Efficacy of a Fixed Combination of Tetracycline, Chloramphenicol, and Colistimethate Sodium for Treatment of Candida albicans Keratitis.

Purpose: To evaluate the antifungal activity of a fixed antibiotic combination (AC) containing tetracycline (TET), chloramphenicol (CAF), and colistimethate sodium (CS). Methods: In vitro: Candida ATCC and clinical strains were used. The minimum inhibitory concentrations (MICs) of AC and of each antibiotic were determined. Fluconazole (FLC) was tested for comparison. Time-killing curves of selected strains were performed. Ex vivo keratitis: corneas were injected intrastromally with the selected strains. After the injection, corneas were divided into groups of treatments: AC, FLC, or saline. Then, the tissues were analyzed for colony-forming units per gram (CFU/g). Propidium iodide (PI) and MitoTracker (MTR) staining were used to investigate the mode of action. Results: Values of MIC required to inhibit the growth of 90% of organisms for the antibiotics alone were higher than FLC. However, their activity was enhanced when used in combination against Candida yeasts. Time-killing curves showed that at 24 hours, AC reduced the load of both strains of approximately 1 Log10 CFU/g compared with the initial inoculum (P < 0.0001). This effect was also significant versus FLC. In ex vivo, AC was effective in decreasing the loads of both strains by 4 Log10 CFU/g with respect to the control. Moreover, it showed higher activity than FLC against Candida albicans ATCC 10231 (1 Log10 CFU/g, P < 0.01 versus control). PI staining demonstrated that CS changed the membrane's permeability, whereas MTR staining demonstrated that TET or CAF altered mitochondrial function. The cells treated with AC and stained showed both effects. Conclusions: In this study, AC showed antifungal efficacy versus Candida spp.; this activity can be due to the synergistic effects of antibiotics in it.

Design, Synthesis, and Antibacterial Activity of a Multivalent Polycationic Calix[4]arene-NO Photodonor Conjugate.

The role of nitric oxide (NO) as an antimicrobial and anticancer agent continues to stimulate the search of compounds generating NO in a controlled fashion. Photochemical generators of NO are particularly appealing due to the accurate spatiotemporal control that light-triggering offers. This contribution reports a novel molecular construct in which multiple units of 3-(trifluoromethyl)-4-nitrobenzenamine NO photodonor are clustered and spatially organized by covalent linkage to a calix[4]arene scaffold bearing two quaternary ammonium groups at the lower rim. This multivalent calix[4]arene-NO donor conjugate is soluble in hydro-alcoholic solvent where it forms nanoaggregates able to release NO under the exclusive control of visible light inputs. The light-stimulated antibacterial activity of the nanoconstruct is demonstrated by the effective bacterial load reduction of Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Escherichia coli ATCC 10536.

Potential Eye Drop Based on a Calix[4]arene Nanoassembly for Curcumin Delivery: Enhanced Drug Solubility, Stability, and Anti-Inflammatory Effect.

Curcumin is an Indian spice with a wide spectrum of biological and pharmacological activities but poor aqueous solubility, rapid degradation, and low bioavailability that affect medical benefits. To overcome these limits in ophthalmic application, curcumin was entrapped in a polycationic calix[4]arene-based nanoaggregate by a simple and reproducible method. The calix[4]arene-curcumin supramolecular assembly (Calix-Cur) appeared as a clear colloidal solution consisting in micellar nanoaggregates with size, polydispersity index, surface potential, and drug loading percentage meeting the requirements for an ocular drug delivery system. The encapsulation in the calix[4]arene nanoassembly markedly enhanced the solubility, reduced the degradation, and improved the anti-inflammatory effects of curcumin compared to free curcumin in both in vitro and in vivo experiments. Calix-Cur did not compromise the viability of J774A.1 macrophages and suppressed pro-inflammatory marker expression in J774A.1 macrophages subjected to LPS-induced oxidative stress. Histological and immunohistochemical analyses showed that Calix-Cur reduced signs of inflammation in a rat model of LPS-induced uveitis when topically administrated in the eyes. Overall, the results supported the calix[4]arene nanoassembly as a promising nanocarrier for delivering curcumin to anterior ocular tissues.

Ocular flora and their antibiotic susceptibility in patients having cataract surgery in Italy.

