University of Puerto Rico

Cayey Campus

2015 RISE Program

Pérez-Ayala, Michelle C.1, Figueroa-Monsanto, Héctor L.2 , Maricelys3 , and

Cruz, Giovanni4

Department of Biology, University of Puerto Rico at Cayey1, Department of

Chemistry, University of Puerto Rico at Cayey2 , Hugher Hughes Program3, and

RISE Program4

Introduction

Microbiology is the field of science that focuses on the study of the morphology,

application, and relevance of microorganisms, such as bacteria with the use of

microscopes and technical procedures. These microorganisms or specimen,belong to

the Bacteria Domain and Monera Kingdom and are found in moist areas. Among these

places, bacteria can be located on epithelial surfaces, saliva, nail grime, as well as on

top of desks, and even within soils. Actually, these diverse localizations have given

these living organisms specific characteristics that permit them to be either heterotrophic

or non-heterotrophic. Non-heterotrophic or autotrophic microorganisms play an eminent

role in processes, such as nitrogen fixation benefiting plants, but not all bacteria have an

optimistic symbiotic relationship or effect on living organisms.

Heterotrophic organisms are adaptable to many organically formed compositions,

such as humans???, acting positively or negatively. In contrast to the autotrophic

individuals (I don’t think it is appropriate to refer to a bacteria as an individual.),

heterotrophic organisms create negative outcomes on humans. For instance, the

species Treponella palidium causes a skin disease called syphilis. This promotes the

development of sores throughout the body by having contact with bodily fluids through

sexual intercourse. Scientists have focused on not only the nourishment preference of

these microorganisms, but also on their cellular wall composition, which makes them

either gram positive or gram negative.

Gram-negative bacteria are more difficult to inhibit with antibiotics compared to gram-

positive ones, due to the double layer of peptidoglycans in their cell wall. Gram-negative

bacteria can be identified under the compound microscope because they retain pink or

red stains from the conservation of safranin during the wet mount and gram staining

methods. In contrast, if the stains are purple, they are classified as gram positive from

crystal violet solution retention followed by the addition and rinse of both iodine and

ethanol. This type of bacteria is more vulnerable to antibiotics because it has only one

layer of peptidoglycan. Surprisingly, uncultured bacteria (gram positive/negative)

methods of recompilation have permitted researchers to find bacterial compounds, such

as teixobactin with the capacity to impede bacteria from emerging (Ling et al. 2015).

Teixobactin is a compound that has shown to be more effective in terms of bacteria

inhibition, compared with other antibiotics, such as ofloaxin (Ling et al. 2015). In addition,

another type of antimicrobial chemical made of lipopeptide is called battacin. It was

isolated from the soil bacterium Paenibacillus tianmuensis and has shown a potent

inhibition effect. For instance, it has the capacity to eradicate bacteria in vitro or in cell

culture, as well as gram-negative bacteria (Qian et al. 2012). Popowska et al. (2012),

have compared the soil bacteria resistance, instead of contrasting antibiotic ability to

inhibit these microrganisms. Specifically, exposing the bacteria to a soil located

underneath animals that contained specific antibiotics with the purpose of testing their

inhibition effects (This sentence lacks clarity.). In other words, uncultured bacterium can

have a wide range of characteristics that can give it the capacity to create resistance to

certain antibiotics.

Both cultured and uncultured bacteria have been the precursors of many antibiotic

chemical compounds that have inhibited disease-causing bacterium. These same

antimicrobials have created resistance or mutations against the same antibiotics. The

resistance effect may occurs 30 years after being placed on the market provoking or

caused serious health issues. For this reason, there is an urgent need to find bacteria-

generated compounds that will serve as long-term effective antimicrobials.

The purpose of this study is to identify if harvested soil collected bacteria will develop

a compound similar in capacity as the teixobactins. Also, in this study, soil cultured

bacteria collected will be grown in order to identify only one type of bacteria genera with

the purpose of discovering an antibiotic that is able to inhibit the division capacity of the

bacteria. expand this section.

