Comparative Metabolomics Reveals Changes in the Metabolic Pathways of Ampicillin- and Gentamicin-Resistant Staphylococcus aureus.

Antibiotic resistance is a major global challenge requiring new treatments and a better understanding of the bacterial resistance mechanisms. In this study, we compared ampicillin-resistant (R-AMP) and gentamicin-resistant (R-GEN) Staphylococcus aureus strains with a sensitive strain (ATCC6538) using metabolomics. We identified 109 metabolites; 28 or 31 metabolites in R-AMP or R-GEN differed from those in ATCC6538. Moreover, R-AMP and R-GEN were enriched in five and four pathways, respectively. R-AMP showed significantly up-regulated amino acid metabolism and down-regulated energy metabolism, whereas R-GEN exhibited an overall decrease in metabolism, including carbohydrate, energy, and amino acid metabolism. Furthermore, the activities of the metabolism-related enzymes pyruvate dehydrogenase and TCA cycle dehydrogenases were inhibited in antibiotic-resistant bacteria. Significant decreases in NADH and ATP levels were also observed. In addition, the arginine biosynthesis pathway, which is related to nitric oxide (NO) production, was enriched in both antibiotic-resistant strains. Enhanced NO synthase activity in S. aureus promoted NO production, which further reduced reactive oxygen species, mediating the development of bacterial resistance to ampicillin and gentamicin. This study reveals that bacterial resistance affects metabolic profile, and changes in energy metabolism and arginine biosynthesis are important factors leading to drug resistance in S. aureus.

Machine Learning-Assisted Portable Microplasma Optical Emission Spectrometer for Food Safety Monitoring.

To meet the needs of food safety for simple, rapid, and low-cost analytical methods, a portable device based on a point discharge microplasma optical emission spectrometer (µPD-OES) was combined with machine learning to enable on-site food freshness evaluation and detection of adulteration. The device was integrated with two modular injection units (i.e., headspace solid-phase microextraction and headspace purge) for the examination of various samples. Aromas from meat and coffee were first introduced to the portable device. The aroma molecules were excited to specific atomic and molecular fragments at excited states by room temperature and atmospheric pressure microplasma due to their different atoms and molecular structures. Subsequently, different aromatic molecules obtained their own specific molecular and atomic emission spectra. With the help of machine learning, the portable device was successfully applied to the assessment of meat freshness with accuracies of 96.0, 98.7, and 94.7% for beef, pork, and chicken meat, respectively, through optical emission patterns of the aroma at different storage times. Furthermore, the developed procedures can identify beef samples containing different amounts of duck meat with an accuracy of 99.5% and classify two coffee species without errors, demonstrating the great potential of their application in the discrimination of food adulteration. The combination of machine learning and µPD-OES provides a simple, portable, and cost-effective strategy for food aroma analysis, potentially addressing field monitoring of food safety.

Metabolomics analysis of the lactobacillus plantarum ATCC 14917 response to antibiotic stress.

BACKGROUND: Lactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites. RESULTS: Using metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP. CONCLUSIONS: The metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment.

Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance.

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.

Solid Phase Photothermo-Induced Chemical Vapor Generation: A New Desorption Method for Mercury Analysis by High-Throughput 20-Fiber Direct Immersion Solid Phase Microextraction.

A simple solid phase photothermo-induced chemical vapor generation (SP-PT-CVG) is described and used as an environmentally friendly desorption method for the sensitive determination of mercury in water by direct immersion solid phase microextraction (DI-SPME) atomic fluorescence spectrometry (AFS). A DI-SPME array equipped with 20 nano-TiO2-coated tungsten fibers was employed to simultaneously preconcentrate mercury from 20 samples, enabling an extraction throughput of 40 samples per hour. Subsequently, the fibers were drawn from the sample solutions and inserted into an inner tube sealed in a specially designed UV lamp in turn for SP-PT-CVG to generate Hg0, which was swept to an AFS detector for its detection. It is worth noting that the tube served as both a vapor generator and a desorption chamber. This proof-of-concept study confirms the feasibility of solid phase CVG. Compared to conventional CVG carried out in the liquid phase, solid phase CVG not only retains the advantages of conventional CVG but also alleviates the matrix interference on vapor generation and preconcentrates analyte prior to vapor generation, improving analytical performance for liquid state samples. DI-SPME-SP-PT-CVG-AFS provides a limit of detection of 2.3 ng L-1 for mercury determination by AFS. In the proposed method, the combination of DI-SPME and SP-PT-CVG eliminates the tedious derivatization steps required in conventional headspace SPME, thus minimizing toxic reagent consumption and improving extraction throughput. The practicality of DI-SPME-SP-PT-CVG-AFS was evaluated by analyzing two different certified reference materials and river water samples with good spike recoveries (98-107%).

