Isolation and Characterization of Bacteria from Tropical SoilsNicole Rivera- Espinal1, Alejandra Ferrer-Díaz1

1Department of Biology, University of Puerto Rico at Cayey. Both authors contributed equally to this project.

Abstract

The purpose of this experiment is to encounter bacteria from tropical soils that have the capability of producing antibiotic. In order to do so, it was necessary to isolate the bacteria, purify it five times and freeze it at a temperature of -80˚C. After purification, gram staining was performed to classify the bacteria as gram positive or negative. The bacteria were characterized by doing purification of genomic DNA and then, a Polymerase Chain Reaction (PCR) with the primers 16sRNA, 16S_1510R and 16S_8F. An electrophoresis was conducted to see if the PCR process went well. The PCR product was purified and analyzed by using Bioinformatics. Finally, both bacteria’s S15UPRCRISENRE30M01 and S15UPRCRISEAFD30M01A had positive results as antibiotic producers for M.luteus.

Introduction:

In the beginning of Earth history, the first thing considered as organisms were bacteria. Even though the first ones found were photosynthetic they functioned as a fundamental base to the understanding of life and evolution (Evert and Eichhorn 2013). Bacteria are prokaryotic microorganisms that can be found practically everywhere, for example, in the soil, water and even in the human body. These can have different types of morphology, and they are classified as: cocci, bacillus, spirillum and spirochetes. Also, bacteria can be differentiated by their peptidoglycan layer located in the cell wall that classifies them as gram-positive or gram-negative. The gram-positive ones are characterized by the thickness of their wall. On the contrary, gram-negative bacteria possess a thinner wall (Beveridge 2001). Due to the properties of these microorganisms, scientists were currently

searching for beneficial ways in which bacteria can contribute to human welfare.

The discovery of penicillin in 1928 by Alexander Fleming indicated that a new transcendental and significant era was about to start (ACS 2015). 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). For example, Rene Dubos of the Rockefeller Institute for Medical Research, in the late 1930’s, discovered and isolated an antibacterial compound—tyrothricin, from the soil microbe Bacillus brevis—capable of destroying Gram-positive bacteria. This discovery revived the stalled interest in penicillin and launched the era of antibiotics (Van Epps 2006). All these contributions have led to a substantial control of powerful diseases that plays an adverse role in human

health. Despite all the important facts discovered about antibiotics, it is essential to know that right now these types of medications are playing an ineffective role on bacteria due to bacterial antibiotics resistance.

Antibiotic resistance is produced when the capability of killing or inhibiting bacterial growth is vanished; in other words, the bacteria are strong enough in comparison to the antibiotic and, as a consequence, they continue to multiply in the presence of therapeutic levels of an antibiotic (APUA 2014). Antibiotic resistance is a natural incident. When an antibiotic is used, bacteria that can confront that antibiotic have a higher chance of survival than those that are "susceptible." Although this effect is natural, the accelerated and worldwide spread of antimicrobial-resistant organisms in recent years has been unparalleled (Vasoo et al. 2014). As a result, the scientific community has been looking for an effective alternative that can counteract bacterial resistance in order to control infectious diseases and its spread. Recent studies have indicated that it is possible to diminish bacterial resistance by manipulating the environment in which the bacteria has been cultured. For example, scientists discovered a new antibiotic which named Teixobactin that can disrupt peptidoglycans located in the cell wall of gram-positive bacteria’s (Ling et al. 2015). The importance of this experiment is the way by which the scientists discovered the antibiotic. They used an apparatus named ichip whose function is to isolate uncultured bacteria by in vivo techniques. This discovery was a major breakthrough because it is virtually certain to be effective for the

multiresistant strains that are now all but impossible to treat (The scientist 2015).

