Effect of Human Maintenance on Soil Microbiome Antibiotic
Resistance in Dry Environments
Background
Soil microbiomes contain diverse antibiotic resistance genes.
The antibiotic resistance genes inside many soil microbiome
communities are crucial in the field of biology, as they can
influence plant growth, human health, and agricultural practices
which affect our food source.1 Antibiotic resistance genes not
only affect our food source, but also influence evolutionary
paths of the microbiome as well as other interacting organisms
in the ecosystem. Since there is sufficient research conducted on
antibiotic resistance in microbiomes, our study aims to analyze
the antibiotic resistance specifically in human-maintained
microbiomes in dry soil and compare our findings to the
antibiotic resistance in non-human maintained microbiomes in
dry soil. Human maintenance introduces factors such as
fertilizer, pesticides, and industrial outputs that can potentially
lead to an increased presence of antibiotic resistance genes
through selection and gene transfer.1 The dryness of the soil is
also important to take into account because previous studies
have found that soil moisture was significantly correlated (p
<0.05) with tetracycline and ciprofloxacin resistance
levels.2 Thus, our research question for this project is, do dry
soil microbiome communities in human-maintained
environments have higher or lower levels of antibiotic
resistance than those in non-human maintained environments?
The findings from this study provides us with critical
information about how to maintain soil microbiomes in
common dry environments, such as residential and industrial
areas, that will influence our agriculture and health.
Objectives & Hypothesis
Objectives:
• To compare the effect of human maintenance on soil
microbiome bacterial diversity and abundance, antibiotic
resistance, nitrogen fixation, and 16S rDNA sequences.
• To understand the impact of human maintenance on soil
microbiome antibiotic resistance.
Hypothesis: We hypothesize that human-maintained
environments will have higher soil microbiome antibiotic
resistance than non-human maintained environments. Multiple
studies have shown that human use and application of manure
significantly increased antibiotic resistance gene presence in dry
soil microbiomes. 3,4 Studies have also revealed
that surrounding human activities such as industry,
manufacturing, and utility use in urban areas increase antibiotic
resistance presence. 5
References
1. Martinez, J. L. “Environmental Pollution by Antibiotics and
by Antibiotic Resistance Determinants.” Environmental Pollution, 2009,
vol. 157(11), pp. 2893-2902. Doi: 10.1016/j.envpol.2009.05.051
2. Negreanu, Y., Pasternak, Z., Jurkevitch, E., & Cytryn, E. (2012).
Impact of Treated Wastewater Irrigation on Antibiotic Resistance in
Agricultural Soils. Environmental Science & Technology, 46(9), 4800-
4808. doi:10.1021/es204665b
3. Chen, Q. L., X. L. An, H. Li, Y. G. Zhu, J. Q. Su, and L. Cui. “Do
Manure-Borne or Indigenous Soil Microorganisms Influence the Spread
of Antibiotic Resistance Genes in Manured Soil?” Soil Biology &
Biochemistry, 2017, vol. 114, pp. 229-237.
4. Pu, Q., L. X. Zhao, Y. T. Li, and J. Q. Su. “Manure Fertilization
Increase Antibiotic Resistance in Soils from Typical Greenhouse
Vegetable Production Bases, China.” Journal of Hazardous Materials,
2020, vol. 391.
5. Yan, Z. Z., Q. L. Chen, Y. J. Zhang, J. Z. He, and H. W. Hu.
“Antibiotic Resistance in Urban Green Spaces Mirrors the Pattern of
Industrial Distribution.” Environmental International, 2019, vol. 132.
Doi: 10.1016/j.envint.2019.105106.
6. Regar, R.K., Gaur, V.K., et al. Science of The Total Environment,
2019; 1(681): p. 413-423.
Manish Bhatta, Olivia He, Arman Ostadazim, Michael Shi, and Anna Yang
1Oxford College of Emory University, Oxford, GA
Methods Results Continued
Sample Collection and Processing
• Soil samples were collected from various sampling locations (Table 1) (Figure 1). 4) Dry, human maintained colonies had higher antibiotic
• Each sample was shaken with 0.5 mL 1X PBS buffer to release bacteria cells from soil particles, resistance levels
and the bacteria was incubated at room temperature for 48 hours on an agar plate.
Analyzing Morphological Traits Analyzing Molecular Traits
Measuring Morphological Diversity (Figure 2) Analyzing Nitrogen-Fixing Genes (Figure 4)
• Colonies were distinguished by pigmentation, • Separate PCRs were conducted for amplifying
whole colony shape, margin shape, and surface nifH genes, amoA genes, and 16S rDNA.
appearance. • A secondary PCR was run on each product for
Calculating Bacterial Abundance (Figure 3) further amplification, which were run on gel
• A transparency grid with 163 squares was laid electrophoresis.
on top of each plate to estimate the percent Sequencing16S rDNA for Molecular Analysis
abundance of each colony type. • The 16S rDNA products were sent to the Conclusions and Future Directions
Measuring Antibiotic Resistance (Figure 4) Georgia Genomics and Bioinformatics Core
• Zone of inhibition was measured using ImageJ facility for sequencing. Conclusions
after two antibiotic disks were placed into agar • The sequences were analyzed using NCBI’s
plates each with one colony type and incubated BLAST and the Ribosomal Database Project •Average zone of inhibition (ZOI) of colonies cultured from
at room temperature for 24-48 hours. (RDP). dry, non-human maintained soil was approximately 0.15 cm
larger than that of dry, human maintained soil.
•All but one human maintained sample had a higher ZOI than
the smallest ZOI of the non-human maintained. The highest
ZOI in our dataset also came from human maintained soil. This
indicates certain non-human maintained bacteria may have
had more resistant antibiotic genes.
•The distribution of colony abundance was lower in the dry,
human-maintained soil than in the dry, non-human maintained
Results soil, suggesting that certain colonies in human-maintained
environments may dominate among other bacterial colonies
1) Dry, human maintained had less distinct colonies and higher colony abundance present.
•Our data is in accordance with a study investigating the
impact of manure-borne microorganisms on antibiotic
resistance; manure-borne microorganisms were found to have
contributed largely to the elevation of antibiotic resistance
genes due to both the addition of “manure-borne antibiotic
resistant bacteria (ARB) in soil and potential horizontal gene
transfer via mobile genetic elements from manure-borne ARB
to indigenous soil microorganisms". 3
•The lack of distribution of colony types in non-human
maintained soil may be attributed to pesticide contamination,
or other substances entering the soil as a result of human
Figure 6. Percent colony abundance for all bacterial colonies identified in dry, Human maintained sample soil maintenance. Studies have found that bacterial communities in
microbiome sites (A) and dry, non-human maintained sample soil microbiome sites (B).
pesticide contaminated soil experience a reduction of
2) Dry, human maintained had more colonies 3) Sequence identification and abundance of microbial diversity, while enriching only
with nitrogen fixation gene presence morphological characteristics of specific communities. Meanwhile, bacterial communities in
bacterial colonies non-pesticide contaminated soil were found to have abundance
among a larger number of phyla. 6
Future Directions
• Comparing the antibiotic resistance of a specific bacterial
colony in non-human versus human maintained soil (bacterial
species is a controlled variable)
• Comparing antibiotic resistance of colonies from similar
environments but different maintenance (nonhuman versus
human maintained) — type of area is a controlled variable
• Further examine trends in percent abundance/distribution of
colonies cultured from human maintained versus non-human
maintained.