Antibiotic sensitivity testing

Antibiotic sensitivity testing is the scientific measurement of how bacteria respond to different antibiotics. This process is vital because many bacteria develop resistance to certain drugs. Resistance means the medicine no longer stops the germ from growing.

Without testing, doctors must use empiric therapy. This is when a doctor chooses a medicine based on clinical suspicion. They guess based on common bacteria that cause specific infections. Sensitivity testing allows a shift to directed therapy. In directed therapy, the doctor chooses a drug based on real knowledge of the specific organism. This ensures the treatment is actually effective against the patient's infection.

To perform these tests, laboratories use two main approaches: phenotypic and genetic methods. Phenotypic testing observes how the bacteria actually grow when exposed to drugs. Genetic testing looks for specific DNA markers that cause resistance.

A key goal of these tests is finding the minimum inhibitory concentration (MIC). The MIC is the lowest concentration of an antibiotic that prevents bacterial growth. Scientists use the MIC to understand exactly how much medicine is needed to fight an infection. Knowing the MIC helps move from general guesses to precise medical treatments.

One common manual method is disc diffusion, often called the Kirby-Bauer method. First, a scientist must prepare an inoculum, which is a sample of bacteria. To ensure accuracy, this sample must be standardized. Scientists compare the turbidity, or cloudiness, of the liquid to McFarland standards.

Once the concentration is correct, they spread the bacteria onto an agar plate. They then place small paper discs soaked in antibiotics onto the agar. If the antibiotic works, a clear ring appears around the disc. This is called a zone of inhibition. The diameter of this ring helps estimate the MIC.

Other phenotypic methods provide different ways to measure growth. The Etest uses a plastic strip rather than individual discs.

This strip is impregnated with a range of antibiotic concentrations. When placed on agar, it creates a teardrop-shaped zone of inhibition. The point where the zone meets the strip indicates the MIC. Another method is broth dilution, which is considered the gold standard for phenotypic testing. In this method, bacteria are placed into multiple tubes containing different antibiotic concentrations. Scientists then use visual inspection or optical tools to find the lowest concentration that stops growth.

Modern laboratories often use automated systems to handle these tasks. Machines such as the VITEK 2, BD Phoenix, and Microscan are common. These systems introduce bacterial suspensions into pre-formulated antibiotic panels. The panels are incubated, and the machines measure growth using light-based methods like spectrophotometry or fluorescence detection. An expert computer system then correlates these measurements to report the MIC. While these machines are faster and more standardized, they can sometimes be less accurate for certain organisms. Because of this, manual disc diffusion remains a useful backup method.

Genetic methods offer a rapid way to identify resistance by looking at DNA. Polymerase chain reaction, or PCR, is a widely used technique. In PCR, the double helix of the bacterial DNA is denatured, meaning the two strands separate. Scientists add primers and DNA polymerase to the solution. If a specific resistance gene is present, the process doubles the amount of that gene every time it runs. This allows scientists to detect genes like mecA in Staphylococcus aureus or vanA in Enterococcus. While these tests are very fast, they can be expensive and require highly trained staff. Additionally, finding a resistance gene does not always match how the bacteria behaves in a living culture.

The history of these tests tracks closely with the history of medicine. The need for sensitivity testing began with the discovery of penicillin, the first beta-lactam antibiotic. Early methods were simple and relied on basic culture or dilution. The Etest strip became available in the 1980s to improve gradient testing. By the early 2000s, genetic methods like PCR became common in labs. Today, researchers are exploring even newer technologies like microfluidics. These advancements aim to make testing faster and more accurate for everyone.