Human Urine Specimens Research Articles

Colorimetric and naked-eye detection of arsenic(iii) using a paper-based microfluidic device decorated with silver nanoparticles.

Arsenic (As) as a metal ion has long-term toxicity and its presence in water poses a serious threat to the environment and human health. So, rapid and accurate recognition of traces of As is of particular importance in environmental and natural resources. In this study, a fast and sensitive colorimetric method was developed using silver nano prisms (Ag NPrs), cysteine-capped Ag NPrs, and methionine-capped Ag NPrs for accurate detection of arsenic-based on transforming the morphology of silver nanoparticles (AgNPs). The generated Ag atoms from the redox reaction of silver nitrate and As(iii) were deposited on the surface of Ag NPrs and their morphology changed to a circle. The morphological changes resulted in a change in the color of the nanoparticles from blue to purple, which was detectable by the naked eye. The rate of change was proportional to the concentration of arsenic. The changes were also confirmed using UV-Vis absorption spectra and showed a linear relationship between the change in adsorption peak and the concentration of arsenic in the range of 0.0005 to 1 ppm with a lower limit of quantification (LLOQ) of 0.0005 ppm. The proposed probes were successfully used to determine the amount of As(iii) in human urine samples. In addition, modified microfluidic substrates were fabricated with Ag NPrs, Cys-capped Ag NPrs, and methionine-capped Ag NPrs nanoparticles that are capable of arsenic detection in the long-time and can be used in the development of on-site As(iii) detection kits. In addition, silver nanowires (AgNWs) were used as a probe to detect arsenic, but good results were not obtained in human urine specimens and paper microfluidic platforms. In this study, for the first time, AgNPs were developed for optical colorimetric detection of arsenic using paper-based microfluidics. Ag NPrs performed best in both optical and colorimetric techniques. Therefore, they can be a promising option for the development of sensitive, inexpensive, and portable tools in the environmental and biomedical diagnosis of As(iii).

A Simple, Fast, and Reliable LC-MS/MS Method for the Measurement of Homovanillic Acid and Vanillylmandelic Acid in Urine Specimens.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are catecholamine metabolites used in the diagnostic workup of neuroendocrine tumors. Here we describe a simple dilute-and-shoot method for simultaneously quantitating HVA and VMA in human urine specimens. The method employs analyte separation on a reverse-phase liquid chromatography column followed by detection using electrospray ionization triple quadrupole mass spectrometry (ESI-MS/MS), wherein qualifier and quantifier ion transitions are monitored. This is a simple and fast analytical method with an injection-to-injection time of 4min.

Protein Kinase A Detection in Human Urine Samples.

Actively involved in tumor maintenance, cAMP-dependent protein kinase A (PKA) has been proposed as a putative biomarker in cancer. Recently, an active PKA form has been identified in human sera and PKA autoantibodies have been detected in cancer patients. However, their serum functions, as well as diagnostic significance, remain largely unknown. Although several PKA detection assays have been developed, none refer to a laboratory diagnostic procedure. Among these, ELISA and Western blotting (WB) assays have been employed in PKA detection. Since, to the best of our knowledge, there are no data showing its presence in human urine samples, herein, we explore the possibility of PKA’s existence in this biological specimen. Interestingly, among the 30 screened urines by quantitative sandwich ELISA, we recognized detectable PKA levels in 5 different samples, and of those two exhibited a considerable high concentration. To corroborate these results, we also evaluated PKA’s presence in both positive and negative ELISA urines by WB. Remarkably, immunoblotting analysis confirmed PKA’s existence in certain, but not in all, human urine specimens. Despite being quite preliminary, these findings firstly identify PKA in urine samples and provide evidence for its potential clinic usage as a diagnostic analyte in laboratory medicine.

