Tetracycline resistant bacterial communities in soils of different

land managements at Furman University

Clayton Bishop

Abstract

This study was to examine the diversity of the tetracycline resistant (tetr) bacterial community in

soils of three different land managements on campus: soil that is chemically managed (Furman

Mall), organically managed (Furman farm), and unmanaged (forested area). We hypothesized

that the diversity of the tetr bacterial community in the organically managed soil of Furman Farm

would be the greatest and that the soil of the chemically managed Furman Mall would be the

lowest. We isolated 50 tetracycline-resistant bacteria from the sites of different land

management. The 16S rRNA gene from each of these isolates was amplified and sequenced, and

the sequences were used to identify these isolates to the family level. The Simpson’s diversity

index results suggested that the tetr bacterial community in the organically managed soil had the

highest diversity, followed by the chemically managed soil, and the unmanaged soil. The

bacterial community of the chemically managed soil became completely homogeneous at 40

µg/mL of tetracycline. We also studied the diversity of tetr genes in soils of different land

managements. We hypothesized that a higher diversity in tetr genes would be present in soil that

was chemically managed. Using total soil DNA and multiplex PCR, we tested for 14 different

tetr genes. We found only three tetr genes with tet(A) being found at all three sites. The

chemically managed and unmanaged soils also contained tet(L), and the organically managed

soil contained tet(K).

Introduction

Soil biodiversity encompasses a great number of different microbes, but can be reduced, for the

sake of experimentation, to the abundance and relative diversity of bacteria present in a soil

sample. These bacteria are present in all soil and play a critical, yet invisible, role. An

understanding of bacteria’s role in soil is deeply important for soil quality and effective

agricultural practices.

The interaction between land management and soil bacteria has also led to the investigation of

antibiotic resistance in soil. Land use often results in changes to the chemistry or composition of

the soil. This, in turn, can have an impact on the community of bacteria present in the soils of

different land managements. Not only can land management affect which microbes are present,

but it often impacts the diversity of the bacterial community (Rampelotto et al. 2013). Soil

bacteria are critical in nutrient cycling, which is directly tied to soil productivity. The

relationship between bacterial diversity and soil quality is inseparable from diversity of plant life

found growing in the soil (Bisset et al. 2011). This has a particular impact for agricultural

practices, which necessitate a high crop yield reliant on land productivity.

The soil is the source from which most antibiotics are discovered. Naturally produced antibiotics

are widely found to be produced by microbes in the soil (Davies and Davies 2010). As

antibiotics become prevalent in the soil, bacteria have evolved to create forms of antibiotic

resistance. Thus, soil bacteria are found to contain a plethora of antibiotic resistant genes

(D’Costa et al. 2006, Riesenfeld et al. 2004). The soil is a large reservoir of antibiotic genes in

both culturable and unculturable bacteria, but research focused on resistance in uncultured

bacteria is lacking (Riesenfeld et al. 2004). Bacteria that cannot be cultured in a laboratory

setting contain antibiotic resistant genes beyond that which can be found in cultured soil bacteria.

This antibiotic resistance in soil bacteria is a result of both antibiotics naturally produced by soil

microbes and the introduction of antibiotics to the soil through human activity (Alekshun and

Levy 2007).

Tetracycline is a broad-spectrum antibiotic derived from chlortetracycline, found as a natural

product of soil fungi. It acts as a bacteriostatic agent that halts bacterial protein synthesis by

allosterically inhibiting binding of the aminoacyl-tRNA to the 30S subunit of the ribosome

(Schnappinger and Hillen 1996). Because of tetracycline’s successful bacteriostatic effects, it is

widely used to treat and prevent bacterial infections in humans, non-human animals, and

agriculture (Aminov et al. 2000). Our use of tetracycline in human and veterinary medicine, as

well as agriculture, has led to tetracycline being introduced at higher amounts in soils, acting as a

selective pressure for resistance (Schmitt et al. 2006). The widespread use, and likely overuse,

has lead to tetracycline resistance being found in almost all bacterial genera (Aminov et al.

