Background: Senior undergraduates isolated a bacterial strain from an anal swab sample and plated on Gram-negative selective plates (EMB). They got the most common gut-associated species, E. coli.

Objective: Determine the anal bacterial isolate’s susceptibility to varying antibiotics with diverse modes of action.

Results: After reviewing and grading 20 students’ lab reports (screenshots of three representative reports), I think there are a few things worth noting here.

1. Penicillin, the first antibiotic discovered, targets the final stage of bacterial cell wall synthesis (peptidoglycan cross-linking), should have a broad-spectrum, but did not work for the E. coli strain across the entire class. It is the “worst drug” in this study and serves as a negative control as most students noticed a 0 Zone of inhibition.

2. Vancomycin also targets the cell wall; it did not inhibit the E.coli across all of the lab reports. It is believed that vancomycin is too large a molecule to efficiently pass through the cell porins.

3. Rifampin is a potent drug for tuberculosis (TB), but did not work for E. coli probably due to the same reason as Penicillin and Vancomycin being hard to pass through the intrinsic resistance in the Gram-negative bacteria’s outer membrane.

What is working?

1. The fluoroquinolone drug works like a charm. Ciprofloxacin is a bactericidal, second-generation fluoroquinolone antibiotic that kills bacteria by inhibiting DNA gyrase (topoisomerase II) and topoisomerase IV. Very effective against Gram-negative bacteria such as urinary tract infection E. coli. In the lab reports, students noticed the greatest zone of inhibition came from Ciprofloxacin!

2. Worth noticing that the 4th-generation, broad-spectrum beta-lactam drug - Piperacillin works the second best. So it is likely the cells did not have a mechanism to break down beta-lactam with beta-lactamase or pump out the beta-lactam. It is more likely due to the physical mechanisms of the cell membrane.

3. Gentamicin is an aminoglycoside drug that targets the 30S ribosomal subunit, thus halting protein synthesis. It is super effective (3rd largest zone of inhibation in many student lab reports) and therefore believed to effectively cross the cell membrane of Gram-negative bacteria and reach the ribosome.

4. Tetracycline, similar to Gentamicin, is a broad spectrum drug that targets on the 30S ribosomal subunit. Highly effective on both G- and G+ bacteria.

5. Polymyxin B is unique as it directly targets the cell membrane but acts as a detergent, forming pores on the outer/inner membrane. Students did not report any case of E.coli’s resistance to Polymycin B, although it is not as effective as Ciprofloxacin, Piperacillin, and Gentamicin.

6. Most students had their E.coli being susceptible to Azithromycin (Macrolide), which is a very large molecule that targets the 50S ribosome subunit.

Mixed results and unclear

The greatest variations among students were seen in two drugs, Amoxicillin and Cephalothin, both of which are classified as beta-lactam antibiotics. Just like Penicillin and Piperacillin, they bind to penicillin-binding proteins (PBPs) to interfere with cell wall synthesis.

1. Amoxicillin is a third-generation beta-lactam drug. Its usage is paired with a beta-lactamase inhibitor, clavulanic acid, which should slow down degradation by the cell’s enzymes. Not surprisingly, it is relatively more effective than Penicillin (0 inhibition) but not as effective as Piperacillin (which is a 4th generation beta-lactam antibiotic). However, some students reported intermediate resistance towards Amoxicillin. Unexpected resistance could be due to real variations among the strains.

2. Cephalothin acts as a bactericidal agent by binding to penicillin-binding proteins (PBPs), which weakens the bacterial cell wall and leads to cell lysis. It is designed to work for G+ cells, and has moderate effects on G- cells. Cephalothin is highly effective against beta-lactamase-producing staphylococci because it differ from penicillins by having this 6-membered ring instead of a 5-membered thiazolidine ring, providing greater stability against beta-lactamases. Not highly effective on G-, many students reported intermediate resistance whereas some reported susceptibility.