Zinc-Driven Antibiotic Resistance Gene Dynamics During Vermicomposting: Insights into Co-Contamination Mitigation for Sustainable Manure Management

TL;DR

This study explores how zinc affects antibiotic resistance genes during vermicomposting, offering insights into reducing their spread in livestock manure.

Contribution

The study reveals zinc's dual role in both reducing and promoting antibiotic resistance genes during vermicomposting.

Findings

Vermicomposting reduced total ARGs by 88.62% over 60 days.

Low zinc levels increased certain ARGs, while high zinc suppressed others but increased horizontal gene transfer.

Vermicomposting outperformed natural composting in reducing ARGs under zinc stress.

Abstract

Background: The coexistence of antibiotic resistance genes (ARGs) and heavy metals in livestock manure poses critical challenges to vermicomposting technology. Objectives: This study aimed to clarify the zinc (Zn)-driven ARG dynamics over 60-day vermicomposting for livestock manure and provide a reference for taking appropriate measures to reduce the spread of ARGs in the environment. Methods: In a vermicomposting system using Eisenia fetida and treated with varying concentrations of Zn, high-throughput sequencing was employed to analyze microbial succession, while quantitative real-time PCR (qPCR) was performed to track the fluctuation patterns of ARG (tet-, erm-, qnr-, str-, chl-, bla-, mcr-ARGs) and mobile genetic element (MGE, intI1 and intI2) abundances over the 60-day treatment period. Results: Generally, sul- (10−3–10−1 copies/16S rRNA), tet- (10−3–10−2 copies/16S rRNA), and str-ARGs (10−3–10−2 copies/16S rRNA) are dominant in dairy manure. Vermicomposting significantly reduced total ARGs (88.62% removal), but Zn stress triggered concentration-dependent shifts. Low Zn (100–250 mg/kg) elevated tet-, erm-, and chl-ARGs via co-selective pressure and disrupted bacterial succession, while high Zn (500–1000 mg/kg) suppressed qnr- and mcr-ARGs but intensified horizontal transfer via cross-resistance. Conclusions: Vermicomposting maintained a greater ARG removal capacity across the Zn gradient (100–1000 mg/kg) than natural composting, proving an effective approach for reducing the threat of antibiotic resistance in bacteria even under high Zn stress. The link between Zn residues and the increased ARG dissemination risks underscores the challenge of co-contaminants, providing essential insights for developing vermicomposting strategies to mitigate ARG risks and ensure sustainable manure management.

Figures

Full text

1. Introduction

In recent years, emerging pollutants have emerged alongside the rapid development of the livestock and poultry breeding industry towards intensification, generating significant environmental pressure and threatening ecological security [1,2]. Particularly, antibiotic resistance genes (ARGs) and heavy metal contamination in livestock and poultry manure have already attracted global attention in agricultural and environmental fields [3,4]. In the breeding process, antibiotics were originally employed to treat inflammatory conditions and prevent infectious diseases [5,6]. However, due to the limited metabolic capacity of animals, substantial amounts of unmetabolized antibiotics and their derived ARGs are ultimately excreted with feces [7], which results in the dissemination of bacterial resistance—particularly via horizontal gene transfer (HGT) [8]. The addition of heavy metals, such as zinc (Zn), copper (Cu), and arsenic (As), to feeds has shown positive effects on the growth and disease prevention of livestock [9]. Nevertheless, heavy metals cannot be degraded like conventional agents. About 60–90% of them are excreted from animals’ gut along with feces, threatening human survival under coexistence conditions with ARGs in the environment [10,11].

Vermicomposting has appeared as a promising technology for resource utilization of livestock manure, converting organic waste into valuable products [12], while decomposing antibiotic-resistant bacteria (ARBs) and ARGs with the enzymes and microorganisms in the earthworm gut [13]. Li et al. [14] reported that earthworms’ participation in cow manure composting led to a significant reduction in the concentrations of tetracycline ARGs (tet-ARGs), β-lactam ARGs (bla-ARGs), and quinolone ARGs (qnr-ARGs) even under high tetracycline stress. Vermicomposting could also reduce the abundance of potential live hosts for ARGs by altering the bio-community structure, diminishing the types and sequences of plasmids and integrons, and thereby lowering the potential risk of ARG spread [15,16]. However, due to the high content and rich types of ARGs in livestock manure, the selective removal of ARGs by vermicomposting leaves a certain amount of ARGs in compost products and earthworm casts, which poses ecological risks for subsequent utilization. For example, qnr-ARGs in sewage sludge could be significantly removed by vermicomposting, while the removal effect on tet-ARGs was relatively weak [17]. A similar phenomenon was observed by Tian et al. [18], that the abundance of blaampc and fexA in cow dung was upgraded after vermicomposting treatment, indicating that the migration and transformation of ARGs in vermicomposting are closely related to process conditions and antibiotic types.

