Smartphone-based fluorescence detection is a promising avenue for biosensing that can aid on-site analysis. However, quantitative detection with fluorescence in the field has been limited due to challenges with robust excitation and calibration requirements. Here, we show that ratiometric analysis with Förster resonance energy transfer (FRET) between dye pairs on DNA aptamers can enable rapid and sensitive kanamycin detection. Since our detection scheme relies on ligand binding-induced changes in the aptamer tertiary structure, it is limited only by the kinetics of ligand binding to the aptamer. Our FRET-based kanamycin binding aptamer (KBA) sensor displays two linear ranges of 0.05-5 nM (detection limit of 0.18 nM) and 50-900 nM of kanamycin. The aptamer displays high specificity even in the presence of the 'natural' background from milk. By immobilizing the aptamer in the flow cell, our KBA sensor design is also suitable for repeated kanamycin detection. Finally, we show that the ratiometric FRET-based analysis can be implemented on a cheap custom-built smartphone setup. This smartphone-based FRET aptamer scheme detects kanamycin in a linear range of 50-500 nM with a limit of detection (LOD) of 28 nM.

The time course of aminoglycoside neurotoxic effect on cochlear nucleus is still obscure. We examined dynamic pathological changes of dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was upregulated in the neurotoxic course of kanamycin on DCN after kanamycin treatment. Rats were treated with kanamycin sulfate/kg/day at a dose of 500mg by subcutaneous injection for 10 days. Dynamic pathological changes, neuron density and neuron apoptosis of the DCN were examined at 1, 7, 14, 28, 56, 70 and 140 days after kanamycin treatment. The expressions of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1 were also detected. Under transmission electron microscopy, the mitochondrial swelling and focal vacuoles as well as endoplasmic reticulum dilation were progressively aggravated from 1 day to 14 days, and gradually recovered from 28 days to 140 days. Meanwhile, both autophagosomes and autolysosomes were increased from 1 day to 56 days. Only few neurons were positive to the TUNEL staining. Moreover, neither the expressions of caspase-3 and DAPK2 nor neurons density of DCN changed significantly. LC3-II was drastically increased at 7 days. Beclin-1 was upgraded at 1 and 7 days. P-Bcl-2 increased at 1, 7, 14 and 28 days. JNK1 increased at 7 days, and Bcl-2 was downgraded at 140 days. LC3-B positive neurons were increased at 1, 7 and 14 days. These data demonstrated that the neurons damage of the DCN caused by kanamycin was reversible and autophagy was upregulated in the neurotoxic course of kanamycin on DCN through JNK1-mediated phosphorylation of Bcl-2 pathway.

Different formulation techniques have been investigated to prepare highly aerosolizable dry powders to deliver a high dose of antibiotics to the lung for treating local infections. In this study, we investigated the influence of the co-amorphization of a model drug, kanamycin, with selected amino acids (valine, methionine, phenylalanine, and tryptophan) by co-spray drying on its aerosolization. The co-amorphicity was confirmed by thermal technique. The physical stability was monitored using low-frequency Raman spectroscopy coupled with principal component analysis. Except for the kanamycin-valine formulation, all the formulations offered improved fine particle fraction (FPF) with the highest FPF of 84% achieved for the kanamycin-methionine formulation. All the co-amorphous formulations were physically stable for 28 days at low relative humidity (25 °C/<15% RH) and exhibited stable aerosolization. At higher RH (53%), even though methionine transformed into its crystalline counterpart, the kanamycin-methionine formulation offered the best aerosolization stability without any decrease in FPF. While further studies are warranted to reveal the underlying mechanism, this study reports that the co-amorphization of kanamycin with amino acids, especially with methionine, has the potential to be developed as a high dose kanamycin dry powder formulation.

In view of the theory that alpha-lipoic acid effectively prevents cochlear cells from injury caused by various factors such as cisplatin and noise, this study examined whether alpha-lipoic acid can prevent kanamycin-induced ototoxicity. To this end, healthy BALB/c mice were injected subcutaneously with alpha-lipoic acid and kanamycin for 14 days. Auditory brainstem response test showed that increased auditory brainstem response threshold shifts caused by kanamycin were significantly inhibited. Immunohistochemical staining and western blot analysis showed that the expression of phosphorylated p38 mitogen-activated protein kinase and phosphorylated c-Jun N-terminal kinase in mouse cochlea was significantly decreased. The experimental findings suggest that phosphorylated p38 and phosphorylated c-Jun N-terminal kinase mediated kanamycin-induced ototoxic injury in BALB/c mice. Alpha-lipoic acid effectively attenuated kanamycin ototoxicity by inhibiting the kanamycin-induced high expression of phosphorylated p38 and phosphorylated c-Jun N-terminal kinase.

