A gene replacement/disruption system of Amycolatopsis mediterranei U32 was developed based on the established electroporation conditions as well as appropriate selective markers. Through two-step selection, ahbas gene in U32 was replaced by a promoterless alpha-amylase gene constructed on the plasmid pDK110 of E. coli. The first single-crossover and the second double-crossover frequencies were approximately 0.5%-0.7% and 2%, respectively. Denaturation of the plasmid pDK110 increased the integration frequency about 7-10 folds, while electric shock treatment of the single-crossover recombinants increased the frequency of second crossover recombination about 5 folds. Employing denatured DNA fragments containing an apramycin-resistance gene flanked with regions of the respective genes, One-step disruption of rifO and amrA genes of U32 was also achieved with an efficiency of 30-50 transformants per microgram of DNA.
Actinomycetales ; drug effects ; genetics ; growth & development ; DNA, Bacterial ; genetics ; Drug Resistance, Microbial ; genetics ; Genes, Bacterial ; genetics ; Mutagenesis ; Nebramycin ; analogs & derivatives ; pharmacology ; Plasmids ; genetics ; Recombination, Genetic

The factors selected as an independent variable to study included antioxidant dinatri EDTA and viscosity enhancers PVP. Value of pH and percent of remained tobramycine contents after 3 months with ageing at 500C compared with primary tobramycine were selected as a dependent variable. Experimental model was designed according to model of quadratic equation including 9 formulas. The relationship between independent variables X and dependent variables Y conformed to quadratic regressive equation. Base on preliminary formulas selected, Modde 5.0 software was used to trace experimentally and deal with the results. Optimal formula of tobramycine eye drop 0.3% had stableness over 3 months at 50oC and over 12 months at normal condition
Ophthalmic Solutions ; Tobramycin

Study on some influencing factors such as buffer, antioxidant substances to build primary formulation of tobramycin eye drops with stability over 12 months. Result: temperature and light have much influence to the stability of tobramycin and pH contents of eye drops solution. High temperature impacts clearly to the decrease of tobramycin pH and contents. The solution was stable with borate buffer more than phosphate and citrate buffer. Product samples without antioxidant substances have low remained tobramycin contents, about 70% after three months ageing. Dinatri EDTA, natri bisulfit and dinatri EDTA-natri bisulfit combination have significant effect to the stable of tobramycin content. However, dinatri EDTA choice compared with dinatri EDTA-natri bisulfit combination is the same because of dinatri EDTA effect. Dinatri EDTA has better effect to the stable of pH and increase anti-bacteria effect of benzalkonium chloride
Ophthalmic Solutions ; Tobramycin ; Drug Stability

To determine whether liposome-encapsulated tobramycin is less toxic than commercial tobramycin and the threshold dose of liposome-encapsulated tobramycin required to produce toxic reactions when it was injected intravitreally in rabbit, we used liposome-encapsulated tobramycin, tobramycin in PBS, mixture of tobramycin and liposome-encapsulated saline, liposome-encapsulated saline and normal saline respectively. After those were injected, we examined the histologic findings and the functional changes of the retina. The final results are summarized as follows; 1. When tobramycin was injected intravitreally alone, there was no toxic reaction of the retina histologically and functionally with dosage 500 micro gram of commercial tobramycin, but dosage more than 750 micro gram produced toxic reaction. 2. When liposome-encapsulated tobramycin was injected intravitreally, there was toxic reaction of the retina histologically and functionally with dosage 1500 micro gram of tobramycin. 3. When a mixture of tobramycin and liposome-encapsulated saline was injected intravitreally, there was similar toxic reaction as tobramycin used alone with dosage more than 750 micro gram of tobramycin. Liposome-encapsulated saline and normal saline did not produce toxic reaction. The above results indicate that liposome encapsulation markedly reduces the ocular toxicity of tobramycin and that as mnch as dosage 1000 micro gram of liposome-encapsulated tobramycin may be tolerated by the intravitreal route in the rabbit eye. Therefore, the results of this study offer some hope that we may use the method of intravitreal injection of liposome-encapsulated tobramycin safely and effeciently for the treatment of bacterial endophthalmitis in near future.
Endophthalmitis ; Hope ; Intravitreal Injections ; Liposomes ; Rabbits* ; Retina* ; Tobramycin*

To evaluate the efficacy of polygexamethylene biguanide(PHMB) in Pseudomonas aeruginosa(P. aeruginosa) keratites model, 10microliter of P. aeruginosa bacterial suspension(1x103 colony-forming unit(cfu)/ml) was injected intrastromally into rabbit corneas. Eighteen rabbits(36 eyes) were divided into three treatment groups: balanced salt solution(BSS) group(N=18 eyes). PHMB(0.02%, 200microliter/ml) group(n=9 eyes), tobramycin(14microliter/ml) group(n=9 eyes). Topical antibiotic drops were given hourly from 12 hours after inoculation. A subconjunctival infection was every 24 hours during the first 72 hours. The Severity of keratitis was scored in basked fashion every 8 hours. Corneal buttons were excised and homogenized at the end of the study to determine the viable bacterial counts. In P. aeruginosa keratitis model, tobramycin was statistically more efficacious than PHMB, according the clinical scores at 58 hours(9.9 vs. 15.1, P<0.0001) and log10 cfu(0.54+/-0.21 vs. 4.87+/-1.07. P<0.0001). No differences were found between the PHMB and BSS groups in either clinical scores or bacterial counts. PHMB appears to be ineffective against P. aeruginosa in experimental keratitis model of rabbit.
Bacterial Load ; Cornea ; Keratitis* ; Pseudomonas aeruginosa* ; Pseudomonas* ; Tobramycin

