Antibiotic resistome determined for microbes during composting
Metagenomic analysis based on the structured SARG database was conducted to determine the antibiotic resistome for microbes during composting. The results showed that ARGs were diverse and abundant in swine feces, even when OTC or Cu was not administered in the CK treatment (Fig. 1). Aerobic composting effectively reduced the abundances and diversity of ARGs. These results are consistent with previous demonstrations [32, 36]. In total, 289 ARG subtypes and 19 ARG types were detected throughout the composting process. On day 0 of the composting process, 235 ARGs were detected but the amount decreased to 135–160 on day 10 and 106–120 on day 42. Sixty ARGs were prevalent throughout the composting process (Figs. 1a and 1b). The total abundances of ARGs were 9.39 ⋅ 10− 1 copies/16S rRNA on day 0, 1.44–2.41 ⋅ 10− 1 copies/16S rRNA on day 10, and 1.08–1.36 ⋅ 10− 1 copies/16S rRNA on day 42. The application of OTC or Cu increased the abundance of ARGs compared with CK, where the total abundances were 43.57%, 39.76%, and 67.17% higher in O, Cu, and OCu on day 10, respectively, and 4.66%, 8.93%, and 26.17% higher on day 42.
The detected ARGs were characterized by three major resistance mechanisms: antibiotic inactivation (37.02%), efflux pump (34.26%), and cell protection (21.45%), which are consistent with the results obtained by Zhu et al. [37] and Qian et al. [32]. The ARG subtypes with antibiotic inactivation mechanisms were the most common with up to 85, but the number decreased to 25–30 in the different treatments at the end of composting. The application of OTC and Cu inhibited the decreases in the abundance of ARGs with the three mechanisms on day 10, and strongly inhibited the removal of ARGs characterized by antibiotic inactivation mechanisms in the compost product. In particular, the abundance of ARGs with antibiotic inactivation mechanisms was 2.10 times that of CK in OCu (Fig. 1c).
The ARGs detected in compost can facilitate resistance to the major antibiotics used in humans and animals, thereby resulting in a high public health risk when employed in agriculture. The ARGs detected in this study mainly conferred resistance to three major classes of antibiotics comprising tetracycline (1.17 ⋅ 10− 2 to 3.12 ⋅ 10− 1 copies/16S rRNA), aminoglycoside (1.73 ⋅ 10− 2 to 2.01 ⋅ 10− 1 copies/16S rRNA) and MLS (1.23 ⋅ 10− 2 to 2.20 ⋅ 10− 1 copies/16S rRNA) resistance genes. The relative abundances of the general ARG types comprising tetracyclines, macrolide–lincosamide–streptogramin (MLS), and aminoglycosides in pig manure compost have been previously reported, and they have also been found in other specific niches [17, 32, 37]. Composting was highly effective at removing these three types of ARGs as well as chloramphenicol and trimethoprim resistance genes, with removal rates above 90.47% (except the removal rate for aminoglycoside resistance genes was 80.47% in OCu) (Fig. 1d). However, multidrug, rifamycin, fosfomycin, and fosmidomycin genes were enriched. Compared with CK, the addition of OTC and Cu increased the abundances of tetracycline resistance genes in the compost product by 16.55% and 41.66%, respectively, and the combined addition of OTC and Cu increased the abundance of aminoglycoside resistance genes by 1.28 times.
PCA showed that during the composting process, the samples were significantly separated at the ARG subtype level, especially in the compost treated with both OTC and Cu (Fig. 2a), possibly because they had the maximum effect on ARGs with different resistance mechanisms. Figure 2b shows the profiles for the 40 ARG subtypes during the composting process with average abundances > 1.0 ⋅ 10− 3 copies/16S rRNA. These ARGs mainly encoded resistance to tetracycline (tetL and tetW), MLS (ermB and ermC), aminoglycosides (aadA, aadE, and aph(3′′)-I), multidrug (multidrug_transporter), sulfonamide (sul1), and chloramphenicol (chloramphenicol exporter). The genes encoding multidrug (multidrug_transporter, multidrug_ABC_transporter, acrB, mexF, and ykkC), MLS (macB and mphA), and rifamycin (rifampin monooxygenase) resistance were enriched in the compost product, whereas the abundances of other genes decreased as the composting process continued compared with day 0. The removal rates of 17 ARGs exceeded 90%, i.e., seven tetracycline, five MLS, four aminoglycoside, and one chloramphenicol resistance gene. TetL is a tetracycline efflux protein found in Gram-negative and Gram-positive bacteria and it was the most abundant ARG subtype in the compost on day 0. At the end of composting, the removal rate for tetL reached 98.44–98.79%. On day 10, the addition of either OTC or Cu alone and both together increased the abundances of all aminoglycoside and tetracycline resistance genes (except for tet44 and tetX in O and OCu) compared with CK. The ARG subtypes in the compost products varied among the different treatments. For example, compared with CK, the abundances of tetracycline resistance genes comprising tetL, tetP, tetX, and tetracycline_resistance_protein tended to increase in the treatments with added OTC and Cu, and their abundances were highest when treated with OTC and Cu combined. However, compared with CK, the abundances of most MLS resistance genes (except for lnuB) decreased in the compost product under the combined treatment, where the ermB, ermA, ermC, ermF, ermX, and ermG genes mainly encode resistance to antibiotics via cell protection.
