Therapeutic effect and immune mechanism of chitosan-gentamicin conjugate on Pacific white shrimp (Litopenaeus vannamei) infected with Vibrio parahaemolyticus.

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.

Green one-pot synthesis of multicomponent-crosslinked carboxymethyl cellulose as a safe carrier for the gentamicin oral delivery.

Multicomponent reactions (MCRs) as a green strategy was used to crosslink carboxymethylcellulose (CMC) via the construction of bioactive carboxamide skeletons for the gentamicin (GM) oral delivery. The prepared multicomponent-crosslinked (MCC) CMC hydrogels (CMC-MCC) were fully characterized using various methods. To explain the productivity of the prepared system, GM delivery tests performed in the simulated gastrointestinal tract (GIT) conditions. CMC-MCC could efficiently protect the loaded-GM against the acidic environment of the stomach and enhance the sustainability of drug dosing with controlling the releases in the GIT circumstances. The antibacterial performance showed that the GM-loaded CMC-MCC has notable activity against S. aureus and E. coli bacteria. Besides, the cytotoxicity study indicated that CMC-MCC toward healthy normal human umbilical vein endothelial cells (HUVEC) has a cytocompatibility nature.

Synthesis of Gentamicin Minor Components: Gentamicin B1 and Gentamicin X2.

The clinical aminoglycoside antibiotic gentamicin is a mixture of several difficult-to-separate major and minor components. The relative inaccessibility of the minor components in particular complicates efforts to separate antibacterial activity from nephro- and/or ototoxicity and to clarify the origin of the potentially therapeutically important read-through activity. With a view to facilitating such studies, the synthesis of a fully and selectively protected garamine-based acceptor has been developed from readily available sisomicin. Glycosylation of this acceptor with a 6-azido-6,7-dideoxy-d-glycero-d-glucoheptopyranosyl donor affords gentamicin B1 after deprotection, whereas employment of a 2-azido-2-deoxy-d-glucopyranosyl donor under N,N-dimethylformamide-directed glycosylation conditions affords gentamicin X2 after deprotection.

Enhancing the antibacterial efficacy of low-dose gentamicin with 5 minute assistance of photothermy at 50 °C.

Implant materials are prone to bacterial infections and cause serious consequences, while traditional antibiotic therapy has a long treatment cycle and even causes bacterial resistance. In this work, a photothermal therapy (PTT) assisted drug release system has been developed on the implant surface for in situ rapid disinfection under 808 nm light irradiation within a short time, in which gentamicin (Gent) is loaded by polyethylene glycol (PEG) modified molybdenum disulfide (MoS2) on Ti surface, and then encapsulated with chitosan (CS) (CS/Gent/PEG/MoS2-Ti). The hyperthermia produced by the coatings irradiated by 808 nm near-infrared (NIR) light can not only accelerate the local release of Gent, but also reduce the activity of bacteria, which makes it easy for these locally released drugs to enter the interior of the bacteria to inhibit the protein synthesis and destroy the cell membrane. When maintained at 50 °C for 5 min under NIR irradiation, this system can achieve an antibacterial efficacy of 99.93% and 99.19% against Escherichia coli and Staphylococcus aureus, respectively. By contrast, even after treatment for 120 min, only a 93.79% antibacterial ratio can be obtained for Gent alone. This is because hyperthermia produced from the coatings during irradiation can assist antibiotics in killing bacteria in a short time. Even under a low dose of 2 µg mL-1, the photothermal effect assisted gentamicin can achieve an antibacterial efficacy of 96.86% within 5 min. In vitro cell culture shows that the modified surface can facilitate cell adhesion, spreading and proliferation. The 7 day subcutaneous infection model confirms that the prepared surface system can exhibit a much faster sterilization and tissue reconstruction than the control group with light assistance. Compared with the traditional drug release system, this photothermy controlled drug-loaded implant surface system can not only provide rapid and high-efficiency in situ sterilization, but also offer long-term prevention of local bacterial infection.

