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Isolation and Identification of Antibiotic-producing Actinomycetes from soil samples in comparison with a known antibiotic producer, Streptomyces griseus Amanda McCune Department of Biological Sciences, York College Streptomyces slide culture Various antibiotic pills Taken from: http://en.wikipedia.org/wiki/Image:Streptomyces_sp_01.p ng Identification results (continued) Taken from: http://library.thinkquest.org/25462/uses.htm l Inclined coverslip slide culture Introduction Antibiotic resistance is a major problem in the medical industry due to the large increases in antibiotic resistant strains of pathogenic bacteria, over the past 20 years. Bacteria are constantly evolving into more efficient pathogens resulting in their ability to resist antibiotics. Many of the current, effective antibiotics in use, such as streptomycin, vancomycin, chloramphenicol, and erythromycin, are synthesized by a specific group of bacteria known as actinomycetes (Strohl, 2003). Actinomycetes are aerobic, grampositive bacteria that form hyphae or branching filaments, and are prolific producers of antibacterial chemicals (Bergey and Holt, 1994). Actinomycetes, typically present in soils, are found in many different areas all over the world from the mountainous regions of the Middle East to caves in Italy (Aghighi et al., 2004 and Laiz et al., 2000). Many researchers have suggested that actinomycetes produce antibacterial chemicals to compete with other microorganisms in the nutrient poor environments in which they best survive (Aghighi et al., 2004). Therefore actinomycetes are ideal candidates in the continuous search for new and better antibiotics. • Test bacteria streaked perpendicular to the isolated bacteria •Coverslip was placed at an angle into an agar well • Biochemical • 0.1ml of test bacteria (E. coli, S. aureus, and B. cereus) were spread over the entire agar plate S. griseus •Distance of inhibition was measured from edge of the disk to the edge of inhibition in mm. Isolate 2 disk Isolate 1 disk tests Table 1: Biochemical test results for gram positive isolates and Citrate Mannitol Lactose Isolate 1 + - - - Isolate 2 + + - - S. griseus - - - + S. griseus disk Isolate 3 disk •Differences in the antibiotic production testing methods are due to the difference in the extraction of the isolate synthesized chemicals. Antibiotic production results •Streak plate is direct contact with the organism and its biosynthesized chemical Streak plate results •Disk diffusion plate uses the chemical extracted from the actual organism Table 2: Measurement of growth based on 0 (no growth) - +3 (complete growth) scale - = orange, + = pink Results Isolated colonies from soil sample obtained along the banks of Tyler Run Isolate 1 Isolate 2 Isolate 3 Literature Cited S. aureus E. coli B. cereus Isolate 1 +3 0 +3 Isolate 2 +3 0 +3 Isolate 3 +3 +2 +3 S. griseus +1 +2 +2 Identification testing Antibiotic production testing Gram stain Streak plate method Inclined coverslip slide culture Disk diffusion method Wet mount Biochemical tests •Laiz, L., Groth, I., Schumann, P., Zezza, F., Felske, A., Hermosin, B., and Saiz-Jimenez, C. 2000. Microbiology of the stalactites from Grotta dei Cervi, Porto, Italy. International Microbiology 3: 25-30. Disk Diffusion results Table 3: Zone of Inhibition (mm) ± SD Identification results • Gram staining • gram (+) purple: isolate 1 and 2, S. griseus • gram (-) pink: isolate 3 S. aureus E. coli B. cereus 0 0.33 ±0.38 0 Unknown 2 1.3±0.95 0.68 ±0.83 0 Unknown 3 0 0.18 ±0.35 0 S. griseus 0 0.83 ±0.95 0 Unknown 1 • Aghighi, S., Shahidi Bonjar, G.H., Rawashdeh, R., Batayneh, S., and Saadoun, I. 2004. First Report of Antifungal spectra of Activity of Iranian Actinomycetes Strains against Alternaria solani, Alternaria alternate, Fusarium solani, Phytophthora megasperma, Verticillium dahliae, and Saccharomyces cerevisiae. Asian Journal of Plant Sciences 3: 463-471. • Bergey, D.H., and Holt, J.G. 1994. Bergey’s Manual of Determinative Bacteriology Vol. 4. 9th ed. Baltimore, MD. All plates were incubated and checked for growth of colonies Colonies isolated •The antibiotic chemical being produced is most effective on gram negative bacteria rather than the gram positive, S. aureus or the spore forming gram positive, B. cereus. •Isolate 2 and S. griseus were most effective against E. coli based on disk diffusion, isolates 1 and 2 were most effective against E. coli based on streak plates. Urea + = blue, - = green Test bacteria spread Plated 10-4, 10-5, and 10-6 soil dilutions on glycerol yeast extract agar (GYE) Antibiotic production • Soak each disk in supernatant obtained from the broth culture of isolated bacteria 1-3 and S. griseus Collected 5 Soil samples from various locations on campus through •Biochemical results point to Rhodococcus for isolate 2, results were inconclusive for isolate 1. Spiral chains of conidia Disk Diffusion method Methods Diluted soil samples from S. griseus B. cereus Mannitol and Lactose •Isolates 1 and 2 were found to be possible actinomycetes from the gram positive results of the gram stain. Isolate 3 was ruled out due to its gram negative results. •Through analysis of mycelium and sporulation structures on the coverslip slide cultures and wet mounts isolates 1 and 2 were narrowed down to either Rhodococcus or Actinomadura genus levels E. coli Biochemical Tests Urea 10-6 Branching filaments Short chains of conidia S. aureus •Isolate and identify actinomycetes from soil samples taken from York College campus 10-2 Identification Isolate 2 Isolated bacteria 1 - 3 or S. griseus Citrate •Test for antibiotic production and compare to a known antibiotic producing actinomycete, Streptomyces griseus Isolate 1 •Growth of test bacteria was recorded as 0 for no growth or complete inhibition through +3 for full growth or no inhibition + = yellow, - = red Objectives •Inclined coverslip slide Streak plate method •Inoculated edge of sterilized coverslip by touching the bacterial colony Conclusions • Strohl, William. 2003. Chapter 31: Antimicrobials. Pages 336-355 in Bull, Alan (ed.). Microbial Diversity and Bioprospecting. ASM Press, Washington D.C. Acknowledgments I would like to thank Dr. Mathur for her advice and support throughout the entire research process and presentation. I would also like to extend my thanks the biology staff for all of their interest and support through the senior thesis process.