Chemical modification of old drugs is an important way to obtain new ones, and it has been widely used in developing new aminoglycoside antibiotics. However, many of the previous modifying strategies seem arbitrary for their lack of support from structural biological detail. In this paper, based on the structural information of aminoglycoside and its drug target, we firstly analyzed the reason that some 2'-N-acetylated products of aminoglycosides caused by aminoglycoside-modifying enzyme AAC(2') can partially retain activity, and then we designed, synthesized, and evaluated a series of 2'-modified kanamycin A derivatives. Bioassay results showed our modifying strategy was feasible. Our study provided valuable structure-activity relationship information, which would help researchers to develop new aminoglycoside antibiotics more effectively.

With a sharp increase in the cases of multi-drug resistant (MDR) bacteria all over the world, there is a huge demand to develop a new generation of antibiotic agents to fight them. As an alternative to the traditional drug discovery route, we have designed an effective antibacterial agent by modifying an existing commercial antibiotic, kanamycin, conjugated on the surface of gold nanoparticles (AuNPs). In this study, we report a single-step synthesis of kanamycin-capped AuNPs (Kan-AuNPs) utilizing the combined reducing and capping properties of kanamycin. While Kan-AuNPs have increased toxicity to a primate cell line (Vero 76), antibacterial assays showed dose-dependent broad spectrum activity of Kan-AuNPs against both Gram-positive and Gram-negative bacteria, including Kanamycin resistant bacteria. Further, a significant reduction in the minimum inhibitory concentration (MIC) of Kan-AuNPs was observed when compared to free kanamycin against all the bacterial strains tested. Mechanistic studies using transmission electron microscopy and fluorescence microscopy indicated that at least part of Kan-AuNPs increased efficacy may be through disrupting the bacterial envelope, resulting in the leakage of cytoplasmic content and the death of bacterial cells. Results of this study provide critical information about a novel method for the development of antibiotic capped AuNPs as potent next-generation antibacterial agents.

The cell-penetrating peptide GV1001 has been investigated as an anticancer agent and recently demonstrated anti-oxidant and anti-inflammatory effects. It has shown a protective effect on a kanamycin (KM)-induced ototoxicity mouse model. In the present study, we administered GV1001 at different time points after inducing hair cell damage, and examined if it rescues hair cell loss and restores hearing. A deaf mouse model was created by intraperitoneal injection of KM and furosemide. First, to test the early temporal change of hearing and extent of hair cell damage after KM and furosemide injection, hearing and outer hair cells (OHCs) morphology were evaluated on day 1, day 2 and day 3 after injection. In the second experiment, following KM and furosemide injection, GV1001, dexamethasone, or saline were given for three consecutive days at different time points: D0 group (days 0, 1, and 2), D1 group (days 1, 2, and 3), D3 group (days 3, 4, and 5) and D7 group (days 7, 8, and 9). The hearing thresholds were measured at 8, 16, and 32 kHz before ototoxic insult, and 7 days and 14 days after KM and furosemide injection. After 14 days, each turn of the cochlea was imaged to evaluate OHCs damage. GV1001-treated mice showed significantly less hearing loss and OHCs damage than the saline control group in the D0, D1 and D3 groups (p < 0.0167). However, there was no hearing restoration or intact hair cell in the D7 group. GV1001 protected against cochlear hair cell damage, and furthermore, delayed administration of GV1001 up to 3 days rescued hair cell damage and hearing loss in KM/furosemide-induced deaf mouse model.

Monoclonal antibody against kanamycin was prepared, and competitive direct ELISA and immunochromatographic assay were developed using the antibody to detect kanamycin in animal plasma and milk. The monoclonal antibody produced was identified to be IgG1, which has a kappa light chain. No cross-reactivity of the antibody was detected with other aminoglycosides, indicating that the monoclonal antibody was highly specific for kanamycin. Based on competitive direct ELISA, the detection limits of kanamycin were determined to be 1.1 ng/ml in PBS, 1.4 ng/ml in plasma, and 1.0 ng/ml in milk. The concentration of intramuscularly injected kanamycin was successfully monitored in rabbit plasma with competitive direct ELISA. Based on the colloidal gold-based immunochromatographic assay, the detection limits of kanamycin were estimated to be about 6-8 ng/ml in PBS, plasma, and milk. The immunochromatographic assay would be suitable for rapid and simple screening of kanamycin residues in veterinary medicine. Screened positives can be confirmed using a more sensitive laboratory method such as competitive direct ELISA. Therefore, the assays developed in this study could be used to complement each other as well as other laboratory findings. Moreover, instead of slaughtering the animals to obtain test samples, these methods could be applied to determine kanamycin concentration in the plasma of live animals.

