Effects of Sub-Inhibitory Antibiotic Concentrations on Genes Associated with Biofilm Formation in Mycobacterium avium

1. Effects of Sub-Inhibitory Antibiotic Concentrations on Genes Associated with Biofilm Formationin Mycobacterium avium Molly Dyan McNab HHMI Summer 2004 Mentor: Dr. Luiz Bermudez OSU College of Veterinary Medicine Department of Biomedical Science

2. What is Biofilm? • A multi-cellular aggregate of bacteria that forms on any surface exposed to the bacteria capable and some amount of water. • Forms to protect the bacteria from host defenses, harsh environmental conditions, and antibiotic agents. • Found almost everywhere in nature, including in rivers, lakes, soil, water pipes, and even inside the human body. • Bacterial biofilms are often a cause of infections associated with medical devices such as catheters and IV lines. A type of bacterial biofilm, responsible for plaque.

3. How is biofilm formed? Surface Surface Planktonic bacteria, in the presence of water, attach to a surface. Bacteria continue to grow, outer cells provide a physical barrier to protect inner cells

4. Biofilm and antibiotic resistance Little or no effect because the bacteria in biofilm are in a different phase than most antibiotics target. Bacteriostatic Antibiotics Surface

5. Mycobacterium avium • A biofilm-forming opportunistic human pathogen found in the environment • Several strains have been isolated from AIDS patients and others with compromised immune systems www.med.sc.edu:85/ fox/mycobacteria.htm

6. Past work • Past work on genes associated with biofilm formation in M. avium has yielded approximately 12 genes that are up-regulated in biofilm formation. • Several of these genes are important for glycopeptidolipid (GPL) biosynthesis, while others play a key role in fatty acid metabolism or the citric acid cycle.

7. Current work • Using primers for previously identified genes associated with biofilm formation, quantify gene expression M. avium strains MAC A5, MAC 101, and MAC 104 in the presence and absence of three different antibiotics at sub-inhibitory concentrations using Real-Time PCR.

8. Antibiotics used • Macrolide antibiotics-Bacteriostatic antibiotics, Do not kill the bacteria, instead they prevent the production of proteins by binding to specific sites on the bacteria’s ribosomes • Claritromycin, fairly low MIC, 2 µg/ml, shown in previous studies to prevent biofilm formation when used early in treatment. • Azithromycin, higher MIC, 16 µg/ml • Fluoroquinolones- syntheticbroad-spectrum bactericidal drugs • Moxifloxycin, MIC is 2 µg/ml

9. Hypothesis • Claritromycin at a concentration of 50% its minimum inhibitory concentration will selectively limit the expression of genes typically up-regulated in biofilm formation • Sub-inhibitory concentrations of Azithromycin and Moxifloxycin will have little or no effect on those genes.

10. Genes / Gene Products Biofilm genes • Glycosyl Transferase, essential for the expression of mature GPLs. • GuaB2 (IMP dehydrogenase), catalyzes the first reaction in GMP biosynthesis • PmmB (Phosphomannose mutase), converts D-Mannose 1-Phosphate TO D-Mannose 6-Phosphate Control gene • 16S RNA, not involved in biofilm formation, to be used as a control

11. Mechanism of M. avium infection When enough bacteria are present, infection spreads across the epithelium. M. Avium bacterium is inhaled and ends up in the lung As the bacteria colony grows, biofilm forms along the epithelial tissue of the airway. Sub-Inhibitory Antibiotic Treatment

12. Procedure 1. Samples of each strain were plated on Polyvinyl Chloride (PVC) with or without the presence of 50% of the minimum inhibitory concentration of each of the three antibiotics and incubated at 37°C for one week.

13. Procedure, cont. 2. After one week, the RNA from each sample was purified, cDNA was made, and Real-Time PCR was performed with primers from the previously identified genes to quantify gene expression in each sample.

14. Results MAC A5

15. Results MAC 101

16. Results MAC 104

17. Discussion / Conclusions • Claritromycin at a concentration of 1 µg/ml selectively disrupts expression in genes associated with biofilm formation in MAC A5 • Azithromycin at a concentration of 16 µg/ml has a small selective effect on the expression on genes associated with biofilm formation in MAC A5 • Moxifloxycin at a concentration of 0.25 µg/ml has little effect on genes associated with biofilm formation in MAC A5

18. Future work • Studying the effects of the treatment on the remaining genes associated with biofilm formation. • Extending this treatment to an animal model. • Developing a protocol for using this concept to prevent biofilm formation, and thus infection, in immunocompromised individuals.

19. Thank you! • Howard Hughes Medical Institute • Dr. Kevin Ahern • Dr. Luiz Bermudez • Dr. Yoshitaka Yamazaki • Bermudez Lab Soccer Team