Tetracycline Resistant Bacterial Communities in Soils of Different Land Managements at Furman University

Clayton Bishop, Neely Wood, Min-Ken LiaoDepartment of Biology, Furman University

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 approximately 30 tetracycline resistant bacterial colonies from each site 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 Reciprocal 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).

Land use often results in changes to the physical and chemical properties of the soil. This, in turn, can have an impact on the composition of bacterial community present in the soil. Additionally, it has been reported that soil, more specifically, soil bacteria, are the reservoir of antibiotic genes (Riesenfeld et al. 2004). In this study, we focus on the tetracycline-resistant bacterial community and tetracycline-resistant genes because of the imprudent use of tetracycline in human and veterinary medicine, as well as agriculture, until recent years. We hypothesized that the different land managements not only influence overall soil bacterial community composition, as has been described abundantly in literature, but that they also influence the tetracycline-resistant bacterial communities.

Here 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 selected for certain tetracycline resistance 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. We hypothesized that the chemically managed soil would have the most diverse tetracycline resistant (tetr) bacterial community, while the unmanaged soil would have the lowest diversity.

Soil Sampling. Soil samples were collected from 3 sites of different land managements at Furman University: unmanaged forested soil, chemically managed soil, and organically managed farm soil. These samples were collected during June and July 2014.Cultured Bacteria. Bacterial colonies were grown on 0.1 TSA media containing concentrations of tetracycline (0, 10, 20, 40 µg/mL) for each site. Colonies were picked and isolated based on morphological differences.Soil DNA Extraction. At each site, soil DNA was extracted using PowerSoil® DNA Isolation Kit (MO BIO, Carlsbad, CA). Total soil DNA was used for tetr gene analysis.PCR Amplification. For each isolated colony, the 16S rRNA gene was amplified for sequencing. All amplicons were sized by gel electrophoresis.Total soil DNA was analyzed using Multiplex PCR for the amplification of tetr genes as described (Ng et al. 2001). Resulting amplicons were analyzed using gel electrophoresis.

Abstract

Materials and Methods

Introduction

Results

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Conclusions

References

AcknowledgementsThis project was funded by Furman Advantage.

Ng LK, Martin I, Alfa M, Mulvey M. 2001. Multiplex PCR for the detection oftetracycline resistant genes. Mol Cell Probes. 15(4):209-215.

Riesenfeld CS, Goodman RM, Handelsman J. 2004. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environ Microbiol. 6(9):981-989.

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Figure 4. 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 1. Aerial map of Furman University (Greenville, SC) shows locations of each sample site. Sample site (a) indicates the unmanaged forested area behind Daniel Chapel. Sample site (b) indicates the Furman Mall, which is chemically managed with pesticides and fertilizers. Sample site (c) indicates the Furman Farm, which contains an organically managed farm treated only with water and organic compost.

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Figures 3a, 3b, 3c. Bacteria from chemically managed soil samples of the Furman Mall (3a) 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. The same procedure was repeated for both the organically managed soil samples from the Furman Farm (3b) and the unmanaged soil samples from the forest behind Daniel Chapel (3c).

• The chemically treated soil of the Furman Mall did not reflect the diversity of tetracycline resistant bacterial community that was hypothesized. At any tested concentration of tetracycline, the community of tetr bacteria remained relatively static.

• The organically managed soil of the Furman Farm exhibited the highest diversity of tetracycline resistant bacteria at the highest tested concentration of tetracycline.

• The unmanaged soil of the forested area behind Furman Chapel had the least diverse tetracycline resistant bacterial community at the highest tested concentration of tetracycline. As concentration of tetracycline increased, the community became completely homogeneous.

• Testing for 14 different tetr genes in the soils of each site, 3 different genes were found. At the Furman Mall and the forest behind the Furman Chapel, both tet(A) and tet(K). In addition to tet(A), tet(L) was also found at the Furman Farm.

Results

Figure 2. Flowchart depicting the experimental method used to test the hypotheses. Soil samples were collected at 3 sites. Bacteria from soil was cultured and isolated. The 16S rRNA was amplified for each colony and sequenced. Using BLAST, colonies were identified at the family level. Total soil DNA was also extracted at each site for analysis of tetr genes. Multiplex PCR was used to amplify tetr genes divided into 4 groups. Resulting amplicons were sized by gel electrophoresis.

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