Development of ophthalmic nanoemulsions of ß-caryophyllene for the treatment of Acanthamoeba keratitis.

Although rare, amoebic keratitis (AK) is a disease caused by Acanthamoeba spp. that can lead to blindness. The drugs currently available for its treatment are very toxic, which has motivated the investigation for more effective and safe therapeutic options. In this study, the in vitro activity of ß-caryophyllene (BCP) was exploited taking into account its action against other protozoans as well as its well-known healing and anti-inflammatory properties (aspects relevant for the AK pathogenesis). On the other hand, high volatilization and oxidation phenomena are found for this compound, which led to its incorporation into nanoemulsions (NEs). Two emulsifying agents were tested, resulting in monodisperse systems with reduced droplet size (<265 nm) and high surface charge (positive and negative for NEs prepared with cetrimonium bromide -CTAB and Phosal® 50+, respectively). NEs prepared with CTAB were shown to be more stable after long-term storage at 4 and 25 °C than those prepared with Phosal®. Pure BCP, at the highest concentration (500 µM), resulted in a level of inhibition of Acanthamoeba trophozoites equivalent to that of reference drug (chlorhexidine). This activity was even greater after oil nanoencapsulation. The reduced droplet size could improve the interaction of the oil with the microorganism, justifying this finding. Changes in surface charge did not impact the activity. Positively charged NEs improved the interaction and retention of BCP in the cornea and thus should be prioritized for further studies.

Supporting data for comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin.

Here we provide the LC-MS/MS data from a comparative analysis of Listeria monocytogenes ATCC 7644 treated and non-treated with a sublethal concentration of nisin (10(-3) mg/mL). Protein samples were analyzed by multidimensional protein identification technology (MudPIT) approach, in an off-line configuration. The raw MS/MS data allowed the detection of 49,591 spectra which resulted in 576 protein identifications. After Scaffold validation, 179 proteins were identified with high confidence. A label-free quantitative analysis based of normalized spectral abundance factor (NSAF) was used and 13 proteins were found differentially expressed between nisin-treated and non-treated cells. Gene ontology analysis of differentially expressed proteins revealed that most of them are correlated to metabolic process, oxidative stress response mechanisms and molecular binding. A detailed analysis and discussion of these data may be found in Miyamoto et al. [1].

Comparative proteomic analysis of Listeria monocytogenes ATCC 7644 exposed to a sublethal concentration of nisin.

Listeria monocytogenes infections have been frequently reported in many food poisoning outbreaks around the world. In this work, the protein repertoires of L. monocytogenes ATCC 7644 cells treated or not with a 10(-3)mg/mL nisin sublethal concentration, established by antimicrobial susceptibility tests, were analyzed by LC-MS/MS. Overall, 179 proteins were identified, 9 of them more abundant in nisin-treated samples, and 4 more abundant in non-treated control samples. In nisin treated cells, proteins associated to oxidative stress response showed higher abundance. Also, the higher abundance of an enzyme related to the production of cell membrane lipids upon nisin exposure is suggestive of both a failure in conventional cell division mechanism and the activation of an alternative L-form mediated division mechanism. Finally, flagellar and motility proteins' overexpression upon nisin exposure is indicative of increased bacterial motility in response to the bacteriocin. Taken together, these results provide new insights on nisin effects on L. monocytogenes cells and on how this bacterium may overcome a bacteriocin-containing environment. BIOLOGICAL SIGNIFICANCE: The antimicrobial mechanism of nisin on target bacterial cells has been extensively studied since discovery of this bacteriocin. The nisin pore-forming mechanism is mediated by its binding to the pyrophosphate portion of membrane lipid II [1], but some evidences point out to alternative mechanisms. Results from assays with mutacin 1140 hybrids [2] showed that the portion of nisin that is not involved with lipid II binding could damage the bacterial cell, independently of pore formation [3,4]. Moreover, there are insufficient data to explain how nisin affects the bacterial survival. In this scenario, proteomics is an interesting approach, as a comparison between treated and untreated cells may provide insights of both antimicrobial mechanisms of action and bacterial response mechanisms [5].