2000). There are three mechanisms by which a bacterium exhibits resistance to tetracycline:

tetracycline-degrading enzymes, efflux pumps to remove tetracycline, and ribosomal protection

to prevent binding of tetracycline (Roberts 1996). The most common mode of resistance is the

efflux of tetracycline. The tetracycline resistant gene encodes for a membrane protein that

actively transports tetracycline out of the bacterium at an equal or greater rate than tetracycline is

able to enter the bacterium (Speer et al. 1992). Bacteria have also developed resistance to

tetracycline through modification of their ribosomes. Tetracycline resistant genes can encode for

various cytoplasmic proteins that block the binding of tetracycline to the ribosome (Roberts

1996). The rare and less-known mechanism of resistance is through tetracycline-degrading

enzymes, which modify tetracycline to an inactive form in the bacterium in the presence of

oxygen and NADPH (Roberts 1996). The only tetracycline resistant (tetr) gene that encodes for

tetracycline-degrading enzymes is tet(X). Tetr genes that result in a tetracycline efflux pump are

tet(A), (B), (C), (D), (E), (G), (L), (M), and (K). Ribosomal protection as a mechanism of

resistance is associated with tet(M), (O), (S), and (Q) (Chopra and Roberts 2001, Ng et al. 2001).

In this study, we examined and compared the diversity of tetracycline resistant bacterial

communities in soils of three different land managements on campus at increasing concentrations

of tetracycline. The overall goal was to determine whether different land managements created

different ecosystems that subsequently supported different tetracycline resistant bacterial

communities or select for certain tetr genes. The effects of organically managed, chemically

managed, and unmanaged soils were tested in regards to the tetracycline resistant communities

using the campus of Furman University as a model. Cultured bacteria determined how

homogeneous or heterogeneous the bacterial communities became at each soil type as the

concentration of tetracycline was increased. We believed that the chemically managed soil

would have the most diverse tetracycline resistant community, while the unmanaged soil would

have the lowest diversity at the highest concentration of tetracycline. We also identified tetr

genes in the bacteria isolated from these three sites. Rather than limiting to cultured bacteria,

total soil DNA was examined for tetr genes. Again, we expected to find that the chemically

managed soil would have the highest diversity of tetr genes.

Materials and Methods

Soil Sampling and Processing

Soil was collected from three different sites on Furman University’s campus (Greenville, SC)

using a 7/8” x 33’ regular soil probe (Forestry Supplies, Jackson, MS). The first sample site was

the manicured mall. The second site was 10-15 feet into the forest behind Furman’s Daniel

Chapel. The third site was at Furman’s organically managed farm. Soil from the farm was

collected at the base of four different types of plants: red sails lettuce (Lactuca sativa), silver

queen corn (Zea mays), zucchini (Cucurbita pepo), and cabbage (Bassica oleracea). Before each

sample was taken, any debris or non-soil material (such as mulch), along with the top layer of

soil, was removed to ensure that bacteria at the surface would not be collected. Probed soil

samples were placed in sterile Whirl-Pak bags.

Soil samples were collected four times at each of the sites over the span of approximately two

months (June/July 2014). Samples were organized by collection site, rather than by date of

collection. Samples were taken from the same approximate location at each site, with the

exception of the organic farm. Soil of different crops had to be sampled due to changes in crop

availability at the farm.

Approximately 0.5g of each soil sample was placed in 25mL of PBS (phosphate buffered saline)

solution for a final concentration of 0.02 g/mL. Each sample was vortexed at maximum speed

for 10 min and the supernatants were subsequently spread on plates. Remaining soil samples

were stored in freezer at -20 degrees Celsius.