Heavy metals could promote the evolution of metal resistance genes (MRGs), exerting persistent selective pressure on bacteria through co-resistance, cross-resistance, and co-regulation mechanisms. Specifically, co-resistance refers to the localization of both ARGs and metal resistance genes (MRGs) on the same genetic element (e.g., plasmids, transposons, or integrons). Cross-resistance occurs when microorganisms employ a single resistance system to confer tolerance to both heavy metals and antibiotics with efflux pump systems being the most prevalent example. The abundance of ARGs in feces was found to be significantly correlated with the concentrations of arsenic and copper, especially under the combined contamination with antibiotics [19]. Co-regulation involves the transcriptional and translational responses activated in microorganisms upon exposure to antibiotics or heavy metals, which modulate the expression of resistance-associated genes. For instance, exposure to As and tetracycline resulted in a more selective and repressive transcriptional profile, causing a slight upregulation of tet-ARGs [20]. Additionally, some metal ions have been proven to stimulate cells to produce reactive oxygen species (ROS) and promote the HGT of ARGs by increasing cell membrane permeability, so that the transfer process could be completed even at low metal concentrations [21]. Therefore, it is of great significance to explore whether the fate of ARGs in the vermicomposting process could be influenced by the coexisting heavy metals in cow manure.

In this study, a vermicomposting system was constructed with varying concentrations of Zn to explore the fate of ARGs and mobile genetic elements (MGEs) in cow manure with 60 days treatment. Quantitative real-time PCR (qPCR) technology was conducted to track the fluctuation patterns of ARG abundance during the composting process, while the succession in bacterial community was investigated via high-throughput sequencing. Physicochemical properties of manure substrates, including total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), pH, conductivity, and moisture content, were determined to evaluate the driving factors for the changes in ARGs, and the evolution of ARGs hosts was further discovered through co-occurrence network analysis. This study aims to reveal the mechanism by which heavy metal zinc affects ARGs during the composting treatment of livestock manure by earthworms, and to provide a reference for taking appropriate measures to reduce the spread of ARGs in the environment.

2. Results and Discussion

2.1. Diversity and Abundance of ARGs and MGEs in Vermicomposting

At different vermicomposting stages, a total of 23 subtypes of ARGs and 2 MGEs were detected, with their relative abundance profiles across treatments and time points detailed in Figure 1. The sul-ARGs (including sul1 and sul2) were consistently among the most abundant gene classes throughout the vermicomposting process, with relative abundances ranging from 10^−3^ copies/16S rRNA copies to 10^−1^ copies/16S rRNA copies across all groups. Owing to the broad-spectrum antibacterial activity of sulfonamides [22], the pervasive presence and high initial abundance of sul-ARGs are consistent with the widespread use of sulfonamides in dairy farming [23], which leads to significant residual contamination of corresponding ARGs in manure [24]. Both subtypes were detected in all treatments, indicating their pervasive presence in dairy manure. Their abundance showed a general declining trend over time in vermicomposting treatments, whereas in the control (CK) group, levels remained more stable. The tet-ARGs exhibited the highest diversity among the detected classes, encompassing six subtypes: tetL, tetM, tetO, tetQ, tetX, tetW. Their relative abundances spanned a wide range from 10^−3^ copies/16S rRNA copies to 10^−1^ copies/16S rRNA copies. The high level and diversity of tet-ARGs reflect the extensive application and subsequent residual contamination of tetracycline antibiotics in intensive farming. Notably, tet-ARGs with different resistance mechanisms, especially tetL (efflux pumps, 10^−5^–10^−4^ copies/16S rRNA copies) and tetX (enzymatic modification, 10^−3^–10^−2^ copies/16S rRNA copies) [25], maintained relatively high abundance at multiple time points, which is related to the vast diversity of host bacteria present in cow manure. The dynamic patterns of tet-ARGs varied by subtype, for instance, tetM and tetW showed a more pronounced decrease over time in earthworm-treated groups compared to the CK group, suggesting the potential of vermicomposting in reducing the residue of ARGs in livestock manure. The str-ARGs, represented by strA, strB, and aadA, were also highly abundant, with levels between 10^−3^ copies/16S rRNA copies to 10^−2^ copies/16S rRNA copies. All three subtypes were consistently detected. The aadA gene often exists on integrons, encoding aminoglycoside adenylate transferase to protect protein synthesis of bacteria [26], and thus its abundance follows a similar trend to that of MGEs. Gene strA and strB are frequently located on plasmids or transposons of Gram-negative bacteria (e.g., Escherichia coli) and are easily transferred through HGT [27], which may contribute to their persistence in the compost. Additionally, strA and strB have been proven to be frequently co-expressed, leading to a significant synergy, which may enhance the bacterial resistance to streptomycin.