Automatic and Matrix Interference-Free Acid-Base Titration by Coupling CO2 Vapor Generation with Microplasma Carbon Optical Emission Spectrometry.

Acid-base titration of complex samples is conducive to the rapid evaluation of the degree and risk of environmental pollution to some extent. However, the traditional titration methods usually suffer from serious interference. Herein, an automatic acid-base titration method coupling miniature point discharge optical emission spectrometry (µPD-OES) with CO2 vapor generation was described for the precise, sensitive, and matrix interference-free acid-base titration of complex samples, particularly those with high color intensity, salinity, and turbidity such as wastewater and soil samples. In this work, acid-base titration was carried out in a chemical vapor generator where CO2 was generated through the addition of HCl or NaHCO3, thus enabling efficient separation of CO2 from a complex matrix. The generated CO2 was subsequently swept into the miniaturized point discharge for excitation and further detection by µPD-OES, where the carbon atomic emission at 193.0 nm was monitored. According to the consumed volume and concentration of HCl, accurate and automatic measurements of OH-, CO32-, and HCO3- can be accomplished. The proposed method possesses a high sensitivity of µPD-OES for the detection of CO2 with a relative standard deviation of below 3.0%. Moreover, the proposed system not only retains several unique advantages of accuracy, simplicity, and elimination of the use of complicated, expensive, and high power-consumption instruments but also alleviates the color and turbid interference from complex samples such as dyeing wastewater samples, oilfield water samples, and soil samples. It retains a promising potential application for titration analysis of other samples such as sludge, sediment, and landfill leachate.

Nozzle Electrode Point Discharge-Enhanced Oxidation and Portable Gas Phase Molecular Fluorescence Spectrometry for Sensitive Field Detection of Sulfide.

It is still a challenge to accurately determine dissolved sulfide due to its susceptibility to contamination and loss during transportation, storage, and analysis in the laboratory, thus highlighting the necessity for its sensitive field analysis. Herein, a robust nozzle electrode point discharge (NEPD) enhanced oxidation coupling with chemical vapor generation (CVG) is described for the highly efficient and flameless conversion of sulfide (S2-) to SO2. Subsequently, a portable and low-power consumption gas phase molecular fluorescence spectrometry (GP-MFS) was constructed for the highly selective and sensitive determination of the generated SO2 via detecting its molecular fluorescence excited by a zinc hollow cathode lamp. Under optimal conditions, a limit of detection (LOD) of 0.1 µM was obtained for dissolved sulfide with a relative standard deviation (RSD, n = 11) of 2.6%. The accuracy and practicability of the proposed method were validated by the analyses of two certified reference materials (CRMs) and several river and lake water samples with satisfactory recoveries of 99%-107%. This work confirms that NEPD enhanced oxidation is a low energy consumption yet highly efficient method for the flameless oxidation of hydrogen sulfide and thus is suitable for the easy field detection of dissolved sulfide in environmental water by CVG-GP-MFS.

Gentamicin resistance to Escherichia coli related to fatty acid metabolism based on transcriptome analysis.

