Isolation and Characterization of Bacteria from Tropical Soils

Jessica Ortiz1 and Carolina Huertas2 1University of Puerto Rico at Cayey, RISE Program, Department of Chemistry 2 University of Puerto Rico at Cayey, RISE Program, Department of and Biology

ABSTRACT

Bacteria had been used to improve our lives in many ways, for example, in healthcare, industry, agriculture, and fuel production. This study aimed to isolate and characterize bacteria from Puerto Rican soil in order to find useful properties; specifically antibiotic resistance and production. Five bacteria were isolated from soils of rural areas of Caguas, Santa Isabel, and Comerío. The bacteria were submitted to Gram staining and antibiotic production and resistance tests. The results of the study showed that all the bacteria isolated were bacillus gram positive, which shows a higher tendency of this type of bacteria in rural areas. Of the five bacteria isolated, two (40%) showed antibiotic resistance against tetracycline, two showed antibiotic production against E.coli, one showed coexistence with E.coli and one showed coexistence with M.luteus. An analysis of the data indicated that these results could be caused by one of the following: bacterial mutation, production of pigments or presence of endospores. However, further experimentation is needed in order to find the specific cause. We believe that the information recorded of the isolated bacteria is highly useful for future investigations based on bacteria from Puerto Rican soils.

INTRODUCTION

One of the most basic and important components of an ecosystem are bacteria. These prokaryotic microorganisms are essential for processes like: obtaining nutrients, geochemical cycles, fermentation processes, etc. Additionally, bacteria can also play harmful roles; such as causing disease. The specific properties and characteristics of each bacterium is what determines its role in the ecosystem. Some bacteria are capable of producing antibiotics, which are chemical substances that inhibit the growth of bacteria. These chemicals are produced by soil bacterium and fungi in order to kill other organisms competing with them for limited resources in their habitat. Penicillin was the first antibiotic introduced to medicine in 1940s. This discovery led to the cure of bacterial infections that were once deadly and began the era of antibiotics

(ACS). Most antibiotics introduced to medicine after the

discovery of penicillin were found by screening soil microorganisms. However, new methods for producing antibiotics are being considered due to the fact that antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice (Ling et al. 2015).

The development of bacterial resistance to antibiotics has become an issue of public health. These circumstances have forced scientists to study the action mechanisms for antibiotic production and resistance. However, these mechanisms have been studied almost exclusively to pathogenic bacteria. It is only in recent years that antibiotic resistance research has focused on the environment, where the

antibiotics were initially extracted: soil microorganisms and their ecosystem. Studies of antibiotic biosynthetic pathways and genome analysis show that antibiotic producing soil bacteria develop resistance proteins or modified cell structures in order to protect themselves from their own chemical defense.

The co-existence of antibiotic production and resistance in this type of bacteria avoids its self- destruction (Walch and Duffy 2013). Other types or resistance seen in antibiotic producing and non-producing bacteria are associated to the physiological characteristics of the bacteria or chromosomal mutation. Bacteria can also develop anthropogenic resistance due to antibiotic residues, detergents, heavy metals, wastewater-treatment plant effluent, agricultural pollution and slurry application (Amos et al. 2015).

All these factors cause resistance development of bacteria in their habitat to increase exponentially. In a recent investigation, Walsh and colleges (2003) isolated 412 antibiotic resistant bacteria from agricultural, urban, and pristine soils. All isolates were multi-drug resistant, of which greater than 80% were resistant to 16–23 antibiotics, comprising almost all classes of antibiotic. It was concluded that there was a high level of multi-drug resistance in soil bacteria to a wide variety of antibiotics, and it was not dependent on the soil use. They point to the importance of soil as a potential reservoir of antibiotic resistance mechanisms requiring thorough investigation.

