Synthesis of kanamycin-azole hybrids and investigation of their antifungal activities.

The World Health Organization (WHO) recognizes Candida albicans and Cryptococcus neoformans as the critical priority fungal pathogens for which therapeutic solutions are needed. Azole-based antifungal agents, including triazoles, diazoles, and thiazoles, are widely used in the treatments for fungal infections. In light of past successes in the transformation of antibacterial kanamycin into antifungal derivatives via chemical modifications, a new library of kanamycin-azole hybrids was synthesized and tested against a panel of azole-resistant and susceptible Candida and Cryptococcus strains. Structure activity relationship (SAR) studies revealed pivotal roles for antifungal activity of the azole ring (imidazole vs triazole) and halogen substituents on the benzene ring (F vs Cl). Most notably, hybrids 13, 14 and 15 were active against resistant C. albicans, C. tropicalis and C. neoformans strains and non-toxic towards mammalian cells. Mode of action investigations using fluorogenic dyes, (SYTOXTM) showed the fungal active compounds could permeabilize fungal membranes even at » MICs. These findings reveal novel azole-based antifungals that could offer new therapeutic options for candidiasis and cryptococcosis.

Fluorescent and polarity-switchable photoelectrochemical dual-mode homogeneous sensing platform for ultrasensitive kanamycin detection based on EXO III-driven signal amplification.

A fluorescent and photoelectrochemical (PEC) dual-mode biosensor based on target biorecognition-triggered cyclic amplification was constructed for Kana detection. With the assistance of the catalyzed reaction of exonuclease III, a Kana-aptamer DNA duplex was designed for conducting the cyclic release of G-rich DNA sequence as well as output DNA S2. The released G-rich sequence triggers the fluorescence (FL) of thioflavin T (ThT), the intensity of which is positively correlated with the Kana concentration. The linear range is 0.2 to 30 nM, and the detection limit reaches 0.07 nM. Simultaneously, the released output DNA S2 was captured by Fe3O4@CdTe-probe ssDNA and then combined with methylene blue to realize the transduction of polarity-reversed PEC signal, leading to the sensitive detection of Kana with a linear range of 0.2 to 40 nM and a calculated detection limit of 0.2 nM. The outstanding performance endows the dual-mode biosensor a promising prospect for practical application.

A universal optical aptasensor for antibiotics determination based on a new high-efficiency Förster resonance energy transfer pair.

A novel "turn-on" aptasensor for kanamycin (Kana) detection based on a new Förster resonance energy transfer (FRET) pair is reported. A new organic small molecule was employed as a high-efficiency quencher for fluorophore. Based on specific interactions between ssDNA and the quencher, an ingenious and amplified strategy was designed. In the absence of the target, the fluorescence of the fluorophore labeled at the end of the aptamer was quenched. After the binding of the aptamer to the target, the fluorescence was recovered and amplified. The proposed aptasensor showed high specificity, selectivity, and stability in complicated systems. With the P3-based strategy, the limit of detection for Kana is estimated to be 10 nM, which is much lower than the maximum allowable concentration in milk. The recoveries of spiked Kana in milk were in the range 99.8 ~ 105.3% (n = 3). Fortunately, this novel method can be easily extended to other antibiotics such as tobramycin by simply replacing the aptamer, showing great potential as a universal platform for selective, sensitive, and rapid detection of hazardous analytes in food samples.

Catalytic hairpin assembly-driven DNA walker to develop a label-free electrochemical aptasensor for antibiotic detection.

An electrochemical aptasensor was developed by utilizing a DNA walker driven by catalytic hairpin assembly (CHA) with kanamycin as the model analyte. Kanamycin bound to the aptamer, causes the release of DNA walker, triggers the CHA reaction, leads to the cyclic movement of the walker's long arm, and results in cascade amplification of the signal. The guanine-rich sequences of the double-stranded products produced by CHA were folded to form G-quadruplex structures, with electrochemical active molecules Hemin embedded, forms G-quadruplex/Hemin complexes in situ on the electrode surface, thereby achieving sensitive, efficient, and label-free detection of kanamycin with a limit of detection (LOD) of 0.27 pM (S/N = 3). Meaningfully, the aptasensor demonstrated high sensitivity and reliability in the detection of kanamycin in milk and livestock wastewater samples, suggesting that it has great potential for application in detecting antibiotics in food products and water samples from the environment.