PURPOSE: To characterize the ocular flora in a consecutive group of patients having cataract surgery and to determine the antibiotic susceptibility profile of isolates to several ophthalmic antibiotics. SETTING: Hospital Di Stefano, Catania, Italy. DESIGN: Observational case series. METHODS: Conjunctival and eyelid cultures from patients were obtained 14 days before surgery and, if positive, repeated the day of the surgery. Antimicrobial susceptibility for aminoglycosides (netilmicin and tobramycin), fluoroquinolones (ofloxacin, levofloxacin, and moxifloxacin), chloramphenicol, and azithromycin was tested using the Kirby-Bauer disk diffusion method. Susceptibility was also tested for oxacillin, cefuroxime, and vancomycin. All positive patients received a 2-day preoperative course of 3 mg/mL netilmicin ophthalmic solution 4 times a day. The recovery rate of microorganisms after antibiotic treatment compared with baseline was calculated. RESULTS: One hundred twenty consecutive patients were included in the study. Cultures were positive in 72.5% of patients; 131 isolates, mainly gram-positive, were identified. Staphylococcus epidermidis (58.0%) and Staphylococcus aureus (15.3%) were the most frequently isolated microorganisms. Methicillin-resistant staphylococci accounted for 3.8% of S epidermidis and 20.0% of S aureus. A high in vitro susceptibility (>90%) for all isolates, including multiresistant coagulase-negative Staphylococcus, was obtained for netilmicin, vancomycin, and cefuroxime. The recovery rate of isolates before surgery was reduced by 93.9% (P < .001). CONCLUSIONS: Conjunctival and lid margin isolates were sensitive to netilmicin, vancomycin, and cefuroxime. Microorganisms were less susceptible to other ophthalmic antibiotics, with the exception of moxifloxacin. A 2-day preoperative course with topical netilmicin reduced most bacteria identified on the conjunctiva and eyelids. FINANCIAL DISCLOSURE: Dr. Papa and Ms. Blanco are employees of Società Industria Farmaceutica Italiana SpA. Dr. Santocono has no financial or proprietary interest in any material or method mentioned.

Ex vivo efficacy of gemifloxacin in experimental keratitis induced by methicillin-resistant Staphylococcus aureus.

In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has been observed in ocular infections. Resistance of MRSA to second- and third-generation fluoroquinolones has increased interest in the fourth-generation fluoroquinolones. In this study, the antibacterial activity of gemifloxacin against MRSA ocular isolates in vitro and in a modified ex vivo rabbit keratitis model was investigated. In vitro susceptibility test results indicated that the minimum inhibitory concentrations (MICs) of gemifloxacin were lower than the MICs of other fluoroquinolones, including moxifloxacin (MIC50 range, 0.016-0.032 µg/mL; MIC90 range, 0.047-0.094 µg/mL). Results from the ex vivo keratitis model showed a statistically significant decrease in MRSA counts (0.5-2 log10 CFU/g; P <0.05) in corneas treated with 0.3% gemifloxacin every 30 min for 7 h. Moreover, the dose-response effect of different concentrations of gemifloxacin (3-3000 µg/mL) demonstrated that a dose of 30 µg/mL had the same efficacy as the highest dose of 3000 µg/mL against all S. aureus strains. Possibly, gemifloxacin reached a steady-state level in the cornea, as the fourth-generation fluoroquinolones have better anterior chamber penetration. This study demonstrated that 0.3% gemifloxacin ophthalmic solution may be an effective topical therapy for the treatment of MRSA keratitis. In addition, this reproducible, ethical and economic ex vivo infection model can be used as a mechanistically-based alternative to in vivo animal testing, bridging the gap between in vitro and in vivo results.

A bactericidal calix[4]arene-based nanoconstruct with amplified NO photorelease.

A hydrophobic N-dodecyl-3-(trifluoromethyl)-4-nitrobenzenamine has been synthesized as a suitable NO photodonor and encapsulated in a nanocontainer based on a polycationic calix[4]arene derivative, leading to a supramolecular micellar-like nanoassembly ca. 45 nm in diameter. Visible light excitation of this nanoconstruct triggers NO generation with an efficiency remarkably higher than that observed for the free NO photoreleaser. This amplified NO release results in considerable antibacterial activity against Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 9027) as representative Gram positive and Gram negative bacteria, respectively.

Photo-antimicrobial polymeric films releasing nitric oxide with fluorescence reporting under visible light.