This research will focus on isolating and characterizing of bacteria collected from tropical soils of Puerto Rico. It is hypothesized that different properties such as bacterial structure, antibiotic resistance and production will be identified. In order to identify the structure of the isolated bacteria

as gram negative or gram positive these will be submitted to gram staining. The antibiotic resistance will be tested by exposing the bacteria to different types of antibiotics and observing bacterial growth. These tests will identify the levels of resistant bacteria in both antibiotic producers and non-antibiotic producers. If the bacteria show resistance to antibiotics this could implicate that the soil from where it was taken promotes natural or anthropogenic resistance. It could also mean that the mechanism of action of the antibiotic used does not affect the growth of the bacteria. The antibiotic production of the bacteria will be tested by exposing them to M.luteus and E.coli and observing bacterial growth. If the bacteria produce antibiotics against these other bacteria it would imply that the DNA of the bacteria codifies a substance that kills or inhibit the growth of that specific organism. After isolating and purifying the bacteria, its 16S rRNA gene will be sequenced in order to determine if the bacteria has been previously identified. This gene is present in almost all bacteria and it is relatively short (1,542 nucleotide bases), which makes it ideal biomarker. In case that the bacteria has not been previously identified it will be named and characterized according to the results of the investigation.

MATERIALS AND METHODS

Soil collection from the RhizosphereSoil was collected from Caguas, Comerío, and Santa Isabel using individually wrapped plastic spoons, gloves, and zip lock bags. The temperature, coordinates, and approximate moisture content of the soil were recorded in each area where each soil sample was collected. Cultivation of Soil Microorganisms: First EnrichmentNote: aseptic technique was used at all times. Gloves were avoided when working

with a Bunsen burner. The next process was used with each soil sample. One gram of the soil sample collected was diluted with 10 mL of sterile distilled water. Dilutions from 100 to and 10-5 were made. Afterwards, 50 μL of each dilution was poured in two plates of different media and spread completely using the spread plate technique. The two media plates used were RDM and ISP4. The plates were stored in an incubator at 30°C for 24-48 hours. Cultivation of Soil Microorganisms: Second EnrichmentThe plates were examined for bacterial growth. Bacteria from the dilutions were chosen and transferred to another plate of the same media using the streak plate technique. The plates were stored in an incubator at 30°C for 24-48 hours.Purification of Microbial IsolatesThe bacteria chosen from the second enrichment were purified by transferring a colony to another plate of the same medium using the streak plate technique. After the incubation time, the new plates were inspected to make sure that the colonies had a uniform morphology. This process was done at least three times to make sure there was only one colony of bacteria in each plate. The Gram stain technique (Cerny 1976) was used to determine the structure of the bacteria. Endospore stain technique was performed with one type of bacteria (Schaeffer and Fulton 1933). Cryogenic freezing The plate with the previously purified isolated bacteria was filled with 1.5 mL of its respective liquid medium to create a cell suspension. This was transferred to a 13 mm sterile test tube that contained 0.5 mL of sterile 80% glycerol. The procedure was repeated twice with each bacteria in order to have both a working and a storage tube. Each cryogenic tube was placed in a box designated for storage at -80°C. Genomic DNA Isolation

The isolated bacteria was cultured in the liquid medium. A total of 500 μL of bacteria cultured was added to a boil-proof microcentrifuge tube. The tube was put for 10 minutes in a bath of hot water (100°C), transferred to a bucket of ice (0°C) for ten minutes, and put in the centrifuge for 10 minutes at 14000 rpm. One hundred and fifty μL of the supernatant were transferred to another microtube to be used for the PCR. PCR of 16S rDNASix μL of DNA, 25 μL of PCR master mix, 2 μL of forward primer, 2 μL of reverse primer, and 15 μL of nuclease free water were added to a PCR tube. This procedure was tried with two different forward and reverse primers. The tubes were put in the PCR machine (Thermocycler). When finished, the PCR tubes were stored at -20°C for the next procedure. Gel Electrophoresis of Amplicons An electrophoresis chamber with the agarose gel and the 1x TAE buffer was set. Two μL of loading dye were added to 7 μL of the DNA prepared in the PCR tubes from the previous procedure. The first well was filled with 2.5 μL of 1 kb DNA ladder, and the bacterial DNA with dye was placed in the subsequent wells. After the electrophoresis is completed, the gel is seen with a UV gel-imaging system. If positive, the PCR sample is used for the next procedure. If not, the procedure is repeated. Purification of PCR productsThe QIAquick PCR purification kit from Qiagen was used for this procedure. The kit’s instructions were followed (Qiagen, 2015). To confirm a successful recovery of the 16S rDNA, a gel electrophoresis was run again with the procedure used in Gel Electrophoresis of Amplicons. DNA Sequencing of 16S rDNA AmpliconsOne μL of the 519R Sequencing Primer are added to 10 μL of the purified PCR products and submitted for sequencing.Testing for Antibiotic Production

M. luteus and E. coli cultures were prepared previously in Luria broth (LB). A sterile swab is placed inside the M. luteus culture and rubbed gently over the agar of a plate. A paper disc is placed inside the bacteria culture. The same procedure is done with E.coli. The plates are incubated for 24-48 hours. If the isolated bacteria produces antibiotic there will be no growth near the paper disc.