Integrating porphyrin-based nanoporous organic polymers with electrochemical aptasensors for ultratrace detection of kanamycin.

The synthesis and characterization of two new porphyrin-based porous organic polymers (POPs) via Sonogashira cross-coupling reaction and leverage the two obtained POPs is reported for the fabrication of electrochemical aptasensors to detect kanamycin at an ultratrace level. The resultant electrochemical aptasensor demonstrates a high linear relationship with the logarithmic value of kanamycin concentration in the range 5 × 10-5-5 µg/L with the limit of detection of 17.6 pg/L or 36.3 fM. During the analysis of real samples from milk and river, a relative standard deviation of less than 4.39%, and good recovery values in the range 97.0-105% were obtained.

Gold/DNA-Cu2+ Complex Nanozyme-Based Aptamer Lateral Flow Assay for Highly Sensitive Detection of Kanamycin.

Aptamer-based lateral flow analysis (Apt-LFAs) has promising applications in many fields. Nanozymes have demonstrated high potential in improving the performance of Apt-LFAs and have been increasingly utilized in recent studies. In this study, we developed a nanozyme-based Apt-LFA for the rapid and sensitive detection of kanamycin by using a novel dual-functionalized AuNPs@polyA-DNA/GpG-Cu2+ nanozyme as a nanoprobe. In the nanoprobe design, the polyA-cDNA strand can discriminate a kanamycin aptamer from the kanamycin/aptamer complex, and the GpG-Cu2+ complex can amplify the detection signal by catalyzing the chromogenic reaction. The nanozyme Apt-LFA can quantify kanamycin in the range of 1-250 ng/mL with an LOD of 0.08 ng/mL, which demonstrated a 4-fold sensitivity improvement and had a wider linear range than the conventional AuNP-based LFA. The Apt-LFA was successfully applied to the detection of kanamycin in honey with good recoveries. Our dual-functionalized AuNP nanoprobe is easily prepared and can be highly compatible with the conventional AuNP-DNA-based LFA platform; thus, it can be extended to the application of Apt-LFAs for other small molecules.

Genome-wide identification of Kanamycin B binding RNA in Escherichia coli.

BACKGROUND: The aminoglycosides are established antibiotics that inhibit bacterial protein synthesis by binding to ribosomal RNA. Additional non-antibiotic aminoglycoside cellular functions have also been identified through aminoglycoside interactions with cellular RNAs. The full extent, however, of genome-wide aminoglycoside RNA interactions in Escherichia coli has not been determined. Here, we report genome-wide identification and verification of the aminoglycoside Kanamycin B binding to Escherichia coli RNAs. Immobilized Kanamycin B beads in pull-down assays were used for transcriptome-profiling analysis (RNA-seq). RESULTS: Over two hundred Kanamycin B binding RNAs were identified. Functional classification analysis of the RNA sequence related genes revealed a wide range of cellular functions. Small RNA fragments (ncRNA, tRNA and rRNA) or small mRNA was used to verify the binding with Kanamycin B in vitro. Kanamycin B and ibsC mRNA was analysed by chemical probing. CONCLUSIONS: The results will provide biochemical evidence and understanding of potential extra-antibiotic cellular functions of aminoglycosides in Escherichia coli.

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Diadenosine tetraphosphate modulated quorum sensing in bacteria treated with kanamycin.