A novel photoresponsive molecular hybrid has been embedded in poly(lactic-co-glycolic acid) (PLGA) to give an antibacterial polymeric film generating nitric oxide (NO) under visible light, with concomitant fluorescence reporting of NO release. The molecular hybrid integrates a nitroaniline NO photodonor and a coumarin latent fluorophore in the same molecular skeleton and results in quite homogeneous distribution in the polymer matrix where it preserves well the photobehavior exhibited in solution. The doped PLGA film shows an excellent optical transparency and can be excited by visible light leading to the production of NO and the parallel fluorescence revival of the coumarin fluorophore, which acts as an optical NO reporter. Photogenerated NO diffuses out of the polymer film, can be transferred to a biological milieu and induces remarkable antibacterial activity against Escherichia coli.

Antioxidant effect of 0.2% xanthan gum in ocular surface corneal epithelial cells.

PURPOSE: Oxidative damage and inflammation are expected to be involved in age-related functional decline of lachrymal gland, that induces lachrymal dysfunction; this resulting in dry eye disease. Therefore, we investigated the potential antioxidant effect of 0.2% xanthan gum (XNT) in human corneal epithelial cells (HCE), in comparison with other widely used tear substitute polymers, such as 0.2% hydroxyethylcellulose (HEC), 0.2% hyaluronic acid (HA) and 0.5% carboxymethylcellulose (CMC). METHODS: Subconfluent (80%) HCE (Human Corneal Epithelial) cultures were treated with the different polysaccharides at the above reported concentrations. The effect of every polymer was investigated with and without 0.5 mM H2O2 In detail, hydrogen peroxide was added 1 hour after the addition of polysaccharides. Twelve hours later, reactive oxygen species (ROS) production (dichlorofluorescein diacetate spectrofluorimetric test) was assessed and their values were normalized versus protein content. Morphological analysis was performed by optical microscopy. RESULTS: No morphological differences in HCE compared to control cells (CTRL, cells treated with the buffer used for polymer solubilization) were observed in any of the tested polymers, whereas, in the presence of 0.5 mM H2O2 HCE clearly showed signs of cytotoxicity. Polymers did protect cultures from oxidative stress with XNT>HA = HEC>CMC, as evidenced by microscopic analysis. These results were confirmed from ROS measurements, which showed XNT as the only polysaccharide to restore the levels of ROS comparable to CTRL, in presence of H2O2. CONCLUSIONS: 0.2% xanthan gum was able to protect HCE by oxidative stress, bringing the ROS level down to CTRL values. Considering that in dry eye syndrome oxidative stress sustains inflammation and apoptotic cell death, the use of xanthan gum in ophthalmic preparations could be beneficial.

Staphylococcal biofilm formation as affected by type acidulant.

Staphylococcal growth and biofilm formation in culture medium where pH was lowered with weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids were studied. The effects were evaluated by biomass measurements, cell-surface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The results demonstrated that the inhibition was related to type of acidulant and pH value. At pH 5.0, the antibacterial effect was more pronounced in the presence of acetic acid (58-60% growth reduction) compared with that in the presence of lactic (7-16% growth reduction) and hydrochloric acids (23-24% reduction). The biofilm biomass of Staphylococcus aureus and Staphylococcus epidermidis was reduced by 92, 85, 63, and 93, 87, 81% after exposition to acetic, lactic, and hydrochloric acids, respectively. Increasing the pH from 5.0 to 6.0 resulted in a noticeable reduction in the effectiveness of acids. A minor cells hydrophobic character was also documented. The SEM and CLSM revealed a poorly structured and thinner biofilm compared with the dense and multilayered control. Acidic environment could have important implications for food-processing system to prevent bacterial colonization and control biofilm formation. The findings of this study lead to consider the rational use of the type of acid to achieve acidic environments.

Susceptibility of methicillin-resistant Staphylococci clinical isolates to netilmicin and other antibiotics commonly used in ophthalmic therapy.

PURPOSE: The aim of this study was to test the activity of selected antimicrobial agents commonly used in the treatment of ocular infections against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE) isolates. METHODS: A total of 43 staphylococci from respiratory tract and ocular infections were characterized for methicillin resistance using the Epsilometer test (E-test), the polymerase chain reaction for mecA gene detection and the PBP2' latex agglutination test. A perfect agreement among them was observed in 20 isolates (8 MRSA and 12 MRSE) which were then employed in the susceptibility test by using the agar disk diffusion test (NCCLS). The antibiotics tested were: netilmicin (NET), tobramycin (TOB), azithromycin (AZM), levofloxacin (LEV), moxifloxacin (MXF), chloramphenicol (C) and vancomycin (VA). RESULTS: All MRSE and most (87.5%) of MRSA isolates tested were NET and VA sensitive. The majority of MRSA were found to be resistant to all the other antibiotics, with the exception of C. In particular, 75%, 87% and 100% of the isolates were resistant to fluoroquinolones (LEV and MXF), AZM and TOB, respectively. As for the MRSE group, 25% of the strains tested were resistant to C and MXF while 33%, 42% and 58% of the strains were resistant to LEV, AZM and TOB, respectively. CONCLUSIONS: Together with VA, NET was the most effective antibiotic tested against both MRSA and MRSE clinical isolates. The exclusive topical use of NET for the treatment of ocular infections may curtail the emergence, spreading and persistence of antibiotic-resistant bacteria.

