Morphology and properties of neutralized chitosan-cellulose nanocrystals biocomposite films.

Chitosan/cellulose nanocrystals (CH-CN) films were obtained by casting of dispersions, and treated with NaOH for neutralization proposes. The composition of films was varied from 1 to 10wt.% of CN. Changes in the morphology of the systems were correlated with the different properties studied. FTIR revealed the presence of a weak interaction between the polymer matrix and nanofiller, confirmed by a slightly increase in thermal stability. SEM images suggested that incorporating CN amounts higher than 3wt.% generates phase-segregated systems, and SAXS showed that CH avoid the typical organization of CN at concentrations below 5wt.%. Improved performance against water was obtained in composite materials comparing to the pure polymer matrix, as well as CH and CH-CN films did not show antibacterial activity demonstrating that remnant acetic acid, when no neutralization step is done, plays an important role in this property.

Treatment of in vitro enterohemorrhagic Escherichia coli infection using phage and probiotics.

AIMS: To assay the combination of phage and probiotics against EHEC in vitro on infected Hep-2 cells. METHODS AND RESULTS: Phage and probiotics treatments on EHEC O157:H7-infected Hep-2 cells were assayed individually or combined. The effect of freeze-drying on phage and probiotic antimicrobial activity was also studied. While treatment with phage alone increased cell detachment caused by EHEC infection, the treatments with MM alone or in combination with phage proved to effectively diminish cell damage caused by EHEC infection. Combined treatment showed a decrease in apoptotic cell count of 57·3% and a reduction in EHEC adhesion to cell monolayer of 1·2 log CFU. The simultaneous use of phage and probiotics showed no antagonistic effect, and freeze-drying did not affect their antipathogenic activity. CONCLUSIONS: The combination of phage and probiotics has great potential for reducing the number of pathogens adhered to epithelial cells during EHEC O157:H7 infection and attenuating the cytotoxic effect derived from it. Further in vivo assays are needed for assessing the actual effectiveness of the treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a freeze-dried formulation of phage and probiotics capable of controlling EHEC infections and reducing epithelial cell damage in vitro.

Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (P<0.05), whereas incubation of cells with MM did not induce the expression of any of the mediators assessed. Interestingly, pre-incubation of HT-29 monolayer with MM produced an inhibition of S. flexneri-induced IL-8, CCL20 and TNF-α mRNA expression. In order to gain insight on the effect of MM (or the individual strains) on this pro-inflammatory response, a series of experiments using a HT-29-NF-κB-hrGFP reporter system were performed. Pre-incubation of HT-29-NF-κB-hrGFP cells with MM significantly dampened Shigella-induced activation. Our results showed that the contribution of yeast strain Kluyveromyces marxianus CIDCA 8154 seems to be crucial in the observed effect. In conclusion, results presented in this study demonstrate that pre-treatment with a microbial mixture containing bacteria and yeasts isolated from kefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.