The effect of statins on aminoglycoside-induced ototoxicity is controversial. This study aimed to explore the role of pravastatin (PV) in kanamycin-induced hearing loss in rats. Adult rats were intraperitoneally treated with 20 mg/kg/day of kanamycin (KM) for 10 days. In the PV- and PV + KM-treated rats, 25 mg/kg/day of PV was intraperitoneally administered for 5 days. The auditory brainstem response (ABR) thresholds were measured before and after drug treatment using a smartEP system at 4, 8, 16, and 32 kHz. Cochlear changes in poly ADP-ribose (PAR) polymerase (PARP), PAR, and caspase 3 were estimated using Western blotting. PV administration did not increase the ABR thresholds. The KM-treated rats showed elevated ABR thresholds at 4, 8, 16, and 32 kHz. The PV + KM-treated rats demonstrated lower ABR thresholds than the KM-treated rats at 4, 8, and 16 kHz. The cochlear outer hair cells and spiral ganglion cells were relatively preserved in the PV + KM-treated rats when compared with that in the KM-treated rats. The cochlear expression levels of PARP, PAR, and caspase 3 were higher in the KM-treated rats. The PV + KM-treated rats showed lower levels of PARP, PAR, and caspase 3 than the KM-treated rats. PV protected cochleae from KM-induced hearing loss in rats. The regulation of autophagy and apoptosis mediated the otoprotective effects of PV.

The dinucleotide alarmone diadenosine tetraphosphate (Ap4A), which is found in cells, has been shown to affect the survival of bacteria under stress.

Aminoglycosides have detrimental effects on the hair cells of the inner ear, yet these agents indisputably are one of the cornerstones in antibiotic therapy. Hence, there is a demand for strategies to prevent aminoglycoside-induced ototoxicity, which are not available today. In vitro data suggests that the pleiotropic growth factor erythropoietin (EPO) is neuroprotective against aminoglycoside-induced hair cell loss. Here, we use a mouse model with EPO-overexpression in neuronal tissue to evaluate whether EPO could also in vivo protect from aminoglycoside-induced hearing loss. Auditory brainstem response (ABR) thresholds were measured in 12-weeks-old mice before and after treatment with kanamycin for 15 days, which resulted in both C57BL/6 and EPO-transgenic animals in a high-frequency hearing loss. However, ABR threshold shifts in EPO-transgenic mice were significantly lower than in C57BL/6 mice (mean difference in ABR threshold shift 13.6 dB at 32 kHz, 95% CI 3.8-23.4 dB, p = 0.003). Correspondingly, quantification of hair cells and spiral ganglion neurons by immunofluorescence revealed that EPO-transgenic mice had a significantly lower hair cell and spiral ganglion neuron loss than C57BL/6 mice. In conclusion, neuronal overexpression of EPO is protective against aminoglycoside-induce hearing loss, which is in accordance with its known neuroprotective effects in other organs, such as the eye or the brain.

In this work, an edible cellulose-based antibacterial material was prepared by cross-linking α-cellulose and kanamycin sulfate via glutaraldehyde to form kanamycin sulfate-glutaraldehyde-cellulose. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and X-ray diffraction results indicated that the kanamycin sulfate molecule was cross-linked with the molecular chain of cellulose. The optimal mass ratio of kanamycin sulfate to α-cellulose was 1:100 and the degree of substitution reached 1.11%. The optimal kanamycin sulfate-glutaraldehyde-cellulose material showed an excellent inhabitation against both Gram-positive and Gram-negative bacteria. Meantime, the optimal kanamycin sulfate-glutaraldehyde-cellulose had a marked resistance to gastric acid and had low cell cytotoxicity. To promote the application of the kanamycin sulfate-glutaraldehyde-cellulose material, the porous microspheres were prepared via the sol-gel method. The particle size of the homogeneous porous microspheres is mainly distributed between 1.5 and 2.0 μm. Therefore, the kanamycin sulfate-glutaraldehyde-cellulose described herein is a potential edible, eco-friendly, potent, stable, inexpensive, and antibacterial carrier material for delivering drugs, proteins, or vaccines.

