With an increase in antibiotic resistance within the medical field the need for novel antibiotics is evident. Through collaboration with the Tiny Earth Project Initiative, we hope to find novel antibiotics through bacteria found in soil. We were able to successfully isolate two microbes that showed broad spectrum antibiotics against both Staphylococcus epidermis and Bacillus subtilis. Through further exploration and collaboration with the Tiny Earth Project Initiative, we might be able to discover novel antibiotics

Jayne, Cory

Scott, Dr. Lori

English

Augustana College, Illinois: Augustana Digital Commons

Augustana College Augustana Digital Commons Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil Biology Winter 1-28-2020 Pseudomonas and Bacillus as Potential Sources of Novel Antibiotics Cory Jayne Augustana College, Rock Island Illinois Dr. Lori Scott Augustana College, Rock Island Illinois Follow this and additional works at: https://digitalcommons.augustana.edu/biolmicro  Part of the Biology Commons, and the Natural Products Chemistry and Pharmacognosy Commons Augustana Digital Commons Citation Jayne, Cory and Scott, Dr. Lori. "Pseudomonas and Bacillus as Potential Sources of Novel Antibiotics" (2020). Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil. https://digitalcommons.augustana.edu/biolmicro/5 This Poster is brought to you for free and open access by the Biology at Augustana Digital Commons. It has been accepted for inclusion in Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil by an authorized administrator of Augustana Digital Commons. For more information, please contact digitalcommons@augustana.edu. Bacillus and Pseudomonas Genera as a Potential Source for Novel AntibioticsCory Jayne and Dr. Lori Scott, Augustana CollegeINTRODUCTIONThe need for new antibiotics is an ever-growing issue in the medical field due to a multitude of issues, most notably the over-prescription of antibiotics and the non-cooperation of patients in following the advised prescription time period.1 This has led to multiple drugs becoming resistant to the various drugs used to treat them. The ESKAPE bacteria are a group of six bacteria that are considered in critical or high need of finding new antibiotics (Figure 1). The six bacteria are Enterococcus faecium, Staphylococcus aureus, Klebsiellapneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Through collaboration with the Tiny Earth Project Initiative, a worldwide database focused on finding novel antibiotics for the treatment of the ESKAPE strains, we hope to discover antibiotics through bacteria found in soil.2Fig 1. The ESKAPE strains are becoming more resistant to the antibiotics used to treat them.5 The graph shows common antibiotics and what percentage of just one strain, Enterococcus faecium, is resistant to them. With the rise of methicillin-resistant Staphylococcus aureus(MRSA), among various other bacteria, hospital-acquired infections (HAI) are becoming a more significant issue.1Staphylococcus aureus and different other strains of the Staphylococcus genus are commonly found in surgical site infections (SSI).3 A study done by Pal et al. showed that 20.8% of patients in a rural hospital in India obtained either a primary or secondary SSI after their procedure. A primary SSI is an infection typically occurring within seven days of the operation, generally while still in the hospital. A secondary SSI is an infection acquired after leaving the hospital. Of the patients who acquired an SSI post-surgery, 64.8% had the bacteria Staphylococcus in the infection 69.9% of which was MRSA, or methicillin-sensitive Staphylococcus aureus(MSSA). The discovery of new antibiotics to treat Staphylococcus aureus is in dire need. Similarly, the genus Bacillus can cause a wide range of issues, including infections, anthrax, and endocarditis.4 Through the use of the safe test strains Staphylococcus epidermidis and Bacillus subtilis, we can use a variety of molecular and biochemical procedures to find and isolate new antibiotic-producing bacteria. These strains, which are evolutionarily similar to the ESKAPE strains, allow for the safe testing and potential discovery of novel antibiotics against Staphylococcus aureus and Bacillus anthracis. Identification and isolation of antibiotic-producing bacteria will be reported back into the global network of the Tiny Earth Project Initiative.METHODSIn collaboration with the Tiny Earth Project Initiative we were able to use many of their procedures described.2 Unless otherwise noted, all of the procedures were followed exactly.