Four universal primers for two tetracycline ARGs (tetW and tetX) and two sulfonamide ARGs (sul1 and sul2) were developed in previous studies, and their prevalence was verified by qPCR. Compared with CK, the abundances of tetracycline ARGs (tetW and tetX) and sulfonamide ARGs (sul1 and sul2) tended to increase in O, Cu, and OCu in a similar manner to the results obtained using metagenomic methods. However, the relative abundances of the genes quantified by qPCR were relatively small, probably due to the great sequence diversity covered by the macrogenes, which could only amplify specific sequences or regions of the target gene [28].
Heavy metal resistome of microbes during composting
The enrichment of ARG in various environments can be caused by co-selective pressure exerted by heavy metals. In water, soil, and fertilizer environments, significant positive correlations between the concentrations of heavy metals and the abundances of ARGs have been widely reported [38–40]. In total, 41 CRGs were detected in the compost samples (Fig. 3) and the total abundance ranged from 4.95 ⋅ 10− 2 to 1.29 ⋅ 10− 1 copies/16S rRNA. Aerobic composting effectively reduced the abundances and diversity of CRGs. In total, 41 CRGs were detected on day 0 and the number decreased to 18–24 in the composting products obtained under different treatments. The total abundance of CRGs before composting was 8.02 ⋅ 10− 2 copies/16S rRNA and it decreased to 4.95–6.27 ⋅ 10− 2 copies/16S rRNA after 42 days. Since these CRGs are often associated with ARGs, it is not surprising that their abundance is reduced [41, 42]. During the composting process, the total abundances of CRGs were always higher in Cu and OCu than CK and O, and the abundance was highest in OCu. Compared with CK, the Cu treatment increased the total abundance of CRGs by 3.41% and 3.08% on day 10 and day 42, respectively, and OCu increased the total abundance of CRGs by 15.05% and 20.82% on day 10 and day 42. These results indicate that the combined effect of OTC and Cu was far greater than that of each single treatment.
In this study, copA was the most abundant CRG in the composting process. CopA encodes the ATPase in the Cu ion efflux pump system, which can excrete copper ions from the cytoplasm into the periplasmic space [43]. The abundance of copA was reduced by 27.74–40.19% in the final compost product compared with day 0. In other CRGs, the periplasmic pco system exists only on plasmids and it provides high resistance to Cu [44]. Composting was effective at removing pco genes, and the removal rates for pco genes reached 100% at the end of composting.
Occurrence and abundances of MGEs
The study also identified the presence of MGEs, including three important integrons and one transposon [45, 46] (Fig. 3), which play important roles in the transfer and acquisition of ARGs in various microorganisms via HGT. Integrons can capture exogenous ARG cassettes and then integrate them into their own gene cassettes through targeted recombination [47]. However, integron genes are defective due to their inability to move, but they are usually linked to transposons, which can serve as a vector for the transmission of genetic material [48, 49]. Aerobic composting reduced the total MGE abundances in the different treatments by 95.11–96.63%. The integrase gene intI1 was the most abundant, where its abundance ranged from 1.38 ⋅ 10− 3 to 4.09 ⋅ 10− 2 copies/16S rRNA during the composting process. Following the tnpA transposon, the abundance range ranged from 3.11 ⋅ 10− 4 to 1.25 ⋅ 10− 2 copies/16S rRNA. The application of OTC and Cu increased the abundances of intI1 and tnpA on day 10. Compared with CK, the abundance of intI1 was 97.38%, 31.99%, and 133.42% higher in O, Cu, and OCu, respectively, and that of tnpA was 56.93%, 43.55%, and 107.33% higher. However, O did not increase the abundances of intI1 and tnpA in the composting products, whereas Cu and OCu increased the abundance of tnpA by 19.07% and 25.90%, respectively. These results indicate that the presence of Cu in pig manure had a more profound impact on ARGs and it was more conducive to increasing the risk of ARGs spreading after the agricultural application of compost.