Since their discovery, bacteria have had an important economic and bio sustainable role. These microorganisms are used in some industries for the production of tobacco, tanning leather, hides, cheese, yogurt, buttermilk, vinegar, and sauerkraut. Also, they are used in the development of antibiotics. Given that bacteria are evolving and creating resistance to many common antibiotics used today, people are suffering strongly from infectious diseases. In this study, the main purpose is to find different types of soil bacteria and characterize them, in order to see if they are capable of producing antibiotics that battle the new strains of infections that are affecting us. Studies have showed that tropical soils are more likely to contain major nutrient availability than other types of soils (Xenos 2014). Due to that fact, we have formulated the following problem: Is it possible to find bacteria with antibiotic properties in the soil of Puerto Rico? Our hypothesis is: Bacteria from tropical soils are capable of developing properties such as antibiotic production. Our hypothesis is sustained by several studies such as the one developed by Dubinsky et al.2010. This investigation attempts to prove how soils in Puerto Rico can provide abundance and activity of microbes varied across the landscape. The significance of this study is that it is determined to encounter bacteria with antibacterial properties, in order to counteract antibiotic resistance. Finally, this investigation attempts to respond an antibiotic worldwide concern by using in vitro techniques.

Materials and Methods

Isolation and purification of the Bacteria:

Soil samples were collected from the tropical soils of Puerto Rico in order to obtain a range of bacteria. Important data of the soil and the place were noted such as temperature, soil composition, texture, and living organisms, percent of humidity, and GPS coordinates from the place. Brief description of the surroundings of the soil was included. After collecting a significant amount of soil, just one gram diluted. The soil was added to a test tube with 8.5 mL of sterile H2O and 90µL of NaCl. The pH of the sample was measured with a strip that has a qualitative and quantitative range that specifies the acidity of the soil. Ninety microliters of Sodium Chloride were added to six micro tubes that were already labeled with numbers 0 to -5. Ten microliters of the soil mix was added to the micro tube marked as zero. In order to finish the dilution process, 30µL of the zero micro tube were collected and poured into the -1 micro tube; this step was followed in a chain action until the -5 micro tubes. Different medium plates were selected to cultivate bacteria from the 0 and -5 micro tubes; these were Rhizobium Medium (RDM) and ISP4. A drop of 30µL was added to the center of the medium plates and spread all over it in the form of a cross. The plates were stored in the incubator at 37˚C.

To obtain one specific colony of bacteria, it is necessary to perform the purification process that consists of the recollection of isolated bacteria and their cultivation in the mediums that facilitated

their reproduction. This process is repeated at least three times. The streak plates with the bacteria were saved in the incubator at 37˚C for a period of 24 hours. Observational data at the macro level, a plate of bacterial culture was taken and observed under the dissecting light microscope to analyze its morphology. After bacteria were completely purified, a cryogenic freezing was completed to store them in the fridge at minimum temperatures.

Bacteria Characterization:

In order to assure that the isolated bacteria were not contaminated and additionally to comprehend the bacteria, the gram staining technique was performed. In the Gram stain, crystal violet, Iodine, alcohol and safranin were used for bacterial classification.Bacteria were tested with E. coli and M. luteus to see if they could serve as an antibiotic agent. E. coli and M. luteus were grown in dish plates that were already divided into two portions, to analyze the antibiotic properties of the follow bacteria S15UPRCRISENRE30M01, S15UPRCRISENRE30P01, S15UPRCRISEAFD30M01A, and S15UPRCRISEAFD30M01B. Then, two paper disks were immersed in the micro tube that contained the supernatant of the bacteria found in tropical soils. After that, the paper disks were placed into each of the portions of the dish plates. This process was performed in a period of 24 hours to see if the bacteria inhibited E.coli and M.luteus.

Isolation of DNA:

For a large concentration of isolated bacteria, it is necessary to complete a bacterial enrichment. This step consists in the mixture of 4.0 ml of RDM broth for the bacteria that are cultured in rhizobium medium and TSB broth for the bacteria that grew in the ISP4 medium. This mixture is saved in a semi-opened test tube for 24 hours in a shaker incubator at a temperature of 37°.