BACKGROUND: The dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress. RESULTS: Here, we labeled Ap4A with biotin and incubated the labeled Ap4A with the total proteins extracted from kanamycin-treated Escherichia coli to identify the Ap4A binding protein in bacteria treated with kanamycin. Liquid chromatography‒mass spectrometry (LCMS) and bioinformatics were used to identify novel proteins that Ap4A interacts with that are involved in biofilm formation, quorum sensing, and lipopolysaccharide biosynthesis pathways. Then, we used the apaH knockout strain of E. coli K12-MG1655, which had increased intracellular Ap4A, to demonstrate that Ap4A affected the expression of genes in these three pathways. We also found that the swarming motility of the apaH mutant strain was reduced compared with that of the wild-type strain, and under kanamycin treatment, the biofilm formation of the mutant strain decreased. CONCLUSIONS: These results showed that Ap4A can reduce the survival rate of bacteria treated with kanamycin by regulating quorum sensing (QS). These effects can expand the application of kanamycin combinations in the treatment of multidrug-resistant bacteria.

High efficient electrochemical biosensor based on exonuclease-â ¢-assisted dual-recycling amplification for ultrasensitive detection of kanamycin.

A target-triggered and exonuclease-Ⅲ-assisted strand displacement, dual-recycling amplification reaction-based biosensor was developed for the rapid, ultrasensitive and accurate detection of kanamycin. The robust profiling platform was constructed using high conductive MXene/VS2 for the electrode surface modification and high active CeCu2O4 bimetallic nanoparticles as nanozyme to improve the sensitivity as well as the catalytic signal amplification of the biosensor. Using the dual supplementary recycling of primer DNA and hairpin DNA, the electrochemical platform could accurately detect kanamycin to as low as 0.6 pM from the range of 5 pM to 5 µM. By profiling five other antibiotics, this platform exhibited high specificity, enhanced repeatability and reproducibility. Based on these intrinsic characteristics and by utilizing milk and water samples, the as-designed biosensor offers a remarkable strategy for antibiotic detection due to its favorable analytical accuracy and reliability, thereby demonstrating potential application prospect for various antibiotic biosensing in food quality control, water contamination detection and biological safety analysis.

Deglycosylation Inactivation Initiated by a Novel Periplasmic Dehydrogenase Complex Provides a Novel Strategy for Eliminating the Recalcitrant Antibiotic Kanamycin.

Biodegradation using enzyme-based systems is a promising approach to minimize antibiotic loads in the environment. Aminoglycosides are refractory antibiotics that are generally considered non-biodegradable. Here, we provide evidence that kanamycin, a common aminoglycoside antibiotic, can be degraded by an environmental bacterium through deglycosylation of its 4'-amino sugar. The unprecedented deglycosylation inactivation of kanamycin is initiated by a novel periplasmic dehydrogenase complex, which we designated AquKGD, composed of a flavin adenine dinucleotide-dependent dehydrogenase (AquKGDα) and a small subunit (AquKGDγ) containing a twin-arginine signal sequence. We demonstrate that the formation of the AquKGDα-AquKGDγ complex is required for both the degradation activity of AquKGD and its translocation into the periplasm. Native AquKGD was successfully expressed in the periplasmic space of Escherichia coli, and physicochemical analysis indicated that AquKGD is a stable enzyme. AquKGD showed excellent degradation performance, and complete elimination of kanamycin from actual kanamycin manufacturing waste was achieved with immobilized AquKGD. Ecotoxicity and cytotoxicity tests suggest that AquKGD-mediated degradation produces less harmful degradation products. Thus, we propose a novel enzymatic antibiotic inactivation strategy for effective and safe treatment of recalcitrant kanamycin residues.

Label-free "signal-off" PEC aptasensor for determination of kanamycin based on 3D nanoflower-like FeIn2S4/CdS Z-scheme heterostructures.

