RESUMEN
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.
Asunto(s)
Mastitis Bovina , Infecciones Estafilocócicas , Animales , Biopelículas , Bovinos , Células Epiteliales , Femenino , Estilo de Vida , Plancton , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureusRESUMEN
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.
Asunto(s)
Quitosano , Mastitis Bovina , Nanopartículas , Animales , Antibacterianos/toxicidad , Bovinos , Quitosano/farmacología , Femenino , Mastitis Bovina/tratamiento farmacológico , Mastitis Bovina/prevención & control , Staphylococcus aureusRESUMEN
Surface water sources are greatly impacted in areas with major agricultural land use. The atrazine quantification in surface waters as well as the spatial-temporal patterns of this herbicide was studied to detect pollution hotspots and to understand the putative factors responsible of its occurrence at the Ctalamochita river basin. The samples were collected on the aeolian fluvial plain of the river basin during five consecutive years. The results showed the high ubiquity of this compound and several sites with hazardous concentration (exceeding 0.1 µg/L international guidelines). The frequencies of quantification range from 67 to 100% in spring and 33%-67% in autumn. The atrazine content in surface water increased during the warm-rainy season, as consequence of atrazine application events (coinciding to the prevalent crop type). Overall, the study highlights the factors that could have favored atrazine pollution in the river basin such as land use, transport by runoff processes and atmospheric deposition.
Asunto(s)
Atrazina , Contaminantes Químicos del Agua , Atrazina/análisis , Monitoreo del Ambiente , Ríos , Estaciones del Año , Agua , Contaminantes Químicos del Agua/análisis , Contaminación del AguaRESUMEN
Intramammary infections (IMI) cause serious economic losses for farmers and the dairy industry. Cases of subclinical mastitis are commonly the result of infection by minor pathogens such as non-aureus staphylococci (NAS), so their correct identification is important for appropriate therapeutic intervention and management. The aim of this study was to assess the reliability of PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) of the groEL and gap genes to discriminate between bovine-associated NAS species, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the reference method. MALDI-TOF MS was able to correctly identify 112 NAS isolates from bovine IMI at species level out of a total of 115 (97.4%). These results were considered definitive and thus compared with those from the PCR-RFLP analyses. Only 50% (56/112) of the samples classified through groEL PCR-RFLP matched the molecular identity determined by MALDI-TOF MS, whereas coincidence rose to 96.4% (108/112) when comparing results from gap PCR-RFLP and the spectral analysis. This study demonstrates that gap PCR-RFLP is a useful and reliable tool for the identification of NAS species isolated from bovine mastitis.
Asunto(s)
Mastitis Bovina , Infecciones Estafilocócicas , Animales , Bovinos , Femenino , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Infecciones Estafilocócicas/veterinaria , Staphylococcus/genéticaRESUMEN
Staphylococcus aureus and coagulase-negative staphylococci (CNS) are important causes of intramammary infection in dairy cattle, and their ability to produce biofilm is considered an important virulence property in the pathogenesis of mastitis. However, the published date on mechanisms and factors involved in infection persistence in the mammary gland remains unclear. The aim of this study was to investigate whether the main Staphylococcus species involved in bovine intramammary infections possess specific characteristics that promote colonization of the udder. We evaluated the biofilm-forming ability and distribution of adhesion- and biofilm-associated genes of Staphylococcus spp. isolated from bovine mastitis infected animals in Argentinean dairy farms. For this purpose, the phenotypic biofilm formation ability of 209 Staphylococcus spp. from bovine mastitis was investigated. All isolates produced biofilm in vitro, being 35,0% and 45,0% of the 127 S. aureus or 51,0% and 29,0% of the 82 CNS strong and moderate biofilm producers respectively. All S. aureus samples were PCR-positive for icaA, icaD, clfA, clfB and fnbpA genes, 76.3% were positive for fnbpB gene and 11.0% were positive for bap gene. In CNS isolates, the positive rates for icaA and icaD were 73.2%, while for clfA, clfB, fnbpA fnbpB and bap genes the percentage were lower. The results demonstrate that in Staphylococcus spp. biofilm formation, the polysaccharide and the adhesion- and biofilm-associated genes are of overall importance on bovine mastitis in Argentina. Therefore, future works should focus on these pathogenic specific factors for the development of more effective therapies of control, being essential to consider the ability of isolates to produce biofilm.