RESUMO
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 (0-200â¯nM) did not affect the viability of MAC-T cells nor that of BoMac cells after 24 and 72â¯h. Concentrations of 0-100â¯mM for 24â¯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 24â¯h decreased the internalization of S. aureus V329 into MAC-T cells (0-100â¯nM), and stimulated the phagocytosis of the same strain and of S. xylosus 4913 (0-10â¯nM). Calcitriol and two conditioned media, obtained by treating the cells with 25-200â¯nM of the metabolite for 24â¯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 (0-200â¯nM for 12 and 24â¯h, respectively). In contrast, the conditioned media (0-100â¯nM for 24â¯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.
Assuntos
Biofilmes , Imunidade Inata , Mastite Bovina , Fagocitose , Staphylococcus , Animais , Bovinos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Feminino , Mastite Bovina/microbiologia , Mastite Bovina/imunologia , Imunidade Inata/efeitos dos fármacos , Staphylococcus/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Calcitriol/farmacologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/tratamento farmacológico , Linhagem Celular , Glândulas Mamárias Animais/microbiologia , Glândulas Mamárias Animais/imunologia , Macrófagos/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Antibacterianos/farmacologia , Quitosana/farmacologia , Mastite Bovina/tratamento farmacológico , Nanopartículas/química , Staphylococcus/efeitos dos fármacos , Animais , Antibacterianos/síntese química , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Bovinos , Quitosana/síntese química , Quitosana/química , Relação Dose-Resposta a Droga , Feminino , Interações Hidrofóbicas e Hidrofílicas , Mastite Bovina/microbiologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Biofilmes/crescimento & desenvolvimento , Mastite Bovina/microbiologia , Staphylococcus/classificação , Staphylococcus/fisiologia , Animais , Bovinos , Sobrevivência Celular , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/microbiologia , Leite/microbiologia , FilogeniaRESUMO
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.