MATERIALS AND METHODS

Soil Collection from Rhizosphere. Samples were collected from two types of

soils rich in nutrients from the rhizosphere. In order to collect both soil samples,

the rhizosphere digging areas were cleared and a small hole was made on each

surface. Both samples were collected with two individual wrapped plastic spoons

until 25% (1/4 parts) of two zip lock bags were filled, respectively. Pictures were

also taken as evidence of the soil collection. The first collection was from a moist

soil located in an urban area at 26˚C at 146˚ SE Caguas, PR 18˚14’25”N 66˚4’7”

W. The area was surrounded by animals such as ducks, and chickens, among

other. A second sample was collected from a rural area approximately 25 ˚C

during the noon at 8:02a.m. in Patillas, PR 17˚53’31”N 65˚53’18” W.

Weight and Dilution of Soil. One gram of the soil samples was weighted and

diluted, in order to obtain a smaller number of bacteria. Each weighted soil was

added to a 10mL saline H2O and NaCl (0.9%) solution containing test tube and

then mixed on a Vortex, respectively. To diminish the concentration inside each

tube and end up with a fewer number of bacteria, 200µL of each concentration

was transferred to an individual 1.5mL centrifuge tube (100) and 20µl of NaCl

were also added. Five different 1.5mL tubes (10-1, 10-2, 10-3, 10-4, 10-5) were filled

with 180µL of NaCl. To complete the dilution, 20µL were transferred from the 100

to the 10-1 and the same process was repeated until 10-5. The TSA and R2A agar

plates were smeared with 30µL of the 100 and 10-5 tubes to grow the bacteria at a

temperature of 30˚C during twenty four hours incubation.

Purification of Bacteria. The medium plates that showed bacterial growth were

purified to eliminate unnecessary debris and to isolate one type of colony,

according to its morphology. In purification #1 to #5, an inoculator loop was used

to swap a group of colonies off the R2A 100 plate onto three areas (North, East,

and South) of a new R2A agar. Using a sterilized loop, the colonies were first

smeared in a zig-zag motion on the North side of the plate. Then, the plate was

closed, the loop was sterilized on the Bunsen burner, the plate cover was

opened, the loop was pressed carefully on a corner of the plate where no

bacteria was present, the bacteria were dragged from the previous section

(North) to the next (East) several times, and at the last movement was zig-zag

motion on the East. The same process was performed for the South. The new

R2A plate was stored inside the incubator at 30˚C and the first R2A 100 plate was

placed in the refrigerator. The same process was performed with the TSA 100

plate, prepared on the previous process using a new TSA plate.

Gram Staining. An inoculator loop was used to swap one colony from a purified

plate and rubbed and mixed with a drop of deionized water onto a microscope

slide. Crystal violet was added followed by water to rinse off the excess purple

dye. The same process was repeated with iodine, ethanol, and saphranin. The

slide was then observed under the microscope to recover an image and

determine the same colony morphology (color, margin, elevation, size, texture,

appearance, pigmentation, and optical property).

Freezing. This process is meant primarily to save a portion of the bacteria for

further use. First, 1.5mL of the broth, equivalent liquid media, and 200µL of

glycerol, which allows the bacteria colony to freeze but not to die at low

temperatures, were deposited in a 2.0mL micro tube. Then the tubes were

labeled for further usage and tests for other parts of investigation.

Electrophoresis. This technique was utilized to characterize the DNA by band

sizes, but in order to have an appreciable amount the DNA must be replicated by

using PCR (polymerized chain reaction). This process seeks to replicate and

amplify the DNA that is to be studied by setting it into different temperatures

specifically: 95˚C which denaturalizes the DNA breaking the chains of DNA, next

a temperature of 60 ˚C for the process of annealing were the base pairing is

taking place and finally extending the DNA chains at about 72 ˚C. After the

replication proceeding to run the PCR adding the replicated DNA with an amount

of dye because the DNA does not exhibit color and wouldn’t be visible in the gel.

