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After performing serial dilutions and picking isolates that showed antibiotic producing potential, these three candidate isolates were chosen and lawn were grown for further testing. Each isolate was beige colored with an irregular form, raised elevation, and undulate margins.

Isolation of Selected Antibiotics Producing Soil Microorganisms

Kylie Cousins, Adia Eleccion, Graeze Nanquil; Advisor: Dr. Olivia George 1University of Hawaii at West Oahu

References: 1. Hernandez, S., Tsang, T., Bascom-Slack, C., Broderick, N., & Handelsman, J. (2018). Tiny Earth -

A Research Guide to Studentsourcing Antibiotic Discovery. XanEdu. 1. Iglewski, B. H. (1996, January 1). Pseudomonas. Retrieved from

https://www.ncbi.nlm.nih.gov/books/NBK8326/. 1. Ventola, C. L. (2015, April). The antibiotic resistance crisis: part 1: causes and threats. Retrieved

from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/.

Antibiotics have helped in achieving major advances in medicine. Many lives have been saved through use of antibiotics. The average lifespan in the 1920’s was 56.4 years old, but because of antibiotics use, U.S. average life span is now nearly 80 years old. There is currently a crisis in treating different infectious diseases. With the overuse of antibiotics, it provides selective pressure and kills sensitive bacteria. This is now a problem because 1/106 cells in bacteria culture carries a mutation that makes it resistant to antibiotics. With sensitive bacteria killed, this gives an opportunity for resistant bacteria to grow. In pharmaceutical companies, few new antibiotics have been discovered in the past decade, while more bacteria are becoming more resistant (Ventola, 2015).

Introduction

Sequence - #37 TCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGATGACGGGAGCTTGCTCCTTGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAATCCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATGCAGAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTCTGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAgTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCTCACAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAA

Each of the bacteria isolate sequences were identical. After running the sequences through the BLAST database, we determined that our bacteria were most likely Pseudomonas taiwanensis. P. taiwanesis had the highest match, but it also matched with other types of Pseudomonas species. Table 1. Summary of sequencing data of three isolates: #37, 41, and 45.

Research Question

Results - Continued DNA Sequencing Results

When testing unknown bacteria against safe ESKAPE pathogens, there was evident antibiotic resistance against S. epidermidis (an opportunistic bacteria found on the skin). Out of 19 different isolates, we each chose three bacterium to grow lawns of for further studies. After choosing the bacteria, gram-stains were performed. Isolates #37 was gram-negative cocci, #41 had both gram-negative and gram-positive bacilli, and #45 gram-positive bacilli. We also extracted genomic DNA from the isolates to sequence the 16s gene. After extraction we also quantitated the DNA (Fig. 5) then performed a PCR reaction quality check for the presence of the 16s gene. The 16s gene was present in each isolate, with #45 amplifying the most clones due to its high concentration of 56.4 μg/mL.

We sequenced the 16s gene of the three bacterium isolated from our original soil sample that showed antibiotic properties against S. epidermidis. However, all three isolates proved to be previously-known Pseudomonas taiwanensis. Pseudomonas bacteria is a genus of gram-negative gammaproteobacteria. It is a harmless bacteria that mildly affects healthy individuals and severely infects those with compromised immune systems. They are opportunistic pathogens commonly found in soil, water, animals, plants, and air; healthy individuals have the bacteria growing on their skin in moist areas such as the axillary or genital regions. P. taiwanesis is a gram-negative, rod-shaped, motile, non-spore-forming bacterial strain.

Within our original soil sample, we saw many colonies that showed antibiotic properties that we did not have the opportunity to isolate and sequence. Further testing will be necessary to determine if these isolates are also antibiotic-producing bacteria.

.

Conclusions

Using local clay soil collected from Pearl City, Hawaiʻi, is it possible to isolate and identify a soil microorganism that can produce antibiotics against human

pathogens?

Results

Figure 2. Bacteria #37 Gram-negative cocci

Figure 3. Bacteria #41: Gram-negative cocci, Gram-positive bacilli

Figure 5 (Left). Genomic Extractions were performed on all three isolates. DNA was quantitated as follows: #41: 4.64 μg/mL; #37: 21.7 μg/mL; and #45: 56.4 μg/mL and the 16s rRNA gene was amplified via PCR to check for purity.

Hypothesis

If there are antibiotic producing bacteria to be found in our soil sample, then possible bacterial candidates can be isolated using serial dilutions, tested, verified through PCR and electrophoresis, and identified through sequencing of the 16s rRNA gene.

Bacteria Genus Most Identical match Origin

#37, #41, #45 Pseudomonas Pseudomonas taiwanensis (99.79%) P. plecoglossicida (99.65%), P. entomophila (99.51%), P. monteilii (99.51%), P. parafulva (99.23%)

Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;

Pseudomonadaceae;

Pseudomonas

Methodology

Isolate #41 Isolate #45 Isolate #37

Schematic 1. Research design and methodology of isolating antibiotic-producing bacteria.

Figure 1.2 Unknown bacteria tested against S. marcescens.

Candidates (circled) showed antibiotic activity.

Figure 1.3 Unknown bacteria tested against E. aerogenes. Candidates (circled) showed

antibiotic activity.

Figure 1.4 Unknown bacteria tested against E. coli,

Candidates (circled) showed antibiotic activity.

Figure 1.5 Unknown bacteria tested against E. faecalis.

Candidates (circled) showed antibiotic activity.

Figure 4. Bacteria #45: Gram-positive bacilli

Gram Staining of Isolates

Polymerase Chain Reaction (PCR)

Patch Testing of Isolates Against Safe Relatives of

ESKAPE Pathogens

Figure 1.1 Unknown bacteria tested against against S.

epidermidis. Candidates (circled) showed antibiotic activity.