Zn2+ Recognition for Pathogenesis of Pick's Disease via a Luminescent Test Kit

AbstractThe pathogenesis of Pick's disease is feasible by detection of the elevated level of Zn2+ in the body fluids. This work aims towards developing a suitable chemosensor towards detection of Zn2+ in human urine specimen and inside eukaryotic cell line. Fluorescence “turn on” in the presence of Zn2+ makes the chemosensor suitable for its utility in Pick's disease diagnosis. Real urine sample analysis together within cell detection of Zn2+ was successful which leads towards the design and fabrication of a “ready‐to‐use” test kit for Zn2+ recognition from human urine specimen.

A highly selective colorimetric assay for the determination of creatinine in biological samples using gluconic acid capped silver nanoparticles after ionic liquid based dispersive liquid phase microextraction

A dispersive liquid-phase microextraction method combined with UV–vis spectrophotometry was utilized to highly selective determination of creatinine in human serum and urine samples. To overcome the interferences in complex matrices, creatinine reacted with 1,4-naphthoquinone-2- potassium sulfonate reagent to produce a red coloured product that could be extracted into a small volume of 1-hexyl-3-methylimidazolium hexafluorophosphate ([HMIM]PF6) ionic liquid solvent. To increase the sensitivity of the assay, gluconic acid capped silver nanoparticles (Ag NPs) were used. On addition of Ag NPs to the red coloured extracted product, the solution turned to blue accompanied with a red shift in wavelength around 620 nm that could be detected by the naked eye. The effective variables on the determination of creatinine such as concentration of the reagent, amount of formic and hydrochloric acids, type and volume of the extractant, and concentration of Ag NPs were investigated. Under the optimal conditions, the calibration plot was bimodal with linear ranges from 0.1 to 1.5 µg mL−1 and 1.5 to 105 µg mL−1 creatinine with a limit of detection 0.1 µg mL−1. The relative standard deviation for five measurements at 35 µg mL−1 concentration level was 3.8%. The newly developed assay was used for the determination of creatinine in human serum and urine specimens with satisfactory results.

Electrical Cartridge Sensor Enables Reliable and Direct Identification of MicroRNAs in Urine of Patients.

Urinary miRNAs are biomarkers that demonstrate considerable promise for the noninvasive diagnosis and prognosis of diseases. However, because of background noise resulting from complex physiological features of urine, instability of miRNAs, and their low concentration, accurate monitoring of miRNAs in urine is challenging. To address these limitations, we developed a urine-based disposable and switchable electrical sensor that enables reliable and direct identification of miRNAs in patient urine. The proposed sensing platform combining disposable sensor chips composed of a reduced graphene oxide nanosheet and peptide nucleic acid facilitates the label-free detection of urinary miRNAs with high specificity and sensitivity. Using real-time detection of miRNAs in patient urine without pretreatment or signal amplification, this sensor allows rapid, direct detection of target miRNAs in a broad dynamic range with a detection limit down to 10 fM in human urine specimens within 20 min and enables simultaneous quantification of multiple miRNAs. As confirmed using a blind comparison with the results of pathological examination of patients with prostate cancer, the sensor offers the potential to improve the accuracy of early diagnosis before a biopsy is taken. This study holds the usefulness of the practical sensor for the clinical diagnosis of urological diseases.

Kinetic Spectroelectrochemical Assay for Uric Acid in Human Urine

AbstractA spectroelectrochemical (SEC) assay for uric acid in human urine has been developed that is essentially reagent‐free and involves relatively short analysis times of ∼12 min per specimen using a single‐point standard addition approach. The SEC method was demonstrated to be selective for uric acid in human urine, avoiding interference through both the usual control parameters (electrolysis potential and measurement wavelength) and the relatively slow kinetics of a post‐electrolysis reaction involving the oxidation product. Results from the SEC analysis of a split set of human urine specimens were well correlated to those obtained using a standard high‐performance liquid chromatography method, suggesting the feasibility of clinical applications.