Hollow Mesoporous Silica@Zeolitic Imidazolate Framework Capsules and Their Applications for Gentamicin Delivery.

We have synthesized hollow mesoporous silica (HMS) at a zeolitic imidazolate framework (ZIF) capsule that can be used as a drug delivery system for gentamicin (GM). The GM is first loaded into HMS. Then, the outer surface of the GM/HMS is coated with uniformed ZIF nanoparticles (denoted as GM/HMS@ZIF). The GM/HMS@ZIF has been successfully prepared and acts as a capsule for GM. The GM/HMS@ZIF shows a good biocompatibility and a good cellular uptake in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. The GM is released slowly within 10 h under acidic conditions, which is used to simulate the pH of the endosome and lysosome compartments. The in vivo assay shows that the signal from fluorescein isothiocyanate (FITC) can be observed after 15 days, when the mice were injected with FITC/HMS@ZIF. This opens new opportunities to construct a delivery system for GM via one controlled low dose and sustained release for the therapy of Ménière's disease.

Electrophoretic Deposition of Gentamicin-Loaded Silk Fibroin Coatings on 3D-Printed Porous Cobalt-Chromium-Molybdenum Bone Substitutes to Prevent Orthopedic Implant Infections.

In addition to customizing shapes of metal bone substitutes for patients, the 3D printing technique can reduce the modulus of the substitutes through the design and manufacture of interconnected porous structures, achieving the modulus match between substitute and surrounding bone to improve implant longevity. However, the porous bone substitutes take more risks of postoperative infection due to its much larger surface area compared with the traditional casting solid bone substitute. Here, we prepared of gentamicin-loaded silk fibroin coatings on 3D-printed porous cobalt-chromium-molybdenum (CoCrMo) bone substitutes via electrophoretic deposition technique. Through optimization, relatively intact, continuous, homogeneous, and conformal coatings with a thickness of 2.30 ± 0.58 µm were deposited around the struts with few pore blocked. The porous metal structures exhibited no loss in mechanical properties after the anode galvanic corrosion in EPD process. The initial osteoblastic response on coatings was better than that on metal surface, including cell spreading, proliferation and cytotoxicity. Antibacterial efficacy experiments showed that the coatings had an antibacterial effect on both adherent and planktonic bacteria within 1 week. These results suggested that the beneficial properties of anode electrophoretic deposited silk fibroin coatings could be exploited to improve the biological functionality of porous structures made of medical metals.

Effects of the sol-gel route on the structural characteristics and antibacterial activity of silica-encapsulated gentamicin.

The effects of sol-gel processes, i.e., acid-catalyzed gelation, base-catalyzed gelation and base-catalyzed precipitation routes, on the encapsulation of gentamicin were investigated. The resulting xerogels were characterized using a series of complementary instrumental techniques, i.e., the adsorption/desorption of nitrogen, small-angle X-ray scattering, Fourier transform infrared spectroscopy, diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy, atomic force microscopy and scanning electron microscopy. The encapsulated gentamicin samples were tested against a series of Gram-positive and Gram-negative bacterial strains. The best antimicrobial activity was observed with the encapsulated gentamicin that was prepared via the precipitation route, even in comparison with the neat antibiotic, especially in the case of the Gram-positive strain Staphylococcus aureus. The gentamicin concentration on the outermost surface and the zeta potential were identified as factors that affected the highest efficiency, as observed in the case of encapsulation via the base-catalyzed process.

Absence of intestinal colonization by vancomycin-resistant enterococci in nonhuman primate / Ausência de enterococos resistentes à vancomicina na microbiota intestinal de primatas não-humanos

