1,25 dihydroxyvitamin D3-mediated effects on bovine innate immunity and on biofilm-forming Staphylococcus spp. isolated from cattle with mastitis.

Mastitis is one the most widespread and serious diseases in dairy cattle. Recurrent and chronic infections are often attributable to certain pathogenicity mechanisms in mastitis-causing pathogens such as Staphylococcus spp. These include growing in biofilm and invading cells, both of which make it possible to resist or evade antimicrobial therapies and the host's immune system. This study tested the effects of active vitamin D3 (i.e., calcitriol or 1,25-dihydroxyvitamin D3) on the internalization and phagocytosis of biofilm-forming Staphylococcus spp. isolated from animals with mastitis. Two established bovine cell lines were used: MAC-T (mammary epithelial cells) and BoMac (macrophages). Calcitriol (0-200 nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72 h. Concentrations of 0-100 mM for 24 h upregulated the expression of 24-hydroxylase in MAC-T cells, but did not alter that of VDR. Pre-treatment of the cells with calcitriol for 24 h decreased the internalization of S. aureus V329 into MAC-T cells (0-100 nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10 nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200 nM of the metabolite for 24 h, were also assessed in terms of their antimicrobial and antibiofilm activity. Neither calcitriol by itself nor the conditioned media affected staphylococcal growth or biofilm formation (0-200 nM for 12 and 24 h, respectively). In contrast, the conditioned media (0-100 nM for 24 h) decreased the biomass of preformed non-aureus staphylococcal biofilms and killed the bacteria within them, without affecting metabolic activity. These effects may be mediated by reactive oxygen species and proteins with antimicrobial and/or antibiofilm activity. In short, calcitriol could make pathogens more accessible to antimicrobial therapies and enhance bacterial clearance by professional phagocytes. Moreover, it may modulate the host's endogenous defenses in the bovine udder and help combat preformed non-aureus staphylococcal biofilms (S. chromogenes 40, S. xylosus 4913, and/or S. haemolyticus 6). The findings confirm calcitriol's potential as an adjuvant to prevent and/or treat intramammary infections caused by Staphylococcus spp., which would in turn contribute to reducing antibiotic use on dairy farms.

Teat-apex colonizer Bacillus from healthy cows antagonizes mastitis-causing Staphylococcus aureus biofilms.

Staphylococcus aureus is the most frequent causal agent of bovine mastitis, which is largely responsible for milk production losses worldwide. The pathogen's ability to form stable biofilms facilitates intramammary colonization and may explain disease persistence. This virulence factor is also highly influential in the development of chronic intramammary infections refractory to antimicrobial therapy, which is why novel therapies that can tackle multiple targets are necessary. Since udder microbiota have important implications in mastitis pathogenesis, they offer opportunities to develop alternative prophylactic and therapeutic strategies. Here, we observed that a Bacillus strain from the teat apex of lactating cows was associated to reduce colonization by S. aureus. The strain, identified as Bacillus sp. H21, was able to antagonize in-formation or mature S. aureus biofilms associated to intramammary infections without affecting cell viability. When exploring the metabolite responsible for this activity, we found that a widespread class of Bacillus exopolysaccharide, levan, eliminated the pathogenic biofilm under evaluated conditions. Moreover, levan had no cytotoxic effects on bovine cellular lines at the biologically active concentration range, which demonstrates its potential for pathogen control. Our results indicate that commensal Bacillus may counteract S. aureus-induced mastitis, and could therefore be used in novel biotechnological strategies to prevent and/or treat this disease.

Chitosan can improve antimicrobial treatment independently of bacterial lifestyle, biofilm biomass intensity and antibiotic resistance pattern in non-aureus staphylococci (NAS) isolated from bovine clinical mastitis.

Bovine mastitis is the most frequent and costly disease that affects dairy cattle. Non-aureus staphylococci (NAS) are currently one of the main pathogens associated with difficult-to-treat intramammary infections. Biofilm is an important virulence factor that can protect bacteria against antimicrobial treatment and prevent their recognition by the host's immune system. Previously, we found that chronic mastitis isolates which were refractory to antibiotic therapy developed strong biofilm biomass. Now, we evaluated the influence of biofilm biomass intensity on the antibiotic resistance pattern in strong and weak biofilm-forming NAS isolates from clinical mastitis. We also assessed the effect of cloxacillin (Clx) and chitosan (Ch), either alone or in combination, on NAS isolates with different lifestyles and abilities to form biofilm. The antibiotic resistance pattern was not the same in strong and weak biofilm producers, and there was a significant association (p ≤ 0.01) between biofilm biomass intensity and antibiotic resistance. Bacterial viability assays showed that a similar antibiotic concentration was effective at killing both groups when they grew planktonically. In contrast, within biofilm the concentrations needed to eliminate strong producers were 16 to 128 times those needed for weak producers, and more than 1,000 times those required for planktonic cultures. Moreover, Ch alone or combined with Clx had significant antimicrobial activity, and represented an improvement over the activity of the antibiotic on its own, independently of the bacterial lifestyle, the biofilm biomass intensity or the antibiotic resistance pattern. In conclusion, the degree of protection conferred by biofilm against antibiotics appears to be associated with the intensity of its biomass, but treatment with Ch might be able to help counteract it. These findings suggest that bacterial biomass should be considered when designing new antimicrobial therapies aimed at reducing antibiotic concentrations while improving cure rates.

Interaction between bovine mammary epithelial cells and planktonic or biofilm Staphylococcus aureus: The bacterial lifestyle determines its internalization ability and the pathogen recognition.