Highly photoactive 3D nanoflower-like FeIn2S4/CdS heterostructures were synthesized by hydrothermal treatment and low-temperature cation exchange. The FeIn2S4/CdS displayed 14.5 times signal amplification in contrast to FeIn2S4 alone. It was applied as a photoactive substrate to construct a label-free photoelectrochemical (PEC) aptasensor for ultrasensitive determination of kanamycin (KAN). Under the optimal conditions, the constructed PEC aptasensor displayed a wide linear range (5.0 × 10-4 ~ 5.0 × 101 ng mL-1) and a low detection limit (S/N = 3) of 40.01 fg mL-1. This study provides some constructive insights for preparation of advanced photoactive materials and exhibits great potential for quantitative determination of antibiotics in foods and environmental samples.

Sub-femtomolar capacitance-based biosensing of kanamycin using screen-printed electrodes coated with redox-active polymeric films.

An ultrasensitive capacitance-based biosensor has been developed capable of detecting the kanamycin (KAN) antibiotic at sub-femtomolar levels. The biosensor was constructed using a potential-pulse-assisted method, allowing for the layer-by-layer deposition of a melanin-like polymeric film (MLPF) on an electrode surface modified with gold nanoparticles (AuNPs). The MLPF was formed through the electrochemical polymerization of dopamine and the specific kanamycin aptamer. By optimizing the operating parameters, we achieved a label-free detection of kanamycin by monitoring the variation of pseudocapacitive properties of the MLPF-modified electrode using electrochemical impedance spectroscopy. The developed biosensor demonstrated a wide linear response ranging from 1 fM to 100 pM, with a remarkable limit of detection of 0.3 fM (S/N = 3) for kanamycin. Furthermore, the biosensor was successfully applied to detect kanamycin in milk samples, exhibiting good recovery. These findings highlight the promising potential of the aptasensor for determination of antibiotic residues and ensuring food safety. In conclusion, our ultrasensitive capacitance-based biosensor provides a reliable and efficient method for detecting trace amounts of kanamycin in dairy products. This technology can contribute to safeguarding consumer health and maintaining high food safety standards.

An unusual complication secondary to kanamycin use in a patient of multidrug-resistant pulmonary tuberculosis.

Gitelman syndrome (GS) is a rare genetic renal disease characterized by hypomagnesemia, hypokalemia, hypocalciuria, and metabolic alkalosis. It usually presents in late childhood or early adulthood. A 30-year-old female diagnosed case of multidrug-resistant (MDR-TB) pulmonary tuberculosis 2 months ago presented to our outpatient department with intermittent painful spasms in all four limb muscles. Her treatment regimen consisted of kanamycin, levofloxacin, cycloserine, and ethionamide. On further evaluation, her investigations revealed hypokalemia, hypocalcemia, hypomagnesemia, metabolic alkalosis with normal serum creatinine level. She was initially treated with intravenous calcium and potassium. However, the electrolyte abnormalities and metabolic alkalosis persisted. All her lab parameters became normal after discontinuing kanamycin and electrolyte replacement for 4 weeks. She was discharged and advised to continue her antituberculosis treatment. There was no recurrence of symptoms on further follow up.

Distribution of Common and Rare Genetic Markers of Second-Line-Injectable-Drug Resistance in Mycobacterium tuberculosis Revealed by a Genome-Wide Association Study.