The animal reservoirs of vancomycin-resistant enterococci (VRE) have important role in the epidemiology of the bacteria and resistant genes. The present work searched fecal samples taken off nonhuman primates for the presence of VRE. Resistance profiles, virulence traits, and genetic variability among enterococci isolates were also analyzed. The samples included Capuchin monkeys (Cebus apella, n=28) and Common marmoset (Callithrix penicillata, n=37) housed in the Primate Center of the University of Brasília, Brazil. Most individuals were captive monkeys from the Central-West and South-East regions of Brazil (n=48). We collected rectal swabs and carried out selective isolation followed by multiplex Polymerase Chain Reaction (PCR) to identify species and resistance genes. No vanA or vanB-containing enterococci were found. The carriage rates ranged from 1.5 percent for the VanC-type E. casseliflavus and E. gallinarum until 12.3 percent (n=8) for Enterococcus faecalis. All E. faecalis isolates showed susceptibility to vancomycin, teicoplanin, ampicillin, gentamicin, and streptomycin. The virulence genes ace and esp were prevalent (100.0 percent, 87.5 percent). Multilocus variable number of tandem repeats (MLVA) revealed diversity in the number of repeats among E. faecalis isolates and targets, which was higher for espC, efa5, and efa6. We identified six different MLVA genotypes that were divergent from those described in human beings. Also, they were clustered into two genogroups that showed host-specificity for the species Cebus apella or Callithrix penicillata. In conclusion, no vanA- or vanB-containing enterococci were found colonizing those primate individuals. This finding suggested that the primate individuals investigated in our study are not directly involved in the epidemiological chain of high-level vancomycin-resistant genes vanA or vanB in Brazil. Our study also showed that E. faecalis isolated from nonhuman primates carry virulence...

Os reservatórios animais de Enterococos Resistentes à Vancomicina (VRE) têm um importante papel na epidemiologia destas bactérias e dos respectivos genes de resistência. O presente estudo examinou a presença de VRE em amostras fecais obtidas de primatas não-humanos. Foram analisados os perfis de resistência, as características de virulência e a variabilidade genética dos isolados. A amostragem incluiu macacos Prego (Cebus apella, n=28) e Sagüis do cerrado (Callithrix penicillata, n=37) alojados no Centro de Primatologia da Universidade de Brasília, Brasil. A maioria dos indivíduos amostrados foram macacos apreendidos na região Centro-Oeste e Sudeste do Brasil (n=48). Assim, foram coletados swabs retais e realizado o isolamento seletivo, seguido da Reação de Polimerização em Cadeia (PCR) multiplex para identificar espécies e genes de resistência. Não foram isolados enterococos contendo os genes vanA ou vanB. A porcentagem de enterococos variou de 1,5 por cento para E. casseliflavus e E. gallinarum VanC até 12,3 por cento (n=8) para Enterococcus faecalis. A totalidade dos isolados da espécie E. faecalis demonstrou sensibilidade aos antimicrobianos vancomicina, teicoplanina, ampicilina, gentamicina e estreptomicina. Os genes de virulência ace e esp foram prevalentes (100 por cento, 87.5 por cento). A análise em multilocus de repetições em tandem de número variável (MLVA) revelou diversidade no número de repetições entre os isolados de E. faecalis, que foi mais alta para espC, efa5 e efa6. Foram identificados seis diferentes genotipos de MVLA, divergindo daqueles já descritos em humanos. Os genotipos foram ainda agrupados em dois genogrupos, demonstrando especificidade de hospedeiro para as espécies Cebus apella ou Callithrix penicillata. Concluindo, não foram isoladas linhagens de enterococos contendo os genes vanA ou vanB colonizando as espécies de primatas analisadas. O presente estudo demonstrou que os isolados de E. faecalis obtidos...

Ternary complexes of gentamicin with iron and lipid catalyze formation of reactive oxygen species.

This study was designed to elucidate the mechanisms underlying the formation of reactive oxygen species (ROS) by aminoglycoside antibiotics which may be causally related to the toxic side effects of these drugs to the kidney and the inner ear. ROS formation by aminoglycosides in vitro requires iron and the presence of polyunsaturated lipids as electron donors. Electron spray ionization mass spectrometry (ESI-MS) confirmed earlier observations that gentamicin strongly binds to L-alpha-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), a membrane lipid rich in arachidonic acid. Studies using lipid-coated membranes (PIP strips) further indicated that iron ions and gentamicin can simultaneously bind to phosphoinositides with at least one phosphate group on the inositol ring, suggesting the existence of ternary complexes among gentamicin, iron, and phospholipids. Peroxidation of PtdIns(4,5)P2 by ferrous ions significantly increased in the presence of gentamicin, and EI-MS measurements indicated that oxidative damage to PtdIns(4,5)P2 was accompanied by the release of arachidonic acid. Arachidonic acid also forms a ternary complex with Fe(2+/3+)-gentamicin, confirmed by ESI-MS, that reacts with lipid peroxides and molecular oxygen, leading to the propagation of arachidonic acid peroxidation.