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Genes Bacterianos , Mastitis Bovina/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/genética , Staphylococcus aureus/fisiología , Adhesinas Bacterianas/genética , Animales , Argentina , Bovinos , Granjas , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Chitin and its derivative chitosan (Q) are abundant structural elements in nature. Q has modulatory and anti-inflammatory effects and also regulates the expression of adhesion molecules. The interaction between cells expressing the αEß7 integrin and E-cadherin facilitates tolerogenic signal transmission and localization of lymphocytes at the frontline for interaction with luminal antigens. In this study we evaluated the ability of orally administered Q to stimulate E-cadherin and CD103 expression in vitro and in vivo. Our findings show that Q promoted epithelial cell migration, accelerated wound healing and increased E-cadherin expression in IEC-18 cells and isolated intestinal epithelial cells (IECs) after Q feeding. The upregulation of E-cadherin was dependent on TLR4 and IFNAR signaling, triggering CD103 expression in lymphocytes. Q reinforced the E-cadherin-αEß7 axis, crucial for intestinal barrier integrity and contributed to the localization of lymphocytes on the epithelium.
Asunto(s)
Antígenos CD , Cadherinas , Quitosano , Cadenas alfa de Integrinas , Mucosa Intestinal , Transducción de Señal , Receptor Toll-Like 4 , Animales , Receptor Toll-Like 4/metabolismo , Quitosano/farmacología , Quitosano/química , Cadherinas/metabolismo , Transducción de Señal/efectos de los fármacos , Cadenas alfa de Integrinas/metabolismo , Ratones , Antígenos CD/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Línea Celular , Intestinos/efectos de los fármacos , Ratas , MasculinoRESUMEN
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.
Asunto(s)
Biopelículas , Inmunidad Innata , Mastitis Bovina , Fagocitosis , Staphylococcus , Animales , Bovinos , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Femenino , Mastitis Bovina/microbiología , Mastitis Bovina/inmunología , Inmunidad Innata/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Calcitriol/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/tratamiento farmacológico , Línea Celular , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/inmunología , Macrófagos/microbiología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismoRESUMEN
Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (â¼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (â¼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.
Asunto(s)
Biopelículas , Luz , Mastitis Bovina , Nanopartículas , Polímeros , Animales , Bovinos , Nanopartículas/química , Mastitis Bovina/microbiología , Mastitis Bovina/tratamiento farmacológico , Biopelículas/efectos de los fármacos , Biopelículas/efectos de la radiación , Femenino , Polímeros/química , Polímeros/farmacología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Porfirinas/química , Porfirinas/farmacología , Staphylococcus/efectos de los fármacos , Staphylococcus/efectos de la radiación , Antibacterianos/farmacología , Antibacterianos/química , Microscopía Electrónica de Rastreo , FotoquimioterapiaRESUMEN
The shortcomings of Staphylococcus aureus vaccines to control bovine mastitis have been attributed to insufficient capacity of the vaccines to induce opsonizing antibodies and to stimulate cellular immune responses. Types of antigen, administration route and adjuvant used in a vaccine formulation have been identified as critical factors for the development of opsonic antibodies. Current commercially available vaccines for Staph. aureus bovine mastitis control are formulated with Al(OH)3 and oil-based adjuvants. The aim of this study was to evaluate the immune response of heifers immunized with a Staph. aureus CP5 whole cell vaccine formulated either with Al(OH)3 or ISCOMATRIX™. Twenty primigravid Holstein dairy heifers in the last trimester of gestation were immunized either with a vaccine formulated with ISCOMATRIX™ (n = 6), Al(OH)3 (n = 7), or saline solution (placebo) (n = 7). Immunization was carried out 38 and 10 d before calving. Heifers vaccinated with Staph. aureus adjuvanted with ISCOMATRIX™ responded with significantly higher levels of anti-bacterin and anti-CP5 IgG and IgG2 in sera than animals in the Al(OH)3 or control groups. Animals in the ISCOMATRIX™ group responded with significantly higher anti-bacterin specific IgG in whey than animals in the Al(OH)3 and control groups, detected from the first week post calving until 60 d of lactation. Sera from animals inoculated with Staph. aureus in ISCOMATRIX™, obtained 7 d post partum, significantly increased both the number of neutrophils ingesting bacteria and the number of bacteria being ingested by the neutrophils, compared with sera obtained from heifers vaccinated with Al(OH)3 or non-vaccinated controls. These features coupled to safety of the ISCOMATRIX™ formulation, warrant additional studies.