Zinc Oxide Nanoparticles Supported on Multi‐walled Carbon Nanotube Modified Electrode for Electrochemical Sensing of a Fluoroquinolone Drug

AbstractA facile, and novel construction strategy invented on electrochemical procedures for the assemblage of zinc oxide nanoparticles (ZnO NPs) with a multiwalled carbon nanotube modified carbon paste electrode (ZnO‐MWCNTMCPE) to ascertain the fluoroquinolone drug‐Moxifloxacin (MOX). The ZnO‐MWCNTMCPE can significantly improve the electron transfer process and lessen the impedance of the surface of the electrode. Besides, the oxidation of MOX has remarkably enhanced on the modified electrode. Under the optimal experimental conditions, the empirical results exhibited that the oxidation peak current was linearly proportional to the concentration of MOX in the range of 1.0×10−7 to 2.4×10−6 mol L−1 with a detection limit of 0.452×10−7 mol L−1. To analyze the practicality of the MOX determination technique in real samples, the intended sensor manipulated for the adjudication of MOX in human urine specimens, and the results authenticated the explore of MOX with the highest accuracy. Further, the proposed sensor system exercised for the simultaneous evaluation of paracetamol (ACOP) and MOX, the DPV technique unveiled the well‐separated peaks among ACOP (0.188 mV) and MOX (0.814 mV) with a peak separation (ΔE) of 626 mV in real samples. Therefore, the suggested system could sense and discover the MOX in the biological systems.

Rapid antibiotic susceptibility testing based on bacterial motion patterns with long short-term memory neural networks.

Antibiotic resistance is an increasing public health threat. To combat it, a fast method to determine the antibiotic susceptibility of infecting pathogens is required. Here we present an optical imaging-based method to track the motion of single bacterial cells and generate a model to classify active and inactive cells based on the motion patterns of the individual cells. The model includes an image-processing algorithm to segment individual bacterial cells and track the motion of the cells over time, and a deep learning algorithm (Long Short-Term Memory network) to learn and determine if a bacterial cell is active or inactive. By applying the model to human urine specimens spiked with an Escherichia coli lab strain, we show that the method can accurately perform antibiotic susceptibility testing as fast as 30 minutes for five commonly used antibiotics.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

Detection of the recently emerged synthetic cannabinoid 4F-MDMB-BINACA in "legal high" products and human urine specimens.

Synthetic cannabinoids (SCs) remain one of the largest groups of new psychoactive substances (NPS) on the European drug market. Although the number of new derivatives occurring on the market has dropped in the last two years, newly emerging NPS still represent a challenge for laboratories performing forensic drug analysis in biological matrices. The newly emerged SC 4F-MDMB-BINACA has been reported by several law enforcement agencies in Europe and the USA since November 2018. This work aimed at revealing urinary markers to prove uptake of 4F-MDMB-BINACA and differentiate from the use of structurally similar SCs. Phase-I metabolites detected in human urine specimens were confirmed by phase-I metabolites generated in vitro using a pooled human liver microsomes (pHLM) assay. Seized materials and test-purchased "legal high" products were analyzed by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole-time-of-flight-mass spectrometry (LC-qToF-MS). Human urine specimens and pHLM assay extracts were measured with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and confirmed by LC-qToF-MS. In January 2019, the Institute of Legal Medicine in Erlangen (Germany) identified 4F-MDMB-BINACA in three herbal blends. During the same time period, the described SC was identified in a research chemical purchased online. Investigation of phase-I metabolism led to the metabolites M10 (ester hydrolysis) and M11 (ester hydrolysis and dehydrogenation) as reliable urinary markers. Widespread distribution on the German drug market was proven by analysis of urine samples from abstinence control programs and by frequent detection of 4F-MDMB-BINACA in "herbal blends" and "'research chemicals" purchased via the Internet.

Shedding and Transmission Modes of Severe Fever With Thrombocytopenia Syndrome Phlebovirus in a Ferret Model.