Sulfated fucan as support for antibiotic immobilization.

Xylofucoglucuronan from Spatoglossum schröederi algae was tested as a support for antibiotic immobilization. The polysaccharide (20 mg in 6 ml) was first activated using carbodiimide, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide methiodide (20 mg in 2 ml), under stirring for 1 h at 25 masculine C and pH from 4.5 to 5.0. After adjusting the pH to 8.0, either gentamicin or amikacin (62.5 mg in 1.25 ml) was then immobilized on this chemically modified polysaccharide with shaking for 24 h in a cold room. Infrared spectra of the activated carbodiimide xylofucoglucuronan showed two bands to carbonyl (C=O at 1647.9 and 1700.7 cm(-1)) and to amide (C-NH2) groups (1662.8 and 1714.0 cm(-1)). Microbial characterization of the derivatives was carried out by the disk diffusion method using Staphylococcus aureus or Klebsiella pneumoniae incorporated in Müller Hinton medium. Inhibition halos of bacterial growth were observed for the antibiotics immobilized on this sulfated heteropolysaccharide before and after dialysis. However, the halos resulting from the samples after dialysis were much smaller, suggesting that dialysis removed either non-covalently bound antibiotic or other small molecules. In contrast, bacterial growth was not inhibited by either xylofucoglucuronan or its activated form or by gentamicin or amikacin after dialysis. An additional experiment was carried out which demonstrated that the sulfated heteropolysaccharide was hydrolyzed by the microorganism. Therefore, the antibiotic immobilized on xylofucoglucuronan can be proposed as a controlled drug delivery system. Furthermore, this sulfated heteropolysaccharide can be extracted easily from sea algae Spatoglossum schröederi.

Mucoadhesive microspheres containing gentamicin sulfate for nasal administration: preparation and in vitro characterization.

In this study, suitable microsphere formulations were designed in order to provide the absorption of a high polar drug through nasal mucosa. For this purpose, gentamicin sulfate (GS) was chosen as a model drug and used at different drug/polymer ratios in the microsphere formulations. The microspheres were prepared by spray drying technique. Hydroxypropyl methylcellulose was used as a mucoadhesive polymer in the formulations to increase the residence time of the microspheres on the mucosa. Sodium cholate was added into the formulations for increasing the absorption of GS through nasal mucosa. The in vitro characteristics of the microspheres were determined. The microspheres were evaluated with respect to the particle size, production yield, encapsulation efficiency, shape and surface properties, drug-polymer interaction, mucoadhesive property, in vitro drug release and suitability for nasal drug delivery.

N-[(2-Sulfo)-9-fluorenylmethoxycarbonyl](3)-gentamicin C(1) is a long-acting prodrug derivative.