Asunto(s)
Adyuvantes Inmunológicos , Bovinos/inmunología , Colesterol/inmunología , Fosfolípidos/inmunología , Saponinas/inmunología , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Combinación de Medicamentos , Femenino , Inmunoglobulina G/sangre , Mastitis Bovina/prevención & control , Leche/inmunología , Neutrófilos/inmunología , Fagocitosis , EmbarazoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Bacillus , Enfermedades de los Bovinos , Mastitis Bovina , Infecciones Estafilocócicas , Femenino , Bovinos , Animales , Staphylococcus aureus , Lactancia , Glándulas Mamarias Animales/patología , Infecciones Estafilocócicas/prevención & control , Infecciones Estafilocócicas/veterinaria , Biopelículas , Mastitis Bovina/prevención & control , Leche , Enfermedades de los Bovinos/patologíaRESUMEN
We evaluated the host metabolic response to chronic varied stress during infection with the fungus Candida albicans. We used four groups of female Wistar rats: normal uninfected and unstressed, stressed, C. albicans infected and infected, and stressed. Infected rats reacted with rapid metabolic adjustments, evident as anorexia and body weight loss, partly mediated by glucocorticoids and TNF-alpha. Higher circulating levels of IL-6 and glucose (p < 0.05) revealed the progress and catabolic effect of the inflammatory response. Infected and stressed rats instead showed anorexia associated with infection and weight loss as the result of reduced food intake. This group exhibited a prompt reduction in circulating leptin on day 3 (p < 0.05), reduction in glucose levels and depletion of hepatic glycogen depots. We also evaluated the contribution of TNF-alpha, glucocorticoids, and food deprivation to liver damage. Lipid peroxidation in liver detected in the infected and infected-stressed groups was exacerbated by the glucocorticoid receptor antagonist RU 486, suggesting the modulatory activity of glucocorticoids, while hepatic fat accumulation and glycogen depletion decreased with anti-TNF-alpha treatment. Food deprivation exacerbated liver injury while the response to stress contributed to greater fungal colonization. Our findings emphasize the impact of metabolic alterations on tissue damage when the host immune activity is modulated by stress mediators.
Asunto(s)
Candidiasis/inmunología , Candidiasis/metabolismo , Estrés Psicológico/inmunología , Estrés Psicológico/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Candidiasis/patología , Ingestión de Alimentos/fisiología , Femenino , Privación de Alimentos/fisiología , Glucocorticoides/fisiología , Antagonistas de Hormonas/farmacología , Leptina/sangre , Hígado/enzimología , Hígado/patología , Glucógeno Hepático/metabolismo , Mifepristona/farmacología , Ratas , Ratas Wistar , Estrés Psicológico/patología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
The yeast Candida albicans belongs to the microflora of healthy individuals, although it can infect a variety of tissues ensuing changes in the host's immune status. To evaluate the effect of neuroendocrine input on the early immune response during the fungal infection, we use a 3-day paradigm of chronic varied stress in Wistar rats infected with C. albicans. We find that stress mediators contribute to the spread of the fungus and downregulate critical functions of phagocytic cells at the infection site. Phenotypic and functional alterations of effector cells account for the decreased resistance to candidiasis and condition the development of the adaptive response. Stressed hosts exhibit a higher fungal burden in kidneys and livers associated with hyphal forms. The hepatic inflammatory reaction is compromised with severe steatosis, increment of functional enzymes, marked lipid peroxidation and hepatocyte apoptosis. Moreover, infection-related sickness symptoms are significantly increased by exposure to stress with anorexia, weight loss, lack of leptin and depletion of glycogen depots. Food deprivation exacerbates the liver injury. Stress mediators perturb the complex immune and metabolic program that operates early during fungal spread and promotes severe tissue damage.