Although most quinolones (e.g., lomefloxacin, pefloxacin, ofloxacin, and norfloxacin) have been banned by regulatory authorities in livestock farming, individual drugs within this class, such as enrofloxacin and ciprofloxacin, are still widely used in this field, leading to the spread of bacterial resistance from livestock manure to the environment [28]. The qnr-ARGs were mainly detected in the CK group (10^−6^–10^−4^ copies/16S rRNA copies), among which qnrS was the predominant subtype. The qnrB encodes quinolone-resistance protein OqxB, which could pump out many bacteriostats (e.g., chloramphenicol, tetracycline) despite quinolones, resulting in multiple resistance. The chl-ARGs, represented by cfr and fexA, were detected at relatively low levels in vermicomposting groups (10^−6^–10^−4^ copies/16S rRNA copies), but abundant in natural composting system. Gene cfr is of particular concern as it encodes a methyltransferase that confers cross-resistance to multiple, critically important antibiotic classes (e.g., lincosamides, oxazolidinones, and pleuromutilins), posing a significant public health risk [29]. The fexA, encoding a chloramphenicol-specific efflux pump, also presents a transmission risk through the food chain despite its narrower resistance profile [30]. As for bla-ARGs, the expression of the blaampC is generally low under normal conditions (10^−5^–10^−4^ copies/16S rRNA copies), while plasmid-borne blaampC is prone to overexpression, leading to enhanced resistance. The blaNDM gene, which encodes metallo-β-lactamases, is a World Health Organization (WHO) critical priority resistance gene as it confers resistance to nearly all β-lactam antibiotics, including carbapenems [31]. The metallo-β-lactamases-producing bacteria (e.g., E. coli, Acinetobacter baumannii) have caused global outbreaks, with livestock manure identified as a key reservoir [32]. Its enzymatic activity requires Zn ions at its active site, probably leading to a higher abundance of blaNDM in Zn-treated groups compared to the control group [33]. Recently, with the emergence and prevalence of carbapenem-resistant gram-negative organisms (CROs), clinical practice has frequently faced a dilemma of limited effective antibiotics [34]. Polymyxins, which exhibit therapeutic efficacy against nearly all CROs, serve as the last line of defense against multidrug-resistant infections [35]. Currently, polymyxin resistance mediated by the plasmid-borne mcr-1 gene has shown a trend of rapid global spread and has been classified as an extremely high-priority threat. Since Chinese scientists first identified bacteria carrying the mcr-1 gene in both livestock and humans in 2015 [36], reports of polymyxin resistance associated with mcr-1 have been documented in more than 50 countries worldwide. In the present study, the detection levels of mcr-1 (10^0^–10^1^ copies/g DNA) in all compost groups were lower than those of other ARGs (Figure S1). However, it remains imperative to strengthen the monitoring, prevention, and control of polymyxin resistance in the application and management of agricultural waste.

Even after vermicomposting, the ARGs remained diverse in cattle manure. Results of qPCR analysis revealed that the MGEs (intI1, intI2) were ubiquitously present in compost samples, with comparable abundance in both the CK group and the Zn-treated groups. As carriers of ARGs, the MGEs facilitate the inhalation and integration of exogenous ARGs into host bacterial genomes through HGT such as conjugation, transduction, and transformation [37]. Microorganisms persistently coexisting with ARGs and MGEs in earthworm guts may evolve antibiotic resistance over time, which is subsequently disseminated into the environment with earthworm excretion.

