Diversity and abundance of ARGs in the animal manure and organic fertilizer
The abundance and diversity of 51 target ARGs in the animal manure and their prepared organic fertilizer were obtained through the HT-qPCR. The result showed that the ARGs diversity in the organic fertilizer samples was relatively lower than those in the animal manure (p<0.05) (Fig. 1A). The absolute abundance of ARGs in the animal manure and organic fertilizer samples was detected in the range from 2.1×105 to 7.8×105 copies/g and from 1.6×105 to 7.3×105 copies/g, respectively, which was similar to the findings by Zhang et al. . Previous study also found that the composting could reduce the abundance and diversity of ARGs in animal manure, likely due to the inactivation of some microorganisms by high temperature .
The types of ARGs among the animal manure from different breeds were significantly different, which was due to the differences in fecal properties and microbial composition (Fig. 1B) . The detection rates of ARG subtypes from a single kind of animal manure to manufactured organic fertilizer samples (JF1→JM1 and NF2→NM2) were reduced by 42% and 34.6%, respectively. However, the detection rates of ARG subtypes from multiple animal manure (JF, YF and NF) to compound organic fertilizer samples (NF1+YF2→NYM1, YF2+JF3→YJM1) increased by 28% and 24%, respectively (Fig. 1A), which was in line with previous observation . These results indicated that composting process could reduce the release of partial ARG subtypes from single manure to organic fertilizer. It is noteworthy that the composite organic fertilizer fermented by multiple manure would increase the diversity of ARGs, which should draw much attention on the importance of manure management concerning the fate of ARGs .
The level of ARGs in chicken manure was detected with the highest absolute abundance (2.8×105~7.8×105 copies/g), which was 2~4 times higher than those in cow manure (2.1×105~3.3×105 copies/g) and sheep manure (2.2×105~5.1×105 copies/g) (Fig. 1C). Previous studies have shown that the difference of ARG levels between poultry manure and cattle manure may be related to the difference of antibiotic use patterns and fecal microorganisms among different species of livestock [32, 33].The removal efficiencies of ARGs in different animal manure and/or different treatment processes were also reported [14, 34]. Most TRGs (tetB–01, tetG–01and tetM–01) decreased by 12%~96% after composting (Fig. 1C), which was confirmed by previous findings [35, 36]. The abundance of tetX was higher than other ARGs in all samples, likely due to the broad range of potential hosts of tetX . Some TRGs decreased, while others persisted or signiﬁcantly increased (such as tetK, tetX) after thermophilic composting .Compared with in response raw of animal manure, the abundance of individual ARGs (tetK) increased 2~216 times in their organic fertilizer. Ezzariai et al. reported that the abundance of tetX in the swine manure reduced exponentially under the anaerobic condition .The reason for this opposite trend was being explored, since the resistance mechanism of tetX was still unknown [14, 38].
The SRGs were predominant in terms of absolute abundance in all samples. From JF1 to JM1, the level of sul1 and sul2 genes decreased from 1.1×105 copies/g to 100 copies/g and from 1.2×105 copies/g to 1.58×101 copies/g, respectively. The results were consistent with the previous study that the level of sul2 dramatically decreased during composting . The abundance of MLSB resistance genes (ermA, ermB, ermF and ermX) significantly decreased by 10%~98% from manure to organic fertilizer (Fig. 1C), lower than TRGs and SRGs. It was related to the low use of macrolides during feeding. The response of ARGs varied in composting due to the ecological complex microbial processes. It suggested that the composting process may cause individual ARGs proliferation.
Fresh manure composting is a feasible approach to decrease the level of certain ARGs before its application to farmland. However, most ARGs would still retain or even proliferate after composting, which may be caused by the difference in external conditions, such as raw materials, environmental factors or microbial community, etc. . The recent study also showed that the variations in microbial communities may have an impact on ARGs in composting .
Microbial communities in the animal manure and organic fertilizer
Figure 2 showed that no significant correlation was observed between the ARGs and the environmental factors in this study, and we focused on the microbial community structures in animal manure and organic fertilizer samples. The results were shown in Fig. 3a, b. It demonstrated that the composition and abundance of microbial communities varied greatly in different sample classifications. The compound organic fertilizer NYJM had the highest microbial diversity (Fig. 3a), and the samples of different animal manure and organic fertilizer were clustered into different categories (Fig. 3b). From the sorted map of the microbial community in Fig. 3b, the fecal samples were partially overlapped with the aggregated organic fertilizer samples (such as NF and NM), suggesting that the bacterial community structures in organic fertilizer were similar with those in manure from the corresponding composting source.
