Staphylococcus aureus causes mastitis in dairy cows, lambs, goats, and skin disorders in pigs and other animals. S. aureus causes localized purulent infections that affect soft tissues, bones, and other organs in humans. Using restriction patterns, the researchers want to isolate and identify methicillin-resistant Staphylococcus aureus (MRSA) strains from cattle and humans. They also hope to assess their genetic relatedness by comparing the mecA1 and mecA2 gene sequence discrepancies. Animals (223 strains) and people have been used to acquire S. aureus strains for study (83). The E-test was used to assess whether or not the bacteria were resistant to methicillin. The mecA1 and mecA2 genes were identified by using pulsed-field gel electrophoresis (PFGE) to analyze DNA restriction patterns. The results were shown. S. aureus strains from animals and men were resistant to methicillin in 32 (14.34 %) and 53 (63.8 %), respectively. PFGE was used to determine the differences between human and veterinary pathology strains. Two strains of bacteria collected from animals were discovered to be identical; nevertheless, microorganisms recovered from humans were found to be significantly similar to the bacteria recovered from animals. Both human and veterinary pathology were implicated in the development of methicillin resistance. The MRSA strains found in humans were much more significant than those found in animals. The strains recovered from animals exhibited a high degree of genetic heterogeneity. Still, the enormous number of indistinguishable bacteria in humans leads one to believe that a dominant clone is present. When it comes to the molecular characterization of MRSA isolates, PFGE might be regarded as the gold standard. Keywords. Animals, Human, MRSA, PFGE, Staphylococcus, mecA genes
Introduction and Aim: Brucellosis is an important zoonotic disease caused by Brucella spp. which is an intracellular gram-negative bacterium. Brucella melitensis lacks the "traditional" virulence factors such as exotoxins or cytolysins, but is capable of persisting intracellularly and evading the immune system. This study aims to identify B. melitensis using PCR and discover genes associated with its severity for early detection and therapy. Materials and Methods: Ten ml of unclotted blood sample was collected from each patient (n=100) suspected to be infected with brucellosis. The Castaneda technique was used to inoculate blood samples onto Brucella Basel agar with a selective supplement and tryptone soy broth in a diphasic flask. Biochemical tests were used in identifying the isolated colonies. B. melitensis isolates were further confirmed by the polymerase chain reaction (PCR) technique using, primers targeting a specific region (IS711 gene) of the genome. Multiplex PCR was used to determine the four virulence related genes (lps B, mgtA, omp25, CBG) in all positive samples. Results: Brucella melitensis was detected in 9% (9/100) of the blood samples. Among the virulence factors, LpsB and mgtA, were detected in all the isolates while, the genes omp25 and CBG were detected in 66.6% and 55.5% of the isolates, respectively. Conclusion: Brucellosis could be diagnosed rapidly using molecular techniques. PCR technique could also be used in identifying the Brucella virulence related genes lpsB, mgtA, CBG, and omp25 that are crucial to the bacterium's pathogenicity in the intracellular environment.
This study involved the use of multiple methods to separate plasmids from bacterial cell DNA for some isolates of pathogenic E. coli through several steps, starting with the analysis of the bacterial cell using lysozymes to remove the outer wall, followed by centrifugation to isolate plasmids found in the solution from the rest of the proteins and other forms of DNA. Many sequential methods were used to separate plasmids. The first method used was the basal denaturation sodium hydroxide-based, which led to the denaturation of the chromosomal DNA without affecting the plasmid DNA, followed by the addition of sodium acetate, which led to the preservation of the shape and structure of the plasmid DNA. Second, using cesium chloride gradient density to isolate the protein cell components and the rest of the DNA forms. The different densities of these components led to the appearance of sequential bundles depending on their different molecular weights. Ethidium bromide, which gave the plasmid bundles a fluorescent dye, was added using ultraviolet rays. The last purification method was using the boiling method using a water bath. Plasmid samples extracted from the previous methods were taken to perform the purification and separation process using the high electrophoresis method. Akarose gel was used to separate the high molecular weight protein fragments. Standard proteins and plasmids were migrated to determine the volumes of purified plasmids.
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