Soil Testing

Overview

Antimicrobial resistance (AMR) poses a significant threat to global health, affecting not just human medicine but also agricultural industries; in fact over 80% of all antimicrobials in the United States are used in agriculture (Am J Public Health, 2015). In Lambert iGEM’s home state of Georgia, over 102 billion livestock are estimated to release over 600,000 kg of antibiotics into the environment every year (Tian et al., 2021). Antibiotics are poorly metabolized in the body, with 70-80% excreted and subsequently washed into water systems (Sharda et al., 2023). From there, they seep into the soil, where selective pressure causes resistant bacteria to proliferate. Runoff from farms carries these antibiotic-resistant organisms into water systems and treatment facilities. These facilities lack specific protocols for removing AMR contaminants from water supplies.

Exacerbating the issue is horizontal gene transfer (HGT), which allows bacteria to share genetic material, including AMR genes, with other bacteria (see Fig. 1). Through HGT, a single bacterium that acquires antimicrobial resistance can rapidly spread these genes to other microbes in the ecosystem. Consequently, a single AMR mutation has the potential to quickly proliferate throughout an entire environmental network, magnifying the scope and impact of the initial contamination.

Figure 1. Three types of Horizontal Gene Transfer

Methodology

To understand the full extent of AMR, our data focused on gathering soil samples downstream of farms along the Chattahoochee River, which serves as the water source for over 80% of the state. Lambert iGEM took action to investigate the prevalence of Tetracycline, a common antibiotic that is now outlawed due to AMR (Granados-Chinchilla & Rodríguez, 2017). We utilized MiniPCR’s Sampling Soil for Antibiotic Resistance kit which provided TetB and TetM primers to assess tetracycline resistance in soil and further highlight the ongoing problem of AMR in our region. However, conversations with Dr. Forsberg, an expert in AMR bacteria present in soil from UT Southwestern, led us to investigate integron PCR primers, specifically Class 1 and Class 2 integron primers. Integrons are genetic elements that allow for site-specific recombination with gene cassettes, some of which encodes for antibiotic resistance. Integrons can be part of either the chromosomal DNA or the plasmid, and contain sequences that rearrange frequently due to a protein called tyrosine recombinase, encoded by the intl gene (see Fig. 2). Because of the constantly changing sequence of the gene cassettes, the integron primers bind to the common promoter (Pc) part of the integron (see Fig. 2). These primers will allow for efficient detection and amplification of the intI gene, which shows the presence of integrons and multiple AMR related genes (Sabbagh et al., 2021).

Figure 2. Structure of an Integron-Integrase Gene

Design

For our initial experimentation, we used MiniPCR’s environmental DNA extraction kit for soil. The kit follows a three-step protocol: eDNA extraction and purification, polymerase chain reaction (PCR) utilizing TetB and TetM primers, and gel electrophoresis. During our procedure, we tested various primers. However, shortly after we began testing, Dr. Forsberg introduced us to Class 1 and Class 2 integron primers, which are associated with agricultural antibiotics. Class 1 integron primers are associated with the detection of the intI-1 gene, likewise Class 2 integron primers are associated with the detection of the intI-2 gene. These primers show the existence of integrons in our bacterial DNA because they allow for the detection and amplification of each intI gene, indicating the presence of either class 1 or class 2 integrons. This allowed for more efficient testing where multiple genes could be detected using one primer rather than testing individual antibiotic primers. The first reaction contained the 16S primer, and reactions B-G contained the selected primers. Tubes D and E acted as the positive control containing control DNA from MiniPCR and reaction F and G were negative controls with deionized water. The 16S primer had an expected product length range of 18-24 bp. For the TetB and TetM primers, we expected nucleotide lengths of 656 bp and 406 bp, respectively. For the class 1 and class 2 integron primer pairs: RB 201/202, RB 317/320, HS 463a/464, and Hep 74/51 the expected product length ranges were 0.3 Kb, 0.9-2.1 Kb, 0.377 Kb, and 2.2 Kb respectively.

