RESUMEN
This study was conducted to investigate the diversity and antimicrobial resistance profiling of Staphylococcus species causing sub-clinical mastitis (SCM) in dairy herds in Bangladesh as well as putative risk factors associated with the infections. Individual quarter milk samples were collected from a total of 284 lactating cows from 30 dairy farms were screened by means of California mastitis test; 178 (62.7%) of them had at least of quarter affected by SCM. After conventional microbiological isolation procedures, PCR tests were used for Staphylococcus species identification and detection of antimicrobial resistance and virulence genes. S. chromogenes (65.7%) was the most predominant species followed by, S. epidermidis (20.2%), S. haemolyticus (19.1%), S. aureus (15.7%), and S. sciuri (5.6%). High levels of antimicrobial resistance to ampicillin and amoxicillin/clavulanic acid were observed in S. aureus (82.1% and 75%) and S. sciuri (80% and 70%), while resistance to cefepime was markedly higher in S. chromogenes (95.7%), S. haemolyticus (94.1%), and S. epidermidis (97.2%). Multidrug resistance isolates were identified in all five species. The mecA gene was detected in S. aureus (32.1%) and S. chromogenes (5.98%). In addition, 20% S. sciuri and 17.7% S. haemolyticus carried the cytotoxin (pvl) gene, while 14.3% S. aureus harbored the toxic shock syndrome toxin (tst) gene. Multivariable logistic regression analysis identified "Old aged" (OR [CI]: 3.5 [1-12.4]); "Early stage of lactation" (OR [CI]: 3.4 [1.2-9.7]) and, "Firm udder condition" (OR [CI]: 4.2 [1.2-14.6]) as risk factors associated with SCM caused by S. aureus, S. chromogenes, and S. haemolyticus, respectively. Moreover, "Use of antimicrobials" (OR [CI]: 10.4 [3.4-32.1] and "History of previous clinical mastitis" (OR [CI]: 4.9 [1.2-19.7] for the carriage of methicillin-resistant Staphylococcus spp.
RESUMEN
Mastitis is one of the most frequent and costly production diseases of dairy cattle. It is frequently treated with broad-spectrum antimicrobials. The objectives of this work were to investigate the prevalence of Staphylococcus aureus and Escherichia coli, find out the antimicrobials used in mastitis treatment, and explore the antimicrobial resistance profile including detection of resistance genes. Bacterial species and antimicrobial resistance genes were confirmed by the polymerase-chain reaction. A total of 450 cows were screened, where 23 (5.11%) and 173 (38.44%) were affected with clinical and sub-clinical mastitis, respectively. The prevalence of S. aureus was 39.13% (n = 9) and 47.97%(n = 83) while, E. coli was 30.43% (n = 7) and 15.60% (n = 27) in clinical and sub-clinical mastitis affected cows, respectively. The highest antimicrobials used for mastitis treatment were ciprofloxacin (83.34%), amoxycillin (80%) and ceftriaxone (76.67%). More than, 70% of S. aureus showed resistance against ampicillin, oxacillin, and tetracycline and more than 60% of E. coli exhibited resistance against oxacillin and sulfamethoxazole-trimethoprim. Selected antimicrobial resistance genes (mecA, tetK, tetL, tetM, tetA, tetB, tetC, sul1, sul2 and sul3) were identified from S. aureus and E. coli. Surprisingly, 7 (7.61%) S. aureus carried the mecA gene and were confirmed as methicillin-resistant S. aureus (MRSA). The most prevalent resistance genes were tetK 18 (19.57%) and tetL 13 (14.13%) for S. aureus, whereas sul1 16 (47.06%), tetA 12 (35.29%), sul2 11 (32.35%) and tetB 7 (20.59%) were the most common resistance genes in E. coli. Indiscriminate use of antimicrobials and the presence of multidrug-resistant bacteria suggest a potential threat to public health.