The main cause of mastitis, one of the most costly diseases in the dairy industry, is bacterial intramammary infection. Many of these bacteria are biofilm formers. Biofilms have been associated with resistance to antibiotics and to the host immune system. Here, we evaluated different experimental models representing bacterial biofilm lifestyle with the aim to study bacterial invasion into bovine mammary epithelial cells and the interaction of these cells with planktonic or biofilm Staphylococcus aureus. Staphylococcus aureus V329, its nonbiofilm-forming mutant and bovine mammary alveolar cells (MAC-T) were used. Bacterial invasion was studied using the gentamicin exclusion test, cell viability by trypan blue exclusion technique, TLR2 expression by flow cytometry, IL1ß/IL6 production by ELISA and IL8/TNFα gene expression by real-time polymerase chain reaction. Biofilm and planktonic S. aureus showed differences in their invasion ability, with the biofilm mode showing a lower ability. Planktonic S. aureus reduced MAC-T viability after 6 h of co-culture, while biofilms did so at 24 h. MAC-T infected with planktonic bacteria showed increased TLR2 expression. Both lifestyles increased IL8 expression and IL1ß/IL6 production but did not modify TNFα expression. Our results demonstrate that the bacterial lifestyle affects the invasion behavior, suggesting that biofilms reduce the bacteria-epithelial cell interaction. Planktonic cultures seem to induce higher cellular activation than biofilms. Further knowledge about the complex host-biofilm interaction is necessary to design more efficient therapies against bovine mastitis.

Chitosan nanoparticles enhance the antibacterial activity of the native polymer against bovine mastitis pathogens.

Staphylococcus is the most commonly isolated genus from animals with intramammary infections, and mastitis is the most prevalent disease that affects dairy cows in many countries. These pathogens can live in biofilms, a self-produced matrix, which allow them evade the innate immune system and the antibiotic therapy, thereby producing persistent infections. The aim of this study was to explore the antimicrobial potential of chitosan nanoparticles (Ch-NPs) obtained by the reverse micellar method. We found that the nanoformulation developed presents antimicrobial activity against mastitis pathogens in a dose-dependent manner. Moreover, different experiments corroborated that the antimicrobial effectiveness of Ch-NP was greater than that shown by the native polymer used in the preparation of these nanocomposites. Ch-NPs caused membrane damage to bacterial cells and inhibited bacterial biofilm formation, without affecting the viability of bovine cells. These findings show the great potential of Ch-NPs as therapeutic agent for bovine mastitis treatment.

Chitosan disrupts biofilm formation and promotes biofilm eradication in Staphylococcus species isolated from bovine mastitis.

Staphylococci are the main pathogens associated with hard-to-control intramammary infections in dairy cattle, and bacterial biofilms are suspected to be responsible for the antimicrobial resistance and persistence of this disease. Biofilms have the ability to resist to higher levels of antibiotics and reduce their efficacy. It is thus necessary to develop strategies targeted to bacterial biofilm infections. Chitosan is a polysaccharide with a proven broad spectrum of antimicrobial activity against fungi and bacteria. The aim of this study was assess the effect of low molecular weight (LMW) chitosan against biofilm hyperproducer Staphylococcus spp. (S. aureus and S. xylosus) strains usually involved in chronic bovine mastitis, and to test their efficacy in biofilm formation and eradication. The results obtained showed that LMW chitosan is able to inhibit S. aureus and S. xylosus planktonic growth in a dose-dependent manner and reduce bacterial viability. LMW chitosan inhibits biofilm formation, reduces biofilm viability and disrupts established biofilm. These results indicate the inhibitory effects of chitosan on biofilm formation, and these effects are observed at lower concentrations for S. xylosus. Our studies show the potential of this biopolymer to be used as an effective antibiofilm agent able to act upon staphylococcal infections.

Commensal coagulase-negative Staphylococcus from the udder of healthy cows inhibits biofilm formation of mastitis-related pathogens.

Bovine mastitis, considered the most important cause of economic losses in the dairy industry, is a major concern in veterinary medicine. Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main pathogens associated with intramammary infections, and bacterial biofilms are suspected to be responsible for the persistence of this disease. CNS from the udder are not necessarily associated with intramammary infections. In fact, some commensal CNS have been shown to have biological activities. This issue led us to screen exoproducts from commensal Staphylococcus chromogenes for anti-biofilm activity against different mastitis pathogens. The cell-free supernatant from S. chromogenes LN1 (LN1-CFS) was confirmed to display a non-biocidal inhibition of pathogenic biofilms. The supernatant was subjected to various treatments to estimate the nature of the biofilm-inhibiting compounds. The results showed that the bioactive compound >5KDa in mass is sensitive to thermal treatment and proteinase K digestion, suggesting its protein properties. LN1-CFS was able to significantly inhibit S. aureus and CNS biofilm formation in a dose-independent manner and without affecting the viability of bovine cells. These findings reveal a new activity of the udder microflora of healthy animals. Studies are underway to purify and identify the anti-biofilm biocompound and to evaluate its biological activity in vivo.

[Molecular aspects of antineoplasic action of vitamin D]. / Aspectos moleculares de la accion antineoplasica de la vitamina D.

The effects of the environment, particularly dietary factors, may influence in the development and prevention of cancer. Vitamin D (colecalciferol) has been associated for years with calcium homeostasis regulation, but many epidemiological, biochemical and genetic studies reveal non classic effects of vitamin D, such as vitamin D involvement in the progression of different types of cancer. The aim of the present article was to give a review about the molecular mechanisms of the antineoplasic action of vitamin D. These effects are still not completely established, but it is well known that vitamin D induces cellular arrest, triggers apoptotic pathways, inhibits angiogenesis and alters cellular adhesion. To maintain suitable vitamin D levels seems to be necessary for many physiological processes, and not only for bone homeostasis. Clinical studies might determine vitamin D levels that can also protect against the cancer development.