2.2. Zn-Dependent ARG Reduction in Cow Manure by Vermicomposting

During the composting process, the total abundance of ARGs in cow manure was effectively reduced (Figure 2). In the CK group, the total ARG abundance decreased from 1.91 × 10^−1^ copies/16S rRNA copies to 1.11 × 10^−1^ copies/16S rRNA copies over time. Specifically, tet-ARGs decreased by 75.82%, erm-ARGs by 92.24%, while sul-ARGs showed a smaller reduction (24.32%). In livestock and poultry manure, the persistent presence of antibiotics imposes selective pressure that may enhance bacterial resistance to the agents, making it a “repository” for ARGs. The composting treatment of cow manure mainly relies on the abundant microorganisms in the matrix to promote the transformation of biodegradable organic matter into stable humus. This method effectively removes the residual antibiotics in animal feces and reduces the microbial exposure, thereby substantially weakening the antibiotic resistance.

Earthworm intervention further reduced the residual levels of ARGs in cow manure. In the CQ group, the total ARG abundance sharply declined from 1.81 × 10^−1^ copies/16S rRNA copies to 2.06 × 10^−2^ copies/16S rRNA copies. Earthworm guts host abundant endogenous bacteria, which were released with the casts, significantly enriching the microbial diversity of the compost substrate. It has been proved that aerobic and anaerobic bacterial counts in earthworm intestine (per gram dry weight) are approximately 12–40 times and 10–4000 times higher than natural soil [38]. These bacteria play critical roles in degrading organic pollutants, antibiotics, and even detoxifying heavy metals in cow manure.

The reduction of ARGs in cow manure by earthworm composting is selective, resulting in a high level of residual ARGs in worm manure, particularly under Zn stress. The qPCR analysis at day 60 revealed that as Zn concentrations increased from 0 mg/kg to 1000 mg/kg, the total ARG abundance in manure fluctuated from 2.06 × 10^−2^ copies/16S rRNA copies to 4.21 × 10^−2^ copies/16S rRNA copies. Heavy metals were considered to exert selective pressure on bacteria, and their coexistence with antibiotics may induce cross-resistance. Bacteria surviving in high-metal environments could evolve adaptive traits, synthesizing metal ion efflux pumps, which can also efflux antibiotics out of the cell [39]. Previous studies have isolated metal-resistant endogenous bacteria from earthworm guts [40]. These bacteria could mitigate the toxicity of heavy metals through their metabolism system, and the MRGs were often detected in plasmids carrying ARGs. In natural ecosystems with severe heavy metal contamination, bacteria generally exhibit heightened antibiotic resistance even at low antibiotic exposure levels.

The trends in the abundance of homologous ARGs varied significantly under different treatment conditions (Figure 3 and Figure S1). For sul-ARGs, relative abundance in the CK group initially increased, peaking at 1.18 × 10^−1^ copies/16S rRNA copies on day 14, followed by a decline and stabilization at 5.80 × 10^−2^ copies/16S rRNA copies (Figure 3a). In contrast, under vermicomposting conditions, sul-ARGs exhibited a continuous decline. After 60 days of composting, their total abundance dropped to 8.62 × 10^−3^~1.86 × 10^−2^ copies/16S rRNA copies, significantly lower than in the CK group. Similarly, the total abundance of str-ARGs under natural compost condition initially rose, reaching a peak of 7.40 × 10^−2^ copies/16S rRNA copies on day 7, then gradually decreased (Figure 3b). It is worth noting that high concentrations of Zn (1000 mg/kg) resulted in an increase in those two types of ARGs at the end of composting compared with the CQ group. Unlike antibiotics and other organic compounds, the heavy metals persist and exert long-term selective pressure on microorganisms. Under 1000 mg/kg Zn stress, the reduction of MGEs (intI1 and intI2) in cow manure by vermicompost was less pronounced than that in low-Zn groups (Figure 3i). The abundance of MGEs in this group reached 6.54 × 10^−4^ copies/16S rRNA copies by day 60, significantly higher than that in the other groups, suggesting that sul- and str-ARGs may rely more on MGEs for HGT, contributing to their prolonged persistence in manure.

Vermicomposting also demonstrated excellent removal efficiency for tet-, erm-, and chl-ARGs (Figure 3c–e), among which the reduction effect on erm-ARGs was the most significant with the abundance decreasing from 4.94 to 5.57 × 10^−3^ copies/16S rRNA copies to 4.18–9.72 × 10^−4^ copies/16S rRNA copies within just 7 days (Figure 3d). However, in later stages, the abundance of those three ARGs in low Zn concentrations (125–250 mg/kg) was significantly higher than that in the CQ group, while the residual amount of ARGs decreased at high Zn treatment (1000 mg/kg). The sub-inhibitory content or ambient concentrations of heavy metals were proven to promote the conjugative transfer of ARGs, while the excessive concentrations may cause serious cell damage, death, or reduction in plasmid, thereby limiting the dissemination of ARGs [41]. Nevertheless, the ecological and health risks of excessive heavy metal residue in compost products cannot be ignored.

