Guarding women's health: Tackling UTIs and diabetes together.
Urinary tract infections (UTIs) are a common bacterial infection that affect any part of the urinary system, such as the bladder, kidneys, ureters, and urethra. Some common symptoms include: a burning sensation while urinating, frequent urination, cloudy/pungent smelling urine, and pelvic discomfort. In recent years, the prevalence of UTIs has surged, solidifying its position as the world's second most common infectious disease. In 2023 alone, UTIs affected an estimated 150 million people globally, with women being 30 times more likely in getting infected than men. This massive difference in rates is due to anatomical differences, where women have a shorter urethra, located close to the anus, that makes it easier for bacteria to reach the bladder. Additionally, pregnancy and menopause also escalate the chances of UTIs due to hormonal changes and urinary incontinence. Diabetes, a chronic health condition that causes high blood glucose levels, has also been steadily increasing around the globe, climbing from 108 million in 1980 to half a billion in 2023. A 2021 study showed that 101 million people in India have diabetes, where poor lifestyle habits and healthcare accessibility will likely increase this number in coming years. Diabetes is a risk factor for UTIs as frequent urination exposes the urinary tract to toilet bacteria, and high glucose levels provide an environment that is conductive to bacterial growth. Ultimately, this intersection underscores a critical health concern that significantly impacts the well-being of rural Indian women.
During my time at PHRII, I had the opportunity to shadow Ms. Kavitha, a Microbiology PhD scholar at JSS Medical College, to learn more UTI infections among diabetic women and antibiotic resistance in urinary bacteria. I also gained further insights into step-by-step lab procedures.
Sterilization:
Her work consisted of sterilizing the nutrient media and plates where the bacteria from urine samples of female UTI diabetic patients would be cultivated. This was an important step in order to prevent contamination from other microorganisms that could compete or interfere with the growth of urine bacteria. The sterilization process involved the use of an autoclave machine, which used high-temperature steam to eliminate undesired microorganisms present on the equipment containing the nutrient media. This process usually took one hour, and the chemical indicators within the autoclave machine signaled when sterilization was finished.
Sterilized agar plates
Nutrient Medias:
Additionally, Ms. Kavitha used solid and liquid nutrient medias for cultivating her bacterial colonies. The reason for this is that solid media is useful for isolating the individual colonies to understand their measurements and morphologies, while liquid media is used for establishing a foundation to conduct further tests on the bacteria. Further tests include DNA extraction, antibiotic resistance profiling, and genotyping.
Inoculation:
Once the nutrient mix and plates had been sterilized, urine bacteria was streaked onto the media in small quantities. This process involved using a sterile inoculation loop to select a well-isolated colony, and spreading it onto the media in four quadrants of the plate. Since a metal inoculation loop was used, it had to be heated over a Bunsen burner after each streak to sterilize it. If a plastic inoculation loop had been used, it would have to be discarded after streaking to prevent cross-contamination.
Inoculating the urine bacteria onto the culture medium
DNA Extraction:
After the bacteria had been cultured, DNA extraction could then be performed. This is an essential step in a microbiology setting as it paves the way for further tests like PCR and genetic studies. These subsequent analyses allow researchers to determine the genetic traits related to virulence and antibiotic resistance. There are several methods to conduct DNA extraction, which include using silica-based membrane technology. This technology is preferred over crude methods as they are easier to use, and produce a better and purer yield of DNA. This method involves:
Lysing the bacterial sample with a lysis buffer to release DNA.
Binding the DNA to the silica membrane of the kit using a binding buffer.
Washing the bacterial sample with buffers to remove impurities.
Eluting the final, purified DNA from the silica membrane using an elution buffer.
Centrifuging is also important when conducting DNA extraction to ensure the DNA mixes with the buffers and binding agents.
Adding buffers to initiate DNA extraction
DNA Quantification:
After extracting the DNA from bacterial samples, Ms. Kavitha then quantified it. This allowed her to assess the quality of her bacterial DNA and ensure that she had enough for downstream tests like PCR, genotyping, and sequence analysis. Like DNA extraction, there are multiple ways to conduct DNA quantification. One of the most widely used and efficient techniques is fluorometry. Fluorometry is able to quantify DNA as it:
Involves fluorescent dyes that bind to specific regions of the DNA.
After the dyes bind to the DNA, they absorb light at a certain wavelength and release light at a longer wavelength known as the fluorescence.
This fluorescence is quantified by a fluorometer, a machine that measures light along the emission spectrum, to determine the amount of DNA. The intensity of the fluorescence is proportional to the quantity of bacterial DNA.
Additionally, this technique is light-sensitive, where the fluorescent dyes degrade under strong light. It is therefore performed in dark conditions.
Fluorometer reading of bacterial DNA
Shadowing Ms. Kavitha in the lab gave me great exposure to the field of microbiology and its application to women's health. It was interesting to see how infections like UTIs, which mainly affect women, are studied and can be better understood. This experience has enriched my research interests and has given me insights on the intersection between UTIs and diabetes in rural India.
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