• Collect soil sample • Serial Dilution of soil samples and isolation of microbes• Media and selective media for isolation (10% TSA, LB, PDA)• Formation of master plates• Testing microbial isolates for the production of secondary metabolites with antibiotics (Staphylococcus epidermidis, Bacillus subtilis, and Erwinia carotovora)• Formation of streak plates to allow for further testing• Differential media for potential identification of isolates (MacConkey, Simmons’ citrate, MSA, TSI slants, and blood agar)• PCR of any antibiotic producing microbes (16S rRNA gene primers are 27F and 1492R)• Gel electrophoresis of PCR products (1% agarose gel)• Sequencing of the DNA of 16S rRNA gene (Iowa Institute of Human Genetics, University of Iowa)DISCUSSIONWe were able to successful identify the genus of isolates CJ-1-TSA and CJ-4-PDA as Pseudomonas and Bacillus. For these two isolates we were able to conclude that they have the potential to be broad spectrum antibiotics against both Staphylococcus epidermidis and Bacillus subtilis.Future directions of this project would include the isolation of the actual secondary metabolite. This would involve organic and biochemical separation techniques. Further directions would include full genome sequencing through the Tiny Earth Project Initiative and the potential identification of the structure of the secondary metabolite. LITERATURE CITED1. Mulani, M. S.; Kamble, E. E.; Kumkar, S. N.; Tawre, M. S.; Pardesi, K. R. Emerging Strategiesto Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review. Frontiers in microbiology 2019, 10, 539.2. Hernandez, S., T. Tsang, C. Bascom-Slack, N. Broderick and J. Handelsman. 2018. Tiny Earth: A research guide to student sourcing antibiotic discovery. XanEdu Publishers, AnnArbor, MI.3. Wright, W. F. Central Venous Access Device-Related Bacillus Cereus Endocarditis: A Case Report and Review of the Literature. Clinical medicine & research 2016, 14, 109-115.4. Pal, S.; Sayana, A.; Joshi, A.; Juyal, D. Staphylococcus aureus: A predominant cause of surgical site infections in a rural healthcare setup of Uttarakhand. Journal of Family Medicine and Primary Care 2019, 8, 3600-3606.5. Silva, D. M.; Menezes, E. M. N.; Silva, E. V.; Lamounier, T. A. C. Prevalence and antimicrobial susceptibility profile of ESKAPE pathogens from the Federal District, Brazil. 2017.6. Altshcul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410. PubMedRESULTSFigure 2. Evidence of antibiotic producing isolates present against both of my Gram-positive strains, Staphylococcus epidermidis and Bacillus subtilis. Halos were present in the first and third columns, Staphylococcus epidermidis and Bacillus subtilis respectively. The middle column was a Gram-negative bacteria, Erwinia carotovora. The plates were grown on LB at 28oC for 24 hours.Figure 3. Successful amplification of the 16S rRNA gene from PCR. We were able to amplify the DNA from the bacteria in lanes 3,5,7, and 9. In these lanes were isolates CJ-1-TSA, CJ-2-TSA, CJ-4-PDA, and CJ-5-PDA, respectively. With the primers we chose, the DNA bands we expected, and produced, were around 1.4Kb. The PCR product was run for 25 minutes on a 1% agarose gel. RESULTS (CONTINUED)Figure 4. 16S rRNA gene sequence data, showing that isolate CJ-1-TSA, and CJ-4-PDA are from the genus Pseudomonasand Bacillus respectively.6 The description section lists the possible strains that our unknown isolates were. Query cover is the percentage of the actual gene that our sequenced data matches up with, in this case we did not amplify the entire gene, but instead about 98%. The E value is the likelihood that we just randomly obtained these strains. Finally, the percent identity is the percent of nucleotides from our isolates that line up exactly with the known strains in the description.  

Pseudomonas and Bacillus as Potential Sources of Novel Antibiotics

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Pseudomonas and Bacillus as Potential Sources of Novel Antibiotics

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