Enhanced activity of carvacrol against biofilm of Staphylococcus aureus and Staphylococcus epidermidis in an acidic environment.

Carvacrol is an antimicrobial monoterpenic phenol which occurs in many plant essential oils. The aim of this study was to investigate its activity at acidic pH on staphylococcal forming and yet established biofilms, with particular focus to improve its effectiveness on Staphylococcus epidermidis biofilm. The results showed that the subinhibitory doses (1/2, 1/4 and 1/8 MIC) of carvacrol determined a higher reduction of S. epidermidis biofilm formation than that observed at neutral pH. A potentiated inhibitory effect was also observed on established biofilm, carvacrol caused either a strong reduction of biomass (>50%) and bacteria attached to polystyrene (>7 log units). The images of scanning electron microscopy and the gas-chromatographic analysis support these results. The development of acidic formulations containing carvacrol could be an important tool to control the staphylococcal biofilm in the medical and food environment.

Effect of alkaline pH on staphylococcal biofilm formation.

Biofilms are a serious problem, cause of severe inconvenience in the biomedical, food and industrial environment. Staphylococcus aureus and S. epidermidis are important pathogenic bacteria able to form thick and resistant biofilms on various surfaces. Therefore, strategies aimed at preventing or at least interfering with the initial adhesion and subsequent biofilm formation are a considerable achievement. The aim of this study was to evaluate the effect of alkaline pH on bacterial adhesion and further biofilm formation of S. aureus and S. epidermidis strains by biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis. The results demonstrated that the amount of biofilm biomass formed and the surface hydrophobicity were significantly less than what were observed at higher levels of pH. SEM and CLSM images revealed a poorly structured and very thin biofilm (2.5-3 times thinner than that of the controls). The inhibiting effect of the alkaline pH on the bacterial attachment impaired the normal development of biofilm that arrested at the microcolony stage. Alkaline formulations could be promising towards the control of bacterial colonization and therefore the reduction of the biofilm-related hazard. In the clinical setting, alkaline solutions or cleaners could be promising to prevent the bacterial colonization, by treating surfaces such as catheters or indwelling medical devices, reducing the risk of biofilm related infections.

Effects of a new lipid tear substitute in a mouse model of dry eye.

PURPOSE: The present investigation is aimed to evaluate the effect of a new lipid artificial tear on tear volume and ocular surface signs in a mouse model of dry eye and to test the hypothesis that the combined application with sodium hyaluronate can improve the performance of the treatments. METHODS: A new oil-in-water emulsion, a 0.2% sodium hyaluronate solution, or their combined administration were given to dry eye mice maintained in a controlled environment chamber and treated with scopolamine (0.75-mg transdermal patch). Mice were treated 4 times a day with (a) sodium hyaluronate, (b) emulsion, and (c) sodium hyaluronate followed by emulsion. A control group of mice exposed to controlled environment chamber remained untreated (CTRL+). Tear volume and corneal damage were assessed after 3 and 7 days of treatment by cotton thread test and fluorescein staining. RESULTS: As regards tear volume, sodium hyaluronate did not show a statistically significant effect at either end point; the emulsion was effective after 7 days, whereas their combined administration counteracted the lacrimal decrease induced by the model both at 3 and 7 days. Corneal damage was reduced in all treated groups with respect to CTRL+. This effect was statistically significant after 3 days when the emulsion alone or in combination with sodium hyaluronate was used, while hyaluronate improved this clinical sign after 7 days. CONCLUSIONS: Our data suggest that the new lipid tear substitute can be used to treat clinical signs of dry eye and that the combined administration with hyaluronate can decrease the lag time before the effect, when the evaporative and the aqueous-deficient components are present.

In vitro activity of carvacrol against staphylococcal preformed biofilm by liquid and vapour contact.