Resistance to antibiotics has become a serious problem for society, and there are increasing efforts to understand the reasons for and sources of resistance. Bacterial-encoded enzymes and transport systems, both innate and acquired, are the most frequent culprits for the development of resistance, although in Mycobacterium tuberculosis, the catalase-peroxidase, KatG, has been linked to the activation of the antitubercular drug isoniazid. While investigating a possible link between aminoglycoside antibiotics and the induction of oxidative bursts, we observed that KatG reduces susceptibility to aminoglycosides. Investigation revealed that kanamycin served as an electron donor for the peroxidase reaction, reducing the oxidized ferryl intermediates of KatG to the resting state. Loss of electrons from kanamycin was accompanied by the addition of a single oxygen atom to the aminoglycoside. The oxidized form of kanamycin proved to be less effective as an antibiotic. Kanamycin inhibited the crystallization of KatG, but the smaller, structurally related glycoside maltose did cocrystallize with KatG, providing a suggestion as to the possible binding site of kanamycin.

TolC is a member of the outer membrane efflux proteins (OEPs) family and acts as an exit duct to export proteins, antibiotics, and substrate molecules across the Escherichia coli cell membrane. Export of these molecules is evidenced to be brought about through the reversible interactions and binding of substrate-specific drug molecules or antibiotics with TolC and by being open for transport, which afterward leads to cross-resistance. Hence, the binding of kanamycin with TolC was monitored through molecular docking (MD), the structural fluctuations and conformational changes to the atomic level. The results were further supported from the steady-state fluorescence binding and isothermal titration calorimetry (ITC) studies. Binding of kanamycin with TolC resulted in a concentration dependent fluorescence intensity quenching with 7 nm blue shift. ITC binding data maintains a single binding site endothermic energetic curve with binding parameters indicating an entropy driven binding process. The confirmational changes resulting from this binding were monitored by a circular dichroism (CD) study, and the results showed insignificant changes in the α-helix and β-sheets secondary structure contents, but the tertiary structure shows inclusive changes in the presence of kanamycin. The experimental data substaintially correlates the RMSD, R g, and RMSF results. The resulting conformational changes of the TolC-kanamycin complexation was stabilized through H-bonding and other interactions.

Kanamycin (KM) and amikacin (AK) are the key aminoglycoside drugs against tuberculosis (TB) and resistance to them severely affects the options for treatment. Many explanations have been proposed for drug resistance to these drugs but still some mechanisms are unknown. Proteins are the functional moiety of the cell and manifest in most of the biological processes; so, these are potential foci for the development of new therapeutics, diagnostics and vaccine. We examined the KM and AK resistant isolates of Mycobacterium tuberculosis using proteomic analysis comprising of two dimensional gel electrophoresis (2DGE), matrix assisted laser desorption ionization time-of-flight/time-of flight (MALDI-TOF/TOF) and bioinformatic tools like BLASTP, InterProScan, KEGG motif scan and molecular docking. Proteins intensities of twelve spots were found to be consistently increased in KM and AK resistant isolates and these were identified as Rv3867, Rv1932, Rv3418c, Rv1876, Rv2031c, Rv0155, Rv0643c, Rv3224, Rv0952, and Rv0440. Among these, Rv3867 and Rv3224 were identified as proteins with unknown function. All the proteins identified were cellular proteins. Molecular docking shows the proper interaction of both drugs with these molecules. Also, Rv1876 and Rv3224 were found to be probably involved in iron regulation/metabolism indicating the role of iron in imparting resistance to second line drugs.

Resistance to anti-tuberculosis drugs is a formidable obstacle to effective tuberculosis (TB) treatment and prevention globally. New forms of multidrug, extensive drug and total drug resistance Mycobacterium tuberculosis (Mtb) causing a serious threat to human as well as animal's population. Mtb shows diverse adaptability under stress conditions especially antibiotic treatment, however underlying physiological mechanism remained elusive. In present study, we investigated Mtb's response and adaptation with reference to gene expression during sub-lethal kanamycin exposure. Mtb were cultured under sub-lethal drug and control conditions, where half were sub-cultured every 3-days to observe serial adaptation under same conditions and the remaining were subjected to RNA-seq. We identified 98 up-regulated and 198 down-regulated responsive genes compared to control through differential analysis, of which Ra1750 and Ra3160 were the most responsive genes. In adaptive analysis, we found Ra1750, Ra3160, Ra3161, Ra3893 and Ra2492 up-regulation at early stage and gradually showed low expression levels at the later stages of drug exposure. The adaptive expression of Ra1750, Ra3160 and Ra3161 were further confirmed by real time qPCR. These results suggested that these genes contributed in Mtb's physiological adaptation during sub-lethal kanamycin exposure. Our findings may aid to edify these potential targets for drug development against drug resistance tuberculosis.