Not all ARGs can be removed through vermicomposting. For instance, qnr-ARGs were partially removed during early composting, but their abundance rebounded over time (Figure 3g). A similar pattern was observed for mcr-ARGs (Figure 3h), where Zn with 0–250 mg/kg resulted in a gradual increase in their abundance at a relatively lower level, and even exceeded the residual amount in the CK group (1.25 × 10^−7^ copies/16S rRNA copies). This may reflect the complex interactions between earthworm gut microbiomes and manure microbial communities. Overall, despite enhanced ARG expression in vermicompost under high Zn stress, their total abundance remained significantly lower than in naturally composted cow manure, thus demonstrating vermicomposting’s effectiveness in reducing ARGs spreading to the environment.

2.3. Perturbation of Zn on Microbial Communities in Vermicomposting

The impact of vermicomposting on ARGs in cow manure was primarily mediated through its effects on host microorganisms. Microorganisms play a crucial role in driving manure humification. However, since these microorganisms also act as ARG carriers, the succession in microbial community structure would directly influence ARG abundance in compost products. To elucidate bacterial community succession patterns under Zn stress during vermicomposting, high-throughput sequencing of 16S rRNA genes was conducted for the analysis of the bacterial communities (Figure 4). Alpha diversity indices (Chao1, Shannon, and Simpson) were calculated to assess microbial community richness and diversity across composting conditions (Figure 4a). Compared to the CK group, vermicomposting significantly enhanced both species richness and diversity, with the Chao index increasing from 1188 to 1214, and the Shannon index rose from 5.34 to 5.71. Concurrently, species evenness decreased, as indicated by a decline in the Simpson index from 0.0161 to 0.0087. Interactions between earthworm gut microbiota and native manure microbes played a critical role in organic matter decomposition, consolidating the dominance of specific bacterial taxa and driving significant divergence in community structure from the CK group during composting (Figure 4b).

Low Zn concentrations (125–250 mg/kg) further increased community richness and diversity, whereas higher Zn levels (500–1000 mg/kg) suppressed microbial activity and reduced species diversity due to severe toxicity [42]. Heavy metals inhibit the activity of certain bacteria within the community, leading to their decline or death, which is the same as the result of a previous study [43]. Species that survive under such stress may evolve dual resistance to Zn and antibiotics, thereby increasing the residual abundance of ARGs in cow manure. Notably, Zn treatment did not fundamentally restructure bacterial communities; instead, all the vermicomposting groups exhibited significant divergence from the CK group, yet maintained structural similarity among themselves.

Changes in the composition of the bacterial community under different treatments were further analyzed as shown in Figure 5. At the phylum level, Proteobacteria, Actinobacteriota, Bacteroidota, Chloroflexi, Firmicutes, and Deinococcota were the dominant phyla during the composting process, collectively accounting for 74.3–93.7% of the total bacterial community [14]. Among all those treatment groups, Proteobacteria remained the most dominant phylum, with its highest relative abundance observed in the CK group (31.18%), slightly higher than that of the vermicomposting groups. Zn stress caused a modest decline in Proteobacteria abundance, though its dominance remained unchallenged. Proteobacteria are widely distributed in soil, aquatic systems, and extreme environments, with certain members participating in nitrogen cycling and organic matter decomposition in animal manure [43]. This phylum has been proved to evolve abundant heavy metal resistance genes, leveraging metal efflux pumps, ion channels, and redox reactions to mitigate metal toxicity. The prominent DNA repair feature also helped it maintain a stable position under Zn stress. Bacteroidota, another key phylum in manure bacterial community, comprises species adept at degrading starch, pectin, and hemicellulose in manure, producing short-chain fatty acids (e.g., propionate, acetate) to optimize carbon metabolism pathway during composting. However, Bacteroidota are sensitive to heavy metals. High concentrations of heavy metals may impair its polysaccharide degradation capacity and interfere with the manure maturation process [44]. The Actinobacteriota phylum thrived in later composting stages as the second-most abundant phylum for decomposing cellulose, lignin, and chitin in cow manure. This phylum excels in decomposing cellulose, lignin, and chitin. Their inherent metal tolerance allowed stable persistence under Zn stress. The heavy metal-resistant species are often detected in this phylum [45], which allows them to maintain a stable proportion under Zn stress. Although the abundance of the Firmicutes phylum declined under vermicomposting, certain species (e.g., Bacillus) exhibited resilience to Zn toxicity by rapidly consuming labile carbon sources and thereby indirectly maintaining their competitive advantage than other metal-sensitive phyla even under 1000 mg/kg Zn treatment with a relative abundance of 8.80%. Notably, the abundance of Chloroflexi markedly increased with composting time, which was mainly participating in decomposing cellulose and lipids in cow manure. Under heavy metal stress, Chloroflexi demonstrated unique survival strengths by adsorbing metal ions and reducing them to less-toxic valence states [46]. Low metal concentrations promoted the enrichment of tolerant strains, inadvertently fostering species with dual resistance and elevating ARG persistence in manure. The sublethal concentration of heavy metals could promote the accumulation of bacteria with dual resistance to be the dominant species in the community, resulting in the higher residual level of ARGs in compost products (Figure 2).

