MATERIALS & METHODS

RESULTSINTRODUCTION

FUTURE DIRECTIONS

Antibiotic Producing Bacteria from Sacramento State University Campus Soil

Linda Acevedo, Elizabeth Kern, Nguyen Vo, and Enid Gonzalez-Orta Ph.D.

Soil Characterization and Alfa-Diversity Colony Morphology

Safe ESKAPE Testing

Table 1. Abiotic factors, soil bacterial population and Alfa-diversity

Sampling Location 38°33'38.9"N 121°25'12.8"W near the American River

Air temperatura & Humidity

16.7 ℃ & 65.5 %

Soil pH 7.0Soil Temperature 12.7 ℃

Soil Depth 12.7 cmBacterial density 8.6 x106 cfu/g soil

Simpson’s index (D) 0.32Shannon-Weaver Diversity

Index (H)1.75

Pielou’s Evenness (e) 0.54

Isolate Strain # Media E. Raffinosus

S. epidermis

E. coli

E. aerogenes

A. baylyi

B. subtilis

11870 S20BIO145LAR21+ R2A Ab+ Ab+

No teste

dNo tested No

tested Ab+

11871 S20BIO145LAR22+ R2A Ab+ Ab+ Ab- Ab- Ab- Ab+

11872 S20BIO145LAR23+ Ab+ Ab- Ab- Ab- Ab- Ab-

12158 S20BIO145LAR24+ R2A No tested Ab- Ab+ No tested No

testedNo

tested

12159 S20BIO145LAR25+ R2A Ab+ Ab- Ab- Ab- Ab- No

tested

Table 2: Antibiotic Producers Table

Antibiotic Producers

ACKNOWLEGMENTS

qThe Tiny Earth Research Guide Program highlights the importance of 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 most abundant source of specialized metabolites that can produce antibiotics. qAccording to the Infectious Diseases Society of America (IDSA,2020), about 2 million Americans per year develop hospital-acquired infections, which results in 99,000 deaths due to antibacterial-resistant pathogens.qThe acronym ESKAPE includes six nosocomial pathogens that exhibit multidrug resistance (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and several species of Enterobacter).qIn this research, we focused on the Safe ESKAPE relatives instead because they have the same physiological features, but they do not possess a health risk in a teaching lab setting. qWe used Enterococcus raffinosus, Staphylococcus epidermis, Escherichia coli, Acinetobacter baylyi, Pseudomonas putida, and Enterobacter aerogenes as the Safe ESKAPE relatives, and we added four additional bacteria to this research, Mycobacterium smegmatis and Bacillus subtilis. As well as Agrobacterium tumefaciens and Erwinia Carotovora that are plant-pathogens bacteria that are agriculturally important.

qTo perform our experiment, we collected our soil samples near the river, right off the bike and walking path at Sacramento State University. We tested the bacteria isolated from the soil samples against those safe ESKAPE pathogens and cultured them on R2A media. We tested nine soil isolates against five different safe ESKAPE relatives that included (Enterococcus raffinosus, Staphylococcus epidermis, Escherichia coli, Acinetobacter baylyi, Enterobacter aerogenes, and one of the additional bacteria added, B. subtilis).

qWe performed:

üSoil characterization & Alfa- diversityüSafe ESKAPE testingü Biochemical testsü DNA ExtractionüPolymerase Chain Reaction (PCR)ü Gel Electrophoresisü Nanodrop on the isolatesüAnalyze the bacteria sequences through different bioinformatic programs( BLAST, Ribosomal Database Project (RDP) that included SeqMatch and Classifier.

We performed four rounds of Safe ESKAPE testing. We Tested nine isolates against the Safe ESKAPE relatives, but only five isolates exhibited antibiotic activity.

qBacteria may sense antibiotics as signaling molecules to trigger different cellular responses for the survival of their polymicrobial community that is why interaction between intra-species and inter-species of bacteria are essential because it’s how the bacteria living in those communities can talk to each other and succeed as a community (Traxler & Kolter., 2015).

qThese studies about the secondary metabolites and how they may affect how our isolates interact with neighboring colonies gives ideas for further testing of our isolates.

DISCUSSION

qThe whole reason for this research was to analyzed soil sample bacteria to find possible antibiotic producers that could inhibit the safe ESKAPE pathogens relatives.q”Terrain matters”, we saw it firsthand with our patching, how specific neighboring isolates triggered others when they were close or apart to specific isolates.qWe were able to control it by modifying were we patched our isolates.

[A] isolate 11870[B] Isolate 11871[C] Isolate 11872[D] Isolate 12158[E] Isolate 12159Four of the isolates inhibited Gram-positive (+) bacteria that included S. epidermis, E. raffinosus, and B. subtilis. Only one of the isolates inhibited Gram-negative bacteria(-), E. coli.

Table 3: Biochemical tests

Being able to work hands-on and discover antibiotic-producing isolates has opened the door to the research world for us, students. Thank you to the Tiny Earth Research Program for giving us this opportunity. We specially thank Dr. Gonzalez-Orta for her guidance and dedication during the project.

REFERENCESAltschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410. Hernandez, J. et al.,2018. Tiny Earth - A Research Guide to Student sourcing Antibiotic Discovery (Print plus e-Book access). Ann Arbor, MI: XanEdu Publishing.Infectious Diseases Society of America. (2020). Facts about Antibiotic Resistance. Traxler, M. F., & Kolter, R. (2015). Natural products in soil microbe interactions and evolution.

Three isolates were classified as Gram-positive species, and the other two were found to be Gram-negative bacteria. Results from KOH tests stated that all five isolates were tested positive for catalase and only isolates 12158 and 12159 were positive for oxidase.

Isolate Name Catalase Oxidase KOH

11870 + - -11871 + - -11872 + - -12158 + + +12159 + + +

Isolate Name

BLASTGenusMax ID

RDP SeqMatch S_ab Score RDP Classifier

11870 Bacillus 99.60% 1.000 Order Bacillales 98%

11871 Streptomyces 99.78% 0.895 Genus Streptomyces 100%

11872 Streptomyces 99.36% 0.995 OrderActinomycetales 98%

12158 Flavobacterium 99.14% 0.934 Genus Nubsella 88%

12159 Flavobacterium 98.99% 0.947 Genus Flavobacterium 100%

Identification of soil isolates by 16s rRNA

Using Bioinformatics we determined that the five isolates chosen belonged to the genera Bacillus, Streptomyces, and Flavobacterium.Table 4: BLAST, RDP (SeqMatch and Classifier ) Table

[A] 11870- The colonies had irregular shapes, mucoid, smooth, and white color. [B] 11871-The colonies were round, with entire margin, flat and smooth. The Back of the plate was mustard color, but the inside was white with a powdery texture. [C]11872- The colonies were a flower-like shape, powdery texture with purple and pinkish, and white colors. [D]12158- The colonies were color pink; the form was round with an entire margin and mucoid. [E]12159- The colonies were a mix of Irregular and round shape, mucoid, and it had a sticky texture, the colonies were whitish-yellow color.

Identification of Soil Isolates by 16s rRNA

Figure 1: Antibiotic Producers isolates /Zone of Inhibitions

Figure 2: Colony morphology of isolates that have antibiotic activity

Figure 3: Patching/Colonies interactions