The Polymerase Chain Reaction (PCR) is a process commonly used in Genetics and Molecular Biology that consists of amplifying specific DNA and producing several copies of it. Before performing this process, it is required to purify the genomic DNA. In order to do so, 300µL of the mixture, that was previously prepared, were transferred to a micro centrifuge tube. Then, the sample was heated at 100˚C for 10 minutes followed by an ice bath for another 10 minutes. To separate the DNA supernatant from the pellet, the micro centrifuge tubes were placed in the centrifuge at 10K RPM for 10 minutes, Then, 150µl of the DNA supernatant was added to a micro tube. Only 6µl of the DNA supernatant was transferred to a PCR tube. Twenty five microliters of master mix, 2µl of forward primer and reverse primer and 15µl of nuclease free water were also added to the PCR tube. The PCR tubes were put in a thermal cycler machine in order to start the PCR process. The first step was denaturalization at 95˚C in order to break the DNA double helix. The second step was annealing at 48˚C that consists of primer attaching. The last one was extension at 72˚C. The first and third step took one minute; the second one is 30 seconds. To test the samples of the PCR

process, the agarose electrophoresis was run. Once the PCR process yielded positive results, DNA purification process was carried out by using a “QIAquick PCR Purification Kit”.

Based on the amount of PCR product used the volume of buffer needed was calculated. To start this protocol, the calculated amount of Buffer PB was added to 1 volume of the PCR reaction; this concoction was mixed in the centrifuge until it turned yellow. It was then centrifuged to allow for the separation of the products by passing the samples through the column. The flow-through was discarded and the column was placed in the same tube. Next, 750µl of Buffer PE was added to wash the QIAquick column. After that, QIAquick was centrifuged one more time to remove the residual wash buffer. In order to elute the DNA, pure H2O (pH 7.0-8.5) was added to the center of the QIAquick membrane and it was centrifuged for 1 minute. Because the purified DNA was going to be analyzed on a gel, 1 volume of Loading Dye was added to 5 volumes of purified DNA.

Results:

A. Gram stainingUpon the completion of this process

it was noticed that all four of the bacteria stained purple, indicating they were gram positive. Likewise, it was noted that two of the bacteria namely, S15UPRCRISENRE30M01(figure 3) and S15UPRCRISENRE30P01(figure 4), were shaped in the form of bacillus. Moreover, S15UPRCRISEAFD30M01A (figure 2) had the form of a cocci bacteria while

S15UPRCRISEAFD30M01B (figure 1) was shaped in the form of a streptobacillus.

Fig 1. Gram stain of bacteria S15UPRCRISEAFD30M01B

Fig 2. Gram stain of S15UPRCRISEAFD30M01A

Fig 3. Gram stain of bacteria: S15UPRCRISENRE30M01

Fig 4. Gram stain of bacteria: S15UPRCRISENRE30P01

Antibiotic producer with M. luteus and E. Coli:

As shown in figure 5 Bacteria S15UPRCRISENRE30M01 had a positive result as an antibiotic producer with M. luteus. A circular edge was underlying the disk plate, which means that the bacteria inhibited the reproduction of M. luteus. The

width of the circular edge observed was broad, but that does not determine the antibiotic strength of S15UPRCRISENRE30M01. This bacterium had negative results when it was tested with E. coli because no edge was observed around the disk plate.

Both bacteria S15UPRCRISEAFD30M01B and S15UPRCRISENRE30P01 presented negative results with bacteria E.coli and M.luteus. As visible in Fig. 5, bacteria S15UPRCRISEAFD30M01A presented an edge around the disk plate giving a positive result as an antibiotic producer for M. Luteus. When bacteria S15UPRCRISEAFD30M01A was tested with E.coli, inhibition was not observed signifying it is not an antibiotic producer for this bacterium. This information is summarized in table 3.