RESUMEN
Two-dimensional capillary electrophoresis was used for the separation of proteins and biogenic amines from the mouse AtT-20 cell line. The first-dimension capillary contained a TRIS-CHES-SDS-dextran buffer to perform capillary sieving electrophoresis, which is based on molecular weight of proteins. The second-dimension capillary contained a TRIS-CHES-SDS buffer for micel1ar electrokinetic capillary chromatography. After a 61 seconds preliminary separation, fractions from the first-dimension capillary were successively transferred to the second-dimension capillary, where they further separated by MECC. The two-dimensional separation required 60 minutes.
RESUMEN
Differential detergent fractionation was used to sequentially extract cytosolic, membrane, nuclear, and cytoskeletal fractions from AtT-20 cells. Extracted components were denatured by sodium dodecyl sulfate (SDS) and then labeled with the fluorogenic reagent 3-(2-furoyl) quinoline-1-carboxaldehyde. Both capillary sieving electrophoresis (CSE) and micellar electrokinetic capillary chromatography (MECC) were used to separate labeled components by one-dimensional (1D) electrophoresis. Labeled components were also separated by two-dimensional (2D) capillary electrophoresis; CSE was employed in the first dimension and MECC in the second dimension. Roughly 150 fractions were transferred from the first to the second capillary for this comprehensive analysis in 2.5 h.
Asunto(s)
Detergentes/química , Electroforesis Capilar/métodos , Electroforesis en Gel Bidimensional/métodos , Animales , Células Cultivadas , Cromatografía Capilar Electrocinética Micelar/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Ratones , Proteínas/análisisRESUMEN
The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by micellar electrokinetic capillary chromatography (MEKC) to obtain protein expression fingerprints. Analytes were detected in a sheath flow cuvette using laser-induced fluorescence. In a 1-h 2D-CE separation, over 100 components were resolved with a spot capacity of 380.
Asunto(s)
Electroforesis Capilar/métodos , Electroforesis en Gel Bidimensional/métodos , Embrión de Mamíferos/química , Proteínas/análisis , Animales , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Ratones , Ratones Noqueados , Reproducibilidad de los Resultados , Cigoto/química , Cigoto/metabolismoAsunto(s)
Bioquímica/métodos , Técnicas de Química Analítica/métodos , Técnicas Citológicas , Electroforesis Capilar/métodos , Neurociencias/métodos , Animales , Carbohidratos/análisis , ADN/análisis , Metilación de ADN , Electroforesis Capilar/instrumentación , Fluorescencia , Humanos , Lípidos/análisis , Espectrometría de Masas/métodos , Poliaminas/análisis , Polimorfismo Genético , Proteínas/análisisRESUMEN
We have developed a two-dimensional capillary electrophoresis method for the study of protein expression in single mammalian cells. The first-dimension capillary contains an SDS-pullulan buffer system to perform capillary sieving electrophoresis, which separates proteins based on molecular weight. The second-dimension capillary contains an SDS buffer for micellar electrokinetic capillary chromatography. After a 6-min-long preliminary separation, fractions from the first capillary are successively transferred to a second capillary, where they undergo further separation by MECC. Over 100 transfers and second-dimension separations are performed over an approximately 3.5-h-long period. We demonstrate this technology by generating protein fingerprints from single native MC3T3-E1 osteoprogenitor cells and MC3T3-E1 cells transfected with the human transcription regulator TWIST. We also present single-cell protein fingerprints from MCF-7 breast cancer cells before and following treatment to induce apoptosis.
Asunto(s)
Cromatografía Capilar Electrocinética Micelar/métodos , Electroforesis Capilar/métodos , Proteínas/aislamiento & purificación , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Ratones , Peso Molecular , Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas/metabolismo , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
Strong and selective binding to a trihistidine peptide has been achieved employing Cu(2+)-histidine interactions in aqueous medium (25 mM HEPES buffer, pH 7.0). When the pattern of cupric ions on a complex matched with the pattern of histidines on the peptide, a strong and selective binding was observed. UV-vis spectroscopic studies show that the cupric ions coordinate to the histidines of the peptides. Thermodynamic studies reveal that the binding process is enthalpy driven over the entire range of working temperature (25-40 degrees C). An enthalpy-entropy compensation effect was also observed.