For the detection of Methicillin-Resistant Staphylococcus aureus (MRSA), Dr. Md. Mosharof Hosen collected milk samples from buffaloes across various buffalo-rearing regions in Bangladesh, Samples were then transported to the Department of Medicine Laboratory at Sylhet Agricultural University for detailed microbiological and molecular analysis. To ensure the accuracy of MRSA detection and maintain sample integrity, he used only properly sterilized and disinfected collection tubes. Throughout the entire process—from collection to laboratory examination—he strictly adhered to aseptic techniques to prevent contamination and ensure the reliability of the research results.

Dr. Hosen ensured that all samples were promptly transferred to the Medicine Laboratory of Sylhet Agricultural University immediately after collection from the research areas. To enhance the growth and isolation of microorganisms from the samples, he prepared various types of culture media. The media frequently used in this study included Nutrient Broth, MacConkey Agar, Eosin Methylene Blue (EMB) Agar, Mannitol Salt Agar, and Soybean Casein Digest Medium. These selective and differential media were essential for enriching and identifying Staphylococcus aureus and other bacteria relevant to the study. Following media preparation

Dr. Hosen cultured the samples on Petri dishes to facilitate the growth and multiplication of microorganisms. Through this process of culture, subculture, and ultimately obtaining pure cultures, the targeted organisms were isolated. Identification of the organisms was based on colony morphology, pigment production, and a series of biochemical tests. This systematic approach ensured accurate recognition and characterization of the bacterial species present in the samples.

Identified the bacterial isolates by performing various biochemical tests to examine their characteristic traits. Among the tests routinely conducted were motility, indole, and urease tests. Additionally, citrate utilization was evaluated using Simmons’ Citrate Agar, and Voges-Proskauer tests were carried out in his laboratory. These biochemical assessments were crucial for the accurate identification and differentiation of the microorganisms involved in the study.

Dr. Hosen utilized the polymerase chain reaction (PCR) technique to amplify specific DNA sequences of the target bacteria, enabling precise identification. After isolating the organisms, molecular confirmation was performed through PCR analysis. He detected key virulence genes (hla, icaA, sea, and fnbA) as well as antimicrobial resistance genes (tetA, strA, aac-3(iv), and sul1) by conducting both uniplex and multiplex PCR assays. This molecular approach provided definitive confirmation of the bacterial isolates and their genetic determinants related to virulence and antibiotic resistance.

Following PCR amplification, the PCR products to gel electrophoresis to separate and analyze the DNA fragments. The gel was then placed under a UV trans-illuminator, allowing the visualization of PCR bands. The results were documented and analyzed using a computer imaging system, facilitating accurate interpretation of the molecular data.

The antibiotic sensitivity of each PCR-positive bacterial isolate was assessed using the Kirby-Bauer disk diffusion method. Twelve commonly used antibiotics were tested to determine the susceptibility patterns of the isolates. This method provided crucial information on antimicrobial resistance profiles to guide appropriate treatment options.