Question

1. Why do different drugs have different ranges of diameters that lead to assignment of Resistant, Intermediate or Susceptible as the outcomes for each test? (For example, why is a strain diagnosed as streptomycin resistant when the diameter is 11 mm or less around a streptomycin disk, but determined to be tetracycline resistant if the diameter is 14 mm or less around the tetracycline disk? See image below for example)
2. What does it mean if there is no zone of inhibition around a disk in the Kirby Bauer test?
3. What is one reason that there is widespread resistance to the quinolone class of antibiotics (also known as fluoroquinolones)?
4. Select a drug that would be best to treat a pregnant woman with a bacteremia (blood) infection from this E. coli, describe why you selected this drug and what it is going to do to the bacteria in her blood.

Answer 1

Answer 1: Let me first state that the image which is mentioned in the question is not uploaded here with the question. Still, I could assume the problem by reading out the question.

The effectiveness of a chemotherapeutic agent against a microbe or pathogen is determined by MIC (Minimal Inhibitory Concentration), that is, the lowest concentration of a drug which is required to prevent the growth of the pathogen. However, MLC (Minimal Lethal Concentration) is the lowest concentration of the drug required to kill the pathogen. There are various methods (like dilution susceptibility test, disk diffusion tests, etest) to determine the susceptibility of a microbe towards a particular drug. Currently, the most used method is disk diffusion test (Kirby-Bauer method), which was developed by William Kirby, A.W Bauer and their group at the University of Washington Medical School.

Principle of this test: Few isolated colonies of the pathogen are mixed well in a liquid broth, which is then used to inoculate an agar plate by a sterile cotton swab. The agar plate containing the pathogen is dried for 5minutes, and the antibiotic disks are placed on the plate with a sterile forcep. The plate is immediately placed in the incubator for 16-18 hours. Post incubation, we observe that some clear zones might appear around the antibiotic disks whose diameter is measured that leads to the assignment of resistant, intermediate or susceptible zones.

Why these zones appear?

When the antibiotic disks are placed on the plate, the antibiotic starts diffusing out radially outward through the agar producing an antibiotic concentration gradient. The antibiotic is present at higher concentrations near the disc, as the diameter of the clear zone increases, the concentration of the antibiotic is lowered to a point where it is unable to kill the microbe anymore. However, if the pathogen is resistant to the drug, no clear zone will be produced. Generally, the wider the clear zone, the more susceptible the pathogen is.

Zone width can also be affected by other factors:

· The solubility and diffusion rate of the drug in the agar

· Antibiotic’s initial concentration

However, the question is why these clear zones are different for individual drugs when tested towards a particular pathogen. Both Streptomycin and Tetracycline bind to the 30S subunit of the ribosome (commonly known as protein synthesis inhibitors). But the structure of the drug is different which might affect zone width. The most important factor is that tetracycline is a broad spectrum drug; it can affect a huge range of gram positive and gram negative bacterium as compared to streptomycin. Therefore, a strain diagnosed as streptomycin resistant when the diameter is 11 mm or less around a streptomycin disk (less susceptible to streptomycin), but determined to be tetracycline resistant if the diameter is 14 mm or less around the tetracycline disk (more susceptible to tetracycline).

Answer 2: If there is no zone of inhibition around a disk in the Kirby-Bauer test, the pathogen is resistant to the antibiotic. The details of Kirby-Bauer test has been explained in Answer 1.

Answer 3: Quinolones are synthetic drugs that contain 4-quinolone ring. They act by inhibiting bacterial DNA gyrase or topoisomerase II and topoisomerase IV, thereby inhibiting DNA strand separation and uncoiling, leading to bactericidal activity. Nalidixic acid, the first member of the group was discovered by George Lesher in 1962 as a byproduct during chloroquine synthesis.

Reasons of widespread resistance:

1. Overuse of the drug

2. Mutation in the A and B subunits of bacterial topoisomerase class II and IV

3. Qnr proteins reduce the binding sites for quinolone drugs

4. Another plasmid encoded protein, aac(6’)-Ib-cr, acetylates the nitrogen of the C7 piperazine ring that is found in norfloxacin and ciprofloxacin, which reduces drug activity

5. Efflux pumps (OqxAB, QepA1 and QepA2) helps in flushing the drug out of the cell