This study outlines a method for isolating and identifying antibiotic-resistant bacteria from water samples. The process involves filtering, culturing on selective media, and molecular techniques to analyze resistance genes.
Begin with a water sample that contains waterborne bacteria, and filter it to remove debris.
Transfer the diluted filtrate onto low-nutrient agar plates, each supplemented with a specific antibiotic. Spread the filtrate and incubate.
Bacteria carrying a specific antibiotic-resistance gene grow on the corresponding antibiotic plates and form colonies.
Count the colonies to determine the colony-forming units (CFUs), representing the number of antibiotic-resistant bacteria.
Select a single colony from each plate and suspend it in sterile distilled water.
Heat the suspension to lyse the cells and release intracellular contents, including bacterial DNA that contains the antibiotic-resistance gene.
Centrifuge the tubes to separate the debris and collect the supernatant containing DNA.
Add amplification reagents to the extracted DNA.
Place the tubes in a thermal cycler and run the program to amplify a specific region of bacterial DNA.
The amplified DNA is ready for sequence analysis to identify the antibiotic resistance gene.
Process the water sample aseptically by filtering it through a sterile muslin cloth to remove any particulate matter. Then, serially dilute the filtered water sample for further analysis. To determine the antibiotic resistant bacterial count, add the antibiotics separately into tubes containing 20 milliliters of sterile molten R2A agar modified approximately at 40 degrees Celsius.
Swirl to mix evenly and pour onto sterile Petri plates before the agar solidifies. Spread 100 microliters of bacterial suspension onto their respective antibiotic containing R2A agar modified plates. Incubate the plates at 35 to 37 degrees Celsius for 48 hours, and then determine the bacterial count.
To prepare the DNA template from the isolates for PCR, using a sterile toothpick, take a single isolated pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 microliters of sterile double distilled water in a micro centrifuge tube and boil it in a boiling water bath or a dry bath for 10 minutes at 100 degrees Celsius. Centrifuge the suspension at 10,000 g for two minutes to pellet the debris and transfer the supernatant to a fresh sterile microcentrifuge tube to use as the crude DNA template.
To amplify the V3 region of the 16S rRNA gene by PCR, prepare 40 microliters of the reaction mixture in a PCR tube. Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler.
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