Tropical Soil Bacteria Isolation & Characterization

Anibal Tornésa,1 & Abneil D. Aliceab,2

a University of Puerto Rico at Cayey, RISE Program, Department of General Sciencesb University of Puerto Rico at Cayey, RISE Program, Department of Chemistry

A B S T R A C TBacteria have been classified as unicellular organisms and have long been conceived to be the cause for many viral infections. The fact that we are studying tropical soil implies that the microorganisms living in it have the capacity to mold themselves and evolve to survive the harsh and spontaneous conditions they live in. We isolated and characterized bacterium taken from various soils, to establish antibiotic production properties, and when sequenced be able to determine more specific traits. To demonstrate these properties, we began the investigation by selecting bacteria from our soil samples and arranged specifics tests, such as; purification, gram staining, cryogenisation, PCR and antibiotic production capacity. Within our test results, we confirmed the presence of 16s rDNA gene in our extracted soil bacterium and classified both samples as gram positive . On the other hand, our hypothesis was partially disproved by the absence of antibiotic production, yet the bio informatics analysis could provide information on production towards other antibiotics and an arsenal of characteristics.

1. Introduction

Bacteria have been classified as unicellular organisms that cause cell damage due to toxin production.

These unicellular organisms have long been conceived to be the cause for many viral infections due to their

proportionally large growth and presence in the thousands over a pencil eraser. Misconceptions of bacteria growth

and the potential threat they hold have been the cornerstone of the public ignorance. These unicellular organisms

can be classified into two main types based on their shape and grouping. Under microscopy after using gram

staining (GS) technique, bacterias can be seen as spherical (cocci), elongated (bacillus), with a comma shape

(vibrio), or spiral shape (spirillum), and these are subdivided into an arsenal of variations in genus. Thanks to the

GS analysis we may determine whether the bacteria is gram positive meaning they lack a cell wall, or Gram

Negative where due to the presence of a cell wall they tend to create antibiotic resistance in a shorter time lapse.

On the other hand, antibiotics are the antagonist chemical solutions that fight against the reproduction of

bacteria and, in other cases, destroy the bacteria by different methods. These methods vary because of the action

mechanism of the antibiotics, since they are designed to attack different parts of the bacteria like cell wall,

ribosomes, inner membrane, DNA, RNA or other proteins, creating different effects in bacteria. The study of

bacterial mechanisms of action and antibiotic resistance has been focused primarily in pathogenic bacteria, instead

of following Alexander Fleming perspective and looking for unique antibiotic in the soil and even vegetation

(Walsh and Duffy 2013). Penicillin was found in the soil, and has, historically, been considered one of the most

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effective antibiotics. This overused medication has recently shown to be less effective in hospitals, since bacteria

have mutated, creating resistance in spite of penicillin antibiotic strength due to the public´s excessive use.

The most pivotal part of this investigation was to characterize and isolate soil microorganisms, principally

bacteria extracted from tropical soil. In order to study bacteria, we needed to understand their growth. There are

four major steps in bacteria growth; beginning with the lag phase, our soil microorganisms would familiarize to

their growth conditions. Secondly, in the exponential phase, bacteria colonies will divide as fast as they can

looking to maximize nutrient feeding. The theoretical growth is determined through a prolonged time according to

nutrient quantity,whilst in the stationary phase, nutrient consumption is maintained according to the medium.

Finalizing this cycle, we have the death phase of the bacteria colonies. We can take, for example, how in Tanzania

a bacteria identified as E. faecium began to be identified in patients at an elevated scale.

The most affected population of nosocomial infection (NI) have been HIV patients due to their immune-

compromised systems, especially the neonates whose mothers may have the active HIV virus. We must not keep

developing antibiotics, but rather, start developing smarter antibiotics and limit them to hospitals. Feltman (2015),

reported that teixobactin (TX), an antibiotic in progress, should have a limited distribution in order to avoid the

drug from gaining resistance too quickly. With all this in mind, we may finally be able to start looking towards the

answer, in the case that antibiotic resistance appeared, the bacteria designated antibiotic might mold itself to avoid

genetic material exchange. If we manage to avoid excessive exposure to the general public, the resistance would

take much more time in order to recur.