The DNA, RNA and proteins at the electrophoresis are measured based on a

sample that runs the entire gel better referred to as the ladder. Once the

electrophoresis is finished, it was read and the length of how far the DNA ran

was determined identifying it by its size or charge.

Antibiotic Resistance Test. Using the corresponding media plate with specific

antibiotics randomly testing upon five of any antibiotics in order to detect if our

bacteria has any resistance against any of those. Striking with the selected

antibiotics and placing a test paper with the bacteria broth for easy identification

of any resistance. It was verified after incubating it at 30 ˚C for a day.

Antibiotic Production Test. The bacteria M.Luteous and E. Coli were each

smeared in a media plate. A test paper was soaked with the bacteria broth and

placed in the plates. The broth is a previously fixed mix with 1.5mL of the

equivalent liquid media and left to stir and maintain an aqueous character. This

test will result in the creation of a ring all around that will mean the bacteria does

produce antibiotic, but if not it means it doesn’t present this particular

characteristic.

Oil Degradation. In order to test the susceptibility of the bacteria, a plate that

has only a media of oil was made. The selected bacterium was streaked into the

oiled plate for testing. The main purpose of this test is to verify if the bacteria will

either feed of the oil surface or repel against it.

An inoculator loop was used to swap one colony from a purified plate and rubbed

and mixed with a drop of deionized water onto a microscope slide. Crystal violet was

added followed by water to rinse off the excess purple dye. The same process was

repeated with iodine, ethanol, and saphranin. The slide was then observed under the

microscope to recover an image and determine the same colony morphology including

(color, margin, elevation, size, texture, appearance, pigmentation, and optical property).

2.5 Freezing

This process was meant primarily to save a portion of the bacteria for further use.

First, 1.5mL of the broth, an equivalentamount of liquid media, and 200µL of glycerol,

which allows the bacteria colony to freeze but not die at low temperatures, were

deposited in a 2.0mL micro tube. Then the tubes were labeled for further usage and

tests for other parts of investigation.

2.6 Electrophoresis DNA was replicated and with the Polymerized Chain Recation (PCR).

Electrophoresis was utilized to characterize the DNA by band sizes The PCR setting

involved different temperatures specifically: 95˚C, which denaturalizes the DNA breaking

the chains of DNA, next a temperature of 60 ˚C for the process of annealing were the

base pairing takes place and finally extending the DNA chains at about 72 ˚C. After the

replication, ethidium bromide was added to the DNA copies to work as the fluorescent

tag. The DNA, RNA, and proteins in the electrophoresis were divided based on size. The

DNA with the tag and the DNA ladder that reflects the base pairs were added the

agarose gel. Once the electrophoresis was finished, the gel was observed to compare

the DNA of the bacteria with the 16S gene that codifies for samples that are bacteria.

2.7 Antibiotic Production Test The bacteria M. luteous and E. coli were tested for antibiotic resistance caused

by mutation by placing each bacterium on agar plates. Four test papers were soaked

with the bacteria broth, previously fixed mix with 1.5mL of the equivalent liquid media

and left to stirred for and maintain an aqueous character. Two papers were placed on

each agar plate with the bacterium.

2.8 Oil Degradation To test if the bacteria would either feed off the oil surface or repel against it, the

bacterium that was positive on the electrophoresis gel was streaked onto a plate of oil

media for an incubation period of seven days at 30 ˚C.

3. Results

3.1 Soil collection from rhizosphere

The first soil collection area is shown in Figure 1. it includes the flora and lake

that surrounds it. The main animals that commonly pass through this area are chickens,

ducks, and dogs. Both this and the parameters included in Table1. show that there is a

probability of finding bacteria in the gathered soil.

Figure 1. a, Shows the tree next to the soil collection area where the animals, including humans tread roughly or calmly. b, The whole at the left of the bag less than a meter away from the tree trunk. c, The whole was about an inch deep surrounded by green grass and it contained roots from the same tree.

