Producing from Sacramento State University Campus Soil Linda Acevedo, Elizabeth Kern, Nguyen Vo, and Enid Gonzalez-Orta Ph.D.

INTRODUCTION RESULTS qThe Tiny Earth Research Guide Program highlights the importance of Soil Characterization and Alfa-Diversity Colony Morphology educating students about the antibiotic crisis by learning about soil which plays host to a rich and diverse community of bacteria that has access to the Table 1. Abiotic factors, soil bacterial population and Alfa-diversity 38°33'38.9"N 121°25'12.8"W near the most abundant source of specialized metabolites that can produce . Sampling Location American River qAccording to the Infectious Diseases Society of America (IDSA,2020), about 2 [A] 11870- The colonies had irregular shapes, mucoid, smooth, and Air temperatura & 16.7 ℃ & 65.5 % white color. million Americans per year develop hospital-acquired infections, which results Humidity [B] 11871-The colonies were round, with entire margin, flat and in 99,000 deaths due to antibacterial-resistant . Soil pH 7.0 smooth. The Back of the plate was mustard color, but the inside was q white with a powdery texture. The acronym ESKAPE includes six nosocomial pathogens that exhibit Soil Temperature 12.7 ℃ multidrug resistance (, , Klebsiella [C]11872- The colonies were a flower-like shape, powdery texture Soil Depth 12.7 cm with purple and pinkish, and white colors. pneumoniae, , , and several 6 Bacterial density 8.6 x10 cfu/g soil [D]12158- The colonies were color pink; the form was round with an species of ). Simpson’s index (D) 0.32 entire margin and mucoid. qIn this research, we focused on the Safe ESKAPE relatives instead because Shannon-Weaver Diversity 1.75 [E]12159- The colonies were a mix of Irregular and round shape, they have the same physiological features, but they do not possess a health Index (H) mucoid, and it had a sticky texture, the colonies were whitish-yellow risk in a teaching lab setting. Pielou’s Evenness (e) 0.54 color. Identification of soil isolates by 16s rRNA qWe used Enterococcus raffinosus, Staphylococcus epidermis, Escherichia coli, Safe ESKAPE Testing Figure 2: Colony morphology of isolates that have antibiotic activity Acinetobacter baylyi, Pseudomonas putida, and Enterobacter aerogenes as We performed four rounds of Safe ESKAPE testing. We Tested the Safe ESKAPE relatives, and we added four additional bacteria to this nine isolates against the Safe ESKAPE relatives, but only five Identification of Soil Isolates by 16s rRNA research, Mycobacterium smegmatis and subtilis. As well as isolates exhibited antibiotic activity. Using Bioinformatics we determined that the five isolates chosen belonged Agrobacterium tumefaciens and Erwinia Carotovora that are plant-pathogens to the genera Bacillus, Streptomyces, and Flavobacterium. bacteria that are agriculturally important. Table 2: Antibiotic Producers Table Table 4: BLAST, RDP (SeqMatch and Classifier ) Table A. E. S. E. E. B. BLAST MATERIALS & METHODS Isolate Strain # Media baylyi Isolate RDP SeqMatch Raffinosus epidermis coli aerogenes subtilis Genus RDP Classifier Name S_ab Score q No Max ID To perform our experiment, we collected our soil samples near the river, right S20BIO145 No 11870 R2A Ab+ Ab+ teste No tested Ab+ off the bike and walking path at Sacramento State University. We tested the LAR21+ tested d 11870 Bacillus 99.60% 1.000 Order Bacillales 98% bacteria isolated from the soil samples against those safe ESKAPE pathogens S20BIO145 11871 R2A Ab+ Ab+ Ab- Ab- Ab- Ab+ and cultured them on R2A media. We tested nine soil isolates against five LAR22+ 11871 Streptomyces 99.78% 0.895 Genus Streptomyces 100% different safe ESKAPE relatives that included (Enterococcus raffinosus, S20BIO145 Staphylococcus epidermis, Escherichia coli, Acinetobacter baylyi, Enterobacter 11872 Ab+ Ab- Ab- Ab- Ab- Ab- Order LAR23+ 11872 Streptomyces 99.36% 0.995 aerogenes, and one of the additional bacteria added, B. subtilis). S20BIO145 No No Actinomycetales 98% 12158 R2A No tested Ab- Ab+ No tested LAR24+ tested tested 12158 Flavobacterium 99.14% 0.934 Genus Nubsella 88% q We performed: S20BIO145 No 12159 R2A Ab+ Ab- Ab- Ab- Ab- Genus Flavobacterium LAR25+ tested 12159 Flavobacterium 98.99% 0.947 üSoil characterization & Alfa- diversity 100% ü Antibiotic Producers Safe ESKAPE testing DISCUSSION ü Biochemical tests [A] isolate 11870 ü DNA Extraction [B] Isolate 11871 qThe whole reason for this research was to analyzed soil üPolymerase Chain Reaction (PCR) [C] Isolate 11872 sample bacteria to find possible antibiotic producers that ü Gel Electrophoresis [D] Isolate 12158 could inhibit the safe ESKAPE pathogens relatives. ü Nanodrop on the isolates [E] Isolate 12159 q”Terrain matters”, we saw it firsthand with our patching, üAnalyze the bacteria sequences through different bioinformatic programs Four of the isolates inhibited Gram- how specific neighboring isolates triggered others when ( BLAST, Ribosomal Database Project (RDP) that included SeqMatch and positive (+) bacteria that included S. they were close or apart to specific isolates. Classifier. epidermis, E. raffinosus, and B. subtilis. Only one of the isolates qWe were able to control it by modifying were we patched our isolates. ACKNOWLEGMENTS inhibited Gram-negative bacteria (-), E. coli. Figure 3: Patching/Colonies interactions Being able to work hands-on and discover antibiotic-producing isolates has Figure 1: Antibiotic Producers isolates /Zone of Inhibitions opened the door to the research world for us, students. Thank you to the Tiny FUTURE DIRECTIONS Table 3: Biochemical tests Three isolates were classified Earth Research Program for giving us this opportunity. We specially thank Dr. q Isolate as Gram-positive species, and Bacteria may sense antibiotics as signaling molecules to trigger Gonzalez-Orta for her guidance and dedication during the project. Catalase Oxidase KOH Name the other two were found to be different cellular responses for the survival of their polymicrobial community that is why interaction between intra-species REFERENCES 11870 + - - Gram-negative bacteria. and inter-species of bacteria are essential because it’s how the bacteria Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. 11871 + - - Results from KOH tests stated Biol. 215:403-410. that all five isolates were tested living in those communities can talk to each other and succeed as a Hernandez, J. et al.,2018. Tiny Earth - A Research Guide to Student sourcing Antibiotic Discovery (Print plus e- 11872 + - - positive for catalase and only community (Traxler & Kolter., 2015). Book access). Ann Arbor, MI: XanEdu Publishing. q Infectious Diseases Society of America. (2020). Facts about Antibiotic Resistance. 12158 + + + isolates 12158 and 12159 were These studies about the secondary metabolites and how they may Traxler, M. F., & Kolter, R. (2015). Natural products in soil microbe interactions and evolution. 12159 + + + positive for oxidase. affect how our isolates interact with neighboring colonies gives ideas for further testing of our isolates.