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