a promising target of anti-MRSA drugs. Here, we report the 3.9-Å cryo-electron microscopy (cryo-EM) structure of a 50% sequence-identical homolog of TarGH from Alicyclobacillus herbarius at an ATP-free and inward-facing conformation. Structural analysis combined with activity assays enables us to clearly decode the binding site and inhibitory mechanism of the anti-MRSA inhibitor Targocil, which targets TarGH. Moreover, we propose a "crankshaft conrod" mechanism utilized by TarGH, which can be applied to similar ABC transporters that translocate a rather big substrate through relatively subtle conformational changes. These findings provide a structural basis for the rational design and optimization of antibiotics against MRSA.IMPORTANCE The wall teichoic acid (WTA) is a major component of cell wall and a pathogenic factor in methicillin-resistant Staphylococcus aureus (MRSA). The ABC transporter TarGH is indispensable for flipping WTA precursor from cytoplasm to the extracellular space, thus making it a promising drug target for anti-MRSA agents. The 3.9-Å cryo-EM structure of a TarGH homolog helps us to decode the binding site and inhibitory mechanism of a recently reported inhibitor, Targocil, and provides a structural platform for rational design and optimization of potential antibiotics. Moreover, we propose a "crankshaft conrod" mechanism to explain how a big substrate is translocated through subtle conformational changes of type II exporters. These findings advance our understanding of anti-MRSA drug design and ABC transporters.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/ultraestructura , Pared Celular/química , Microscopía por Crioelectrón , Ácidos Teicoicos/química , Alicyclobacillus/química , Alicyclobacillus/genética , Antibacterianos/farmacología , Pared Celular/ultraestructura , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Unión ProteicaRESUMEN
Overexpression of phosphopantetheinyl transferase (PPtase)-encoding genes sfp and svp in the marine-derived Verrucosispora sp. SCSIO 40062 led to the production of two new aminofuran monomers, proximicin F (1) and proximicin G (3) and a new dimer diproximicin A (2), along with two known compounds, proximicins B (4) and C (5). Their structures were unambiguously elucidated on the basis of detailed NMR spectroscopic analysis and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Proximicin B (4) showed moderate antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus, and Bacillus subtilis.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Micromonosporaceae/química , Netropsina/análogos & derivados , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Netropsina/química , Netropsina/farmacología , Espectrometría de Masa por Ionización de Electrospray , Staphylococcus aureus/químicaRESUMEN
Differentiating bacteria strains using biophysical forces has been the focus of recent studies using dielectrophoresis (DEP). The refinement of these studies has created high-resolution separations such that very subtle properties of the cells are enough to induce significant differences in measurable biophysical properties. These high-resolution capabilities build upon the advantages of DEP which include small sample sizes and fast analysis times. Studies focusing on differentiating antimicrobial resistant and susceptible bacteria potentially have significant impact on human health and medical care. A prime example is Staphylococcus aureus, which commonly colonizes adults without ill effects. However, the pathogen is an important cause of infections, including surgical site infections. Treatment of S. aureus infections is generally possible with antimicrobials, but antimicrobial resistance has emerged. Of special importance is resistance to methicillin, an antimicrobial created in response to resistance to penicillin. Here, dielectrophoresis is used to study methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA) strains, both with and without the addition of a fluorescent label. The capture onset potential of fluorescently-labeled MRSA (865 ± 71 V) and thus the ratio of electrokinetic to dielectrophoretic mobility, was found to be higher than that of fluorescently-labeled MSSA (685 ± 61 V). This may be attributable to the PBP2a enzyme present in the MRSA strain and not in the MSSA bacteria. Further, unlabeled MRSA was found to have a capture onset potential of 732 ± 44 V, while unlabeled MSSA was found to have a capture onset potential of 562 ± 59 V. This shows that the fluorescently-labeled bacteria require a higher applied potential, and thus ratio of mobilities, to capture than the unlabeled bacteria.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina/clasificación , Separación Celular/métodos , Técnicas Electroquímicas/métodos , Colorantes Fluorescentes/química , Staphylococcus aureus Resistente a Meticilina/química , Rodaminas/químicaRESUMEN
New antibiotics and innovative approaches to kill drug-resistant bacteria are urgently needed. Metal complexes offer access to alternative modes of action but have only sparingly been investigated in antibacterial drug discovery. We have developed a light-activated rhenium complex with activity against drug-resistant S. aureus and E. coli. The activity profile against mutant strains combined with assessments of cellular uptake and synergy suggest two distinct modes of action.
Asunto(s)
Antibacterianos/farmacología , Complejos de Coordinación/química , Escherichia coli/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Renio/química , Antibacterianos/química , Complejos de Coordinación/farmacología , Escherichia coli/química , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus/efectos de los fármacosRESUMEN
Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.
Asunto(s)
Antibacterianos/uso terapéutico , Bacterias/clasificación , Infecciones Bacterianas/diagnóstico , Aprendizaje Profundo , Espectrometría Raman/métodos , Bacterias/química , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiología , Técnicas de Tipificación Bacteriana , Candida/química , Candida/clasificación , Enterococcus/química , Enterococcus/clasificación , Escherichia coli/química , Escherichia coli/clasificación , Humanos , Klebsiella/química , Klebsiella/clasificación , Modelos Logísticos , Staphylococcus aureus Resistente a Meticilina/química , Staphylococcus aureus Resistente a Meticilina/clasificación , Pruebas de Sensibilidad Microbiana , Redes Neurales de la Computación , Análisis de Componente Principal , Proteus mirabilis/química , Proteus mirabilis/clasificación , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/clasificación , Salmonella enterica/química , Salmonella enterica/clasificación , Análisis de la Célula Individual , Staphylococcus aureus/química , Staphylococcus aureus/clasificación , Streptococcus/química , Streptococcus/clasificación , Máquina de Vectores de SoporteRESUMEN
The objective of the present study was to reveal the antibacterial mechanism of lactobionic acid (LBA) against methicillin-resistant Staphylococcus aureus (MRSA) using quantitative proteomics by sequential window acquisition of all theoretical mass spectra (SWATH-MS) to analyze 100 differentially expressed proteins after LBA treatment. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis including reactive oxygen species (ROS), virulence-associated gene expression, and the relative quantification of target proteins and genes by parallel reaction monitoring and quantitative real-time PCR. Combining the ultrastructure observations, proteomic analysis, and our previous research, the mode of LBA action against MRSA was speculated as cell wall damage and loss of membrane integrity; inhibition of DNA repair and protein synthesis; inhibition of virulence factors and biofilm production; induction of oxidative stress; and inhibition of metabolic pathways. These results suggest potential applications for LBA in food safety and pharmaceuticals, considering its multitarget effects against MRSA.