The previously mentioned bacteria is present naturally in our metabolic pathway; nevertheless, an increase

of its concentration from a genetically mutated version has taken its toll mainly in children at Muhimbili National

Hospital. This hospital bacteria cleansing is based on vancomycin and penicillin withal, this spread has made E.

faecium, not only Vancomycin-resistant enterococci (VRE), but also high level gentamicin-resistant (HGLR)

(Aamodt et al. 2015). Resistance, prevalence, and even genetic variations occur between bacteria and their growth

rates, since these organisms are exposed for an extended amount of time to their antibiotics, they suffer a genetic

material exchange. Therefore, the hypothesis for our tropical soil analysis is that our characterization will have

antibiotic production properties, and when sequenced we will be able to determine more specific traits.

There is an arsenal of possibilities from the study of these soil microorganisms, principally related to the

medical field, for example, avoid the propagation of NI. We aimed to identify the levels of culturable resistant

bacteria, both antibiotic producers and non-antibiotic producers, and identify the roles of the different mechanisms

of resistance (Yuan et al. 2015). With all of this is mind we will determine selective variables which will influence

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our experimental protocols due to the great fluke that the tropical soil microorganisms due to their constant

adaption to spontaneous conditions.

2. Materials & Methods2.1 Soil Analysis

Our research began with fieldwork to subsequently analyze soil microorganisms in the lab. We obtained soil

samples from different points throughout the island of Puerto Rico while documenting the surroundings. The

collected specimen had to be handled with extreme precautions in order to avoid cross-contamination with bacteria.

We initially weighed a 1.0 g sample of the soil and afterwards we diluted the mother solution to 10 -5 in order to

measure the pH. We dispensed the 100 and 10-5 solutions into our R2A and ISP4 agar mediums with a spreader to

promote bacterial colony growth and set the incubator at room temperature (27º C).

2.2 Bacteria Selection . Purification , Cryogenization , Gram Staining & PCR

Once the dilutions were done and the spread plates were analyzed, the aseptic technique protocol was used to avoid

cross-contamination before and after our bench work. We passed on to choose the bacteria of preference, whilst

taking into consideration that the more secluded the bacteria colony seemed on the agar plate, the more effective

our isolation and purification would be. A streaking protocol was repeated until homogeneous morphological

characteristics were determined. After obtaining desired bacteria, we cryogenized our samples, this process is done

in order to help preserve bacteria for future test trials.. In spite of the bacteria being frozen, 0.5mL of an 80%

glycerol solution would conserve them and avoid their crystallization through the freezing time interval.

After obtaining a working purified cryogenization, we began gram staining. Occasionally, however, isolates are

encountered in the clinical laboratory that appear to be gram variable or gram negative (Carlone et al. 1983). To

aid our investigation, we took into consideration the generic gram stain protocol, applied by Cerny in 1976 for a

microscopic understanding of the studied bacteria. After understanding that we were dealing with bacterium from

the microscopic analysis, we went on to PCR & Electrophoresis. For PCR we used 16S rDNA found in an arsenal

of bacteria, this gene aided our tests to confirm bacterial assimilation and avoid sequencing a protein. We followed

the protocol that aided our investigation with the use of PCR, as refered by Mullis (1994) procedure. Nevertheless,

a protocol variation consisted of pipetting 17 microliters into the gel, 15 microliters of which were bacteria DNA

and 2 microliters consisting of a dye in order to help observe the movement of the gel bands.

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2.6 Antibiotic Production Test

Bacteria in their natural environment compete for resources like any other organism, therefore they create

resistance and produce natural antibiotics to overcome their competitors. After following aseptic protocol,we took

two new mediums and streaked them with a sterile inoculation loop containing M.luteus and E.coli. Next, we took

forceps and placed our disc inside our broth. The broth would then be placed in the agar of the medium

2.7 PCR Product Purification

The protocol instructions were given by the manufacturer, moreover, calculations were made to determine 5

volumes of PB buffer which turned out to be 190 microliters. They were placed within the PCR micro tube with

the DNA. Afterwards, we pipetted this volume into a QIAquick column in order to centrifuge and filter all the

impurities. Finally, the pure DNA was used to run a second electrophoresis and sent out to sequence.

