Addressing Antibiotic Resistance by Isolation and Characterization of Genus Lysobacter and Genus Unknown Antibiotic-producers in Pittsburgh Soil Miriam Bols and Emma Wylie Table 2-Zones of Antibiotic Producers from Soil Sample Tested Against Various ESKAPE Safe Relatives RESULTS: INTRODUCTION: Over the past few decades, the antibiotic resistance crisis has dramatically escalated proving the urgency to find new antibiotics. Antibiotics are needed most in fighting the six most commonly resistant pathogens, also known as the ESKAPE pathogens. These pathogens include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. In this study, bacterial isolates were cultured from soil around the University of Pittsburgh campus and tested for antibiotic activity against ESKAPE pathogen safe relatives to find potential new antibiotics. *Producers Zones of Inhibition (mm) Erwinia carotovura Bacillus subtillis Staphylococcus epidermis Escherichia coli Pseudomonas putida Enterococcus raffinosus MB-LBA-22 2 MB-LBA-15 3 Lysobacter antibiotics 5 MB-PDA-51 1 MB-LBA-52 MB-PDA-15 MB-PDA-12 Table 1- Soil Type, pH, Sample Site, and Percent Organic, Inorganic, and Water Content for Wet Soil Soil Site Soil Texture pH % Water content % Inorganic content % Organic content MB Soil Sample 40° 26' 47"N 79° 57'9" W Silt Loam 5.5 55 23 22 EW Soil Sample 40° 26’ 47”N 79° 57’7” W Sandy Clay Loam 0.78 94 OBJECTIVES: To obtain a soil sample and analyze it for antibiotic activity by isolating single bacterial colonies and testing them against ESKAPE pathogen safe relatives. Examine soil samples pH, organic composition, and frequency of antibiotic production. Identify and characterize the physical and chemical properties of found antibiotic producers through organic extract activity, cell morphology testing, and genus classification. Contribute to the Small World Initiative by documenting research in the Small World Initiative Database and ultimately adding to the efforts of resolving the antibiotic resistance crisis. *EW sample did not display producers against strains. Isolates were tested against S. cohnii, E.coli, E. rafinosus, P. putida, and A. baylyi. Bold face isolates are those chosen to study further. Figure 4- Antibiotic Assays of Isolates Displaying Zone of Inhibition A B C METHODS: Figure 1- Methods Flow Diagram for All Experiments Done to Identify and Characterize Antibiotic Producer from Soil Sample Figure 4A and 4B- MB-LBA-5 2 soil isolate displaying antibiotic growth against Staphylococcus epidermis and Enterococcus raffinosus respectively. Figure 4C- MB-LBA-15 isolate showing antibiotic production against Bacillus subtillis. Table 3- Soil Isolate Characterization by Gram Staining, PCR Blast Sequencing, BioLog , and Organic Extract Testing Chosen Soil Isolate Cell Wall Composition Cell Shape Organic Extract Zone of Inhibition Genus Possible Species MB-LBA-52 Gram-positive Coccus Inconclusive Lysobacter Sp. 1011TES3C9,31 or PB-6250 MB-LBA-15 Positive Figure 2 - Bacterial Abundance (CFU/g) for Soil Samples on Different Media Types To obtain this data, one gram of soil samples were diluted with nine millimeters of water. 100 micro liters of vortexed mixture was then put in another 900 micro liters of water. Serial dilutions were done up to four times to reach the several dilutions that were plated in order to count the colonies for calculations. LBA , TSA, and PDA for MB sample were incubated for five days at 30℃ and LBA, R2A, and TSA for EW sample were incubated at 20 ℃ for 7 days. Plate dilutions on variety of media types Characterize Soil Sample Perform Serial dilutions of sample Collect soil sample CONCLUSIONS: Testing revealed two antibiotic producers from one soil sample site. MB-LBA-52 produced antibiotics against Staphylococcus epidermidis and Enterococcus raffinosus and MB-LBA-15 produced against Bacillus subtilis. Both antibiotic producers from this soil sample inhibited the growth of Gram-positive bacteria. Further characterization revealed that soil isolate MB-LBA-52 was a circular, Gram-positive organism from the genus Lysobacter. Similarly, the isolate MB-LBA-15 was also Gram-positive with a circular structure, and its organic compounds were found to be antibiotic producers. Future directions include using this soil sample’s six antibiotic producers that could each be tested further using the same experimentation previously described. Also performing further testing on both isolates to determine their exact makeup and validity as an antibiotic. Create library of isolates Perform PCR with soil antibiotic producer AGCGCCCC Perform 16S rDNA sequencing Screen isolates for antibiotic production against ESKAPE pathogen relatives Acknowledgements and References: Special thanks to Elia Crisucci, the undergraduate teaching assistants, and all of our fellow Small World classmates for their continued encouragement, guidance, and teamwork. Hernandez S., Tsang T., Handelsman J. 2015. A:  Small World Initiative Research Protocol. Hernandez S., Tsang T., Handelsman J. 2015. B:  Small World Initiative Research Guide. Figure 3- Media Type Used For Soil Isolates with Frequency of Antibiotic Production Frequencies displayed on graph were calculated by dividing the total number of antibiotic producers on one media type by the total number of isolates patched on the same media. Producers were identified by patching isolates on top of the tester strain using the spread/patch method and incubating the plate at 30 ℃. After incubation, isolates that produced a zone of inhibition were identified as antibiotic producers. PDA and LBA were used for the MB soil sample and TSA was used for the EW sample. If PCR invalid, perform Bio Log Extract organic compounds Gram stain