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Isolation and Characterization of Bacteria from Tropical Soils
Alejandra Ferrer1
Nicole S. Rivera1
Jessica Ortiz1
Carolina Huertas1
Dr. Michael Rubin Date: May 15, 2015
RISE Program Biology and Chemistry DepartmentUniversity of Puerto Rico at Cayey
1 This author contributed equally to this project
Background • Bacteria are prokaryotic microorganisms that can be differentiated by
their peptidoglycan layer located in the cell wall. • This aspect classifies them as gram-positive or gram-negative.
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Antibiotic Era • The discovery of penicillin in 1928 was by Alexander Fleming.
• Since 1940’s scientists, using Fleming’s research as a source, have been studying and developing antibiotics in order to reduce illness and death from infectious diseases (CDC 2014).
• Rene Dubos- discovered and isolated an antibacterial compound called tyrothricin.
• Recently, scientists have discovered a new antibiotic, from the soil, which name is Teixobactin (Ling et al. 2015).
http://www.pharmamicroresources.com/2015/01/new-penicillin-mechanism-revealed.html
• Antibiotic resistance is produced when the capability of killing or inhibiting bacterial growth is eliminated. The bacteria can grow in the presence of the antibiotic (APUA 2014).
• In a recent investigation Walsh and colleagues (2003) isolated 412 antibiotic resistant bacteria from agricultural, urban, and pristine soils.
Significance● The importance of this study is that it attempts to find new
bacteria from tropical soils with antibacterial properties.
● This investigation responds to the need for new antibiotics that can inhibit bacterial reproduction.
Problem
• Can we find bacteria in soil that have useful properties such as antibiotic production and antibiotic resistance?
Hypothesis• It is hypothesized that different properties of the
isolated soil bacteria will be characterized such as bacterial structure, antibiotic resistance and antibiotic production .
• There will be more bacteria found in tropical soils capable of antibiotic production and resistance . (Dubisnky et al. 2010)
Goals• Isolation, purification and freezing of soil bacteria
• Gram staining and colony morphology
• Test for antibiotic production and resistance • Generation of genomic DNA, PCR, gel electrophoresis
and purification
• Sequencing and Bioinformatics Analysis
Experimental Design
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1 gram of soil
mix
Label the tubes 0→ -5
water (8.5ml)
NaCl(90µl)
streak the plate NaCl
medium plates
collect the colony
II. Purification
III. Cryogenic Freezing
10 mL pipette
S15UPRC-RISEInitials30M01S :Spring15 :2015UPRC: UPR-CayeyRISE: RISE ProgramInitials: Your Initials (MRR)30: Incubation Temperature in CelsiusM: Media (T = Tryptic Soy Agar; N = Nutrient Agar; L = LB Agar; M = RDM; R = R2A; P = 1SP4)
01: Soil Sample Numberspread the bacteria
medium broth
80% glycerol
micro tube
cultured medium plate
pipette
scratch the plate
collect the mix
test tube mix
identify the tubes
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IV. Gram staining
A drop of water in the center of the slide.
Collect bacteria and add them to the slide
For 1 minute and remove it with water
safranin
ethanol
crystal violet
bacteria
slides
water
inoculation loop
V. Genomic DNA and Polymerase Chain Reaction (PCR) centrifuge
dry bath ice bath
liquid broth with bacteria
PCR tubecollect the supernatant
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VI. Electrophoresis
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VII. Antibiotic Production and Resistance Tests
M. LuteusE. Coli
Grow bacteria in separate plates
Divide the plates in two portions
Dip the paper disk in the bacteria culture and place it
in a side of the petri dish
Incubator 24 hours
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VIII. Purification of PCR Products for Sequence Determination
QIAquick PCR Purification kit
Calculate the volume of the
buffer required
add calculated amount to PCR product
Transfer into a QIAquick column
Centrifugeadd buffer PEPlace the column in a
clean microtube
Pure waterAnalyze the sample
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Geolocation of Bacteria
Alejandra Ferrer Nicole Rivera Carolina Huertas Jessica Ortiz
Soil Collection
Santa Isabel
Caguas
Comerío
Soil Collection