Carvacrol is an important component of essential oils and recently has attracted much attention as a result of its biological properties, such as a wide spectrum of antimicrobial activity. The aim of this study was to evaluate the effect of carvacrol in liquid and vapour phase on preformed biofilms of Staphylococcus aureus and Staphylococcus epidermidis by determining biofilm biomass and cultivable cell numbers, and by using epifluorescence and scanning electron microscopy. Carvacrol was able to reduce biofilm biomass and cell viability more effectively when used with liquid contact rather than with vapour phase. The efficacy of treatment with carvacrol vapour was found to be dependent on exposure time. The predominance of red fluorescence using a LIVE/DEAD BacLight Viability kit (Molecular Probes) and the partially destroyed biofilm architecture as determined by microscopy in treated samples provided evidence for the efficacy of carvacrol. The findings of this investigation suggest a potential application for carvacrol in the inactivation of staphylococcal biofilms.

Effects of oregano, carvacrol and thymol on Staphylococcus aureus and Staphylococcus epidermidis biofilms.

The aim of this study was to evaluate the effect of oregano essential oil, carvacrol and thymol on biofilm-grown Staphylococcus aureus and Staphylococcus epidermidis strains, as well as the effects of the oils on biofilm formation. For most of the S. aureus (n=6) and S. epidermidis (n=6) strains tested, the biofilm inhibitory concentration (0.125-0.500 %, v/v, for oregano, and 0.031-0.125 %, v/v, for carvacrol and thymol) and biofilm eradication concentration (0.25-1.0 %, v/v, for oregano and 0.125-0.500 %, v/v, for carvacrol and thymol) values were twofold or fourfold greater than the concentration required to inhibit planktonic growth. Subinhibitory concentrations of the oils attenuated biofilm formation of S. aureus and S. epidermidis strains on polystyrene microtitre plates.

Epigallocatechin gallate inhibits biofilm formation by ocular staphylococcal isolates.

Epigallocatechin gallate (EGCg), the main polyphenol component of green tea, has several antibacterial properties. Here we show that sub-MICs of EGCg appear to decrease slime production, therefore inhibiting biofilm formation by ocular staphylococcal isolates previously characterized for the presence of ica genes by the Congo red agar plate assay and for adhesion to microtiter plates.

Epigallocatechin-gallate enhances the activity of tetracycline in staphylococci by inhibiting its efflux from bacterial cells.

Epigallocatechin-gallate (EGCg), the major catechin present in green tea extracts, has been shown to have several antibacterial activities, limiting bacterial growth and invasion and acting in synergy with beta-lactam antibiotics. In this article, we report that EGCg at doses half and below its calculated MIC of 100 microg/ml, is able to reverse tetracycline resistance in staphylococcal isolates expressing the specific efflux pump Tet(K) and appears to improve the MICs of tetracycline for susceptible staphylococcal isolates as well. The visible effect of EGCg is an increased accumulation of tetracycline inside bacterial cells. This effect is likely due to the inhibition of pump activity, and it is evident not only for Tet(K) pumps but also for efflux pumps of a different class [Tet(B)]. In summary, our data indicate that the observed dramatic enhancement by EGCg of tetracycline activity for resistant staphylococcal isolates is caused by impairment of tetracycline efflux pump activity and increased intracellular retention of the drug, suggesting a possible use of EGCg as an adjuvant in antibacterial therapy.

Susceptibility of methicillin-resistant staphylococci to oregano essential oil, carvacrol and thymol.

The aim of this study was to evaluate the susceptibility of methicillin-susceptible and methicillin-resistant staphylococci (MSS, MRS) to oregano essential oil, carvacrol and thymol. The commercial aerial parts of Origanum vulgare L. were hydrodistilled and the essential oil analysed by gas- chromatography/electron impact mass spectrometry. The inhibition efficacy of this essence and its major components was assayed against 26 MSS and 21 MRS, using an agar dilution method. The methicillin resistance was thoroughly typed by Epsilometer test (E-test), polymerase chain reaction for mecA gene detection and PBP2' latex agglutination test. The results clearly demonstrated that the comparison between the susceptibility of MSS and MRS to oregano oil, carvacrol and thymol showed no significant differences (Fisher's exact test, P > 0.05). The best minimum inhibitory concentration values were reported for carvacrol (0.015-0.03%, v/v) followed by thymol (0.03-0.06%, v/v) and oregano oil (0.06-0.125%, v/v).