Cultured Bacteria

Bacteria from soil samples were cultured on both R2A (Reasoner’s 2A agar) and 0.1 TSA

(tryptic soy agar) media (Fisher Scientific, Pittsburgh, PA). The R2A medium was prepared

according to supplier’s instructions. The 0.1 TSA media was prepared according to supplies

instructions. Both types of media were supplemented with three different concentrations of

tetracycline: 10 µg/mL, 20 µg/mL, and 40 µg/mL.

For each suspended soil sample, 100 µl of supernatant was spread on the following plates: 2 0

µg/mL tetracycline R2A plates, 2 0 µg/mL tetracycline 0.1 TSA plates, 2 10 µg/mL tetracycline

0.1 TSA plates, 2 20 µg/mL tetracycline 0.1 TSA plates, and 2 40 µg/mL tetracycline 0.1 TSA

plates. Plates with no tetracycline were incubated at 27 degrees Celsius overnight, and plates

with tetracycline were incubated for 48 hours. After incubation, all morphologically different

colonies on each plate were selected to be isolated. For soil samples behind the Chapel, 15

colonies were isolated at 0 µg/mL tetracycline, 6 colonies were isolated at 10 µg/mL, 5 colonies

were isolated at 20 µg/mL, and only 1 colony was isolated at 40 µg/mL. For soil samples of the

Mall, 19 colonies were isolated at 0 µg/mL tetracycline, 7 colonies at 10 µg/mL, 5 colonies at 20

µg/mL, and 3 colonies at 40 µg/mL. For Farm soil samples, 41 colonies were isolated at 0

µg/mL tetracycline, 10 colonies at 10 µg/mL, 5 colonies at 20 µg/mL, and 4 colonies at 40

µg/mL.

Bacterial and Soil DNA Extraction

A small amount of an isolated colony was picked and placed in 50µL nuclease-free water. The

mixture was briefly vortexed. Exactly 2µl of the bacterial mixture was used as the PCR

template.

To analyze unculturable bacteria, soil DNA was extracted using PowerSoil® DNA Isolation Kit

(MO BIO, Carlsbad, CA). Procedure strictly adhered to the protocol provided for the kit.

Extracted DNA was placed in the freezer at -20 degrees Celsius for future analysis.

PCR

Each PCR reaction contained 1µM for each primer (universal forward and universal reverse), 2µl

bacterial mixture, 2x GoTaq® Green Master Mix (Promega, Madison, WI). PCR was set for

denaturation at 94 degrees Celsius for 5 min; 30 cycles of 94 degrees Celsius for 1 min, 55

degrees Celsius for 2 min, and 72 degrees Celsius for 3 min; and final extension at 72 degrees

Celsius for 10 min. Final PCR products of 16S rRNA gene were subjected to gel electrophoresis

and a portion of this collection was used for DNA sequencing. All amplicons were sized by gel

electrophoresis.

Total soil extracted DNA was analyzed using multiplex PCR following protocol of Ng et al.

(2001). Tetracycline genes were divided into four groups. Group I amplified tet(B), (C), and

(D). Group II amplified tet(A), (E), and (G). Group III amplified tet(K), (L), (M), (O), and (S).

Group IV amplified tetA(P), (Q), and (X). PCR condition to amplify tetracycline-resistance

genes is as follows: initial denaturation at 94 degrees Celsius for 5 min; 35 cycles of 94 degrees

Celsius for 1 min, 55 degrees Celsius for 1 min, and 72 degrees Celsius for 1.5 min; and final

extension at 72 degrees Celsius for 7 min. Same protocol was also used for isolated bacterial

colonies grown on media containing 10 µg/mL, 20 µg/mL, and 40 µg/mL of tetracycline (Ng et

al., 2001). Resulting amplicons were analyzed using gel electrophoresis.

DNA Sequencing

16S rDNA amplicons were purified using ExoSAP-IT® (Affymetrix, Santa Clara, CA) according

to supplier’s instruction. Purified amplicons were shipped overnight to Arizona State University

(Tempe, AZ) for sequencing. The16S rDNA sequences were analyzed using BLAST® (NCBI,

Washington, DC). Each BLAST entry was identified to the genus level.