Latitude: 18° 8’ 38.6” NLongitude: 66° 14’ 45.8” W
Aguas Buenas
Aibonito
Results
Results
Gram Staining Endospore Staining
Bacteria: S15UPRCRISEJCOR30P01AJessica Ortiz
Purified bacteria Coexistence with E.coli
Bacteria: S15UPRCRISEJCOR30P01AResistance to Tetracycline
Clusters of bacteria Repetition Repetition with broth of clusters
Bacteria: S15UPRCRISEJCOR30P01A
Agarose gel #1: PCR Product Agarose gel #2: Purified PCR Products
Bacteria: S15UPRCRISEJCOR30P02A Jessica Ortiz
Changes in color according to incubation period at 30 C
1 day 2 days 3 days
Gram Staining
Bacteria: S15UPRCRISEJCOR30P02AResistance to Tetracycline
Clusters of bacteria Repetition with broth of clusters
Bacteria: S15UPRCRISEJCOR30M02BJessica Ortiz
Purified Bacteria
Gram Staining Circles of resistance against tetracycline
Coexistence with M.luteus
Bacteria: S15UPRCRISEJCOR30M02B
Agarose gel #1: PCR Product
Agarose gel #2: Purified PCR Products
Bacteria:S15UPRCRISECHA30P01Carolina Huertas
Morphology of Bacteria Gram Stain of Bacteria
Antibiotic Production of Bacteria
Bacteria: S15UPRCRISECHA30M01Carolina Huertas
Morphology of Bacteria Gram Stain of Bacteria
Antibiotic Production of Bacteria -->
Bacteria S15UPRCRISEAFD30M01A
Gram stain
Colony morphologyGram stain
Antibiotic production for M. Luteus
Bacteria S15UPRCRISEAFD30M01A
16S_1510R and 16S_8F primers
Alternative Primers
Bacteria S15UPRCRISEAFD30M01B. Alejandra Ferrer
Colony morphology Gram stain
Antibiotic production
Bacteria: S15UPRCRISENRE30M01A
Latitude: 18° 8’ 38.6” NLongitude: 66°14’ 45.8” W
Nicole S. Rivera EspinalAntibiotic production
Gram stain
Colony morphology
S15UPRCRISENRE30M01A
16S_1510R and 16S_8F primers
Bacteria: S15UPRCRISENRE30P01B
Nicole S. Rivera Espinal
Gram stain
Colony morphologyAntibiotic production
Conclusion: Morphology
Conclusion: Phenotype
Conclusion • Nine bacteria were isolated from tropical soils of Puerto
Rico and characterized.• All bacteria are gram positive and seven of nine are
bacilli.• Four produced antibiotics, two bacteria coexisted with
Escherichia coli or Micrococcus leteus, two bacteria showed antibiotic resistance to tetracycline.
• Bacteria with antibiotic resistance and antibiotic production are found in Puerto Rican (tropical) soil.
Future Experiments• Test bacteria for cellulose and oil
degradation capability• Test antibiotic production with other bacteria • Repeat the investigation by isolating new
bacteria in order to have a better geographical distribution
Acknowledgments• RISE Program (Proposal:5R25GMO59429-16)
University of Puerto Rico at Cayey
• Dr. Michael Rubin Department of Biology, University of Puerto Rico at Cayey
• Dra. Eneida Diaz RISE Program, Department of Biology, University of Puerto Rico at Cayey
• Dra. Elena González RISE Program, Department of English , University of Puerto Rico at Cayey
Acknowledgments• Giovanni Cruz
RISE Program, University of Puerto Rico at Cayey
• Lizbeth Pérez RISE Student, University of Puerto Rico at Cayey
• José Pabón Department of Biology, University of Puerto Rico at Cayey
• Cristopher Quintanal Howard Hughes Program, University of Puerto Rico at Cayey
• Laura Ramos Department of Biology, University of Puerto Rico at Cayey
Cited literature • [ACS] American Chemical Society[Internet][2015] Washington (DC): American Chemical Society;
[cited 2015 Jan 8]. Available in: http://
www.acs.org/content/acs/en/education/whatischemistry/landmarks/flemingpenicillin.html#alexander-
fleming-penicillin
• Alanis AJ. 2005. Resistance to antibiotics: Are we in the post-antibiotic era? Arch. Med. Res. 36:697-
705.
• [APUA] Alliance for the prudent use of antibiotics.[Internet].[2014]. Boston (MA): Alliance for the
prudent use of antibiotics;[Cited 2015 Jan].
Available:http://www.tufts.edu/med/apua/about_issue/about_antibioticres.shtml
• Azvolinsky A. 2015 January 7. New Antibiotic from Soil Bacteria. The Scientist; [Cited 2015 Jan 8].
Available in: http://www.the-scientist.com/?
articles.view/articleNo/41850/title/New-Antibiotic-from-Soil-Bacteria/
• Beveridge T.J. 2001. Use of the Gram stain in microbiology. Biotech Histochem 76 (3): 111–8
Isolation and Characterization of Bacteria from Tropical Soils
Alejandra Ferrer1
Nicole S. Rivera1
Jessica Ortiz1
Carolina Huertas1
Dr. Michael Rubin Date: May 15, 2015
RISE Program Biology and Chemistry DepartmentUniversity of Puerto Rico at Cayey
1 This author contributed equally to this project