BackgroundAlthough human-to-human transmission of severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) via direct contact with body fluids has been reported, the role of specific body fluids from SFTSV-infected hosts has not been investigated in detail.MethodsTo demonstrate the virus transmission kinetics in SFTSV-infected hosts, we adapted the ferret infection model and evaluated the virus shedding periods, virus titers, and transmission modes from various specimens of infected ferrets.ResultsLarge amounts of infectious SFTSV are shed through nasal discharge, saliva, and urine from SFTSV-infected ferrets. Virus could be detected from 2 dpi and persisted until 12 dpi in these specimens, compared with the relatively short virus-shedding period in sera. Further, transmission studies revealed that SFTSV can be transmitted to close direct and indirect contact naïve animals through various mediums, especially through contact with serum and urine. Further, ferrets contacted with human urine specimens from SFTSV-positive patients were successfully infected with SFTSV, suggesting that urine specimens could be a source of SFTSV infection in humans.ConclusionsOur results demonstrate that the SFTSV can be shed in various body fluids for more than 12 days and that these specimens could be a source for direct or indirect transmission through close personal contact.

Electrocatalytic Study of Low-Cost Bimetallic Cobalt/Iron Catalyst on Carbon for Non-Enzymatic Glucose Sensor in Human Urine

This study investigated an electrocatalytic behavior of non-enzymatic glucose detection in urine by using low-cost non-precious metal CoFe catalyst on carbon (C) supported. The bimetal catalyst was prepared by the reduction of oleic acid and loaded 10% wt. metal onto the activated carbon. Due to the synergistic effect, CoFe exhibited its intrinsic electrocatalytic property, suitable for the chemisorption of glucose molecule and the d-electron of metal. For morphology and elemental composition, CoFe/C was characterized by Transmission Electron Microscopy (TEM), and X-ray Energy Dispersive Spectroscopy (EDS) and X-ray Diffraction (XRD), exhibiting Co (111) and Fe2O3(104) with the nanocluster average diameter of 25 nm. More importantly, electrocatalytic behaviors of CoFe/C were assessed by cyclic voltammetry (CV) and Differential Pulse Voltammetry (DPV) on the glassy carbon rotating disk electrode for glucose detection (0-3 mM) in modified artificial urine (mAUM), and human urine specimens. In particular, excellent sensitivities from the lower range of glucose level (< 1 mM) and the higher level by DPV in mAUM were estimated to be 318.42 and 82.20 μA.cm-2.mM-1 with the correlation coefficient (R2) values of 0.90 and 0.94, respectively. Furthermore, the as-prepared CoFe/C biosensor also demonstrated practical measurement in human urine sample with the sensitivity of 59.72 μA.cm-2.mM-1 (R2 = 0.99) without any electron facilitators (e.g. sodium hydroxide), thereby providing a promising cost-effective catalyst design for future technology of non-enzymatic glucose sensing applications in urine.

Detection and quantification of 5F-ADB and its methyl ester hydrolysis metabolite in fatal intoxication cases by liquid chromatography–high resolution mass spectrometry

5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl]amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market. It has been classified as an illicit drug in most European countries, and also in Turkey, Japan, and the United States. In this study, 5F-ADB and its methyl ester metabolite were determined in the blood and urine samples taken from fatal cases using liquid chromatography–highresolution mass spectrometry (LC–HRMS). The extraction of samples was performed using a solid-phase extraction method, followed by LC–HRMS analysis. The method was fully validated for linearities, limits of detection (LODs), limits of quantification (LOQs), recoveries, matrix effects, process efficiencies, accuracies, precisions, and stabilities and was applied to 70 blood and 36 urine samples from fatal cases where 5F-ADB was the only drug detected. The LODs were between 0.08 and 0.10ng/mL, and LOQs were between 0.10 and 0.12ng/mL for both blood and urine samples. 5F-ADB and its methyl ester hydrolysis metabolite were found at the blood concentrations ranging from 0.10 to 1.55ng/mL (mean=0.40ng/mL) and 0.15 to 23.4ng/mL (mean=2.69ng/mL), respectively. 5F-ADB was not detected in any urine samples. 5F-ADBmethyl ester hydrolysis metabolite was detected in 35 urine samples with a detection range of 0.28–72.2ng/mL and a mean of 9.02ng/mL. The synthetic cannabinoid 5F-ADB and its methyl ester metabolite were identified and quantified in authentic human blood and/or urine specimens obtained from 70 fatal cases. The method was successfully applied to postmortem blood and urine samples.

