Relative innocuity of different forms of chitosan in cows.

<p>Each quarter of cow’s udder has received 500 mg of chitosan (2.6 kDa or 4.0 kDa) or saline as the negative control. Milk samples and somatic cell counts (SCC) were determined 12 and 3 hours before the instillation of chitosan. After the intramammary instillation, milk samples were aseptically collected from cows at several points in time to evaluate inflammation by determining the SCC (A) and milk yields (B). Symbols represent the means and vertical lines the standard deviation. Data were analyzed by ANOVA using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). For the first experiment, time was used as a repeated effect and treatment (cow) was used as the subject. Orthogonal contrasts were performed to compare the effect of each treatment to control. No difference was observed between saline and the 2.6 kDa chitosan (SCC and quarter milk yield). Significant differences were observed between saline and 4.0 kDa for SCC: *, <i>P</i><0.05; **, <i>P</i>< 0.01; ***, <i>P</i><0.0001.</p

Preformed biofilms of <i>S</i>. <i>aureus</i> strains exposed to the 2.6 kDa chitosan.

<p>(A) Reduction of a preformed biofilm on pegs following exposure to increasing concentrations of chitosan. (B) Bactericidal effect of chitosan on preformed biofilms. The CFU/peg after 24 h of biofilm formation was evaluated for control pegs (CTRL 24 h) and this represented the inoculum at the onset of treatment, which occurred at 24 h for another 24 h of incubation. The CFU/peg obtained for the untreated pegs after the total incubation period served as the reference (CTRL 48 h) for treatment efficacy. Data were obtained from three independent experiments. Significant differences in comparison to the untreated controls (0 mg/ml in A, and CTRL 48 h in B) are shown. Statistical analysis was performed using non-parametric one way ANOVA: **, <i>P</i><0.005; ***, <i>P</i><0.001; ****, <i>P</i><0.0001.</p

Antibiofilm activity of the different forms of chitosan against MRSA 1158c and the biofilm hyperproducer strain 2117.

<p>(A) CHOS, (B) 1.3 kDa, (C) 2.6 kDa, and (D) 4.0 kDa chitosan. Bars represent the means and vertical lines the standard deviation (SD). Data were obtained from three independent experiments. Significant differences in comparison to the untreated control (0 mg/ml) are shown by asterisks. Statistical analysis was performed using Kruskal-Wallis test (non-parametric one way ANOVA) with Dunn’s multiple comparison test: ns, non significant; *, <i>P</i><0.05.</p

MIC values of antibiotics and chitosan (2.6 kDa) in a checkerboard assay against <i>S</i>. <i>aureus</i> ATCC 29213.

<p>MIC values of antibiotics and chitosan (2.6 kDa) in a checkerboard assay against <i>S</i>. <i>aureus</i> ATCC 29213.</p

Inflammation scores obtained after administration of chitosan by intramammary injection in mice.

<p>Inflammation scores obtained after administration of chitosan by intramammary injection in mice.</p

Antibiofilm and antibacterial effects of specific chitosan molecules on <i>Staphylococcus aureus</i> isolates associated with bovine mastitis - Fig 4

<p><b>Time-kill experiments showing the viability of <i>S</i>. <i>aureus</i> untreated (Ctrl) or in the presence of increasing concentrations of the 2.6 kDa chitosan, (A) for strain MRSA 1158c, and (B) for the biofilm hyperproducer strain 2117.</b> The CFU detection limit is 10 CFU/ml. Data are presented as means with standard deviations from three independent experiments.</p

MIC values of the various forms of chitosan against <i>S</i>. <i>aureus</i> strains.

<p>MIC values of the various forms of chitosan against <i>S</i>. <i>aureus</i> strains.</p

Effect of the 2.6 kDa chitosan on LDH release from MAC-T bovine mammary epithelial cells.

<p>Cells were exposed to the 2.6 kDa chitosan at different concentrations (0.5 mg/ml, 2 mg/ml and 8 mg/ml). Triton x-100 and culture medium were used as cytotoxic positive and negative (Ctrl) controls, respectively. The amount of formazan formed was normalized to the amount formed by the non-treated cells (Ctrl). Bars represent the means and vertical lines the standard deviation (SD). Significant differences in comparison to the negative control (Ctrl) are shown. Statistical analysis was performed using the Kruskal-Wallis test (non-parametric one way ANOVA) with Dunn’s multiple comparison test: *, <i>P</i><0.05; ***, <i>P</i><0.0001.</p

Efficacy of the 2.6 kDa chitosan used alone or in combination with tilmicosin on <i>S</i>. <i>aureus</i> in a mouse mastitis model.

<p>(A) Chitosan was administered twice at (0 h and 4 h post-inoculation) using 0.2, 2 or 10 mg/gland for the challenge against <i>S</i>. <i>aureus</i> strain Newbould. (B) A challenge with <i>S</i>. <i>aureus</i> strain 2117 was treated with the 2.6 kDa chitosan or tilmicosin (TIL) or a combination of both. Chitosan was administered twice at (0h and 4 h post-inoculation) whereas tilmicosin was administered 4 h post-inoculation. The middle bars indicate median values for each group of glands (n = 5–8) whereas the boxes specify quartiles Q1-Q3. The detection limit was 200 CFU (dotted line), below that, the glands were considered uninfected. Statistical analysis was performed using the Kruskal-Wallis test: *, <i>P</i><0.05; ***, <i>P</i><0.001.</p