Fig 5. Positive result for bacteriaS15UPRCRISENRE30M01

Fig 6. Minimal positive result for bacteria S15UPRCRISEAFD30M01A

PCR product:

After completing the PCR process for the first time with the primer 16sRNA, the results were negative. As shown in figure 8, it was not possible to appreciate the DNA amplification bands in comparison to the KB ladder rail. After repeating the procedure with alternative primers, the DNA amplification appeared positive in bacteria S15UPRCRISENRE30P01 and S15UPRCRISEAFD30M01A. Conversely, it was not possible to observe the DNA amplification bands in bacteria S15UPRCRISEAFD30 and S15UPRCRISENRE30M01M01B, The results from these bacteria were considered negative. These results are shown in table 3.

Fig 7. Rail #1 and #4 represents theamplification of bacteria’s S15UPRCRISENRE30P01 and S15UPRCRISEAFD30M01A, respectively.

Fig 8. Negative results for bacterial DNAamplification with primer 16sRNA

Table 1: Soil collection

Date of Collection

Site of Collection Air temperature

Moisture Coordinates pH of soil

January 28, 2015

Aguas Buenas Caverns

82˚F Moist 18° 25' 86" N66° 01' 32'' W

5.5

February 1, 2015

Aguas Buenas Caverns

82˚F Moist 18° 25' 86" N66° 01' 32'' W

5.5

February , 2015 Aibonito shooting club

75° F Moist Latitude: 18° 8’ 38.6” NLongitude: 66° 14’ 45.8” W

6.0

February 1, 2015

Aibonito shooting club

75° F Moist Latitude: 18° 8’ 38.6” NLongitude: 66° 14’ 45.8” W

6.0

Table 2: Bacteria Isolated

Bacterial Designator Soil Number of purificatio

ns

Form Surface

Elevation

Color

S15UPRCRISEAFD30M01A

“dots”

Aguas Buenas 5   Circular Smooth

Raised White

S15UPRCRISEAFD30 M01B

Aguas Buenas 5 lines with projectio

ns

Rough Flat White

S15UPRCRISENRE30M01

 Aibonito shooting club

5 Punctiform

Rough Flat Cream with a cover of

dense white

S15UPRCRISENRE30P01

Aibonito shooting club

5 Circular Smooth

Raised Cream and yellowish

Table 3: Results to multiple testes

Bacterial Designator Gram Staining Antibiotic Resistance

Antibiotic Production

PCR Product

S15UPRCRISEAFD30M01A Cocci, Gram Positive Not determined Positive, M.luteus

Positive

S15UPRCRISEAFD30 M01B

Streptobacillus, Gram Positive

Not determined Negative Negative

S15UPRCRISENRE30M01 Bacillus, Gram Positive

Not determined Positive, M.luteus

First time: negative

Positive

S15UPRCRISENRE30P01 Bacillus, Gram Positive

Not determined Negative Negative2nd time: negative

Discussion:

Due to the results obtained during the completion of this research it is concluded that

the hypothesis established was correct. The hypothesis was proven because the bacteria obtained from tropical soils had antibacterial properties. From the four bacteria encountered

from different environments, two developed antibacterial properties. These two represent 50% of the bacteria examined. It is important to emphasize that the bacteria that did not developed antibiotic properties, for either M.luteus or E.coli, are not discarded from developing these properties with another bacteria. Also, 50% of the bacteria had positive results on the PCR product which means that bacteria from tropical soils can be characterized and tested in Bioinformatics.

For further studies, bacteria are going to be tested with cellulose to determine if they have the potential to break it down into monomers and ingest it. Also, an endospore stain can be performed to observe if bacteria exhibit a prolonged dormancy stage. This is relevant to the study because it implies that the bacteria encountered can live and reproduce in a hostile environment.

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Acknowledgments

RISE ProgramUniversity of Puerto Rico at Cayey

Dr. Michael RubinDepartment of Biology, University of Puerto Rico at Cayey

Lizbeth PérezRISE Student, University of Puerto Rico at Cayey

Mr. Giovanni CruzRISE lab technician

Dr. Eneida DíazDepartment of Biology, University of Puerto Rico at Cayey

Dr. Elena GonzálezDepartment of English, University of Puerto Rico at Cayey