Quinolone resistance screening

Inexpensive, rapid testing of Quinolone-resistant bacteria

Shaun Lee, Department of Biological Sciences (UND); Marya Lieberman, Department of Chemistry and Biochemistry (UND)

Although quinolone antibiotics have been used as effective treatments for bacterial infections since their discovery in 1962, rapid resistance to this class of antibiotics has been observed in numerous Gram-positive and Gram-negative pathogens. Resistance to quinolones is high in such organisms as Pseudomonas aeruginosa and other significant pathogens such as Neisseria gonorrhoeae. Acquisition of quinolone resistance in already multiple-resistant 'superbugs' such as methicillin-resistant Staphylococcus aureus  has increased rapidly in recent years.[i] Culturing and susceptibility testing of potentially antibiotic resistant isolates have recently given way to non-cultured diagnostic methods to detect antibiotic-resistant strains. These methods include sequencing, microarrays, and other molecular and PCR-based techniques. A significant limitation of these techniques is that quinolone resistance is often determined via a single mutation (ser91) in the gyrA gene of N. gonorrhoeae DNA gyrase.[ii]It is likely that as rates of resistant isolates increase, other not yet identified mutations are likely to arise that will go undetected unless first reported through primary research literature.

The REU student will assist in the development of an inexpensive in vitro assay to detect the activity of bacterial DNA gyrase as an indication of quinolone resistance. Methods to assay DNA gyrase activity have been established[iii] and primarily involve determining gyrase activity through observation of DNA supercoiling and cleavage by gel electrophoresis. We have recently developed a method to measure DNA migration using a paper test. Samples that display gyrase activity (as shown by DNA migration) in the presence of fluoroquinolones would be indicative of quinolone resistant bacteria. Thus, mutants that are resistant to quinolones that might be missed using molecular methods can still be identified with this approach. Furthermore, susceptibility to different quinolone variants, as well as the detection of counterfeit quinolone drugs can be assayed by determining whether the drugs are able to shut down the bacterial gyrases. The student will develop the project with guidance from faculty mentors and will be able to present his or her results at local and national scientific conferences.

[i] Ruiz, J. Mechanisms of resistance to quinolones: Target alterations, decreased accumulation and DNA gyrase protection. The Journal of Antimicrobial Chemotherapy. 2003; 51(5),:1109-1117.

[ii]  Siedner, M.J. et al.  Real Time PCR Assay for the Detection of Quinolone-Resistant Neisseria gonorrhoeae in Urine samples. J Clin Microbiol. 2007; 45(4):1250-1254.

[iii]   Fisher, M.L. et al. Methods to Assay Inhibitors of DNA Gyrase and Topoisomerase IV activities. Meth Molec Med. 2007; 142:11-23.