The structure of the bacterial community also significantly changed at the genus level (Figure 5c). Under different composting conditions, the dominant bacterial genera in the community were similar. The top three most abundant species were Truepera, norank_f_Xanthomonadaceae, and norank_f_JG30-KF-CM45, all of which were shared between natural composting (CK) and vermicomposting systems (Figure 5b). However, composting conditions significantly influenced community structure. The bacterium, such as Paeniclostridium, Pelagibacterium, Muricauda, and Luteimonas, were predominantly detected in naturally composted manure but rarely observed in vermicomposting groups. Conversely, norank_f_norank_o_Actinomarinales, norank_f_Microscillaceae, Ilumatobacter, norank_f_norank_o_norank_c_KD4-96, and Demequina were primarily found in the substrate treated under earthworm’s participation.

Truepera, a member of the Deinococcota phylum, thrives during the thermophilic phase of composting (50–65 °C). It participates in the degradation of complex organic matter (e.g., cellulose, lignin) and is closely associated with nitrification and nitrogen fixation in manure [47], exhibiting a positive correlation with TN content (Figure 5c). As shown in Figure 5d, its relative abundance also significantly correlated with the content of organic matter (p = 0.013), yet the Zn stress inhibited its activity. Similarly, the abundances of norank_f_Xanthomonadaceae, Romboutsia, unclassified_f_Rhodobacteraceae, Paeniclostridium, Pelagibacterium, Membranicola, and Fermentimonas declined over time. These genera primarily contributed to the decomposition of carbohydrate, lipid, and polysaccharide during early composting stages. The genus Paeniclostridium potentially facilitated the release of ions (e.g., K^+^, Na^+^), correlating positively with manure electrical conductivity [48]. Romboutsia, unclassified_f_Rhodobacteraceae, and Pelagibacterium are closely related to the nitrogen cycle in manure, mainly acting in the denitrification reaction, potentially mitigating nitrate accumulation but increasing nitrogen loss [49].

There were also some species that accumulated during the later stages of composting. For instance, as a typical denitrifying bacterium, norank_f_norank_o_Actinomarinales was identified as another contributor to nitrogen loss in compost products [50]. In contrast, norank_f_norank_o_norank_c_KD4-96, while also a denitrifier, demonstrated a robust capacity to metabolize recalcitrant organic compounds [51]. Consistent with previous studies, Ilumatobacter showed a significant negative correlation with organic matter content in cow manure [52]. Additionally, unclassified_f_Flavobacteriaceae, Demequina, and Altererythrobacter thrived in low-to-moderate temperature environments, becoming particularly active in the maturation phase, where they further promoted the humification degree.

With increasing Zn stress, the abundance of certain species declined significantly, including Truepera, norank_f_Xanthomonadaceae, Romboutsia, Paeniclostridium, Pelagibacterium, and Membranicola. Most of the nitrogen and phosphorus in the substrate were significantly increased after vermicomposting compared to the initial cow manure, resulting from the enrichment effect of the transformation by earthworms. As shown in Figure 5d, the Zn stress showed no significant impact on nitrogen-fixing microorganisms, but it likely inhibited the activity of specific bacteria involved in phosphorus fixing and organic phosphorus mineralization. For instance, the abundance of Membranicola exhibited a positive correlation with TP content in the manure, suggesting its functional role in phosphorus cycling. On the contrary, the taxa such as norank_f_JG30-KF-CM45, Ilumatobacter, norank_f_norank_o_Actinomarinales, norank_f_Microscillaceae, norank_f_norank_o_norank_c_KD4-96, Demequina, norank_f_Vicinamibacteraceae, and Chryseolinea became enriched under high Zn stress, with most of these species predominantly occurring in the final composting stages. Species surviving long-term heavy metal contamination would evolve outstanding metal tolerance. For example, Microscillaceae sp. has been demonstrated to decompose carbohydrates through several metabolic pathways, such as sulfur metabolism, glycolysis, and pyruvate metabolism [53]. Under cadmium exposure, its abundance in earthworm guts increased by 3.56% compared to controls [54]. Bacteria resist heavy metal toxicity through efflux pump systems, transformation, intracellular sequestration, and extracellular binding. some of which also confer antibiotic resistance. This overlap facilitates the emergence of dual-resistance genes, rendering these species potential vectors for resistance.

