A comparative study of antimicrobial activity of differently-synthesized chitosan nanoparticles against bovine mastitis pathogens.

The greatest concern in dairy farming nowadays is bovine mastitis (BM), which results mainly from bacterial colonization of the mammary gland. Antibiotics are the most widely used strategy for its prevention and treatment, but overuse has led to growing antimicrobial resistance. Pathogens have also developed other mechanisms to persist in the udder, such as biofilm formation and internalization into bovine epithelial cells. New therapies are therefore needed to reduce or replace antibiotic therapies. In a previous study, we found that chitosan nanoparticles (Ch-NPs) have considerable potential for the treatment of BM. The aim of the present study was to evaluate the antimicrobial activity of differently-synthesized Ch-NPs against BM pathogens and their toxicity in bovine cells in vitro, to further explore the attributes of Ch-NPs for the prevention and treatment of intramammary infections. We also looked into their ability to inhibit biofilm formation and prevent the internalization of S. aureus into mammary epithelial cells. Finally, since an interesting approach for BM prevention is to enhance the host's immune response, we studied whether Ch-NPs could promote the release of pro-inflammatory cytokines in mammary epithelial cells. The results reveal that the bactericidal effect of Ch-NPs on BM pathogens and their ability to inhibit biofilm formation are size-dependent, with smaller particles being more efficient. In contrast, their effect on the viability of the cell lines is not size-dependent and all samples tested were non-toxic. The smallest Ch-NPs successfully prevented the internalization of S. aureus into the cells, but did not promote the production of pro-inflammatory cytokines. These findings make it possible to conclude that Ch-NPs are a great bactericidal agent which can prevent the main mechanisms developed by BM pathogens to persist in the udder.

AOT reverse micelles as versatile reaction media for chitosan nanoparticles synthesis.

It is known that Chitosan (Ch) can be used in several applications, such as antimicrobial agent or as drug delivery agent. However, being its water dispersibility very low at physiological pH it is necessary to find a way to improve it. One attractive strategy is to synthesize Chitosan Nanoparticles (Ch-NPs). In this work, a versatile method to obtain Ch-NPs with different and controlled sizes, that were successfully prepared by cross-linking reaction of glutaraldehyde and native chitosan inside of n-heptane/sodium 1,4-bis-2-ethylhexylsulfosuccinate (AOT)/water reverse micelles (RMs) is presented. Highly monodisperse NPs were synthesized as confirmed by Dynamic Light Scattering (DLS) and Scanning Electron Microscopy (SEM) techniques. The particle size was dependent on the reactants concentration, cross-linking degree and mainly the amount of water inside of the AOT RMs used as nanoreactors. While the cross-linking is quite difficult to control in bulk water, the reaction inside the RMs is more manageable and efficient.