Compared with their derived fertilizer, the abundance of microorganisms decreased in cow manure, conversely, an increasing of the abundance was observed in chicken manure, which might be related to composting conditions and manure nutritional structure (Fig. 3a) [40, 41]. Especially, the abundance of microorganisms varied dramatically among JM samples, which might originate from the condition of livestock farm as well as individual difference in animals (such as age, species) . As shown in Fig. 3b, the composition of the microbial community was significantly different between animal manure and their derived organic fertilizer (e.g. YF→YM, and JF→JM). Interestingly, the Bray−Curtis distance between YF and YM and between JF and JM was closer than YF or JF samples to other organic fertilizer samples. The overlap between NF and NM indicated that the structures of microbial communities between NF and NM were more alike than the others. The intersections between YJM and JM, JF, YF samples suggested there was a correlation of microbial composition between animal manure and their derived organic fertilizer.
Relationship between the bacterial communities and ARGs
Result from Mantel test indicated that ARGs abundance was substantially correlated with bacterial community structures based on Bray−Curtis distance. Procrustes analysis showed the clustering based on the abundance of ARGs and 16S OTUs exhibited a goodness-of-fit test (sum of squares M2 = 0.476, r = 0.799, p<0.01, 999 permutations), indicating that the bacterial community structures exerted significant influence on ARGs abundance. This finding was consistent with the previous study that the changed structure of microbial communities was the major factor driving the variation of ARGs profile in the animal manure and organic fertilizer [43, 44].PCA result also confirmed that ARGs within different animal manure and organic fertilizer showed a similar distribution pattern of the bacteria communities (Fig. 4). It was concluded that the ARGs variation from animal manure to organic fertilizer was strongly correlated with the microbial community. The shift of the bacterial communities played key roles in the direct change of the ARGs patterns . It is noted that the presence of pathogens in the microbial community would not only decrease the productivity of livestock and poultry breeding, but also increase the risk of ARGs spread from organic fertilizer to agricultural environment.
The fate of pathogens from animal manure to organic fertilizer
The relative abundance variability of the top 20 genera was selected to evaluate the risk of pathogens from manure to organic fertilizer (Fig. 5).It is worth noting that most of the top 20 genera were pathogens. Corynebacerium–1, Virgibacillus, Streptomyces and Actinomadura were the major genera in animal manure and organic fertilizer samples. After composting, the relative abundance of pathogens was altered, but the dominant genus was still Corynebacerium–1 which was usually a heterogeneous population composed of human and animal pathogens that could cause disease in livestock . The relative abundance of Virgibacillus was significantly reduced from animal manure to prepared organic fertilizer (especially in JF-JM). Besides, the abundance of Actinomadura in organic fertilizer was 2~300 times higher than those in animal manure. These two genera were the carriers of antibiotic resistance genes (such as ermX, tetPA), and their abundances were found to have a similar reduction trend as ARGs after composting. The Actinomadura and Virgibacillus genera belong to the phylum Actinobacteria, which are opportunistic pathogen that would cause disease in animals and plants . Lv et al. identified that Actinobacteria were prominent in the thermophilic stage and the groups probably could carry and disseminate ARGs .The abundance of Pseudomonas (phylum Proteobacteria) which was found as opportunistic pathogen carrying most ARGs with multiple resistance was signiﬁcantly increased from animal manure to organic fertilizer [27, 48]. The network analysis was conducted to determine the correlations between ARGs and the top bacterial genera, which may judge the potential host bacteria for ARGs. As illustrated inFig. 6, there were significant correlations between ARGs and the potential host bacteria in the animal manure and their derived organic fertilizer samples (p<0.05 and R>0.60). In addition to the host bacteria obtained above, Atopostipes from Firmicutes were found to contain potential host bacteria for ermA, ermB, ermX, tetK, tetM–01.
Pathogens could not be completely removed from animal manure composting to organic fertilizer, but would cause partial proliferation. Significant correlations between pathogens and ARGs occurred, suggesting that the pathogens might become the important hosts of ARGs . Therefore, once antibiotic-resistant pathogens are ubiquitous in organic fertilizer, they are bound to pose a threat to the health of farm land soil and crops.