Percentage of Antibiotic Producers per Isolates Tested Antibiotics Isolated from Soil Located in Areas Around Arden Hills, Minnesota Katie Walsh, McKenzie DeKam, Jordan Heser, Kealey Neuville, Brittany Pederson Department of Biological Sciences, Bethel University, St. Paul, Minnesota 55112 Background Solving the global health crisis of antibiotic resistance requires an international call to action. As the number of cases of antibiotic resistance continues to increase worldwide, the contributions of the U.S. pharmaceutical industry to the development of novel antibiotics has declined steadily since the mid 1980s (Figure 1). In addition, there has been a shift in the US Food and Drug Administration’s focus from antibacterial entities to other pharmaceutical agents (Figure 2). For this reason, there is an international need for the discovery of new antibiotic producing entities. In partnership with the Small World Initiative (Yale University), the purpose of this study was to crowd-source the discovery of new antibiotics in local habitats surrounding Bethel University, St. Paul, MN. We hypothesized that since most of the current antibiotics in clinical use originated from soil bacteria, screening microbes from local habitats might uncover novel strains and antimicrobial compounds. Chemical Extraction of Metabolites     Organic Extraction Prepare Plates Chop Up Plates Figure 1. New antibacterial molecular entities approved by the US Food and Drug Administration in a 5-year period. A B C B Figure 7. Flow chart depicting method of organic extraction. In week 1, we plated each of our 13 isolates of interest on TSA plates to produce a bacterial lawn. In week 2, we chopped up each of the plates and scooped them in labeled tubes. In week 3, we performed our extraction using ethyl acetate. Under UV light, 7 of 13 isolates fluoresced, confirming we extracted the strain’s metabolites. Figure 5. Images of selected isolates tested against ESKAPE pathogens. Each plate tested 24 tester strains which were isolated from the Soil Sample Plates 1—7 against a particular pathogen. Each of the 24 isolates were tested against 8 ESKAPE pathogens. For example, Plate A tested against M. luteus, Plate B tested against B. subtilis, and Plate C tested against B. cereus. We recorded 13 unique strains which were exhibiting zones of inhibition. Figure 2. Antibacterial versus anti-HIV new molecular entities (NMEs) approved by the US Food and Drug Administration in a 5-year period. Soil Sample Number Isolates Patched Tester Strains Antibiotic Producers Percentage of Antibiotic Producers per Isolates Tested 1 6 8 16.7 % 2 3 37.5 % 0.00 % 4 25.0 % 5 9 33.3 % 7 Figure 3. Diagram of the research workflow. Serial Dilution of Soil Samples Pick & Patch Diverse Colonies Classify Antibiotic Producing Strain by Gram Staining Test Antibiotic Producing Isolates against ESKAPE Pathogens Organic Extraction of Metabolites Obtain Pure Cultures of Antibiotic Producing Isolates Characterize Antibiotic Producer   Future Experimentation 16S rRNA gene sequence analysis Test organic extracts for tolerance against Chlamydomonas Test antibiotics against plants Purification of organic extracts Table 2. Table summarizing antibiotic resistance of isolated colonies from Samples 1-7 against eight ESKAPE pathogens. A variety of colonies from the 7 soil sample plates at various concentrations were selected and plated onto a Master Plate. Only colonies which appeared to be exhibiting antibiotic activity against other colonies in the plate were selected for the Master Plate. Next, colonies from the Master Plate were picked and patched onto eight different TSA Plates which had been spread with 8 different ESKAPE pathogens. All isolates of interest were tested against both gram positive and gram negative pathogens. After 24 hours of incubation at 37˚C, pictures were taken of each plate and colonies which appeared to be exhibiting antibiotic activity against a specific pathogen were recorded. Soil Samples 2 and 6 had the greatest percentage of antibiotic producers per isolates tested.   