Testing for Antibiotic ResistanceA sterile cotton swab is dipped into the liquid culture of the isolated bacteria, and rubbed over the agar. After letting it dry for 15 minutes, antibiotic discs are placed in the plate. The plates are incubated for 24-48 hours. The nearer the bacteria grew around the antibiotic disc, the stronger the resistance.

RESULTS

A total of five soil bacteria were successfully isolated and characterized. As seen in table 1 all the soil samples were collected from rural areas with low to moderate temperatures and slightly acidic soil. The areas of recollection included Caguas, Santa Isabel and Comerío. Distant areas were chosen in order to have a better geographical analysis of the data.

Table 1: Soil Collection

Date of Collection

Site of Collection

Air temperature

Moisture Coordinates pH of soil

January 28, 2015

Plantain Farm in Caguas

82˚F Dry 18˚12’33.6”N66˚01’53.1”W

7.56

February 1, 2015

Millennium Genetics Corp. in

Santa Isabel

86˚F Moist 17˚57’52.1”N66˚23’23.7”W

6.50

February 1, 2015

Comerío 77˚F Moist 18˚14’46”N66˚11’50”W 6.50

Bacteria: S15UPRCRISEJCOR30P01A(JCOR30P01A)

The first bacteria was isolated from a plantain farm in Caguas. This bacteria was characterized by having an irregular form and an ivory color (Figure 1). The surface of the bacteria was flat and smooth. Figure 2 presents the result from the gram staining, in which the bacteria was identified as a gram positive bacillus. Figure 3 shows the result from an endospore staining. The green dots seen in the picture are endospores. Endospores are usually developed as a

response to nutrient deprivation. It allows the bacterium to produce a dormant and highly resistant cell to preserve the cell's genetic material in times of extreme stress.  The extraordinary resistance properties of endospores make them of particular importance because they are not readily killed by many antimicrobial treatments (Recee et al. 2013).

Figure 1: Morphology of JCOR30P01A

Figure 2: Gram Staining of JCOR30P01A

Figure 3: Endospores Staining of JCOR30P01A

The results of the test of antibiotic resistance for this bacteria show how the bacteria has the ability of creating multiple circles of resistance against tetracycline (Figure 4). These circles are very uncommon in the scientific community and could not be explained by previous investigations. A liquid culture of the clusters of bacteria closer to the antibiotic was made and the test

was repeated with this culture. The results show how the diameter of the clearing around tetracycline (Te30) decreases significantly (Figure 5). Based on the results, it is hypothesized that the bacteria closer to the antibiotic developed some type of resistance associated with bacterial mutation or the presence of endospores. Further experimentation is needed in order to test this hypothesis. The test for the production of antibiotics showed that the bacteria did not produce antibiotics against E.coli and M.luteus. However, as shown in figure 6, the E.coli did not stop the growing of the bacteria, meaning that they can coexist.

Figure 4: Multiple rings of resistance against Te30

Figure 5: Resistance against TE30 with closer clusters of bacteria

Figure 6: Coexistence of E.coli and JCOR30P01A

Bacteria: S15UPRCRISEJCOR30P02A(JCOR30P02A)

This bacteria was isolated from Millennium Genetics Corp. in Santa Isabel. It was one of the most interesting bacteria because it grew as a white spongy bacterium at first, but with time its surface turned red. Figure 7 shows the pigment gained by the bacteria after increasing the incubation period. In bacteria, pigment formation is associated with morphological characteristics, cellular activities, pathogenesis, protection and survival (Reece et al. 2013). The antibiotic resistance test showed clusters of bacteria

that were able to grow very close to tetracycline. A liquid broth is prepared with these bacteria and the test is repeated. The results showed a significant decrease in the clearing zone around the antibiotic (Figure 8). It is concluded that those clusters were able to develop some type of resistance against the antibiotic. This resistance can be a result of mutation or the pigmentation produced by the bacteria. As in the previous case, further experimentation is needed in order to test this hypothesis. The gram staining shows that the bacteria is a gram positive bacillus (Figure 9).

