Federation of American Scientists Case Studies in Dual Use Biological Research Module 5.0: Antibiotic Resistance Case Study
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To test antibiotic resistance, plates were streaked with bacteria and disks saturated with different antibiotics were placed on them. Susceptible bacteria are unable to grow near the disks, forming a clear halo around them, but E. coli and Y. pestis expressing MarA47YP were not affected by the antibiotics and were able to grow right up to the disks. Bacteria expressing YPO1737 did not become resistant to the antibiotics, meaning that despite the difference in size, marA47YP was the most likely marA gene in Y. pestis. By overexpressing a single gene, Y. pestis was created that showed resistance to tetracycline, rifampicin, chloramphenicol, doxycycline, nalidixic acid and norfloxacin.

MarA47YP was able to cause antibiotic resistance like MarA, but to confirm that its mechanism of action was the same, an analysis of transcription by real-time PCR (RT-PCR) was done. When MarA47YP was overexpressed in Y. pestis, the transcription of the efflux pump also increased, showing that this is a conserved mechanism of resistance. A further experiment was done
whereY. pestis was grown in the presence of low levels of tetracycline to induce spontaneous mutations that caused the bacteria to be resistant. Analysis of the resistant mutants showed that 4 out of 5 harbored mutations causing MarA overexpression and suggests that it is a frequent mechanism for bacteria to acquire resistance.

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