Identification of potential urinary exposure markers for the toxicant diisononyl phthalate in rubber worker urine specimens by high-resolution mass spectrometry-based metabolomics

Abstract Diisononyl phthalate (DiNP) is commonly used as a plasticizer in polyvinyl chloride (PVC) products. However, DiNP can interfere with the human endocrine system and is listed as a chemical with reproductive toxicity. It has a short half-life and can be metabolized into water-soluble substances that are excreted in the urine, and thus, DiNP exposure markers can be identified in urine, a noninvasive and easily accessible specimen. In a previous study, we established an isotope tracing strategy involving an in-house computational signal mining algorithm with isotope tracing (SMAIT) to filter probable metabolite signals from complex LC-MS data. In this study, we used SMAIT to filter the probable metabolite signals, which were verified as potential DiNP exposure markers by analyzing human urine specimens from rubber workers who had occupational exposure to DiNP. We filtered 16 probable metabolite signals out of approximately 1400 signals by SMAIT. Fourteen of the 16 probable metabolite signals were confirmed as DiNP structure-related metabolites according to the fragment ion at m/z 121.029, which is a benzoate ion (C6H5CO2), identified in D0-MMOP mass spectra by ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) in the full-scan mode. The 14 structure-related metabolite signals were further verified as potential DiNP exposure markers by analyzing rubber worker urine specimens. Three DiNP structure-related metabolite signals at m/z 305.139 (MOINP), 307.155 (MHINP), and 375.142 (derivatives of MHINP) were identified as potential exposure markers of DiNP. We need more research to validate whether these metabolites could be used as specific biomarkers for human exposure to DiNP. The 3 structure-related metabolite signals could provide the basis for future exposure and risk assessment for DiNP.

In vitro and in vivo human metabolism of a synthetic cannabinoid EAM-2201 detected by LC–quadrupole-ion trap-MS/MS and high-resolution LC–Orbitrap-MS/MS

The aim of this study is to characterize the metabolites of EAM-2201 in human hepatocytes obtained in vitro and those in liver and urine specimens obtained in vivo from the autopsy of an EAM-2201 abuser. EAM-2201 was incubated with human hepatocytes for 3 h in a CO2 incubator and the metabolites of EAM-2201 were produced. The human liver specimen was homogenized and the metabolites were extracted. The urine specimen was hydrolyzed first with β-glucuronidase and the metabolites were extracted. The tentative detection and identification of those metabolites were performed using liquid chromatography (LC)–quadrupole-ion trap tandem mass spectrometry (MS/MS) and high-resolution LC–Orbitrap-MS/MS, respectively. Twelve metabolites of EAM-2201 could be characterized in hepatocytes, liver and urine. They were produced by defluorination, hydroxylation, carboxylation, dehydrogenation, N-dealkylation and/or glucuronidation of EAM-2201. The most detectable metabolite obtained from the in vitro hepatocyte incubation was that monohydroxylated at the 4-ethylnaphthalene moiety of EAM-2201 after dehydrogenation. However, only a trace amount of the same metabolite was detected in the liver tissue obtained postmortem from an EAM-2201 abuser. In the liver tissue, the highest metabolite was 5-carboxypentyl derivative of EAM-2201, which was monohydroxylated at the 4-ethylnaphthalene moiety, followed by monohydroxylpentyl and monohydroxy-4-ethylnaphthalenyl derivative of EAM-2201 without dehydrogenation. Twelve metabolites of a synthetic cannabinoid, EAM-2201, were tentatively identified in human hepatocytes in vitro, and in human liver and urine specimens in vivo for the first time. There was a distinct difference in metabolism profile between the in vitro and in vivo results.