The authers investigated the effct of liposome encapsulation on the pharmacokinetics of tobramycin after subconjuctival in jnjection in rabbits. Tobramycin was encapsulated into neutral liposomes of phosphatidylcholine and cholesterol. The final liposomal suspension contained tobramycin, 5mg/ml. One of the each rabbit received a subconjunctival injection(2.5mg/0.5ml) of liposome-encapsulated tobramycin or tobramycin alone. Tobramycin levels in the sclera measured 3 and 24 hours after injection were marked higher with the liposome-encapsulated drug than with the another preperation. The difference between liposomeencapsulated drug and the another preperation was not significant for cornea.
Cholesterol ; Cornea* ; Liposomes ; Pharmacokinetics ; Phosphatidylcholines ; Rabbits* ; Sclera* ; Tobramycin*

Bacterial keratitis is a common ophthalmic disease. Recently, topical and subconjunctival therapy were equally effective in reducing the number of viable bacteria in experimental corneal ulcers. Subconjunctival injection produced high but transient concentrations followed by persistent low levels. In contrast, eyedrops produced moderate but sustained concentrations throughout the treatment period. Liposomes are small, biodegradable lipid vesicles with an aqueous core. Incorporation of drugs into liposomes provides a convenient way to retard their release from a relatively inert depot without changing the intrinsic characteristics of the agents.
Bacteria ; Cornea* ; Keratitis ; Liposomes* ; Ophthalmic Solutions ; Rabbits* ; Sclera* ; Tobramycin* ; Ulcer

Green nail syndrome (chromonychia) is a nail disorder characterized by onycholysis and green-black discoloration of the nail bed. This condition is often associated with chronic paronychia. Pseudomonas aeruginosa is the most commonly identified organism in cultures from the affected area. Despite the various treatment options available, removal of the nail is still necessary in many cases. A 35-year-old man presented with dark-greenish discoloration of the nail plate and onycholysis on the left thumbnail. He had been treated with oral antifungal and antibiotic agents for several months; however, the lesion showed no improvement. The diagnosis of green nail syndrome was established after a positive bacterial culture, and on the basis of the antibiotic sensitivity test result, tobramycin eye drop (Tobrex(R)) was then prescribed. Three weeks later, the nail discoloration almost vanished but the onycholysis remained. Herein, we recommend the application of tobramycin eye drop as an easy and safe treatment option for green nail syndrome.
Adult ; Diagnosis ; Humans ; Onycholysis ; Paronychia ; Pseudomonas aeruginosa ; Tobramycin*

PURPOSE: Relapsing polychondritis is an uncommon systemic autoimmune disorder which is characterized by recurrent and often progressive inflammatory episodes involving multiple organ systems, including the ophthalmic, otorhinolaryngeal, respiratory, musculoskeletal, renal, cardiovascular, and dermatologic systems. The most common ocular manifestations are episcleritis and scleritis. Uveitis, especially the nongranulomatous type, has been reported in 3% to 22% of relapsing polychondritis cases. We report uncommon hypopyon uveitis as an ophthalmic finding associated with relapsing polychondritis. CASE SUMMARY: A 56-year-old woman with known relapsing polychondritis presented with ocular pain and redness in the right eye which had developed two months before and was managed for scleritis. However, she developed blurred vision, and hypopyon and vitreous opacity was found. The patient presented to our clinic and we diagnosed her with hypopyon uveitis associated with relapsing polychondritis. The patient was started on systemic steroid therapy consisting of 1% prednisolone acetate, 0.5% moxifloxacin, and 0.5% tobramycin in the right eye. Hypopyon disappeared 8 days following the initiation of treatment, and all symptoms had resolved after 14 days.
Aza Compounds ; Eye ; Female ; Humans ; Middle Aged ; Polychondritis, Relapsing ; Prednisolone ; Quinolines ; Scleritis ; Tobramycin ; Uveitis ; Vision, Ocular

Bacterial endophthalmitis, which is a devastating complication of intraocular surgery or eye trauma, has a poor prognosis. Intravitreal injection of antimicrobial agents has become a part of the standard treatment of endophthalmitis. The authors investigate the pharmacokinetics of intravitreal liposome-encapsulated tobramycin as a possible method of prolonging the duration of therapeutic concentrations. Tobramycin was encapsulated into liposomes of phosphatidylcholine, phosphatidic acid, and alpha-tocopherol by the reverse phase evaporation method. The final liposomal suspension contained tobramycin, 7.0 mg/ml, 60.5% encapsulated. One eye received an intravitreal injection of either liposome-encapsulated tobramycin (LET), tobramycin phosphated-buffered saline (TS) or a mixture of tobramycin and liposome-encapsulated saline (TEL), and the results were as follows: 1. Concentrations of free tobramycin were significantly lower with LET than with TS or TEL at 1 hour after intravitreal injection. 2. Concentrations of free and total tobramycin were significantly higher with LET than with TS or TEL at 5 and 8 days after intravitreal injection. Concentrations of free tobramycin with TS were lower than the minimal inhibitory concentration(MIC) of tobramycin for Pseudomonas aeruginosa at 8 days after intravitreal injection, while those with LET were higher than the MIC of tobramycin for Pseudomonas aeruginosa 18 days after injection.
Animal ; Delayed-Action Preparations ; Injections ; Liposomes ; Rabbits ; Tobramycin/administration & dosage/*pharmacokinetics ; Vitreous Body/*metabolism