B C A Conclusions In our team’s response to the antibiotic resistance crisis, we have successfully isolated 13 potentially unique antibiotic producing strains. We have demonstrated that crowdsourcing is a successful method for isolating these potentially novel strains right in our own backyard. By harvesting soil samples from seven novel locations on our university campus, we confirmed the that there are antibiotic producing microbes in the soil of our local habitats. Further experimentation will involve 16S rRNA gene sequence analysis. From this, we will determine if these s are unique microbial strains. In addition, we will test our organic extracts for tolerance against Chlamydomonas in order to confirm if it is harmful against eukaryotic organisms. We will also test the organic extracts for tolerance against plants. Our research has opened the doors for several different avenues of experimentation. We look forward to continuing in this national crowdsourcing effort in order to combat the global health crisis of antibiotic resistance. 0.1 mg soil 1 mg soil 0.01 mg soil 100 µL mL 100 µL 1 mL Weigh 1.00 g Soil Sample   TSA Agar Plates 10 mL PBS Figure 4. Diagram illustrating the method for serial dilution. Researchers used original soil samples to perform a series of dilutions with the goal of ensuring a countable plate of microbial colonies. This exponential dilution allowed researchers to identify possible zones of inhibition and select colonies to re-culture and examine. Figure 6. Images of gram stains of isolated antibiotic producers. After we isolated our antibiotic producing strains, we performed a gram stain on each strain. Image A is strain, A17, a gram positive rod, Image B was B6, gram positive spore, and Image C was A6, a gram positive rod. Soil Sample Number GPS Location Concentration of Sample Shannon-Weiner Index CFU/g   1 45 ˚ 3' 24'' N 93 ˚ 9' 58'' W 1 mg 1.412 9.6E+04 0.1 mg 1.678 6.0E+05 0.01 mg 1.074 8.0E+05 2 93 ˚ 9' 51'' W .9995 1.8E+05 1.197 4.7E+05 1.618 2.3E+05 3 45 ˚ 3' 25'' N 2.0E+03 1.072 1.3E+06 4 45 ˚ 3' 21'' N 93 ˚ 9' 44'' W 1.312 7.4E+04 1.404 6.1E+05 1.426 1.9E+06 5 45 ˚ 3' 29'' N 93 ˚ 9' 48'' W 1.308 2.1E+05 1.6E+06 1.605 6.9E+06 6 1.580 1.1E+06 2.026 6.9E+05 2.047 7 45 ˚ 3' 46'' N 93 ˚ 9' 45'' W 1.703 8.7E+04 1.615 2.7E+05 1.055 1.4E+06 Location on Master Plate Original Soil Plate Number Antibiotic Production Against (Gram + or - ): Morphology Gram Positive or Gram Negative A 2 1 B. cereus (+) Rod A 6 6 B. subtilis (+), S. aureus (+) A 16 2 B. subtilis (+) A 17 B. subtilis (+), S. aureus (+), B. cereus (+) A 19 B. subtilis (+), B. cereus (+) A 20 B 1 4 M. luteus (+), S. aureus (+) Coccus B 3 M. luteus (+), B. cereus (+) B 6 5 M. luteus (+), B. subtilis (+), B. cereus (+) Spore B 11 M. luteus (+) B 15 B 19 B 20 M. luteus (+), B. subtilis (+), S. aureus (+) Literature Cited French GL. 2010. The continuing crisis in antibiotic resistance . International Journal of Antimicrobial Agents [Internet]. [cited 2014 Apr 11] (36): Supplement 3 S3-S7. Available from: http://www.sciencedirect.com/science/article/pii/S0924857910700030 Neu H. 1992. The Crisis in Antibiotic Resistance. Science Magazine [Internet]. [cited 2014 Apr 11] (257): (5073) 1064-1073. Available from: http://www.sciencemag.org/content/257/5073/1064.short Spellberg B, Guido R, Gilbert D, Bradley J, Boucher H, Scheld M, Bartlett J, Edwards J. 2008. The Epidemic of Antibiotic-Resistant Infections: A Call to Action for the Medical Community from the Infectious Diseases Society of America. Clinical Infectious Diseases [Internet]. [cited 2014 Apr 11] (46): (2): 155-164.. Available from: http://cid.oxfordjournals.org/content/46/2/155.full Table 1. Table summarizing initial data collected from Soil Samples 1-7. All samples were collected from various locations around Bethel University Campus in St. Paul, MN. A serial dilution was performed on each sample and then, concentrations of 1 mg, 0.1 mg and 0.01 mg of soil were plated on TSA Plates. After 48 hours of incubation at 37˚C, pictures of all samples were taken and the number of colonies and diversity of colonies were recorded on 2/14/2014. From this data, Shannon-Weiner Index and CFU/g were calculated. Table 3. Table summarizing the characteristics of each of our 13 isolated antibiotic-producing strains. The majority of our isolates came from Soil Sample 2 and the majority of our strains were gram positive rods.