The occurrence of opportunistic local infections and improper integration of metallic implants results in severe health conditions. Protective and tunable coatings represent an attractive and challenging selection for improving the metallic devices' biofunctional performances to restore or replace bone tissue. Composite materials based on hydroxyapatite (HAp), Kanamycin (KAN), and fibroblast growth factor 2 (FGF2) are herein proposed as multifunctional coatings for hard tissue implants. The superior cytocompatibility of the obtained composite coatings was evidenced by performing proliferation and morphological assays on osteoblast cell cultures. The addition of FGF2 proved beneficial concerning the metabolic activity, adhesion, and spreading of cells. The KAN-embedded coatings exhibited significant inhibitory effects against bacterial biofilm development for at least two days, the results being superior in the case of Gram-positive pathogens. HAp-based coatings embedded with KAN and FGF2 protein are proposed as multifunctional materials with superior osseointegration potential and the ability to reduce device-associated infections.

Aeromonas hydrophila is a widespread opportunistic pathogen of aquatic fishes in freshwater habitats. The current emergence of antimicrobial-resistant A. hydrophila has been reported in the world while the bacterial antibiotics adaptive mechanism remains poorly explored. In this study, using quantitative proteomics technology, the behavior of A. hydrophila was investigated by comparing the differentially expression proteins between with and without kanamycin (KAN) treatment. A total of 374 altered proteins including 184 increasing and 190 proteins decreasing abundances were quantified when responding to KAN stress. The bioinformatics analysis showed that stress related proteins were hub proteins that significantly increased to reduce the pressure from the misreading of mRNA caused by KAN. Moreover, several metallic pathways, such as oxidative phosphorylation and TCA cycle pathways may affect KAN resistance. Finally, eight selected genes were deleted and their antibiotics susceptibilities to kanamycin were valued, respectively. Results showed that OmpA II family protein A0KI26, and two-component system protein AtoC may involve in the KAN resistance in this study. In general, our results provide an insight into the behaviors of bacterial responding to KAN stress, and demonstrate the intrinsic antibiotics adaptive mechanism of A. hydrophila. BIOLOGICAL SIGNIFICANCE: In this study, the differentially expressed proteins (DEPs) of A. hydrophila strain between with and without kanamycin (KAN) were compared by using a data-independent acquisition (DIA) - based quantitative proteomics method. Bioinformatics analysis showed that stress - related proteins are hub proteins that significantly increased under KAN stress. Moreover, several metallic pathways, such as oxidative phosphorylation and citrate cycle (TCA cycle) pathways, can affect KAN resistance. Finally, our antibiotics susceptibility assay showed that the protein A0KI26 of the OmpA II family, and the AtoC of the two-component system may involve in KAN resistance in this study. These results provide insights into the antibiotics adaptation mechanism of A. hydrophila when responding to KAN stress.

Kanamycin, an injectable agent, is currently used to treat drug-resistant tuberculosis (TB). Parenteral kanamycin causes high systemic toxicity which could be avoided by direct delivery to the lungs. This study focused on producing a highly aerosolizable dry-powder of hygroscopic kanamycin by spray-drying with l-leucine.

The structure-switching aptamers are designed for the simple and rapid detection of kanamycin based on the signal transduction principle of fluorescence resonance energy transfer (FRET). The structure switch is composed of kanamycin-binding aptamers and the complementary strands, respectively labeled with fluorophore and quencher, denoted as FDNA and QDNA. In the absence of kanamycin, FDNA and QDNA form the double helix structure through the complementary pairing of bases. The fluorophore and the quencher are brought into close proximity, which results in the fluorescence quenching because of the FRET mechanism. In the presence of kanamycin, the FDNA specifically bind to the target due to the high affinity of aptamers, and the QDNA are dissociated. The specific recognition between aptamers and kanamycin will obstruct the formation of structure switch and reduce the efficiency of FRET between FDNA and QDNA, thus leading to the fluorescence enhancement. Therefore, based on the structure-switching aptamers, a simple fluorescent assay for rapid detection of kanamycin was developed. Under optimal conditions, there was a good linear relationship between kanamycin concentration and the fluorescence signal recovery. The linear range of this method in milk samples was 100-600 nM with the detection limit of 13.52 nM (3σ), which is well below the maximum residue limit (MRL) of kanamycin in milk. This method shows excellent selectivity for kanamycin over the other common antibiotics. The structure-switching aptamers have been successfully applied to the detection of kanamycin spiked in milk samples with the satisfying recoveries between 101.3 and 109.1%, which is well-consistent with the results from LC-MS/MS. Due to the outstanding advantages of facile operation, rapid detection, high sensitivity, excellent specificity, and low cost, the application and extension of this strategy for rapid determination of antibiotics in food samples may greatly improve the efficiency in food safety and quality supervision.