Point mutations in the rrs gene and the eis promoter are known to confer resistance to the second-line injectable drugs (SLIDs) amikacin (AMK), capreomycin (CAP), and kanamycin (KAN). While mutations in these canonical genes confer the majority of SLID resistance, alternative mechanisms of resistance are not uncommon and threaten effective treatment decisions when using conventional molecular diagnostics. In total, 1,184 clinical Mycobacterium tuberculosis isolates from 7 countries were studied for genomic markers associated with phenotypic resistance. The markers rrs:A1401G and rrs:G1484T were associated with resistance to all three SLIDs, and three known markers in the eis promoter (eis:G-10A, eis:C-12T, and eis:C-14T) were similarly associated with kanamycin resistance (KAN-R). Among 325, 324, and 270 AMK-R, CAP-R, and KAN-R isolates, 274 (84.3%), 250 (77.2%), and 249 (92.3%) harbored canonical mutations, respectively. Thirteen isolates harbored more than one canonical mutation. Canonical mutations did not account for 103 of the phenotypically resistant isolates. A genome-wide association study identified three genes and promoters with mutations that, on aggregate, were associated with unexplained resistance to at least one SLID. Our analysis associated whiB7 5'-untranslated-region mutations with KAN resistance, supporting clinical relevance for this previously demonstrated mechanism of KAN resistance. We also provide evidence for the novel association of CAP resistance with the promoter of the Rv2680-Rv2681 operon, which encodes an exoribonuclease that may influence the binding of CAP to the ribosome. Aggregating mutations by gene can provide additional insight and therefore is recommended for identifying rare mechanisms of resistance when individual mutations carry insufficient statistical power.

Target-mediated competitive hybridization of hairpin probes for kanamycin detection based on exonuclease III cleavage and DNAzyme catalysis.

Based on aptamer recognition and target-mediated competitive hybridization of hairpin probes, we developed a fluorescence sensor for kanamycin (KAN) detection. The aptamer and KAN binding will open hairpin H1 to release the trigger DNA fragment, which can initiate the competitive hybridization between hairpins H2 and H3. Then, exonuclease III (Exo III) can cleave H2 and H3 to produce numerous DNA3 and DNA4. Through the synergetic hybridization among DNA1, DNA2, DNA3, and DNA4, an active Mg2+-DNAzyme can be formed. The cleavage reaction toward FAM-BHQ-modified DNA2 will produce a high fluorescence signal for KAN assay. Through Exo III-guided cleavage and Mg2+-DNAzyme-based catalysis, the sensor exhibits high sensitivity, with a detection limit of 3.1 fM. This method is robust and has been applied to the detection of KAN in milk and water samples with good accuracy and reliability. Our developed fluorescence sensor exhibits the advantages of simple operation, high sensitivity, and good robustness, which are beneficial for KAN detection in food samples.

Development of label-free fluorescent biosensor for the detection of kanamycin based on aptamer capped metal-organic framework.

The abuse of antibiotics has caused serious threat to human health, so it is of great significance to develop a simple and sensitive method for the detection of trace residues of antibiotics in the environment and food. Herein, a novel label-free fluorescent biosensing platform based on the fluorescence change of aptamers-capped zeolitic imidazolate framework-8 (ZIF-8) @ 2,2',2″,2‴-((ethene-1,1,2,2-tetrayltetrakis (benzene-4,1-diyl)) tetrakis (oxy)) tetraacetic acid (TPE) through ATP-assisted competitive coordination reaction was designed for such an end. ZIF-8@TPE/Aptamer (Apt) emits strong fluorescence at 425 nm in HEPES buffer due to the aggregation induced luminescence properties of TPE molecules in confined state. Once kanamycin was added, the conformation of aptamer capped on the surface of ZIF-8@TPE changes because of the specific recognition of kanamycin with aptamer, leading to the collapse of ZIF-8 and release of TPE, accompanied with a dramatic decrease of fluorescence intensity. Under the optimal conditions, a good correlation was obtained between the fluorescence intensity of ZIF-8@TPE/Apt and the concentration of kanamycin ranging from 10 to 103 ng/mL with a detection limit of 7.3 ng/mL. The satisfactory analytical performance of the assay for kanamycin detection suggests good prospect for its application in food safety analysis.

Aminoglycoside treatment alters hearing-related genes and depicts behavioral defects in Drosophila.

