Soil Sample Analysis

Brieanna Bell, Kally Morozin, Alex Trubey, and Mako Yanagida

Rose-Hulman Institute of Technology

Introduction Results Conclusions

Antibiotic resistance is a medical crisis that must be address within Table 1. Soil Characterization of the Collection Site This research resulted in four antibiotic
● Four soil samples were collected
the upcoming years. Since their development in the 1930’s, people (Table 1), microbial colonies were producing isolates. Based on metabolic testing
have completely relied on antibiotics to keep human populations isolated on a patch plate (Figure 1). the isolates were classified as:
safe from infectious disease. The CDC estimates that nearly 154
■ Isolate 1: Micrococcus luteus
million antibiotic prescriptions are given out by doctors each year to
combat dangerous diseases. However, the effectiveness of these ■ Isolate 2: Klebsiella pneumoniae
antibiotics is at risk due to microbes rapidly adapting resistant or ■ Isolate 3: Bacillus lentus
immunity to these treatment. Antibiotic resistance is now being ■ Isolate 4:
acquired at a rate far faster than antibiotic development. As a result
many people are dying from diseases that were once easily treated Based on BLAST sequencing isolate 2 was
by antibiotics. identified most likely to be Klebsiella
pneumoniae. BLAST sequencing for the other
To combat antibiotic resistance,
three isolates failed. The reason why can't get
soil microbes can be analysis
to determine if they have the DNA sequence
potential to combat dangerous ● Based on gram staining (Figure 2) ○ Low production of purpose
pathogens. Microorganisms in metabolic testing the four soil isolates were DNA template
the soil can be bacteria, actino- Figure 1. Patch plate up close: Formation of classified as Micrococcus Luteus, ○ Insufficient removal of
mycetes, viruses, fungi, algae, Micrococcus luteus, & Bacillus lentus (Table
an isolate colony with irregular morphology. protein, RNA, salt and other
and protozoa; each microbe 2). Some of the metabolic test conducted
include the hemolysis test for which isolate chemicals, the sequence re-
has diversity properties and
characteristics. Many soil micro- 3 showed complete hemolysis (Figure 3) action enzyme was inhibited.
Table 3. Analysis of Metabolic Testing
organisms have produce anti-
biotic compounds against pathogenic bacteria, thus these soil The microbial identification of isolate 4 based on
microbes could be used as antibiotics. metabolic testing and BLAST sequencing
Figure 2. (Left)
matched up accurately.
In this research, microorganisms from a sample of soil were Viewing a
collected, isolated, and tested for antibiotic effectiveness against bacterial isolate 1 More research should be conducted on the
ESKAPE pathogens. The objective of this study was to data under a isolates to further investigate their potential as
produced from identify microorganism with antibiotic potential. microscope after antibiotics to combat ESKAPE pathogen.
gram staining it:
Looks like
Gram +!

Materials and Methods References

Biochemical/Metabolic Testing Method
● Gram stain
● SIM tube (sulfide,indole,motility)
● TSI slant
(Triple sugar: Glucose, Lactose, Sucrose)
● Isolate 2 was the only one
of the four isolates
sucessfully sequenced and
identified using BLAST
sequencing. The isolate
had an 88% consensus
sequence with Klebsiella
pneumoniae (Figure 4)
● All of the collected isolates
produced a zone of
inhibition in response to a
given ESKAPE pathogen,
which represents the
production of antibiotics by
● Simmons Citrate tube the isolate (Table 1)
● MSA plate / fermentation
● MR-VP broth
etc
Figure 3. (Right) ● Hernandez, S., et al, Tiny Earth: A Research Guide
Testing the to Studentsourcing Antibiotic Discovery. (Oct
hemolysis of red 2018).
blood cells with ● “Improve Antibiotic Use to Combat Antibiotic
the Blood Agar Resistance.” Centers for Disease Control and
Test of isolate 3: Prevention, Centers for Disease Control and
complete, β Prevention,
hemolysis www.cdc.gov/media/releases/2016/p0503-
unnecessary-prescriptions.html.
● Bergey, D. H., et al. Bergey's Manual of
Determinative Bacteriology; a Key for the
Figure 4. Results of BLAST sequencing: Identified as 88% Identification of Organisms of the Class
Klebsiella pneimoniae Schizomycetes. 9th ed., Williams & Wilkins Co.
Acknowledgements
We would like to thank Dr. O’Connor for her continual guidance
throughout this project. We would also like to acknowledge Shannon
Tieken for lab preparation and Tom Rogge for poster printing. This
research was conducted through the Tiny Earth Initiative.
Each base of wavelength in the PCR product
Green : Adenine, Red: Thiamine, Blue: citrine, Black: Guanine