2.4. Zn-Mediated Selection of ARGs via Microbial and Environmental Interactions

In order to further reveal the mechanism by which Zn affects the persistence of ARGs during vermicomposting, the Mantel test was conducted in this study to analyze the correlation between environmental factors (bacterial diversity, physicochemical properties of cow manure, and Zn content) and the abundance of ARGs, as shown in Figure 6a. The Zn content demonstrates a significant impact on the abundance of some ARGs, such as sul2, tetX, oqxB, qnrB, and cfr. Especially under high concentrations of Zn stress, bacteria may maintain their resistance to antibiotics by enhancing the expression of such ARGs. Meanwhile, the variations in the physicochemical properties of manure substrates also affect the occurrence level of ARGs, especially sul- and chl-ARGs.

The RDA was further conducted on the linkage between ARGs, MGEs, and manure physicochemical properties (Figure 6b and Figure S2). Totally 83.38% of the ARG changes were explained by RDA1 and RDA2. For sul-, tet-, and str-ARGs, which have the highest pollution levels during the vermicomposting process, their abundances were mainly affected by the conductivity, pH, and organic matter content of manure substrates with significant positive correlations. The organic matter was decomposed through the synergistic effect between the microorganisms in earthworm intestines and cow manure. Under the influence of high concentrations of the heavy metal Zn, some bacteria have evolved stronger antibiotic resistance to maintain their key role in the maturation process. The moisture content was positively correlated with the abundances of sul1, tetO, tetM, tetQ, tetX, tetW, blaNDM, ermB, and aadA. At the same time, it also significantly affected the horizontal transfer of ARGs and increased the residual levels of intI1 and intI2. Therefore, in the actual breeding process, it is best to reduce the moisture content in the manure substrate under the condition of ensuring the survival of earthworms to inhibit the accumulation and transformation of such ARGs. Zn is positively correlated with mcr-1, tetW, and blaNDM, indicating that the presence of Zn is a key factor causing the increase in the residual levels of these ARGs. This result is consistent with previous studies that heavy metals can exert continuous selective pressure on ARGs because they are difficult to degrade in the environment. The abundance of ARGs in the environment would be increased by co-selection and cross-selection effects of antibiotics and heavy metals [55]. Therefore, if the heavy metals accumulated in cow manure are not treated, the commercial organic fertilizer produced from the composting process will inevitably affect the health of humans through the food chain when applied. The succession of microbial community structure and function, especially the proportion of species involved in the nitrogen–phosphorus conversion process, also affected the conversion of ARGs in compost products, resulting in certain correlations between ARG abundance and contents of TN and TP. Additionally, the change in pH value may affect the self-replication of DNA within bacterial cells [56]. After 60 days of vermicomposting treatment, the pH of the substrate showed a downward trend. The abundance of most ARGs was affected and decreased, while the expressions of genes such as qnrB, qnrS, blaampC, and ermC were negatively correlated with it, resulting in the residual bacterial resistance in the later composting stage.

2.5. Co-Occurrence Network Analysis of ARGs and Bacterial Hosts Under Zn Stress

Microorganisms serve as the carriers of ARGs. The alteration in bacterial diversity would influence the persistence of ARGs in manure substrates, while the succession in community structure would further drive changes in the fate of ARGs [11]. Based on Spearman’s rank correlation coefficient, the relationships between bacterial genera, ARGs, and MGEs in manure substrates were demonstrated using the co-occurrence network to infer the shift in potential ARG host under varying composting conditions Figure 7. The threshold of r ≥ 0.8 was selected to identify strong and potentially ecologically relevant associations between ARGs and bacterial taxa, while minimizing the inclusion of weak or spurious correlations that could obscure interpretable network patterns. This stringent criterion is commonly employed in microbial co-occurrence network studies to ensure biological relevance of the inferred connections [57,58]. The co-occurrence network in vermicomposting groups exhibited greater complexity than that in the natural composting group, with 164 and 200 positively correlated edges identified between ARGs and bacteria, respectively (Table S3). Earthworms inevitably introduce gut microorganisms into the substrate during their metabolism, which is associated with changes in microbial community composition and functionality [14,59]. Their feeding and activities also lead to substrate homogenization, which co-occurred with a lower diversity of potential ARG host bacteria and a reduction in ARG persistence in composting products based on network inference. Notably, the ARGs such as tetL, tetW, ermC, oqxB, qnrS, qnrB, cfr, blaampC, blaTEM-1, and blaNDM showed no significant host associations, proving that vermicomposting is an effective way to reduce ARGs in the resource utilization treatment of cow manure.