Asunto(s)
Tolerancia Inmunológica/inmunología , Huésped Inmunocomprometido/inmunología , Micosis/inmunología , Sistemas Neurosecretores/inmunología , Inmunidad Adaptativa/inmunología , Animales , Caquexia/inmunología , Caquexia/metabolismo , Caquexia/fisiopatología , Modelos Animales de Enfermedad , Hepatitis/inmunología , Hepatitis/metabolismo , Hepatitis/fisiopatología , Humanos , Inmunidad Innata/inmunología , Inmunocompetencia/fisiología , Micosis/fisiopatología , Ratas , Estrés Psicológico/inmunologíaRESUMEN
In this study, water-soluble chitosan (Ch) derivatives were synthesized by the Maillard reaction between Ch and lactose. The Ch derivatives were characterized by FT-IR, 1H-NMR and SLS to determine their structure, degree of deacetylation (DD), and molecular weight (Mw). The solubility at physiological pH, the in vitro antioxidant activity against hydroxyl radical, anion superoxide radical and ABTS cation radical, and the cytotoxicity against epithelial cells of the rat ileum (IEC-18) were also evaluated. The Maillard reaction, derivatives with lower Mw and DD and greater solubility than Ch were obtained. The biological properties of the derivatives were dependent on the concentration, Mw and DD, with antioxidant activity greater than or equal to that of Ch and non-cytotoxic in a wide range of concentrations. The results indicate that Ch derivatization with lactose produces new water-soluble polysaccharides, with antioxidant activity and non-cytotoxic, which can be used as biomaterials for food and pharmaceutical applications.
Asunto(s)
Fenómenos Químicos , Quitosano/química , Citotoxinas/química , Depuradores de Radicales Libres/química , Lactosa/química , Agua/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citotoxinas/toxicidad , Depuradores de Radicales Libres/toxicidad , Ratas , SolubilidadRESUMEN
Inoculation practice with plant growth-promoting bacteria (PGPB) has been proposed as a good biotechnological tool to enhance plant performance and alleviate heavy metal/metalloid stress. Soybean is often cultivated in soil with high arsenic (As) content or irrigated with As-contaminated groundwater, which causes deleterious effects on its growth and yield, even when it was inoculated with rhizobium. Thus, the effect of double inoculation with known PGPB strains, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39 was evaluated in plants grown in pots under controlled conditions and treated with As. First, the viability of these co-cultivated bacteria was assayed using a flow cytometry analysis using SYTO9 and propidium iodide (PI) dyes. This was performed in vitro to evaluate the bacterial population dynamic under 25⯵M AsV and AsIII treatment. A synergistic effect was observed when bacteria were co-cultured, since mortality diminished, compared to each growing alone. Indole acetic acid (IAA) produced by A. brasilense Az39 would be one of the main components involved in B. japonicum E109 mortality reduction, mainly under AsIII treatment. Regarding in vivo assays, under As stress, plant growth improvement, nodule number and N content increase were observed in double inoculated plants. Furthermore, double inoculation strategy reduced As translocation to aerial parts thus improving As phytostabilization potential of soybean plants. These results suggest that double inoculation with B. japonicum E109 and A. brasilense Az39 could be a safe and advantageous practice to improve growth and yield of soybean exposed to As, accompanied by an important metalloid phytostabilization.