Antibiotic overuse and misuse have promoted the emergence and spread of antibiotic-resistant bacteria. Increasing bacterial resistance to antibiotics is a major healthcare problem, necessitating elucidation of antibiotic resistance mechanisms. In this study, we explored the mechanism of gentamicin resistance by comparing the transcriptomes of antibiotic-sensitive and -resistant Escherichia coli. A total of 410 differentially expressed genes were identified, of which 233 (56.83%) were up-regulated and 177 (43.17%) were down-regulated in the resistant strain compared with the sensitive strain. Gene Ontology (GO) analysis classifies differential gene expression into three main categories: biological processes, cellular components, and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the up-regulated genes were enriched in eight metabolic pathways, including fatty acid metabolism, which suggests that fatty acid metabolism may be involved in the development of gentamicin resistance in E. coli. This was demonstrated by measuring the acetyl-CoA carboxylase activity, plays a fundamental role in fatty acid metabolism, was increased in gentamicin-resistant E. coli. Treatment of fatty acid synthesis inhibitor, triclosan, promoted gentamicin-mediated killing efficacy to antibiotic-resistant bacteria. We also found that exogenous addition of oleic acid, which involved in fatty acid metabolism, reduced E. coli sensitivity to gentamicin. Overall, our results provide insight into the molecular mechanism of gentamicin resistance development in E. coli.

Sensitive and Environmentally Friendly Field Analysis of Waterborne Arsenic by Electrochemical Hydride Generation Microplasma Optical Emission Spectrometry.

To avoid polluting the environment, it is desirable to develop methods consuming as few chemicals as possible for field elemental analysis. In this work, a lithium-ion battery supplied, compact handheld optical emission spectrometer (OES) (0.3 kg, length 18 cm × width 5 cm × height 10 cm) was fabricated for the sensitive field analysis of waterborne arsenic by utilizing electrochemical hydride generation (ECHG) and miniaturized ballpoint discharge (µPD) as sample introduction means and excitation source, respectively. The high ECHG efficiency of arsenic was obtained using a superior cathode of Fe@PbO/Pb and the generated arsine was separated from an aqueous phase and further swept to the µPD microplasma for detection. It is worth noting that the Fe@PbO/Pb cathode not only retains advantages of large specific surface area, robust stability, and excellent reproducibility for the ECHG of arsenic but also accomplishes the preconcentration of As(III), thus improving the kinetics of the surface chemistry at the cathode, alleviating the corrosion of the electrode, and minimizing the release of Pb. A limit of detection of 1.0 µg L-1 was obtained with a relative standard deviation of 4.2% for 20 µg L-1 As(III). Owing to the advantages of ECHG and µPD-OES, the system retains a promising potential for the sensitive, cost-effective, and environmentally friendly field analysis of waterborne arsenic.

Learning from Headspace Sampling: a Versatile High-Throughput Reactor for Photochemical Vapor Generation.

Inspired by the headspace sampling (HS) device, a versatile high-throughput photochemical reactor with twenty vessels serving as both the photochemical vapor generator and the HS device was developed for the rapid and sensitive determination of mercury, nickel, and selenium by coupling photochemical vapor generation (PVG) to atomic fluorescence spectrometry (AFS) or point discharge optical emission spectrometry (µPD-OES). The photochemical reactor utilized a specially designed annular UV lamp around which the vessels containing sample solution were automatically rotated and irradiated to yield gaseous analyte species. Subsequently, the species escaped into the headspace of vessels prior to introduction to the atomic spectrometer. Compared with the conventional flow injection (FI) or continuous flow (CF) PVG, the developed PVG-HS method possesses several unique advantages including high throughput (260 pcs h-1), high sensitivity, and the elimination of matrix interference from transition metal ions and the memory effect associated with the quantification of mercury. Limits of detection (LODs) of 0.002, 0.007, and 0.01 µg L-1 were obtained for Hg (II), Ni (II), and Se (IV) by PVG-HS-AFS, respectively, and 0.02 and 0.2 µg L-1 were obtained for Hg (II) and Ni (II) by PVG-HS-µPD-OES, respectively. The practicality of the reactor was evaluated by the detection of Hg (II), Ni (II), and Se (IV) in five certified reference materials, including water (GBW08603, GBW08607, and GBW(E)080395), National Research Council Canada dogfish liver (DOLT-5), fish protein (DORM-4), and three river water samples with good recoveries (92-106%).

Promoting effect of Fe3+ on gentamicin resistance in Escherichia coli.

