Analysis of Soil Microbes for Antibiotic PropertiesJake Kieserman & Linda Zheng

There is an upcoming crisis in modern medicine in which pathogenic strains of bacteria are becoming resistant to the antibiotics that physicians commonly prescribe. Infections that were once commonly treatable are now developing into severe infections increasing the mortality rate of humans across the globe. In this study, bacterial isolates were cultured from soil samples around the University of Pittsburgh’s campus and were analyzed for antibiotic producing properties against ESKAPE pathogen (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacterbaumannii, Pseudomonas aeruginosa and Enterobacter spp.) in hopes of developing new medications to combat this multi-drug resistant organisms.

Introduction

Objectives1. To obtain bacterial samples from soil taken from around Pitt’s

campus and culture the bacteria as single colonies to determine which bacterial specimens are antibiotic producers against ESKAPE pathogens

2. To analyze and characterize soil isolates by their physical and chemical properties

3. To contribute to the Small World Initiative database to work collaboratively toward addressing the antibiotic crisis

Results

Conclusions

ReferencesHernandez, S.A., et al. 2013. Introduction. In: The Small World Initiative

Student Laboratory Manual. Yale University. 7.Reynolds, J. 2005. Small World Initiative Protocols. Yale University. 70-

106.

Methods Flow Diagram

Test Jake’s Sample Linda’s Sample

Soil Type Silty loam Sandy clay loam

pH of Soil (deionized water) 8 6

Percent Water Content 33.45% 30.7%

Percent Organic Content (Wet Weight) 11% 8.16%

Optimal Agar Medium NA R2A

Optimal Dilution Ratio 1:1000 1:1000

CFU/g from Optimal Dilution 9.4x105 1.8x106

Test Jake’s Results Linda’s Results

Starch Hydrolysis Positive for amylase Positive for amylase

Catalase Reaction Negative for catalase Negative for catalase

SIM (Sulfide/Indole/Motility)

Negative for sulfide/indole, Positive for motility

Negative for sulfide /indole, Positive for motility

Fluid Thioglycollate Aerotolerant anaerobe Facultative anaerobe

Triple Sugar Iron Ferments glucose, produces acid

Ferments glucose/sucrose/lactose, produces acid

MacConkey Agar No growth (Gram +) No growth (Gram +)

DNA match (with BLAST)

Bacillus funiculus (99% identity)

Paenibacillus xylanexedens (99% identity)

Table 2. Results of biochemical characterization testing

Table 1. Results of soil characterization and optimization testing

Our testing resulted in two antibiotic producers, one against Staphylococcus epidermis and the other against Escherichia coli and Enterobacter aerogenes, proving that antibiotic-producing microbes can be found and isolated from soil samples. From the various characterization testing, we see that the antibiotic producing bacterial isolates were diverse, exhibiting many different physical and chemical properties. Although we were not able to extract the antibiotic organic compounds, this may have been because the antibiotic compounds were not organic or difficulties with using ethyl acetate. Further testing may yield other antibiotic-producing microbes, such as if we continued to test other isolates from our soil samples using the same process.

AcknowledgementsSpecial thanks to Elia Crisucci and Jean Schmidt for their commitment and assistance to our research, our classmates for their continued encouragement and teamwork, and the University of Pittsburgh for funding and creating this opportunity for us to become part of the Small World Initiative.

Identify antibiotic

producers (zones of

inhibition)

Collect soil

sample

Analyze soil

sample

Serial dilution of

soil

Perform PCR reaction

to amplify antibiotic-

producing isolate DNA Gel electrophoresis to

confirm PCR results

Repeat if PCR unsuccessful

ATGTCAATASend DNA to be

sequenced and identify

isolate

Test various agar

typesIsolate

microbes

Test against safe

“ESKAPE”

pathogen relatives

Repeat if unsuccessful

LB

PDA R2A

NA

Perform gram

staining on isolateExtract antibiotic

organic compounds

Biochemical

characterization of isolates

(e.g. starch hydrolysis test)

• Two antibiotic-producing isolates found and identified, Bacillus funiculus produced a zone of inhibition against E. coli (Fig. 1) and E. aerogenes (Fig. 2), and Paenibacillus xylandexedensagainst S. epidermis (Fig. 3), although attempts to extract the antibiotic organic compounds were unsuccessful

• Each soil sample was initially thoroughly analyzed (Table 1) and later biochemically characterized (Table 2)

Figure 3. Results from spread/patch screen for soil isolate antibiotic production against S. epidermis, incubated at 30°C for seven days.

Figure 2. Results from broad spectrum screen for soil isolate #1 antibiotic production against E. aerogenes, incubated at 30°C for two days.

Figure 3. Results from spread/patch screen for soil isolate antibiotic production against E. coli, incubated at 30°C for five days.