The second soil collection area is shown in Figure 2. it, which includes the flora

and shows absence of no water sources around. Only domestic animals such like dogs,

cats, chickens, iguanas and humans pass through by this area. Figure 2 Both this and

the parameters included in Table1. show that there is a probability of finding bacteria.

Figure 2. a, Shows the soil collection area approximately near to many types of trees. b,

The hole was a fairly deep one with an approximate radius of three inches. c, The hole

within had grass roots, dirt pebbles, and small organisms worm like.

Characteristics First soil collection Second soil collection

1. Temperature (˚C) 26˚C 25˚C

2. Coordinates 18˚14’25”N 66˚4’7” W 17˚53’31”N 65˚53’18” W

3. City, State 146˚ SE at Caguas, PR Patillas, PR

4. Soil Description It had a moist texture with visible grass and roots mixed with the soil particles

It was moist during the morning, it lacked roots, and it had little organisms.

5. Surroundings The area was surrounded by animals such as ducks and chickens, among other

The area included avocado cannon trees and a dog

Table 1. These parameters reflect the circumstances of the collection area, as well as its location.

a, b, c,

3.2 Weight and dilution of soilApproximately, 1.033±0.001 grams of the soil sample was weighted and diluted in six

tubes (100, 10-1, 10-2, 10-3, 10-4 and 10-5) to obtain a smaller number of bacteria. Only the

100 tube previously spread in the TSA and R2A agar plates shown in Figure 2.

Demonstrated showed bacteria colonies, after a weekend incubation at about 23˚C.

These bacteria might be either registered in the DNA database or they may ight be a

new genus or species. The 10-5 plate shown in Figure 3. had no bacteria growth and the

plates were discarded. These might have been due to a higher presence of the aqueous

NaCl.

a, b,

Figure 2. a, Shows the TSA 100 colonies with an extensive morphology explained in the Purification bacteria results. b, R2A 100 bacteria colonies were different from the TSA’s.

a, b,

Figure 3. a, Shows that the TSA 10-5 solution had no bacteria. b, R2A 10-5 had the same

effect.

3.3 Purification of bacteria From the first soil collection, The TSA 100 colony morphology is shown in Table

2. and Table 3. shows the morphology for R2A 100 bacteria.

TSA 100 morphology Purr.#1 Purr.#2 Purr.#3

1. Shape Circular Circular Circular

2. Margin Curled Curled Entire

3. Elevation Flat Flat Convex

4. Size Moderate Punctiform, small and moderate

Moderate

5. Texture Smooth Smooth Smooth

6. Appearance Dull Dull Dull

7. Pigmentation Cream Cream-yellow Cream

8. Optical property Opaque Opaque Opaque

Table 2. TSA colony morphology.

R2A 100

morphologyPurr.#1 Purr.#2 Purr.#3 Purr.#4 Purr.#5

1. Shape Circular Circular Circular Circular and irregular

Circular

2. Margin Entire and curled

Curled Undulated and entire

Curled Rhizoid

3. Elevation Flat Flat Raised Flat Flat

4. Size Punctiform and small

Small or punctiform

Punctiform, small, moderate and large

Punctiform, small, moderate and large

Punctiform, small and moderate

5. Texture Smooth Smooth Smooth Smooth Smooth

6. Appearance Dull Dull Dull Dull Dull

7. Pigmentation

White White White White White

8. Optical property

Opaque Opaque Opaque Opaque Opaque

Table 3. R2A colony morphology.