3. Results and Discussion

Since we are going to analyze and isolate soil bacteria, we took specific types of soils from which we made various

types of analysis. The data we recorded during fieldwork was tabulated in Figure 1.1..

Fieldwork Data

Sample Extraction Date & TimeCollected

Site Features Coordinates Humidity Temperature pH

Sample A -Medical Center

01/28/155:42 pm

Urban 18°23'45.3"N 66°04'27.0"W

Moist 302 K 7.33

Sample B- Spout 01/29/156:45 am

Semi Rural 18°12'26.9"N 66°03'10.4"W

Moist 296 K 6.5

(Fig 1.1)

We proceed to analyze the characteristics of isolated soil microorganisms which were identified as sample

A and sample B. In the case of sample A, it was extracted from the first floor of our Medical Center. Beside the

soil extraction point, there was a parking lot, a steady leakage from the surgery floor, and needles from drug

addicts in the area. The weather was mostly clear, with a humidity of 62% the day the soil samples were collected.

Sample B was collected near a spout that began at the Turabo River. The morning of the soil collection extraction

was partly cloudy with humidity of 79% and pressure of 29.97 as show in Figure 1.3. People from the area

believed that the water flow adjacent to the soil extraction point is very contaminated due to the waste houses from

upstream dispose. The water flow includes bathroom pipelines that could end up in the river mouth.

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Google Earth soil extraction location

Sample A Sample B

(Fig 1.2)

Soil Analysis w/Coordinates

Sample A - 18°23'45.3"N 66°04'27.0"W Sample B - 18°12'26.9"N66°03'10.4"W

(Fig 1.3)

Once the soil samples were collected, we took the pH of the solution in the lab. The soil was diluted as part

of series of analyses to complete this task. The pH test helped us detect if our extracted soil samples were acid,

base or neutral. In the case of sample A, the pH was slightly basic with 7.33, whilst for sample B, the 6.5 measure

demonstrated it to be quite acid as show in Figure 1.4 and proved with a potentiometer as well.

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pH Test

Sample A Sample B

(Fig 1.4)

After the selection and analysis of the soil sample, we diluted the samples from 10 0 to 10-5 and tested these

dilutions with 2 different kinds of agar mediums: R2A and ISP-4. As a result, the positive mediums and dilutions

were 100 and R2A for both samples, A and B. This occurred because the solution 100 was much more concentrated

than its dilutions. Therefore, this protocol let us start to know the numerous amount of bacteria in our samples, and

gave us the chance to selected specific bacterias of interest in order to begin working with them.

Purifications - R2A Mediums

Sample A Sample B

Mother Medium Mother medium

ATB 30R01A

ADAP 30R01A

ADAP 30R01B

(Fig 1.5)

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The gram stain protocol helps identify whether a bacteria is gram positive or gram negative due to the cell

wall reaction to the dyes. This meant that both, the chemical and physical properties of our bacteria varied with

sample B being coccus/cocci, and sample A being bacillus/bacilli as shown in Figure 1.6. These preliminary

suppositions were established due to their morphology in our mediums. Sample A presented a round to form and

entire to margin. Sample B proved to be round according to margin and convex to elevation, this was determined

after a comprehensive visual analysis to morphology formation in our plates according to Figure 1.5.

Gram Stains

ATB 30R01A

ADAP 30R01A

ADAP 30R01B

(Fig 1.6)The antibiotic production, according to Fig 1.7, demonstrated that our samples did not cause any inhibition

to M. luteus, and E. coli. Since none of our bacteria had the capacity to inhibit the growth of other bacterias like M.

luteus and E. coli, the discs placed in the petri plate developed no resistance.The lack of antibiotic production

against M. luteus and E. coli bacterias, may have occured since their chemical composition and the conditions in

their immediate environment weren't optimal to provide an antibacterial space. According to our hypothesis we

were expecting to have positive results in this tests, which unproved our predictions.