Kinds of antibiotics are used to prevent and control bacteria infections, unfortunately, the overuse and misuse of antibiotic have promoted the emergence and spread of antibiotic-resistant bacteria. Therefore, understanding the mechanism of antibiotic resistance is very important. This study explores the combined effection of metal ions and antibiotic to the drug resistance of Escherichia coli. Our results found that the minimum inhibitory concentration (MIC) increased as the ammonium ferric citrate concentration increased, especially for gentamicin antibiotic. When the Fe3+ concentration reached 300 µM, the survival of E. coli was stably restored with the increased gentamicin concentration. Exogenous Fe3+ could decrease intracellular gentamicin concentration. On the other hand, Fe3+ treatment together with gentamicin could reduce reactive oxygen species (ROS) production, characterized by decreased levels of NADH and ATP. Furthermore, ROS-scavenging enzymes of superoxide dismutase (SOD) and catalase (CAT) were up-regulated and H2O2 plus gentamicin-mediated killing was restored by Fe3+. These results may have significant implications in understanding bacterial antibiotic-resistant mechanisms based on the external Fe3+ concentration.

The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis.

BACKGROUND: WRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata. RESULTS: In this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five ß-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants. CONCLUSION: These results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

Metabolites in the TCA Cycle Promote Resistance to Chloramphenicol of Edwardsiella tarda.

Antibiotic-resistant bacteria are a serious threat to human and animal health. Metabolite-enabled eradication of drug-resistant pathogens is an attractive strategy, and metabolite adjuvants, such as fumarate, are used for restoring the bactericidal ability of antibiotics. However, we show that metabolites in the TCA cycle increase the viability of Edwardsiella tarda against chloramphenicol (CAP), based on the survival assay of differential metabolites identified by LC-MS/MS. Furthermore, NADPH promotes CAP resistance in the CAP-resistant strain, while oxidants restore the bactericidal ability. Finally, we show that the intracellular redox state determines the sensitivity to CAP, and the total antioxidative capacity is decreased significantly in the antibiotic-resistant strain. Considering that the metabolites promote CAP resistance, metabolite adjuvants should be applied very cautiously. Overall, our research expands on the knowledge that the redox state is related to the bactericidal ability of CAP.

Methanol-Enhanced Liquid Electrode Discharge Microplasma-Induced Vapor Generation of Hg, Cd, and Zn: The Possible Mechanism and Its Application.

Despite increased interest in microplasma-induced vapor generation (µPIVG) over the past several years, applications in real sample analyses remain limited due to their relatively low vapor generation efficiency and ambiguous mechanism. In this work, a novel method using methanol for significantly enhancing the liquid electrode discharge µPIVG efficiency was developed for the simultaneous and sensitive determination of Hg, Cd, and Zn by atomic fluorescence spectrometry (AFS). It is worth noting that the possible enhancement mechanism was investigated via the characterizations of volatile products by AFS, microplasma optical emission spectrometry, online gas chromatography, and gas chromatography-mass spectrometry, which involved the reductive species such as electrons, hydrogen radicals (·H), methyl radicals (·CH3), and other intermediates in the argon plasma adding methanol. Under the optimized conditions, the limits of detection of 0.007, 0.05, and 0.5 µg L-1 were obtained for Hg, Cd, and Zn, respectively, with relative standard deviations of 3.1, 3.7, and 5.2% for these elements, respectively. Vapor generation efficiencies of 90, 83, and 55% were achieved for Hg, Cd, and Zn, respectively, and improved 2.7-, 4.8-, and 7.9-fold, respectively, compared to those obtained in the absence of methanol. The accuracy and practicability of the proposed method were validated by the determination of Hg, Cd, and Zn in a certified reference material (CRM, Lobster hepatopancreas, TORT-3) and crayfish samples collected from three different provinces of China.

Three-Dimensional Printed Dual-Mode Chemical Vapor Generation Point Discharge Optical Emission Spectrometer for Field Speciation Analyses of Mercury and Inorganic Selenium.

