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 (n56) and S. epidermidis (n56) 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. INTRODUCTIONStaphylococci are important nosocomial pathogens. Eradication of these micro-organisms is not always successful due to their ability to form biofilms. Experimental evidence has shown that micro-organisms in biofilms are less susceptible to conventional treatment (Brown & Gilbert, 1993) than their planktonic counterparts. Many factors contribute to the lowered susceptibility of bacteria enclosed in a biofilm, and include the induction of a biofilm phenotype, the stress response and failure of the antimicrobial agents to penetrate the biofilm (Mah & O'Toole, 2001).As such, alternative strategies or more effective agents exhibiting activity against biofilm-producing micro-organisms are of great interest. Natural drugs could represent an interesting approach to limit the emergence and the spread of these organisms, which currently are difficult to treat. Recently, there has been considerable interest in the study of plant materials as sources of new compounds for processing into therapeutic agents. One approach may be the use of essential oils that have been shown to be potential agents in the treatment of infections, and are safe in terms of human and animal health. In this context, oregano oil and its major phenolic components, carvacrol [2-methyl-5-(1-methylethyl)phenol] and thymol (2-isopropyl-5-methylphenol), are known for their wide spectrum of antimicrobial activity, which has been the subject of several investigations in vitro (Dorman & Deans, 2000;Lambert et al., 2001) and in vivo (Adam et al., 1998;Manohar et al., 2001). They possess multiple biological properties such as anti-inflammatory, anti-leishmanial, antioxidant, hepatoprotective and anti-tumoral activities (Aeschbach et al., 1994; Alam et al., 1999; Robledo et al., 2005;Skold et al., 1998;Weber & de Bont, 1996;Zeytinoglu et al., 2003).Previously, we have shown the efficacy of oregano oil, carvacrol and thymol against planktonic Staphylococcus aureus and Staphylococcus epidermidis, including meticillinresistant strains (Nostro et al., 2004). The objective of this study was to extend the research to evaluate the activity of oregano oil, carvacrol and thymol on biofilm-grown S. aureus and S. epidermidis strains, as well as the effects of oils on biofilm formation....

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).

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 ␤-lactam antibiotics. In this article, we report that EGCg at doses half and below its calculated MIC of 100 g/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 (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.Biofilm formation is a three-stage process (6) that significantly contributes to the pathogenesis of staphylococcal infections. The first stage, docking, is mainly due to hydrophobic interactions, whereas the next two stages, locking and maturation, are mediated by capsular polysaccharide adhesins, PIA (polysaccharide intercellular adhesin) and PNAG (poly-Nacetylglucosamine polysaccharide), both of which are synthesized by the gene products of the ica operon (icaADBC), with the main contribution coming from an N-acetylglucosaminyltransferase encoded by the icaA gene (5, 12). The expression of the icaA gene alone results in low enzymatic activity, but coexpression with icaD leads to a significant increase in activity (8).Green tea polyphenols and, more specifically, epigallocatechin gallate (EGCg) are known to possess both direct bactericidal activity (18,19) and the ability to potentiate the effects of certain antibiotics (15,16,17,21). Moreover, they have already been shown to have at least an indirect influence on biofilm production, in that they can retard the formation of dental plaque (9,11,20).In this study, we have investigated the effects of sub-MICs of EGCg (99% pure; Sigma) ( Fig. 1A, inset) on biofilm formation by 20 different ocular staphylococcal isolates derived from patients with community-acquired ocular infections and belonging to our private collection. The different isolates included 8 Staphylococcus aureus isolates and 12 Staphylococcus epidermidis isolates. Moreover, two American Type Culture Collection strains (S. epidermidis ATCC 35984 and S. epidermidis ATCC 12228) were used as reference controls. Table 1 summarizes the characterization of bacterial isolates for biofilm production. The MIC of EGCg for each strain was determined in tryptic soy broth (TSB) by a broth dilution method according to the guidelines of the CLSI (formerly the NCCLS) (13). The quality of the lot of EGCg was controlled by reverse-phase high-pressure liquid chromatography (HPLC) and showed just one single, sharp peak, suggesting a very high degree of purity (Fig. 1A). The MICs obtained ranged from 125 to 500 mg/ml, similar to that which we reported recently (17); however, they were slightly higher than those already described by other authors (22, 24).Each ocular isolate was then characterized for biofilm-related properties. Biofilm-forming ability was tested by determination of adhesion to microtiter plates (5) and was quantitated by safranin staining and reading of the absorbance at 492 nm. The biofilm production of the different strains was then arbitrarily classified as strong (optical density [OD], Ͼ0.9 OD), medium (0.6 Ͻ OD Ͻ 0.9),...

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.