Since integron primers alone cannot determine the specific AMR genes present in bacteria, we submitted the positive samples to GeneWiz (Azenta Life Sciences) for sequencing. However, the initial sequencing attempt was unsuccessful, as the bands from the gels we sent appeared to be primer dimers rather than the expected gene cassettes. We are currently preparing new PCR samples for resubmission, to obtain a usable sequence. Once acquired, the sequence will be analyzed using BLAST to identify matches with known antibiotic-resistance genes. We compared the DNA sequences with a database of known antimicrobial-resistant genes. Using the database, we designed a map of Georgia, displaying the resistant genes (see Figure 3).

Results

With the help of MiniPCR’s Sampling Soil for Antibiotic Resistance kit, we were able to extract DNA from 15 soil samples and successfully test them with chosen primers. Our first set of experiments aimed to extract DNA from the soil. The extraction protocol (see Experimentation) includes homogenizing soil samples, lysing cells, and purifying the DNA. The resulting DNA was assessed using a NanoDrop spectrophotometer, with concentration values ranging from 10 ng/μl - 150 ng/μl.

We compiled our data (see Table. 1) and created a map displaying the results. Our samples exhibited the widespread presence of the tetracycline resistance gene TetM across the state (see Fig. 3). Initially, there appears to be a correlation between positive Tet resistance and proximity to the Chattahoochee River, though more samples will be needed to confirm this observation. The detection of TetM resistance sequences across these sites suggests that soil can act as a reservoir for resistant bacteria, posing a potential public health risk. These findings underscore the importance of continued surveillance and proactive measures to address the spread of AMR in environmental settings.

Overall Tetracycline Results

Figure 3. Map showing tetracycline resistance

The results from testing for the presence of integrons were invalid due to persistent primer dimerization during gel electrophoresis (see Notebook). With primer dimers persisting after multiple tests, our team believes that initial positive results may have been false positives. The absence of proper band lengths suggests the sampling area may have been too limited to capture a representative portion of the microbial population. This better aligns with our expectations, as integrons are relatively rare in soil environments. Statistically, it is expected that only around 15% of environmental samples would test positive for integrons (Byrne-Bailey et al., 2011), meaning a more extensive and geographically diverse sampling strategy may be required to detect integrons in this region.

Integron Results

Gel Results

After the extraction, the samples were run on a gel to determine what type of resistance was present in the sample. These samples were run on a 2% agarose gel along with a positive and negative control utilizing tetracycline primers, TetB and TetM (see Fig. 7). Well A consists of a 16s primer, wells B, D, & F consisting of TetB primer, and wells C, E, & G consisting of TetM primer (see Experimentation). The positive gel results displayed 4 positive TetM samples, 2 positive TetB samples and 1 sample carrying both TetB and TetM resistance.

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Figure 4. Slide 1: Chattahoochee Point (CP6) showing positive fluorescent band for TetM. Slide 2: Vogel State Park (V): showing positive fluorescent band for TetM. Slide 3: Chattahoochee River National Recreation Center (NR) showing positive fluorescent band for TetM. Slide 4: Windermere Park (W) showing positive fluorescent band for TetM. Slide 5: Lambert High School (3F) showing positive fluorescent band for TetB. Slide 6: Villa Rica (S1) showing positive fluorescent band for TetB. Slide 7: Nichols View (NV) showing positive fluorescent band for TetM and TetB.

Future Implementation

This year, Lambert iGEM investigated the prevalence of AMR across the state of Georgia by testing soil from various locations to gather a broad expanse of data. Next year, we aim to expand our map beyond the state of Georgia by sending kits to both national and international iGEM teams to evaluate the problem on a global scale. We also plan to continue testing with integron primers with a bigger sample size. Through collaboration with other groups, we hope to contribute valuable data to the fight against AMR across the world.

References