Due to the large size and high energy consumption of instruments, field elemental speciation analysis is still challenging so far. In this work, a portable and compact system device (230 mm length × 38 mm width × 84 mm height) was fabricated by using three-dimensional (3D) printing technology for the field speciation analyses of mercury and inorganic selenium. The device comprises a cold vapor generator, photochemical vapor generator, and miniaturized point discharge optical emission spectrometer (µPD-OES). For mercury, inorganic mercury (IHg) was selectively reduced to Hg0 by cold vapor generation, whereas the reductions of both IHg and methylmercury (MeHg) were obtained by photochemical vapor generation (PVG) in the presence of formic acid. For selenium, Se(IV) and total inorganic selenium were converted to their volatile species by PVG in the presence and the absence of nano-TiO2, respectively. The generated volatile species were consequently detected by µPD-OES. Limits of detection of MeHg, IHg, Se(IV), and Se(VI) were 0.1, 0.1, 5.2, and 3.5 µg L-1, respectively. Precision expressed as the relative standard deviations (n = 11) were better than 4.5%. The accuracy and practicality of the proposed method were evaluated by the analyses of Certified Reference Materials (DORM-4, DOLT-5, and GBW(E)080395) and several environmental water samples with satisfactory recoveries (95-103%). This work confirms that 3D printing has great potential to fabricate a simple, miniaturized, easy-to-operate, and low gas and power consuming atomic spectrometer for field elemental speciation analysis.

Cloning and characterization of KoOsmotin from mangrove plant Kandelia obovata under cold stress.

BACKGROUND: Low temperature is a major abiotic stress that seriously limits mangrove productivity and distribution. Kandelia obovata is the most cold-resistance specie in mangrove plants, but little is known about the molecular mechanism underlying its resistance to cold. Osmotin is a key protein associated with abiotic and biotic stress response in plants but no information about this gene in K. obovata was reported. RESULTS: In this study, a cDNA sequence encoding osmotin, KoOsmotin (GenBank accession no. KP267758), was cloned from mangrove plant K. obovata. The KoOsmotin protein was composed of 221 amino acids and showed a calculated molecular mass of 24.11 kDa with pI 4.92. The KoOsmotin contained sixteen cysteine residues and an N-terminal signal peptide, which were common signatures to most osmotins and pathogenesis-related 5 proteins. The three-dimensional (3D) model of KoOsmotin, contained one α-helix and eleven ß-strands, was formed by three characteristic domains. Database comparisons of the KoOsmotin showed the closest identity (55.75%) with the osmotin 34 from Theobroma cacao. The phylogenetic tree also revealed that the KoOsmotin was clustered in the branch of osmotin/OLP (osmotin-like protien). The KoOsmotin protein was proved to be localized to both the plasma membrane and cytoplasm by the subcellular localization analysis. Gene expression showed that the KoOsmotin was induced primarily and highly in the leaves of K. obovata, but less abundantly in stems and roots. The overexpressing of KoOsmotin conferred cold tolerance in Escherichia coli cells. CONCLUSION: As we known, this is the first study to explore the osmotin of K. obovata. Our study provided valuable clues for further exploring the function of KoOsmotin response to stress.

Pyruvate cycle increases aminoglycoside efficacy and provides respiratory energy in bacteria.

The emergence and ongoing spread of multidrug-resistant bacteria puts humans and other species at risk for potentially lethal infections. Thus, novel antibiotics or alternative approaches are needed to target drug-resistant bacteria, and metabolic modulation has been documented to improve antibiotic efficacy, but the relevant metabolic mechanisms require more studies. Here, we show that glutamate potentiates aminoglycoside antibiotics, resulting in improved elimination of antibiotic-resistant pathogens. When exploring the metabolic flux of glutamate, it was found that the enzymes that link the phosphoenolpyruvate (PEP)-pyruvate-AcCoA pathway to the TCA cycle were key players in this increased efficacy. Together, the PEP-pyruvate-AcCoA pathway and TCA cycle can be considered the pyruvate cycle (P cycle). Our results show that inhibition or gene depletion of the enzymes in the P cycle shut down the TCA cycle even in the presence of excess carbon sources, and that the P cycle operates routinely as a general mechanism for energy production and regulation in Escherichia coli and Edwardsiella tarda These findings address metabolic mechanisms of metabolite-induced potentiation and fundamental questions about bacterial biochemistry and energy metabolism.

Metabolic mechanism of colistin resistance and its reverting in Vibrio alginolyticus.