The hearing organ of Drosophila is present within the second segment of antennae. The hearing organ of Drosophila (Johnston's organ [JO]) shares much structural, developmental, and functional similarity with the vertebrate hearing organ (Organ of Corti). JO is evolving as a potential model system to examine the hearing-associated defects in vertebrates. In the vertebrates, aminoglycosides like gentamicin, kanamycin, and neomycin have been known to cause defects in the hearing organ. However, a complete mechanism of toxicity is not known. Taking the evolutionary conservation into account the current study aims to test various concentrations of aminoglycoside on the model organism, Drosophila melanogaster. The current study uses the oral route to check the toxicity of various aminoglycosides at different concentrations (50, 100, 150, 200, and 250 µg ml- 1 ). In Drosophila, many foreign particles enter the body through the gut via food. The aminoglycoside treated third instar larvae show defective crawling and sound avoidance behavior. The adult flies release lower amounts of acetylcholine esterase and higher amounts of reactive oxygen species than control untreated animals, accompanied by defective climbing and aggressive behavior. All these behavioral defects are further confirmed by the altered expression level of hearing genes such as nompC, inactive, nanchung, pyrexia. All the behavioral and genetic defects are reported as a readout of aminoglycoside toxicity.

Single-step antibiotic-mediated synthesis of kanamycin-conjugated gold nanoparticles for broad-spectrum antibacterial applications.

Widespread and irrational use of antibiotics results in the development of antibiotic-resistant bacteria. Thus, there is a need to develop novel antibacterial agents in order to replace conventional antibiotics and to increase the efficacy of already existing antibiotics by combining them with other materials. Herein, a single-step antibiotic-mediated synthesis of antibiotic-conjugated gold nanoparticles is reported. In this single-step method antibiotic Kanamycin, an aminoglycoside itself plays the role of reducing as well as capping agent by reducing gold salt into gold nanoparticles. The kanamycin-conjugated gold nanoparticles (Kan-AuNPs) were confirmed by UV-Visible spectroscopy and further physico-chemically characterized by various instrumental techniques. Synthesized Kan-AuNPs showed broad-spectrum antibacterial activity against Gram-positive Staphylococcus aureus as well as Gram-negative Escherichia coli bacterial strains. They are also found to be effective against Pseudomonas aeruginosa and pathogenic E. coli isolated from urinary tract infections (UTIs) patients, which are responsible to cause hospital-acquired infections like nosocomial, burn wound and UTIs. The minimum inhibitory concentration (MIC) of Kan-AuNPs is 50 µg ml-1 for S. aureus and E. coli, 125 µg ml-1 for P. aeruginosa and 100 µg ml-1 for E. coli isolated from UTIs patients. It is also evident that the MIC of Kan-AuNPs for antibacterial activity is lower as compared to antibiotic kanamycin alone for all bacterial strains. Hence, the one-step strategy of synthesis for Kan-AuNPs is a suitable strategy for fighting infectious bacterial strains in hospitals, healthcare and the pharmaceutical industry.

Alarmone Ap4A is elevated by aminoglycoside antibiotics and enhances their bactericidal activity.

Second messenger molecules play important roles in the responses to various stimuli that can determine a cell's fate under stress conditions. Here, we report that lethal concentrations of aminoglycoside antibiotics result in the production of a dinucleotide alarmone metabolite-diadenosine tetraphosphate (Ap4A), which promotes bacterial cell killing by this class of antibiotics. We show that the treatment of Escherichia coli with lethal concentrations of kanamycin (Kan) dramatically increases the production of Ap4A. This elevation of Ap4A is dependent on the production of a hydroxyl radical and involves the induction of the Ap4A synthetase lysyl-tRNA synthetase (LysU). Ectopic alteration of intracellular Ap4A concentration via the elimination of the Ap4A phosphatase diadenosine tetraphosphatase (ApaH) and the overexpression of LysU causes over a 5,000-fold increase in bacterial killing by aminoglycosides. This increased susceptibility to aminoglycosides correlates with bacterial membrane disruption. Our findings provide a role for the alarmone Ap4A and suggest that blocking Ap4A degradation or increasing its synthesis might constitute an approach to enhance aminoglycoside killing potency by broadening their therapeutic index and thereby allowing lower nontoxic dosages of these antibiotics to be used in the treatment of multidrug-resistant infections.