Anna Rita Blanco

Epigallocatechin-Gallate Enhances the Activity of Tetracycline in Staphylococci by Inhibiting Its Efflux from Bacterial Cells

ABSTRACT 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 β-lactam antibiotics. In this article, we report that EGCg at doses half and below its calculated MIC of 100 μg/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.

Epigallocatechin Gallate Inhibits Biofilm Formation by Ocular Staphylococcal Isolates

ABSTRACT 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.

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.

(-)Epigallocatechin-3-gallate inhibits gelatinase activity of some bacterial isolates from ocular infection, and limits their invasion through gelatine.

The objective of this paper is to assess the gelatinase production by some ocular pathogenic bacterial strains, and evaluate the ability of (-)epigallocatechin-3-gallate (EGCg) to inhibit this gelatinase activity and thus limit bacterial invasion. The effect of EGCg on bacterial gelatinase activity was tested by classic zymography methods, while its effect on bacterial invasion was evaluated through the ability of growing bacteria to liquefy and thus penetrate a semisolid gelatine substrate. It was found that EGCg inhibits bacterial gelatinases with an IC(50) of about 0.2 mM, and limits invasion of gelatinase-positive bacteria at concentrations above 2 mM. These results show for the first time that EGCg, as well as having direct antibacterial activity, can also inhibit bacterial gelatinases, thus limiting their invasion on gelatine. Possible use of EGCg is thus suggested as an adjuvant in antibacterial chemotherapy.

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.

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.

Antioxidant Effect of 0.2% Xanthan Gum in Ocular Surface Corneal Epithelial Cells

Abstract 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.

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

Abstract 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.

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.

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.