Effects of Composting Different Types of Organic Fertilizer on the Microbial Community Structure and Antibiotic Resistance Genes

Abstract

Organic fertilizer is a major carrier that stores and transmits antibiotic resistance genes (ARGs). In the environment, due to the application of organic fertilizers in agriculture, the increasing diversity and abundance of ARGs poses a potential threat to human health and environmental safety. In this paper, the microbial community structure and ARGs in different types of organic fertilizer treated with composting were examined. We found that the abundance and diversity of ARGs in earthworm cast organic fertilizer were the lowest and the highest in chicken manure organic fertilizer. Interestingly, the abundance and diversity of ARGs, especially beta-lactam resistance genes, sulfonamide resistance genes, and macrolide-lincosamide-streptogramin B (MLSB) resistance genes, in organic fertilizers were reduced significantly, while composting caused no significant change in mobile genetic elements (MGEs), where antibiotic deactivation and the use of efflux pumps were the two most dominant mechanisms. It was clear that removal of ARGs became more efficient with increasing reduction in the bacterial abundances and diversity of potential ARG hosts, and integron-mediated horizontal gene transfers (HGTs) played an important role in the proliferation of most ARG types. Therefore, the reduction in ARGs was mainly driven by changes in bacterial community composition caused by composting. Furthermore, rather than HGTs, the diversity and abundance of bacterial communities affected by compost physical and chemical properties were the main drivers shaping and altering the abundance and diversity of ARGs, which was indicated by a correlation analysis of these properties, antibiotic residues, microbial community structure, and ARGs. In general, high-temperature composting effectively removed antibiotic residues and ARGs from these organic fertilizers; however, it cannot prevent the proliferation of MGEs. The insights gained from these results may be of assistance in the safe and rational use of organic fertilizers by indicating the changes in microbial community structure and ARGs in different types of organic fertilizer treated with composting.

Keywords: ARGs; MGEs; antibiotics; composting; microbial community structure; organic fertilizer.

Figure 1

Richness of bacterial communities in organic fertilizers using the Chao1 estimator. Samples from compost were denoted CDC, CMC, SMC, and ECC. Uncomposted manure samples were denoted CDU, CMU, SMU, and ECU.

Figure 2

The effect of composting on the composition of organic fertilizer microbial community. The composition of the microbial community at the class level. Only the most abundant taxa (>1% genus) are displayed. The order of the genes is based on their relative abundance (mean, n = 3).

Figure 3

Redundancy analysis (RDA) of the correlations between physicochemical properties of organic fertilizer samples before and after composting and major microbial phyla (>1%) (Actinobacteria, Firmicutes, Proteobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Acidobacteria, Deinococcus-Thermus, Bacteria, Nitrospirae). TN: total nitrogen; TC: total organic carbon.

Figure 4

The detected number of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in the four sets of organic fertilizer samples, includes CMU and CMC, ECU and ECC, CDU and CDC, SMU and SMC. MLSB: macrolide-lincosamide-streptogramin B.

Figure 5

Distribution of each ARG type in eight organic fertilizer samples. The data were visualized via Circos software (http://circos.ca/). The length of the bars of each sample on the outer ring represents the percentage of ARGs in each sample.

Figure 6

Log number of absolute gene copy number (copies per gram) of ARGs and MGEs. The histogram showing the distribution of different types of ARGs (classified by the classes of antibiotics that they resisted) and MGEs in the four groups of organic fertilizers before and after composting. ** (p < 0.01) on the bar indicates a statistically significant difference. * (p < 0.05) on the bar indicates a statistically significant difference.

Figure 7

Resistance gene profile from eight organic fertilizer samples. Each column is labeled with a sample name, and each row is the result from a single primer set. Resistance profiles that confer resistance to all major classes of antibiotics included resistance to aminoglycoside, beta-lactamase, chloramphenicol, MLSB, multidrug, tetracycline, vancomycin, and sulfonamide.

Figure 8

Heatmap analysis of ARGs in organic fertilizer samples. The vertical axis lists the detected ARGs found in this study. The order of the genes was based on their similarity abundance.

Figure 9

Network analysis of cooccurrence between ARGs, MGEs, and bacteria. Relationships between ARGs, MGEs (relative gene copy number), and bacteria (at the phylum level, 16S rRNA gene sequence data) based on Pearson’s correlation coefficients (p < 0.05). The nodes are colored according to ARG class and phylum, and the node size is dependent on the number of connections to other nodes (degree). Each connection represents a significant correlation (p < 0.05), and the edge line width represents the corresponding Spearman’s correlation coefficient.

Figure 10

(a) Canonical correspondence analysis (CCA) illustrating relationships between microbial phyla, ARGs and environmental factors, including total nitrogen, total carbon, and pH. The percentage of variation explained by each axis is shown, and the relationship is significant (p < 0.01) based on 999 permutations. (b) Variation partitioning analysis (VPA) differentiates the effects of bacterial communities, environmental factors, and mobile genetic elements (MGEs) on ARG profile alterations. TN: total nitrogen; TC: total organic carbon; and MGEs: mobile genetic elements.