Most low-molecular-weight drugs are short-lived species in the circulatory system, being rapidly eliminated by glomerular filtration in the kidney. However, binding to human serum albumin (HSA) can slow clearance and prolong lifetime profile in vivo. In this study, we have engineered a gentamicin derivative with affinity to albumin by linking three (2-sulfo)-9-fluorenylmethoxycarbonyl (FMS) to three amino groups of gentamicin C(1). FMS(3)-gentamicin associates with HSA with a K(a) value of (1.31 +/- 0.2) x 10(5) M(-1). It has less than 1% the antibacterial potency of native gentamicin. Upon incubation at pH 8.5 and 37 degrees C, the FMS moieties from FMS(3)-gentamicin undergo slow hydrolysis (t(1/2) = 8.0 +/- 0.2 h), leading to a linear regeneration of the antibacterial potency with a t(1/2) value of 11 +/- 0.7 h. FMS(3)-gentamicin is a long-lived species in the rat circulatory system. Following a single subcutaneous or intravenous administration, it maintains a prolonged pharmacokinetic profile with a peak and a "through" concentration of immuno/antibacterial active gentamicin exceeding 4-5 times the duration obtained by administered native gentamicin. To sum up, an approach aimed at elongating the lifetime of low-molecular-weight drugs in vivo has been examined here with gentamicin. Two to three FMS per mole of compound are to be introduced to obtain an albumin associating affinity of K(d) = 7.6-9.2 microM and, hence, to significantly extend the drug's lifetime in situ following administration. By use of this technology, the loss of pharmacological potency with derivatization is of no consequence, since FMS moieties are hydrolyzed and activity is generated at physiological conditions.

Aminoglycoside-arginine conjugates that bind TAR RNA: synthesis, characterization, and antiviral activity.

Regulation of HIV gene expression is crucially dependent on binding of the trans-activator protein, Tat, to the trans-activation response RNA element, TAR, found at the 5' end of all HIV-1 transcripts. Tat-TAR interaction is mediated by a short arginine-rich domain of the protein. Disruption of this interaction could, in theory, create a state of complete viral latency. A new class of small-molecule peptidomimetic TAR RNA binders, conjugates of aminoglycosides and arginine, was recently designed [Litovchick, A., Evdokimov, A. G., and Lapidot, A. (1999) FEBS Lett. 445, 73-79]. Two of these compounds, the tri-arginine derivative of gentamicin C (R3G) and the tetra-arginine derivative of kanamycin A (R4K), bind efficiently and specifically to TAR RNA. These compounds display negligible toxicity while being transported and accumulated in cell nuclei. Here we present a detailed synthesis and chemical characterization of the aminoglycoside-arginine conjugates R3G and R4K as well as GB4K, the tetra-gamma-guanidinobutyric derivative of kanamycin A. Their binding sites on TAR RNA were assigned by RNase A, uranyl nitrate, and lead acetate footprinting. The conjugates interact with TAR RNA in the widened major groove, formed by the UCU bulge and the neighboring base pairs of the upper stem portion of TAR, the binding site of Tat protein, and Tat-derived peptides (e.g., R52). Our results suggest an additional binding site of R4K and R3G compounds, in the lower stem-bulge region of TAR. The antiviral activity of the conjugates in cultured equine dermal fibroblasts infected with equine infectious anemia virus, used as a model system of HIV-infected cells, is also presented.

[Mechanical properties of new low viscosity bone cements--can be improved by vacuum mixing]. / Mechanische Eigenschaften neuer niedrigvisköser Knochenzemente--verbesserbar durch Evakuierung.

With the objective of improving mechanical properties by using a new catalyst, a new bone cement (Sulfix-60) and a new low-viscosity cement with added gentamycin (Allofix-G) were developed. Dynamic weakness was the major disadvantage of the older Sulfix-6 cement, especially in comparison with high-viscosity bone cement. Although static strength could be improved, investigations into improved fatigue strength and the effect of vacuum-mixing on the static and fatigue strength had yet to be carried out. To investigate fatigue stability specimens of the new bone cements were tested using loads under wet conditions until breakage or 20 million cycles. In a first series the cement was hand-mixed in a second series vacuum-mixed alone, and in a third series vacuum-mixed under pressure. Additionally, the ultimate static bending strength was investigated using the standard four-point bending test for the three series of bone cement. Vacuum-mixing brought about an improvement in the dynamic strength of Sulfix-60 from 6.3 MPa, to 9.1 MPa and for Allofix-G from 6.3 MPa to 8.2 MPa; additional pressure brought no further improvement. A 200% increased fatigue stability was detectable in comparison with the older cement. The ultimate bending strength was also significantly improved by vacuum-mixing for both bone cements.