Results

Simpson’s reciprocal index (1/D) was used to determine the bacterial diversity at each of the

three sites for each concentration of tetracycline. Sequenced isolates identified at the genus level

were grouped into respective families, and family diversity was determined at each site. When

no tetracycline was present in the growth media, the soil of the Chapel’s wooded area was the

most bacterially diverse (1/D = 3.42), followed by the Furman Farm (1/D = 2.15) and the

Furman Mall (1/D = 1.57) (Figure 1). The only bacterial family found at all three sites was

Bacillaceae.

The diversity pattern of tetracycline resistant bacterial communities is different from the one of

unselected communities. At a tetracycline concentration of 10 µg/mL, the variance between

Simpson’s reciprocal index values decreased. The soil at the Farm had the highest diversity of

bacteria (1/D = 2.17). The soils behind the Chapel (1/D = 2.00) and at the Mall (1/D = 1.96) had

a similar level of bacterial diversity. While the family Bacillaceae was present at each site

without the presence of tetracycline, it only appears in the soil of the Farm at 10 µg/mL.

Although the tetracycline resistant Bacillaceae were not isolated from the Chapel and Mall soil,

Burkholderiaceae was present in the soils of each site.

At a concentration of tetracycline at 20 µg/mL, the pattern of relative diversity varied. The soil

behind the Chapel again had the highest Simpson’s reciprocal index value (1/D = 2.27). Both the

soils of the Mall and the Farm had the same bacterial diversity at 20 µg/mL (1/D = 1.92). No

bacterial family was found in all three soil types, but Burkholderiaceae was present in the soils

of both behind the Chapel and at the Mall.

When the concentration of tetracycline was doubled from 20 to 40 µg/mL, the pattern of relative

bacterial diversity again changed. The unmanaged soil behind the Chapel went from having the

highest diversity to having the least (1/D = 1). Only Burkholderiaceae was present in the soil

(Figure 2). The bacterial diversity of the Mall soil decreased slightly (1/D = 1.8). At the highest

level of tetracycline, the Farm exhibited its highest diversity seen at any tetracycline

concentration (1/D = 2.67). Thus, when tetracycline was at its highest tested concentration, the

most diverse tetracycline resistant bacterial communities were in the soil at the Furman Farm,

followed by the Mall soil and then behind the Chapel soil.

The composition of the bacterial communities from unmanaged soil behind the Chapel changed

as the concentration of tetracycline increased. As tetracycline increased from 0 µg/mL to 10

µg/mL, all families were absent with the exception of Burkholderiaceae. At 10 µg/mL,

Sphingobacteriaceae and Pseudomonadaceae became present. Sphingobacteriaceae remained

present at 20 µg/mL, while Pseudomonadaceae became absent and Flavobacteriaceae became

present. At the highest concentration of tetracycline, 40 µg/mL, only Burkholderiaceae was

present, as in all concentrations of tetracycline (Figure 2).

The chemically managed soil of the Mall had a very consistent pattern of bacterial families

present at various concentrations of tetracycline. As tetracycline was introduced at 10 µg/mL, all

families at 0 µg/mL were absent except Flavobacteriaceae, while Burkholderiaceae was present.

There was no further change observed as tetracycline concentration increased to 40 µg/mL, with

Burkholderiaceae and Flavobacteriaceae present in 10, 20, and 40 µg/mL tetracycline (Figure

3).

The organically managed Farm soil had a very different pattern of families. At 10 µg/mL

tetracycline, all families present at 0 µg/mL were absent with the exception of Bacillaceae and

Flavobacteriaceae. Burkholderiaceae was observed at 10 µg/mL. As tetracycline was increased

from 10 µg/mL to 20 µg/mL, all families observed without the selection of tetracycline were not

observed. At 20 µg/mL, Xanthomonadaceae was present and Comamonadaceae was observed

for the first time. At 40 µg/mL, both Xanthomonadaceae and Comamonadaceae were observed

with the addition of Alcaligenaceae, which was absent in selection of lower tetracycline

concentrations (Figure 4).