The bacteria did not produce antibiotics against M.luteus and E.coli.

Figure 7: Morphology of JCOR30P02A after 1 and 3 days in the incubator

Figure 8: Resistance of bacterial clusters to Te30

Figure 9: Gram Staining of JCOR30P02A

Bacteria:

S15UPRCRISEJCOR30P02B(JCOR30P02B)

This bacteria was also isolated from Milenium Genetics Corp. in Santa Isabel. As seen in figure 10 the bacteria is punctiform and has an orange color. The results of the gram staining showed that the bacteria was a gram negative bacillus (Figure 11). This bacteria showed multiple circles of inhibition against tetracycline similar to the ones showed by bacteria JCOR30P01A (Figure 12). In this case, the test could not be repeated with the bacterial clusters closer to the antibiotics due to lack of materials. However, it is inferred that the results should be similar to the ones shown in the first bacteria. The bacteria showed a coexistence with M.luteus, but no antibiotic production against it (Figure 13). The multiple circles of resistance and the coexistence with M.luteus might be a direct result of the production of pigment or mutation.

Figure 10: Morphology of JCOR30P02B

Figure 11: Circles of resistance against Te30

Figure 12: Gram Staining of JCOR30P02B

Bacteria:

S15UPRCRISECHA30P01 (CHA30P01)

The bacteria was isolated from soil located in Comerío. As seen in Figure 13 the bacteria has light pink pigmentation, circular form, a smooth surface, and it is elevated.

The Gram-Staining test (Figure 14) showed that the bacteria are bacillus and gram positive. This bacterium, when tested, resulted positive for antibiotic production with E. coli and negative with M.luteus. This result lets us assume that the antibiotic production is generated in the presence of gram-negative bacteria (E. coli), which contains a thin layer of peptidoglycan that conforms the cell wall, making it easier for the antibiotic to enter the cell wall and inhibit the formation of E. coli. On the contrary, gram-positive bacteria like CHA30P01 does not have an antibiotic effect. The reasoning for this could be that because gram-positive bacteria has various layers of peptidoglycan making the cell wall thicker (Beveridge 1999), any antibiotic produced will not be able to enter the cell wall of M. luteus.

Figure 13: Morphology of CHA30P01

Figure 14: Gram Staining of CHA30P01

Figure 15: Antibiotic Production of CHA30P01 with E.coli.

Bacteria:

S15UPRCRISECHA30M01 (CHA30M01)

This bacteria was also collected from soil located in Comerío. It does not have an exact form, yet tends to be flat, smooth, and colorless (Figure 16). The Gram Staining test, Figure 17, confirmed that the bacteria is bacillus and gram positive. When testing for antibiotic production, it was positive for E. Coli and negative with M. luteus. This result lets us assume that the antibiotic production of CHA30M01 has an effect in presence of gram negative bacteria (E. coli), but does not have an effect if the bacteria is gram positive. The reasoning behind this could be

the layer of peptidoglycan conforming the cell wall: for gram positive bacteria it is thick, and for gram negative bacteria it is thin (Beveridge 1999). For the gram negative bacteria, the antibiotic produced could easily enter the cell and affect it; on the other hand, the antibiotic produced cannot enter the thick cell wall of gram positive bacteria and therefore not have an effect on it.

Figure 16: Morphology of CHA30M01

Figure 17: Gram Staining of CHA30M01

Figure 19: Antibiotic Production of CHA30M01 with E.coli.

DISCUSSION

The major findings of this study were the multiple clearing circles of antibiotic resistance produced by some bacteria and the antibiotic production against E.coli. We were able to prove our hypothesis by identifying the different properties of all the bacteria, such as bacterial structure, antibiotic resistance and antibiotic production. All the bacteria isolated were gram positive bacillus, which shows a higher tendency of this type of bacteria in rural areas. Table 2 shows how the physical properties of the bacteria varied according to region.