Synthesis and antimicrobial and toxicological studies of amino acid and peptide derivatives of kanamycin A and netilmicin.

Amino acid and peptide derivatives of aminoglycosides have been obtained by substitution of the 1-N or 6'-N amino functions of kanamycin A and netilmicin via the temporary complexation of vicinal and nonvicinal amino and hydroxy functions by copper ion [1-N kanamycin A derivatives: L-Ala (6a), D-Ala (6b), Gly (6c), L-Asp (6d), L-Ala-L-Ala (6e). 6'-N kanamycin A derivatives: L-Ala (3a), D-Ala (3b), Gly (3c), L-Ala-L-Ala (3e), L-Leu (3f). 6'-N netilmicin derivatives: L-Ala (9a), D-Ala (9b), Gly (9c), L-Asp (9d), L-Ala-L-Ala (9e)]. Characterization was made by FAB-MS, IR, 1H-NMR, and 13C-NMR. All derivatives were essentially inactive. The nephrotoxic potential of the derivatives obtained in sufficient quantities (3b,e and 9a-e) was assessed by measuring their inhibitory potential toward the activity of lysosomal phospholipase A1 acting on phosphatidylcholine embedded in negatively-charged membranes. One compound, 6'-N-L-Ala-netilmicin (9a), showed a 2-fold decrease of inhibitory potency compared to its parent drug. A conformational analysis revealed that it adopts two equally probable conformations and orientations when interacting with phosphatidylinositol. The first in which the drug lies parallel to the hydrophobic-hydrophilic interface, is similar to that of netilmicin. The second, in which the drug inserts itself in the bilayer across the hydrophilic/hydrophobic interface, is similar to that described for streptomycin, an almost non-nephrotoxic aminoglycoside.

Role of the 1-amino group in aminocyclitol antibiotics: synthesis of 1-deaminogentamicin C2.

The synthesis of 1-deaminogentamicin C2 described here, uses 3,2',6',3"-tetrakis-N-tert-butoxycarbonylgentamicin C2 (2) as intermediate. N-Formylation of 2 followed by per-O-acetylation and dehydration furnished the isocyanide 5. Radical-induced deamination of the latter using tri-n-butylstannane and removal of the protecting groups afforded the target 1-deaminogentamicin C2 (7). Its in vitro antibacterial activity is less than that of the parent gentamicin C2. The behaviour of 7 towards aminoglycoside-inactivating enzymes was also examined; interestingly, it was found to be neither substrate nor inhibitor for such enzymes. These results strongly suggest that the substitution pattern of the 1-position determines the biological properties of the aminoglycoside antibiotics.

Synthesis of a tight-binding, multisubstrate analog inhibitor of gentamicin acetyltransferase I.

Gentamicin acetyltransferase I will catalyze acyl transfer from chloroacetylcoenzyme A to form 3-N-chloroacetylgentamicin. This product can be linked to coenzyme A to form a multisubstrate analog by nucleophilic displacement of the chlorine by the sulfur of coenzyme A. The analog can be purified by selective binding to cationic and anionic ion exchange resins. Kinetic analysis of a time-dependent onset and reversal of inhibition of gentamicin acetyltransferase I by the purified multisubstrate analog yields an inhibition constant of 5 approximately 20 x 10(-10) M. The inhibitor does not potentiate antibiotic activity against resistant Escherichia coli. Nevertheless, the effectiveness of the tight-binding between the enzyme and the multisubstrate analog demonstrates that inhibitors of resistance can be designed and prepared by specific enzymatic synthesis.

The syntheses and biological properties of 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B and 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B.

The syntheses of 1-N-(S-4-amino-2-hydroxybutyryl)-gentamicin B and 1-N-(S-3-amino-2-hydroxypropionyl)-gentamicin B, designated sch 20287 and Sch 21420, respectively, by procedures similar to those developed by KAWAGUCHI and co-workers for the transformation of kanamycin A to amikacin are described. The in vitro microbiological properties of Sch 20287 and Sch 21420 are compared with amikacin, gentamicin and tobramycin.