We tested the presence of 14 different tetr genes and only detected three. Soil from each land

management contained two different tetr genes, with the tet(A) gene being found in at all three

sites. In both the Mall and Chapel soil, tet(K) genes were detected. There was no difference in

tetr genes between the Mall and Chapel soil bacteria, both tet(A) and tet(K) were detected. At

the Farm, tet(L) gene and tet(A) genes were detected.

Discussion

Unmanaged Forest Soil

Without any tetracycline present, the forested soil behind the Chapel had the most diverse

bacterial community, while the soil of the Mall had the least diverse community. As the tested

concentration of tetracycline increased to 40 µg/mL, the community in the Farm soil became

more heterogeneous. The bacterial community in the chemically Mall soil remained relatively

stable in regards to diversity, and the community in the unmanaged soil behind the Chapel

became more homogeneous.

The Simpson’s Reciprocal Index is highest in the Chapel soil at 0 µg/mL, indicating the highest

diversity before tetracycline was introduced. Unmanaged soil behind the Furman Chapel is the

soil least changed by human activity of the three sites tested. Based on our hypotheses, this

bacterial community was expected to have a higher relative diversity, comparing to communities

from Mall soil and Farm soil, because bacteria do not have to respond to any selective pressures

of human influence (Popowska et al. 2012). As concentrations of tetracycline increased, the

relative bacterial diversity decreased, as was hypothesized. Without experiencing a changing

bacterial community or any outside selective pressure, there is little pressure for selection of

tetracycline resistance in the bacteria.

Unmanaged soil behind the Chapel also contained tet(A) and tet(K) genes. Both tetr genes are

efflux pumps that provide general antibiotic resistance. These pumps can also be used for

removal of other toxins including heavy metals (Allen et al. 2010). Without the selective

pressure of antibiotics in the Forest soil, resistance could have been selected in response to other

selective pressures, such as toxic heavy metals. With our approach of multiplex PCR and gel

electrophoresis, we did not detect many tetracycline resistant genes, which we had hypothesized

to find.

Chemically Managed Mall Soil

The chemically managed soil samples of the Furman Mall provide the most stable detectable

bacterial diversity among increasing concentrations of tetracycline. Although there is an overall

decrease in diversity as tetracycline becomes more concentrated in the growth media, the decline

is largely expected due to selection for tetracycline resistant bacteria. It was predicted that the

Furman Mall would have a high relative bacterial diversity at 40 µg/mL due to selective

pressures introduced through land management. Treatment with pesticides and growth aids, such

as manure, was expected to cause an overall pressure to adapt to toxins and antibiotics. The Mall

soil is, on occasion, treated with manure. It is the only soil type we tested that would have been

treated with manure. The research of Heuer and Smalla (2007) found that soil treated with

manure had a higher amount of antibiotic resistant bacteria due to the introduction of resistant

populations and antibiotics found within manure. In addition, soil treated with pesticides was

found to have an affected ecosystem due to high levels of both the pesticide itself and

intermediate metabolites of pesticide degradation (Cycoń et al. 2010). The presence of

pesticides in the soil may introduce a new selective pressure for adaptation, but it is also possible

as a source of reduction in the number of different soil bacteria, reducing the overall diversity

(Nicholson and Hirsch 1997). When both manure, which can increase tetracycline resistant

bacterial diversity, and pesticides, which can decrease bacterial diversity, are both introduced to

the Mall soil, the result may be the stagnant level of diversity that is found in our data. It is

therefore believed that the ecosystem of the Mall soil produces a homogeneous bacterial

community that does not fluctuate greatly as tetracycline was introduced.