ARGs carried by HPB during composting
Composting effectively reduced the abundances of HPB in pig manure. Compared with day 0, 58.16–65.43% and 77.61–83.71% of the total HPB in compost were removed on day 10 and day 42, respectively, under different treatments. The ARGs carried by HPB comprised 37 subtypes and the variations in three ARG types mainly affected the resistance profiles of HPB during the whole composting process. Tetracycline and multidrug resistance genes were the most common ARG subtypes with 11 and 12 genes, respectively. Only seven subtypes of MLS resistance genes were detected in HPB but they were present in 9 HPB species, where macB was carried by nine HPB (Fig. 4).
ARGs were harbored by 20 different pathogenic hosts. These pathogenic hosts were dominated by bacteria such as Escherichia coli, Clostridium botulinum, Enterococcus faecium, and Corynebacterium jeikeium, which belong to Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Chlamydiae. These findings are consistent with previous studies of cow feces, which found that the ARGs originated from Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria [50]. Among the HPB that carried ARGs, Escherichia coli was the main host bacteria and it harbored the highest diversity ARGs, including genes encoding resistance to tetracycline, MLS, multidrug, fosmidomycin, quinolone, bacitracin, and chloramphenicol. A previous survey of surface water in aquaculture areas found that the frequency of resistant Escherichia coli isolates increased as the distance decreased from farms [51]. Corynebacterium is an opportunistic pathogen that settles on human skin and it has recently been implicated as the cause of various infections [52]. Corynebacterium infections are resistant to multiple antibiotics [53]. However, in the present study, adding OTC and/or Cu increased the abundance of Corynebacterium jeikeium by 11.17%, 61.78%, and 17.90% in the composting products obtained with O, Cu, and OCu, respectively, compared with CK. Enterococcus faecalis and Enterococcus faecium are increasingly important hospital pathogens worldwide, and they are mainly related to specific multi-drug resistance clone lineages in hospital environments [54]. However, other studies found that tcrB was located on a conjugating plasmid in Enterococcus faecium. The addition of the heavy metal Cu and copper tolerance may help to select for or maintain multi-drug resistant Enterococcus [55]. Enterococcus exhibited multidrug, MLS, and bacitracin resistance in the present study, and the abundances of Enterococcus faecalis and Enterococcus faecium in the compost products were 21.99% and 38.76% higher, respectively, in Cu treatment compared with CK. In addition, Pseudomonas aeruginosa is recognized as a well-known difficult to treat HPB and its efflux mechanism responsible for antibiotic resistance represents a great challenge for the treatment of human diseases [56]. In the present study, the addition of OTC and Cu increased the abundance of Pseudomonas aeruginosa in the compost products by 8.82%, 32.28%, and 2.98% in O, Cu, and OCu, respectively, and it was mainly resistant to tetracycline (tetB and tet32) and bacitracin (bcra).
Factors that affected ARG profiles during composting
The ranges of ARGs carried by the microorganisms were determined in this study. Various factors affected the abundances of ARG but the key factors related to the responses of the dominant ARGs to composting should be emphasized. RDA and SEMs were used to separate and order the factors related to changes in ARGs [57, 58]. The first two axes for HPB and CRGs were extracted by PCA before RDA because the number of environmental variables (related factors) cannot exceed the number of species variables (ARGs). The results showed that the factors considered in this study could explain 96.8% of the fate of ARGs (Fig. 5), where HPB_1, MRG_1, tnpA, intI1, and intI2 were the main factors that influenced the variations in ARGs, where they explained 19.10%, 19.03%, 18.89%, 18.09%, and 17.54% of the variations in the ARG profiles, respectively. Microorganisms are carriers of genes so the reductions in the abundances of HPB during composting contributed to the reductions in the abundances of ARGs. However, the increases in the abundances of potential pathogens after composting were concerning, such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Bordetella bronchiseptica. In this study, the ARGs in these HPB encoded tetracycline, bacitracin, and multidrug resistance.
SEMs were used to assess the direct and indirect effects of the five main drivers (HPB_1, MRG_1, tnpA, intI1, and intI2) on three major antibiotic resistance profiles (Fig. 6). HPB was the most important positive factor for shaping the ARG profiles. HPB can impose an indirect effect on ARGs by strongly affecting the abundance of MGEs and MRGs in the composting process. MGEs had strong and direct impacts on the abundances of ARGs in the composting process. HPB_1 had the highest standardized total effect on the ARG profiles (0.98–0.99), followed by intI1 (0.78–0.85). In addition, the co-selective effect of MRGs on ARGs cannot be ignored. In particular, MRGs significantly increased the abundance of ARGs by positively affecting intI1. Similar results were obtained in previous studies, where Rosewarne et al. [59] and Wright et al. [60] found intI1 abundance was significantly increased due to the presence of heavy metals.