Analysis of Soil Microbes for Antibiotic Properties Jake Kieserman & Linda Zheng There is an upcoming crisis in modern medicine in which pathogenic strains of bacteria are becoming resistant to the antibiotics that physicians commonly prescribe. Infections that were once commonly treatable are now developing into severe infections increasing the mortality rate of humans across the globe. In this study, bacterial isolates were cultured from soil samples around the University of Pittsburgh’s campus and were analyzed for antibiotic producing properties against ESKAPE pathogen (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) in hopes of developing new medications to combat this multi-drug resistant organisms. Introduction Objectives 1. To obtain bacterial samples from soil taken from around Pitt’s campus and culture the bacteria as single colonies to determine which bacterial specimens are antibiotic producers against ESKAPE pathogens 2. To analyze and characterize soil isolates by their physical and chemical properties 3. To contribute to the Small World Initiative database to work collaboratively toward addressing the antibiotic crisis Results Conclusions References Hernandez, S.A., et al. 2013. Introduction. In: The Small World Initiative Student Laboratory Manual. Yale University. 7. Reynolds, J. 2005. Small World Initiative Protocols. Yale University. 70- 106. Methods Flow Diagram Test Jake’s Sample Linda’s Sample Soil Type Silty loam Sandy clay loam pH of Soil (deionized water) 8 6 Percent Water Content 33.45% 30.7% Percent Organic Content (Wet Weight) 11% 8.16% Optimal Agar Medium NA R2A Optimal Dilution Ratio 1:1000 1:1000 CFU/g from Optimal Dilution 9.4x10 5 1.8x10 6 Test Jake’s Results Linda’s Results Starch Hydrolysis Positive for amylase Positive for amylase Catalase Reaction Negative for catalase Negative for catalase SIM (Sulfide /Indole/Motility) Negative for sulfide/indole, Positive for motility Negative for sulfide /indole, Positive for motility Fluid Thioglycollate Aerotolerant anaerobe Facultative anaerobe Triple Sugar Iron Ferments glucose, produces acid Ferments glucose/sucrose /lactose, produces acid MacConkey Agar No growth (Gram +) No growth (Gram +) DNA match (with BLAST) Bacillus funiculus (99% identity) Paenibacillus xylanexedens (99% identity) Table 2. Results of biochemical characterization testing Table 1. Results of soil characterization and optimization testing Our testing resulted in two antibiotic producers, one against Staphylococcus epidermis and the other against Escherichia coli and Enterobacter aerogenes, proving that antibiotic-producing microbes can be found and isolated from soil samples. From the various characterization testing, we see that the antibiotic producing bacterial isolates were diverse, exhibiting many different physical and chemical properties. Although we were not able to extract the antibiotic organic compounds, this may have been because the antibiotic compounds were not organic or difficulties with using ethyl acetate. Further testing may yield other antibiotic-producing microbes, such as if we continued to test other isolates from our soil samples using the same process. Acknowledgements Special thanks to Elia Crisucci and Jean Schmidt for their commitment and assistance to our research, our classmates for their continued encouragement and teamwork, and the University of Pittsburgh for funding and creating this opportunity for us to become part of the Small World Initiative. Identify antibiotic producers (zones of inhibition) Collect soil sample Analyze soil sample Serial dilution of soil Perform PCR reaction to amplify antibiotic- producing isolate DNA Gel electrophoresis to confirm PCR results Repeat if PCR unsuccessful A TGTCAA T A Send DNA to be sequenced and identify isolate Test various agar types Isolate microbes Test against safe “ESKAPE” pathogen relatives Repeat if unsuccessful LB PDA R2A NA Perform gram staining on isolate Extract antibiotic organic compounds Biochemical characterization of isolates (e.g. starch hydrolysis test) • Two antibiotic-producing isolates found and identified, Bacillus funiculus produced a zone of inhibition against E. coli (Fig. 1) and E. aerogenes (Fig. 2), and Paenibacillus xylandexedens against S. epidermis (Fig. 3), although attempts to extract the antibiotic organic compounds were unsuccessful • Each soil sample was initially thoroughly analyzed (Table 1) and later biochemically characterized (Table 2) Figure 3. Results from spread /patch screen for soil isolate antibiotic production against S. epidermis, incubated at 30°C for seven days. Figure 2. Results from broad spectrum screen for soil isolate #1 antibiotic production against E. aerogenes, incubated at 30°C for two days. Figure 3. Results from spread/patch screen for soil isolate antibiotic production against E. coli, incubated at 30°C for five days.