Regulation of Antibiotic Resistance Genes on Agricultural Land Is Dependent on Both Choice of Organic Amendment and Prevalence of Predatory Bacteria

Abstract

Antibiotic resistance genes (ARGs) are widespread in the environment, and soils, specifically, are hotspots for microorganisms with inherent antibiotic resistance. Manure and sludge used as fertilizers in agricultural production have been shown to contain vast amounts of ARGs, and due to continued applications, ARGs accumulate in agricultural soils. Some soils, however, harbor a resilience capacity that could depend on specific soil properties, as well as the presence of predatory bacteria that are able to hydrolyse living bacteria, including bacteria of clinical importance. The objectives of this study were to (i) investigate if the antibiotic resistance profile of the soil microbiota could be differently affected by the addition of cow manure, chicken manure, and sludge, and (ii) investigate if the amendments had an effect on the presence of predatory bacteria. The three organic amendments were mixed separately with a field soil, divided into pots, and incubated in a greenhouse for 28 days. Droplet digital PCR (ddPCR) was used to quantify three ARGs, two predatory bacteria, and total number of bacteria. In this study, we demonstrated that the choice of organic amendment significantly affected the antibiotic resistance profile of soil, and promoted the growth of predatory bacteria, while the total number of bacteria was unaffected.

Keywords: BALO; antibiotic resistance; manure; sewage sludge; soil; tetracycline; vancomycin.

Figure 1

Schematic overview of the study design. Soil from organic production was added to the pots and complemented with different types of organic amendments: chicken manure, cow manure, and sludge. All pots were watered, covered with black plastic, and incubated in a greenhouse for 28 days. Samples were taken at four time points with four biological replicates. This image was created with BioRender.com accessed on 4 July 2024.

Figure 2

Prevalence of antibiotic resistance genes in soil exposed to different organic manure. DNA was extracted from soil exposed to different manure (cow, chicken, or sludge) for a time span of 0, 1, 7, or 28 days. Gene copies of (A) tetA, (B) tetM, and (C) vanA were determined through ddPCR and reported as copies per gram soil. Samples from day 0 represent the additive only (e.g., manure). Each mark (i.e., black dot, red square, and green or blue triangle) represents a true biological sample for which three technical replicates were conducted. For each sample, four biological replicates were taken (i.e., four marks per time point), and is displayed as the four different time points for each amendment on the x-axis, and number of gene copies on the y-axis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Prevalence of predatory bacteria in soil exposed to different organic manure. DNA was extracted from soil exposed to different manure (cow, chicken, or sludge) for a time span of 0, 1, 7, or 28 days. Gene copies of (A) 16S rRNA of the total bacterial population, (B) 16S rRNA gene copies of Bdellovibrio, and (C) 16S rRNA gene copies of Bacteriovorax were determined through ddPCR and reported as copies per gram soil. Samples from day 0 represent the additive only (e.g., manure). Each mark (i.e., black dot, red square, green or blue triangle) represents a true biological sample for which three technical replicates were conducted. For each sample, four biological replicates were taken (i.e., four marks per time point), and are displayed as the four different time points for each amendment on the x-axis, and number of gene copies on the y-axis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Prevalence of antibiotic resistance genes in relation to 16S. Gene copy number per gram soil (log10) of antibiotic resistance genes and 16S from all soil samples were collected at 0, 1, 7, and 28 days, and relative values are shown for (A) tetA, (B) tetA, and (C) vanA. Each mark (i.e., black dot, red square, and green or blue triangle) represent a true biological sample for which three technical replicates were conducted. For each sample, four biological replicates were taken (i.e., four marks per time point), and are displayed as the four different time points for each amendment on the x-axis, and number of gene copies on the y-axis. * p < 0.05, ** p < 0.01.

Figure 5

Correlations between the prevalence of antibiotic resistance genes and the 16S rDNA gene of predatory bacteria. Absolute quantities (gene copy number per gram soil) of predatory bacteria and antibiotic resistance genes from all soil samples were analyzed for correlative values between (A) tetA and Bdellovibrio, (B) tetA and Bacteriovorax, (C) tetM and Bdellovibrio, (D) tetM and Bacteriovorax, (E) vanA and Bdellovibrio, and (F) vanA and Bacteriovorax.