Antibiotic Production

Sample A Sample B

(Fig 1.7)

Polymerase Chain Reaction (PCR) was imperative in our bioinformatic analysis, because it helped us

multiply our DNA results. This cycling process begins with 4 DNA strands as it multiplies itself by 2

progressively, obtaining million of DNA strands over a short lapse of time (Yuan Q-B et. al 2015). Then we did

electrophoresis, a test that helped us determine if there was a proper amplification with the 16S rDNA genes.

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Electrophoresis Samples

Sample A is the last visible. #24

Sample B consists of the first two visible. #1, #2

Purified ( + ) PCR Samples A & B

(Fig 1.8)PCR purification was the last step of our lab work , the main purpose of the PCR purification process was

to take the amplified DNA and purify it to obtain positive electrophoresis product as shown in Figure 1.8. The

bioinformatic process let us confirm that we were effectively dealing with a bacteria and not some other

microorganisms, besides showing us what kind of bacteria we were dealing with.

Characterization ResultsBacteria Designator

CodeSamples Antibiotic

ProductionPCR &

ElectrophoresisBioinformatic

AnalysisATB 30R01A Sample A - + N/A

ADAP 30R01ASample B

- + N/A

ADAP 30R01B - + N/A

(Fig 1.9)

Regarding our hypothesis, we couldn’t prove many of our propositions, since the time factor limited the

analysis of other tests. This led us to list a series of future work, such as antibiotic resistance test, oil and cellulose

degradation in application to bio diesel. Further studies like these will be done for future research investigations,

understanding how these isolated bacteria work was just the beginning, since more concrete experimentation will

be done when sequenced results arrive.

Cited Literature:

Aamodt H, Mohn S, Maselle S, Manji K, Willems R, Jureen R, Langeland N, Blomberg B. 2015. Genetic relatedness and risk factor analysis of ampicillin-resistant and high level gentamicin-resistant enterococci causing bloodstream infections in Tanzanian children. BioMedCentral 15:107 doi.org 10.1186/s12879-015-0845-8/BMC

Åkerman B. 1999. Affinity Gel Electrophoresis of DNA. American Chemistry Society (121) [Internet]. [cited 2015 May 1] 7292-7301. doi.10.1021/ja984154e

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Carlone GM, Valadez MJ, Pickett MJ. 1983. Methods for Distinguishing Gram-Positive from Gram Negative Bacteria. JOURNAL OF CLINICAL MICROBIOLOGY. [1983 dec 2, cited 1982 Aug 2] Vol. 16(6):1157-1159. Available from: http://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC272558&blobtype=pdf

Cerny, G. 1976. Method for the distinction of gram negative from gram-positive bacteria. Eur. J. Appl. Microbiol. 3:223-225.

Feltman R. 2015. New class of antibiotic found in dirt could prove resistant to resistance. New York City(NY):Washington Post; [2015 January 7, cited 2015 March 11] . Available from: http://www.washingtonpost.com/news/speaking-of-science/wp/2015/01/07/new-class-of-antibiotic-found-in-dirt-could-prove-resistant-to-resistance/

Mullis KB, Ferre F, Gibbs RA,. 1994. The Polymerase Chain Reaction [Internet]. First edition. Boston(MA):Birkhauser; [2012 Feb 2, cited 2015 May 1] Available from: https://books.google.com.pr/books?id=gjrTBwAAQBAJ&printsec=frontcover&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false

Walsh F, Duffy B. 2013. The Culturable Soil Antibiotic Resistome: A Community of Multi-Drug Resistant Bacteria. Plos One [Internet]. [2013 April 26, cited 2013 Feb 28 ] 8(6): e65567. doi:10.1371/ journal.pone.0065567.

Yuan Q-B, Guo M-T, Yang J (2015). Fate of Antibiotic Resistant Bacteria and Genes during Wastewater Chlorination: Implication for Antibiotic Resistance Control. Plos One. [ 2015 March 4, cited 2015 April 27] 10(3): e0119403. doi:10.1371/journal.pone.0119403

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