TSA 10-5 and colonies demonstrated different morphology when had a different

purification.Purification #1 for TSA revealed a circular shape, curved margins , flat

elevation, small, smooth texture, dull appearance, cream pigmentation, and an opaque

optical property. The R2A 10-5 bacteria had more defined shape circular, curved with flat

elevation, punctifor, with a smooth texture, dull appearance, white pigmentation, and the

same optical property. For purification #2 the TSA 10-5 plate showed bacteria with

irregular shape, curled margin, flat elevation with sizes that vary from a type of cloth that

covers the whole plate, , smooth texture, dull appearance, a cream-yellow pigmentation,

and an opaque optical property. The R2A colonies showed the same shape, margin, and

elevation. The sizes were small or linear due to the streaking. They , smooth texture, dull

appearance, white pigmentation, and an opaque optical property. The R2A (puur3)

bacteria colonies had a cloch like appearence, an undulated and entire margin, flat

elevation, punctiform at the edges, small, moderate and large sizes, smooth texture, dull

appearance, white pigmentation, and opaque optical property. The R2A (puur.4) showed

irregular shape, curled margin, flat elevation, all sizes, smooth texture, dull appearance,

white pigmentation and opaque optical property. R2A (purr.5) presented linear shape, an

entire margin, flat elevation, punctiform size, smooth texture, dull appearance, cream

pigmentation and opaque optical property.

3.4 Gram staining

From the first soil collection, both the TSA and R2A colonies showed purple

pigmentations. The colonies were Streptobacillus gram negative, according to Figure 4.

a, b,

c,

d,

Figure 4. a,b, Shows that the R2A 100 colonies are gram negative and Streptobacillus.

c,d, sShows the Streptobacillus gram negative bacteria for TSA 100.

From the second soil sample according to Figure 5. when doing Gram staining the

TSA Streptococcus colored in purple therefore tested to be gram positive. Also the R2A

was proven to be Streptobacillus pink therefore tested to be gram negative.

Figure 5.

3.5 Freezing

From the first soil sample two tubes Figure 6. each with 1.5 mL of broth, 0.2 mL of

glycerol and the R2A colonies.

a, b,

c, d,

Figure 6. a, Freezing of TSA bacteria. b, TSA back up. c, R2A frozen bacteria. d, R2A back up.

3.6 Electrophoresis

The R2A copies of the PCR were positive for the 16S gene, according to the

electrophoresis gel in Figure 7. This shows that the DNA copies belonged to bacteria.

Figure 7. a, The R2A DNA in line 4, without counting the DNA ladder, was positive to the

16S gene.

3.7 Antibiotic production test

M. Luteous was inhibited only by TSA 100, according to Figure 6 a,b,. E. coli on Figure

8. c,d, was not inhibited neither by TSA nor by R2A. This shows that TSA bacteria might

have an antibiotic property on M. luteous.

a, b,

c, d,

Figure 8. a, b, Show that TSA bacteria had an antibiotic resistance on M. luteus, but R2A did not. c, d, E. coli was had no inhibition.

3.8 Oil degradation

The oil degradation test for the bacteria was negative, according to Figure 9. This

shows that the bacteria will not be useful to degrade oil in the development of oil-derived

products.

Figure 9. The plate was from another oil test. It shows the same negative result that displayed both soil collection bacteria.

4. Cited Literature

Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A,

Sch¨aberle TF, Hughes DE, Epstein S et al. 2015. A new antibiotic kills pathogens

without detectable resistance. Nature 517, 455–459. doi: 10.1038/nature14098.

Popowska M, Rzeczycka M, Miernik A, Krawczyk-Balska A, Walsh F, Duffy B. 2012.

Influence of soil use on prevalence of tetracycline, streptomycin, and erythromycin

resistance and associated resistance genes. Antimicrobial Agents and Chemotherapy

53 (3): 1434-43. doi: 10.1128/AAC.05766-11.

Qian CD, Wu XC, Teng Y, Zhao WP, Li O, Fang SG, Huang ZH, Gao HC. 2012. Battacin

(octapeptin b5), a new cyclic lipopeptide antibiotic from Paenibacillus tianmuensis

active against multidrug-resistant gram-negative bacteria. Antimicrobial Agents and

Chemotherapy 56(3): 1458-1465. doi: 10.1128/AAC.05580-11.