The same tetr genes detected in soil behind the Chapel were also found in the Mall soil. These

tetr genes could have arisen for similar reasons as in the soil behind the Chapel, as a general

mechanism to efflux any toxins, especially in the Mall soil were toxins and pesticides are

abundant (Allen et al. 2010). A more likely explanation is that these two tetr genes are simply

more prevalent in soil bacteria.

Organically Managed Farm Soil

The organically managed soil of the Furman Farm presented a fairly consistent pattern of

diversity at 0, 10, and 20 µg/mL, but exhibited a sharp increase in diversity at the highest

concentration of tetracycline, 40 µg/mL. It was predicted that soil from the Farm would have a

high diversity as tetracycline increased due to the number different bacteria introduced to the soil

through organic compost. The soil of the Farm is treated with organic compost that can generate

an increase in the bacterial diversity (Bending et al. 2002). Addition of organic compost to the

soil has been found to stimulate microbial soil diversity beyond simply introducing new bacterial

by altering the physical and chemical aspects of the ecosystem. Compost introduces high

amounts of necessary nutrients such as carbon, nitrogen, and phosphorus (Saison et al. 2006). It

was unexpected, however, to find that biodiversity was not as consistently high in low-to-none

concentrations of tetracycline. Antibiotic resistance could also be conferred through the water

supply used to water the farm, as antibiotics are often found in the water supply (Allen et al.

2010). This pathway of tetracycline introduction to the environment can expose soil bacteria to

tetracycline and select for resistance. However, this mechanism of introduction is not guaranteed

and depends on the level of tetracycline found in the water supply.

The Farm soil contained tet(L), in addition to tet(A) found at all 3 sites. Both tetr genes are

efflux resistance mechanisms that are most often to be found in soil bacteria. Future research

would investigate particular differences in bacterial diversity between different crops and the

components of the organic compost used in the soil.

References

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0 10 20 400

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Forest

Forest

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Mall MallMall

Farm FarmFarm

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Concentration of Tetracycline (µg/mL)

Sim

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/D)

Figure 1. Simpson’s Reciprocal Index (1/D) values were determined at each soil site, unmanaged forest behind

Chapel, chemically managed Mall, and organically managed Farm, at each concentration of tetracycline, 0 µg/mL,

10 µg/mL, 20 µg/mL, and 40 µg/mL. Bacterial families at each site and for each tetracycline concentration were

determined through amplification and sequencing of 16S rRNA, followed by BLAST of sequenced 16S rRNA gene

region. Differences in bacterial families were used to determine Simpson’s index, which represents the relative

diversity of the bacteria in the soil.

Figure 2. Bacteria from unmanaged soil samples behind the Furman Chapel were cultured and isolated on 0.1 TSA

containing concentrations of tetracycline: 0 µg/mL, 10 µg/mL, 20 µg/mL, and 40 µg/mL. The 16S rRNA gene of

each colony was amplified and sequenced. Identification at the family level was based on BLAST analysis of the

sequence of the amplified 16S rRNA gene region.

Figure 3. Chemically managed soil samples containing soil bacteria from the Furman Mall were cultured and

isolated on 0.1 TSA. Media contained increasing concentrations of tetracycline: 0 µg/mL, 10 µg/mL, 20 µg/mL,

and 40 µg/mL. Colonies were isolated, and 16S rRNA gene of each colony was amplified and sequenced. Blast

analysis of the amplified 16S rRNA gene region indentified each colony at the family level.

Figure 4. Bacterial colonies found in the organically managed soil samples of the Furman Farm were grown on 0.1

TSA, which contained concentrations of tetracycline: 0 µg/mL, 10 µg/mL, 20 µg/mL, and 40 µg/mL. For each

isolated colony, the 16S rRNA gene was amplified and then sequenced. BLAST analysis of the sequenced 16S

rRNA gene region identified each colony at the family level.