presentation

Question 1

As efflux pump overexpression is a primary driver for DLX resistance, do you think combining DLX with an inhibitor (like the PaβN) would be a strategy, or are there safety concerns?

Answer 1

Combining them is a good strategy in a lab setting, but right now it is not a usable strategy. Most known efflux pump inhibitors, including PaβN, are toxic to mammalian cells, often causing neurological issues.

Question 2

The resistance evolution was tracked over 30 days. Would we expect different results if the test ran longer?

Answer 2

If the test ran like 30-90 days, I think that some strains might acquire secondary mutations that have a much higher level of resistance. So the 30 days could have shown us the initial path to resistance and the 90 days would show us the ceiling of that resistance.

Question 3

When E. coli overexpresses efflux pumps to resist DLX, is there a fitness cost to the bacteria?

Answer 3

Yes, there is. Efflux pumps need a lot of ATP to work. When a bacterium overexpresses these pumps, it is taking the energy away from growth and reproduction. So resistance often fades once the antibiotic pressure is gone because those original wild-type strains outcompete the mutants.

Question 4

Did any mutations provide resistance to one drug while causing sensitivity to the other?

Answer 4

We didn’t see sensitivity to other drugs but we did see cross-resistance, because both drugs share the same target like in the gyrA gene a mutation that blocks CIP usually reduces the effectiveness of DLX as well.

Question 5

Since DLX is non-zwitterionic (helping it penetrate membranes at low pH) are there other antibiotics that could be modified to be non-zwitterionic to improve their performance in low pH?

Answer 5

Theoretically yes, but chemically pretty difficult. Many antibiotics, like Beta-lactams or other Fluoroquinolones, rely on their specific ionic charges to bind to their targets and might no longer be able to bind properly.

Question 6

If we successfully make more drugs non-zwitterionic to bypass the membrane, would bacteria just evolve a different way to block them?

Answer 6

Yes, bacteria are very adaptable through other common resistance mechanisms, like enzymatic inactivation of the drug. Since the non-zwitterionic structure only solves the problem of getting into the cell, the bacteria can evolve to prevent the drug from binding using enzymes.

Question 7

What makes DLX unique? How is it non-zwitterionic?

Answer 7

Unlike the CIP, which contains both a basic group (N. NH square ring) and an acidic carboxylic acid. With the basic group, it picks up extra protons, giving it a net positive charge. Because cell membranes are hydrophobic, this positive charge makes it harder for CIP to go through. DLX was made, replacing that basic group with a weakly acidic heteroaromatic ring and adding chlorine and fluorine atoms to the core structure, making it non-zwitterionic. This allows it to stay neutral at low pH.

Question 8

What is the biggest hurdle or first step you think they should take to start tests in humans?

Answer 8

The first step would be to test on human tissue samples collected from actual infection sites, like abscesses, to see if the drug accumulates as effectively in complex human biology as it did in the lab.