A comparative study of non-enzymatic glucose detection in artificial human urine and human urine specimens by using mesoporous bimetallic cobalt-iron supported N-doped graphene biosensor based on differential pulse voltammetry

Development of non-invasive glucose measurement allows future advancement of smart sensing platform for diagnostic technology. Especially for endocrine disorders, advanced kidney diseases, and diabetes, monitoring excessive glucose level in urine can provide invaluable information for clinical prognosis and preventive healthcare. Herein, we present a comparative electrochemical study of cobalt/iron (CoFe) catalyst on nitrogen-doped graphene (NG) for non-enzymatic glucose detection, carried out in physiological pH urine including (i) modified artificial urine medium (mAUM), (ii) commercial standard urine (Surine), and (iii) human urine specimens. With no requirement of strong alkaline addition, catalytic properties of CoFe-NG were assessed by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) on a glassy carbon rotating disk electrode. Upon successive glucose additions from 0 to 3 mM, DPV results revealed two anodic peaks at +0.18 V and +0.42 V versus Ag/AgCl, corresponding to Co3+ and Co4+ as a result of glucose binding in urine. By evaluating at +0.18 V, the sensitivities of CoFe-NG were estimated to be 16.77 (R2 = 0.987), 45.36 (R2 = 0.988), and 20.26 (R2 = 0.991) μA mM−1 cm−2 with the limit of detection of 0.25, 0.07, and 0.19 mM in mAUM, Surine, and human urine specimen with low serum creatinine, respectively. Furthermore, the effects of CoFe on graphene (G) and carbon Vulcan XC-72 (C) were also studied in comparison of NG on the bimetal. Interestingly, CoFe-C showed a good electrochemical trend in glucose detection in urine. However, negligible catalytic activity was presented in CoFe-G. Thus, electrochemical responses of CoFe-C were also further studied in the comparison of CoFe-NG in each type of urine. Overall, CoFe-NG outperformed CoFe-C in all types of urine and exhibited an excellent anti-interference property toward uric acid, thereby suggesting great potential for the next generation of glucose sensing platform in urine.

Nitrogen and chlorine co-doped carbon dots as probe for sensing and imaging in biological samples.

A facile one-step hydrothermal synthesis approach was proposed to prepare nitrogen and chlorine co-doped carbon dots (CDs) using l-ornithine hydrochloride as the sole precursor. The configuration and component of CDs were characterized by transmission electron microscopy and X-ray photoelectron and Fourier transform infrared spectroscopies. The obtained CDs (Orn-CDs) with a mean diameter of 2.1 nm were well monodispersed in aqueous solutions. The as-prepared CDs exhibited a bright blue fluorescence with a high yield of 60%, good photostability and low cytotoxicity. The emission of Orn-CDs could be selectively and effectively suppressed by Fe3+. Thus, a quantitative assay of Fe3+ was realized by this nanoprobe with a detection limit of 95.6 nmol l−1 in the range of 0.3–50 µmol l−1. Furthermore, ascorbic acid could recover the fluorescence of Orn-CDs suppressed by Fe3+, owing to the transformation of Fe3+ to Fe2+ by ascorbic acid. The limit of detection for ascorbic acid was 137 nmol l−1 in the range of 0.5–10 µmol l−1. In addition, the established method was successfully applied for Fe3+ and ascorbic acid sensing in human serum and urine specimens and for imaging of Fe3+ in living cells. Orn-CD-based sensing platform showed its potential to be used for biomedicine-related study because it is cost-effective, easily scalable and can be used without additional functionalization and sample pre-treatment.

The plasmid-encoded transcription factor ArdK contributes to the repression of the IMP-6 metallo-β-lactamase gene blaIMP-6, leading to a carbapenem-susceptible phenotype in the blaIMP-6-positive Escherichia coli strain A56-1S.