Asunto(s)
Arsénico/farmacología , Azospirillum brasilense/metabolismo , Bradyrhizobium/metabolismo , Glycine max/crecimiento & desarrollo , Glycine max/microbiología , Estrés Fisiológico/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Quitosano/farmacología , Mastitis Bovina/tratamiento farmacológico , Nanopartículas/química , Staphylococcus/efectos de los fármacos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Biopelículas/efectos de los fármacos , Bovinos , Quitosano/síntesis química , Quitosano/química , Relación Dosis-Respuesta a Droga , Femenino , Interacciones Hidrofóbicas e Hidrofílicas , Mastitis Bovina/microbiología , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Quitosano/farmacología , Mastitis Bovina/microbiología , Staphylococcus/crecimiento & desarrollo , Staphylococcus/aislamiento & purificación , Animales , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/efectos de los fármacos , Bovinos , Femenino , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Peso Molecular , Staphylococcus/efectos de los fármacos , Staphylococcus/ultraestructuraRESUMEN
We used water-soluble Chitosan obtained by Maillard reaction with glucosamine to microencapsulate soy genistein (Ge) and preserve its biological activity for oral administration. Release of Ge was pH dependent with a super Case II mechanism at pH 1.2 and an anomalous transport with non-Fickian kinetics at pH 6.8. Microencapsulated Ge retained its antioxidant properties in vitro and its daily administration to mice attenuated clinical signs of acute colitis, limited inflammatory reaction and reduced oxidative stress and tissue injury as well. Remarkably, after feeding microencapsulated Ge the production of IL-10 in colonic tissue was restored to levels of untreated controls. According to statistical multivariate analysis, this cytokine was the parameter with the highest influence on the inflammatory/oxidative status. Microencapsulation of Ge with derivatized Chitosan becomes an interesting alternative to develop therapeutic approaches for oxidative inflammatory diseases; our findings suggest that the soy isoflavone could be incorporated into any functional food for application in intestinal inflammation.
Asunto(s)
Antioxidantes/administración & dosificación , Colitis/dietoterapia , Genisteína/administración & dosificación , Administración Oral , Animales , Antioxidantes/química , Antioxidantes/farmacología , Quitosano/química , Colitis/inducido químicamente , Colitis/metabolismo , Citocinas/metabolismo , Suplementos Dietéticos , Modelos Animales de Enfermedad , Composición de Medicamentos/métodos , Femenino , Genisteína/química , Genisteína/farmacología , Interleucina-10/metabolismo , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Solubilidad , Glycine max/químicaRESUMEN
Bovine mastitis affects the health of dairy cows and the profitability of herds worldwide. Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens in bovine intramammary infection. Based on the wide range of antimicrobial, mucoadhesive and immunostimulant properties demonstrated by chitosan, we have evaluated therapy efficiency of chitosan incorporation to cloxacillin antibiotic as well as its effect against different bacterial lifestyles of seven CNS isolates from chronic intramammary infections. The therapeutic effects of combinations were evaluated on planktonic cultures, bacterial biofilms and intracellular growth in mammary epithelial cells. We found that biofilms and intracellular growth forms offered a strong protection against antibiotic therapy. On the other hand, we found that chitosan addition to cloxacillin efficiently reduced the antibiotic concentration necessary for bacterial killing in different lifestyle. Remarkably, the combined treatment was not only able to inhibit bacterial biofilm establishment and increase preformed biofilm eradication, but it also reduced intracellular bacterial viability while it increased IL-6 secretion by infected epithelial cells. These findings provide a new approach to prophylactic drying therapy that could help to improve conventional antimicrobial treatment against different forms of bacterial growth in an efficient, safer and greener manner reducing multiresistant bacteria generation and spread.
Asunto(s)
Antibacterianos/uso terapéutico , Quitosano/uso terapéutico , Cloxacilina/uso terapéutico , Mastitis Bovina/tratamiento farmacológico , Infecciones Estafilocócicas/veterinaria , Staphylococcus/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Bovinos , Quitosano/administración & dosificación , Quitosano/farmacología , Cloxacilina/administración & dosificación , Cloxacilina/farmacología , Femenino , Mastitis Bovina/microbiología , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus/aislamiento & purificación , Staphylococcus/fisiologíaRESUMEN
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.