Colistin is a last-line antibiotic against Gram-negative multidrug-resistant bacteria, but the increased resistance poses a huge challenge to this drug. However, the mechanisms underlying such resistance are largely unexplored. The present study first identified the mutations of two genes encoding AceF subunit of pyruvate dehydrogenase (PDH) and TetR family transcriptional regulator in colistin-resistant Vibrio alginolyticus (VA-RCT ) through genome sequencing. Then, gas chromatography-mass spectroscopy-based metabolomics was adopted to investigate metabolic responses since PDH plays a role in central carbon metabolism. Colistin resistance was associated with the reduction of the central carbon metabolism and energy metabolism, featuring the alteration of the pyruvate cycle, a recently characterized energy-producing cycle. Metabolites in the pyruvate cycle reprogramed colistin-resistant metabolome to colistin-sensitive metabolome, resulting in increased gene expression, enzyme activity or protein abundance of the cycle and sodium-translocating nicotinamide adenine dinucleotide-ubiquinone oxidoreductase. This reprogramming promoted the production of the proton motive force that enhances the binding between colistin and lipid A in lipopolysaccharide. Moreover, this metabolic approach was effective against VA-RCT in vitro and in vivo as well as other clinical isolates. These findings reveal a previously unknown mechanism of colistin resistance and develop a metabolome-reprogramming approach to promote colistin efficiency to combat with colistin-resistant bacteria.

Simple Universal Strategy for Quantification of Carboxyl Groups on Carbon Nanomaterials: Carbon Dioxide Vapor Generation Coupled to Microplasma for Optical Emission Spectrometric Detection.

The physicochemical properties and applications of carbon nanomaterials are remarkably dependent on the amount of carboxyl group on their surfaces. Unfortunately, it is challenging to determine the carboxyl group on carbon nanomaterials at an ultralow density not only due to the low sensitivities of conventional techniques, but also because there are no matrix-matched certified reference materials available. In this work, a novel strategy comprising coupling carbon dioxide vapor generation to a microplasma optical emission spectrometer was developed for the sensitive and accurate quantification of surface carboxyl groups on carbon nanomaterials. The carboxyl group on multiwall carbon nanotubes (MWCNTs), graphene (G), or its oxide (GO) was converted to carboxylic acid using concentrated hydrochloric acid prior to quantification. The generated carboxylic acid was purified and then reacted with sodium bicarbonate to generate CO2, which was swept into a miniaturized point discharge optical emission spectrometer (µPD-OES) for the detection of carbon atomic emission lines. Potassium hydrogen phthalate (KHP) served as a calibration standard for quantification of the carboxyl group on G/GO/MWCNTs, thus, overcoming the lack of CRMs. Owing to the high sensitivity of µPD-OES for the detection of CO2, a limit of detection of 0.1 µmol g-1 (1 nmol) was obtained for the carboxyl group based on a sample mass of 10 mg G/GO/MWCNTs, superior to that obtained using conventional methods. Moreover, the proposed method not only retains several unique advantages of good accuracy and elimination of the use of complicated, expensive, and high power-consumption instruments, but was also applicable to the quantification of the carboxyl group on other nanomaterials such as carboxylated magnetic microspheres.

A nanocomposite prepared from copper(II) and nitrogen-doped graphene quantum dots with peroxidase mimicking properties for chemiluminescent determination of uric acid.

Nitrogen-doped graphene quantum dots (N-GQD) were employed along with Cu(II) ions under alkaline conditions and room temperature to synthesize nanocomposites of type Cu(II)/Cu2O/N-GQDs. These nanocomposites exhibit excellent stability and dispersity, and also display a peroxidase-like activity that is superior to pure Cu2O nanoparticles and natural peroxidase (POx). A chemiluminescence (CL) method was designed that is based on the use of uricase which oxidizes uric acid under formation of H2O2. The nanocomposites were used as a POx mimic in the luminol-H2O2 CL system. Under optimized conditions, a linear relationship between CL intensity and the uric acid (UA) concentration in the range of 0.16-4.0 µM, and a detection limit of 0.041 µM (at S/N = 3) were obtained. The CL method was applied to the determination of UA in spiked serum and urine, and recoveries ranged from 85.0 to 121.3%. Graphical abstract Schematic presentation of synthesis strategy of Cu(II)/Cu2O/N-GQDs and the CL method based Cu(II)/Cu2O/N-GQDs for H2O2-meidated uric acid detection. The method can be used for the determination of uric acid (UA) with the detection limit of 0.041 µM.