Table 2: Morphology of Bacteria

Bacterial Designator Soil Form Surface Elevation Color Gram Staining

S15UPRCRISEJCOR30P01A Caguas Irregular Smooth Flat Ivory Bacillus, Gram Positive

S15UPRCRISEJCOR30P02A Santa Isabel

Circular Spongy Convex Top: redBack: white

Bacillus, Gram Positive

S15UPRCRISEJCOR30M02B Santa Isabel

Punctiform Smooth Flat Orange Bacillus, Gram Positive

S15UPRCRISECHA30P01A Comerío Circular Smooth Convex Pink Bacillus, Gram Positive

S15UPRCRISECHA30M01B Comerío Irregular Smooth Flat White Bacillus, Gram Positive

Of the physical properties of the bacteria, the production of pigmentation was associated with antibiotic resistance and production since from the three bacteria that produced pigments one showed antibiotic production against E.coli (CHA30P01A), one showed coexistence with M.luteus (JCOR30P02B) and two showed resistance against tetracycline (JCOR30P01A and

JCOR30P02A). As seen in table 3, 40% of the isolated bacteria showed resistance against tetracycline. An investigation performed in 2015 by Kyselkova and colleagues concluded that fecal pollution increases the abundance of tetracycline resistance genes in soil. Another investigation showed that tetracycline resistance bacteria is more abundant in polluted environments (Harnisz et al. 2015) Considering that the bacteria that showed resistance to Te30 were isolated from farm soil, there is a possibility that these had experienced fecal pollution or other types of pollution caused by fertilizers. All the bacteria that showed resistance against tetracycline were bacillus. This result correlates with an investigation performed in China where Bacillus was the most dominant bacterial genus in tetracycline-resistant bacteria (Gao et al. 2012).

Table 3: Results of multiple tests

Bacterial Designator Antibiotic Resistance Antibiotic Production PCR ProductS15UPRCRISEJCOR30P01A Positive for Tetracycline Coexistence with E.coli PositiveS15UPRCRISEJCOR30P02A Positive for Tetracycline Negative NegativeS15UPRCRISEJCOR30M02B NP Coexistence with

M.luteus and E.coliPositive

S15UPRCRISECHA30P01A NP Positive, E. coli NegativeS15UPRCRISECHA30M01B NP Positive, E. coli Negative

The two bacteria that showed antibiotic production were taken from a mountain known as La Tiza Peak in Comerío. This mountain, as established by Parker (1962), contains two types of minerals: alunite in its natural form and natroalunite. Multiple sources, like mindat.org, the Hudson Institute of Mineralogy (2015) and Hildebrand and Smith (1959) agree that this is the only place in Puerto Rico with this type of mineral. Additionally, the pH being slightly acidic (6.5) reinforces the presence of minerals because the decrease in pH typically means that the soil contains weathered kaolin minerals (alunite coexists with it ), iron and aluminum oxides (University of Hawai’i 2015). It is known that bacteria weathers soil minerals. It can be because it uses minerals as a direct source of energy or it may be a consequence of acid production, like sulfuric acid (FAO 2015). Bacteria can also produce minerals, a process known as bio-mineralization that can be classified as: Biologically controlled mineralization (BCM) and Biologically induced mineralization (BIM). The difference between BCM and BIM is that the latter form the minerals outside of the cell (exytracellular), and BCM produces it inside the cell under certain conditions (Dhami et al. 2013). That being said, it is possible to also hypothesize that the production of antibiotics can be a response or effect of the presence of minerals (especially of alunite) in the soil and/or the process of bio-mineralization caused by the bacteria. In the future, more experiments can be done to test this hypothesis.

One of the objectives of the investigation was to sequence the 16S rDNA gene of the bacteria. However, as seen in table 3 only two bacteria showed a positive PCR product. The DNA of these two bacteria was purified and sent for sequencing, but the results are not available yet. In the future, the procedure will be repeated in order to sequence the DNA of all the bacteria. The results of this investigation can be used to increase the understanding of the emergence of antibiotic resistance from the natural reservoir, which may aid the development of inhibitors of resistance mechanisms and resistant bacteria. In addition, the cases of antibiotic production against E.coli can be investigated further in order to see if these antibiotics have the potential of being introduced to medicine.

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