Carbapenemase-producing Enterobacteriaceae (CPE) are a global concern because these bacteria are resistant to almost all β-lactams. Horizontal interspecies gene transfer via plasmid conjugation has increased the global dissemination of CPE. Recently, an Enterobacteriaceae strain positive for carbapenemase gene but showing a carbapenem-susceptible phenotype was identified, suggesting that these susceptible strains may be challenging to detect solely via antimicrobial susceptibility tests without molecular analysis. Here, we isolated a blaIMP-6 carbapenemase-gene positive but imipenem- and meropenem-susceptible Escherichia coli (ISMS-E) strain A56-1S (imipenem and meropenem minimum inhibitory concentration, ≤ 0.125 mg/L), from a human urine specimen in Japan. A56-1S was carbapenemase negative by the Carba NP test, suggesting that IMP-6 production was low or undetectable. Thus, to characterize the mechanism of this phenotype, a meropenem-resistant E. coli A56-1R strain was obtained using meropenem-selection. A56-1R was positive for carbapenemase production by the Carba NP test, and blaIMP-6 transcription in A56-1R was 53-fold higher than in A56-1S, indicating that blaIMP-6 in A56-1S is negatively regulated at the transcriptional level. Comparative genomic analysis between the two strains revealed that the alleviation of restriction of DNA (ardK) gene encoding a putative transcription factor is disrupted by the IS26 insertion in A56-1R. A cotransformation assay of ardK and the regulatory element upstream of blaIMP-6 showed repression of blaIMP-6 transcription, indicating that ArdK negatively modulates blaIMP-6 transcription. ArdK binding and affinity assays demonstrated that ArdK directly binds to the regulatory element upstream of blaIMP-6 with dissociation constant values comparable to those of general transcription factors. The IMP-6 carbapenemase showed low hydrolytic activity against imipenem, resulting in an imipenem-susceptible and meropenem-resistant (ISMR) phenotype (previously reported as a stealth phenotype). However, the low expression of IMP-6 in the A56-1S strain could be a typical characteristic of ISMS-E due to gene repression, indicating that conventional antimicrobial susceptibility tests might be unable to detect such strains even when using both imipenem and meropenem. Bacteria that exhibit the ISMS phenotype could play a potential role as undetectable reservoirs and might facilitate gene transfer to other organisms while avoiding detection.

Sensitive quantification of BB-22 and its metabolite BB-22 3-carboxyindole, and characterization of new metabolites in authentic urine and/or serum specimens obtained from three individuals by LC\u2013QTRAP-MS/MS and high-resolution LC\u2013Orbitrap-MS/MS

PurposeA synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole have not yet been quantified in human urine. The aim of this study is to establish a sensitive analytical method for the quantification of BB-22 and its 3-carboxyindole in human serum and urine specimens, and the characterization of the unreported metabolites of BB-22 in authentic urine specimens from three individuals.MethodsThese compounds were extracted from β-glucuronide-hydrolyzed and unhydrolyzed urine and/or serum via liquid–liquid extraction. The identification and quantification were performed using liquid chromatography (LC)–QTRAP-tandem mass spectrometry (MS/MS) and the characterization of the new metabolites was made by high-resolution LC–MS/MS.ResultsThe limits of detection of BB-22 and BB-22 3-carboxyindole were 3 and 30 pg/mL in urine, respectively. The devised method was applied to quantify these compounds in authentic serum and urine obtained from two drug abusers and in urine from one drug abuser. The serum levels of BB-22 were 149 and 6680 pg/mL, and those of BB-22 3-carboxyindole were 0.755 and 38.0 ng/mL in cases 1 and 2, respectively. The urine levels of BB-22 were 5.64, 5.52 and 6.92 pg/mL and those of BB-22 3-carboxyindole were 0.131, 21.4 and 5.15 ng/mL in cases 1, 2 and 3, respectively. New monohydroxyl metabolites retaining the structure of BB-22 were found in the urine specimens.ConclusionsThe synthetic cannabinoid BB-22 and its metabolite BB-22 3-carboxyindole were identified and quantified in authentic human serum and urine specimens for the first time, and new metabolites of BB-22 were tentatively identified in authentic urine specimens obtained from three drug users in this study.