HPLC Fingerprinting of Biologically Active Extracts from Streptomyces sp

Transcription

1 HPLC Fingerprinting of Biologically Active Extracts from Streptomyces sp Brooke Vatthauer 1, Rochelle Herzog 1, Jason Brantner 2, Venugopal Mukku 1 1 University of Minnesota Crookston (UMC) 2 Northwest Research and Outreach Center (NWROC)

2 Introduction High rates of antibiotic resistance in hospitals MRSA methicillin-resistant Staphylococcus aureus VRSA vancomycin-resistant S. aureus MDR Multi-drug resistant PDR Pan-drug-resistant MDR-TB and XDR (extensively-drug-resistant)-tb (Mycobacterium tuberculosis) References 1. Fischbach, Michael A., and Christopher T. Walsh. "Antibiotics For Emerging Pathogens." NIH Public Access Science (2009): Web. 12 Apr

3 Introduction, Continued Pharma companies are content with tweaking the structures of existing classes of drugs New drugs need to be developed to combat MDR and PDR We have been working on prioritizing a series of Streptomyces strains with the ability to produce antibiotics. References: 1. Science. Author manuscript; available in PMC 2010 January 7 2. MacDougall, C., Powell, J., Johnson, C. K., Edmond, M. B., & Polk, R. E. Clin. Infect. Dis., 2005, 41, De Kraker, M. A., Davey, P. G., & Grundmann, H. Plos Med., 2011, 8, Ling, Losee L., Tanja Schneider, Aaron J. Peoples, Amy L. Spoering, and Ina Engels. "A New Antibiotic Kills Pathogens Without Detectable Resistance." Nature 517 (2015). Web. 12 Apr <

4 Hypothesis One of us was working on sugar beet root rot and observed that lime treatment increased crop yields and reduced the root rot caused by A. cochlioides. It was also noted that lime treatment increased number of Streptomyces isolates. A total of 61 strains were isolated in early It was hypothesized that these Streptomyces strains are involved in the observed effect.

5 Isolating the Strains Thirty six strains isolated from the soil were grown on GYEA plates

6 Materials and Methods

7 Shaker Culture One strain (NWROC -11B) was prioritized Grown on a shaker for a week at 28 C vacuum filtered organic compounds were extracted using ethyl acetate

8 Concentration Ethyl acetate was rotavapped at 39 C and the residue was transferred to a vial using DCM-MeOH (9:1) The extract was allowed to dry and was tested against B. subtilis and C. albicans using disc diffusion assay

9 Bio-assay C. albicans and B. subtilis were both grown in 10mL of nutrient broth Stored in incubator at 30 C Optical densities were recorded using spectrophotometer Diluted to approx. 0.50

10 Bio-assay Original Dilution (OD): 0.02 g NWROC-11B dissolved in Dimethylsulfoxide (DMSO) (250 µl) Dilution One (D1): 125µL OD + 125µL DMSO Dilution Two (D2): 125µL D µL DMSO OD D1 D2

11 Bio-assay 100µL of C. albicans or B. subtilis was transferred onto the MHA plates Spread using sterile L-shaped glass rod Separated petri-dish into 4 quadrants OD D1 B D2

12 Bio-assay 6mm blank paper discs Dissolved extracts OD, D1, D2 and DMSO (Blank (B)) 10µL onto each blank paper disk OD: 800 µg per disc Dried Placed on MHA plate in appropriate quadrant Incubated for 3 days in incubator at 30 C Three total replicates were made of each OD B D1 D2

13 NWROC-11B Procedure was completed twice Sample was also grown on a 20L scale Biotechnology Resource Center (BRC) University of Minnesota

14 Bio-assay Results: Part I

15 Bio-assay Results: First Shaker Culture NWROC-11B: First Analysis B. subtilis OD D1 D AVG Results in millimeters NWROC-11B: First Analysis C. albicans OD D1 D AVG Results in millimeters

16 Bio-assay Results: Second Shaker Culture NWROC-11B: Second Analysis B. subtilis OD D1 D AVG Results in millimeters NWROC-11B: Second Analysis C. albicans OD D1 D AVG Results in millimeters

17 Bio-assay Results: Fermentation NWROC-11B: Fermentation B. subtilis OD D1 D AVG Results in millimeters NWROC-11B: Fermentation C. albicans OD D1 D AVG Results in millimeters

18 HPLC

19 HPLC Gradient HPLC Gradient Injections 1mL Flow Rate 1 ml/min Time (minutes) Percent Methanol 0-5 min min min min min min min 10

20 Shaker Culture cf. Fermentation Fermentation Extract 2 nd Extract

21 Shaker Culture cf. Fermentation Fermentation Extract 2 nd Extract

22 First cf. Second Shaker Cultures 2 nd Extract 1 st Extract

23 First cf. Second Shaker Cultures 2 nd Extract 1 st Extract

24 HPLC Separation by polarity Materials and Methods: Part II

25 HPLC Gradient HPLC Gradient Injections 0.5mL and 1.5mL Flow Rate 10 ml/min Time (minutes) Percent Methanol 0-5 min min min min min min min 10

26 HPLC Collection Samples were collected in separate flasks minutes minutes minutes minutes min min min minutes minutes minutes 0-10 min min min min

27 Bio-assay Original Dilution (OD): 800µL Dilution One (D1): 400µL Dilution Two (D2): 200µL Time Product Weight (mg) Tested 0-10 min 41.4 Yes min 3.2 Yes min 3.2 Yes min 2.1 Yes min 0.7 NO min 1.8 NO min 2.6 Yes X(250) 20 = µl OD D1 D2

28 Bio-assay Results: Part II

29 Bio-assay Results: Part II None of the Preparative Column timed samples showed resistance Not enough sample to complete the Bio-assay with 800µg of product Procedure will be repeated with a larger starting sample

30 Conclusion All three extracts had peaks at similar retention times Preparative Column Bio-assay will be repeated using a larger amount of starting material

31 Questions