At Zn concentrations of 100–250 mg/kg, the co-occurrence network exhibited greater complexity compared to the CQ group, with the number of positive correlations increasing to 286. Over prolonged composting, certain species evolved dual resistance to antibiotics and heavy metals, with confirmed ARGs with definite hosts from 15 (CQ) to 20 (T1). Resistance re-emerged for tetW, ermC, oqxB, qnrS, qnrB, and blaTEM-1, linked to 1, 2, 2, 1, 7, and 13 host species, respectively. Conversely, under high Zn concentrations (≥500 mg/kg), the co-occurrence network showed a decrease in the diversity of bacterial nodes (genera) linked to ARGs, likely due to irreversible impacts of Zn on metabolic activity and cell structure of functional microbes. Some species with relatively high proportions gradually evolved multi-drug resistance (e.g., Truepera, norank_f_Xanthomonadaceae, and Romboutsia), enabling their ecological dominance under harsh conditions. The number of bacterial genera showing strong co-occurrence with MGEs was higher under 1000 mg/kg Zn (T4) than in the CQ group (39 vs. 28). This pattern suggests a potential enhancement of horizontal gene transfer (HGT) under high Zn stress, which may contribute to ARG dissemination and persistence in composting products.

2.6. Responses of ARG to Different Composting Factors

Structure equation model (SEM) analysis revealed the effects of earthworm activity and zinc stress on compost characteristics, bacterial community structure and MGEs, as well as the interference pathway on the abundance of ARGs in the compost substrate (Figure 8). The SEM explained the variance of 77% of ARGs in the composts (Figure 8a). Compared with natural composting, earthworm activities have a certain positive reshaping effect on the bacterial community structure (0.348). During the plowing and feeding processes of earthworms in cow manure, the microenvironment of the substrate was gradually optimized, which had a significant and direct negative effect (−0.355) on the physicochemical properties (e.g., pH, moisture content, electrical conductivity). This promoted the improvement in bacterial diversity and functional differentiation (−0.416), and even enriched some organic matter degradation species such as Truepera. In addition, bacterial diversity negatively and remarkably affected MGEs (−0.559), with the total abundance of intI1 and intI2 in the CQ group was 50.96% lower than that in the CK group, which indirectly reduced the HGT risk of ARGs. The addition of Zn showed a positive impact on the content of nutrients (e.g., TN, TP, and TOC) in the compost (0.068), while causing toxic inhibition of the activity of certain functional microorganisms and had a negative impact on species diversity (−0.063) at higher levels (500–1000 mg/kg). The surviving species had the opportunity to evolve co-resistance to antibiotics and Zn, and directly promote the spread of ARGs in the bacterial community through HGT.

Earthworm activities and Zn did not act independently but rather influenced the decomposition or diffusion of ARGs through the synergistic regulation of the microenvironment and the bacterial community structure. During the vermicomposting process, Zn showed a significant dose-effect on ARGs, while the activities of earthworms can exert a certain abirritation on the Zn stress by optimizing physicochemical properties and community structure, making vermicompost an outstanding measure in the application of controlling the spread of antibiotic resistance in livestock manure than traditional natural compost.

3. Materials and Methods

3.1. Experimental Materials

The cow manure used in vermicomposting was collected from a dairy farm in Tianjin. Before use, the moisture content of cow manure was first determined via the vacuum oven method (GB/T 8576-2010). Subsequently, all the manure was thoroughly mixed with distilled water to uniformly adjust its initial moisture content to 70 ± 1% for subsequent composting experiments. Eisenia fetida, which is globally recognized as one of the most efficient and widely used earthworm species for the vermicomposting of livestock manure and other organic wastes [14], was employed in the present study, with an average individual weight of 0.5 ± 0.2 g. The earthworms with obvious clitellum were selected after 30 days of domestication with cow manure. After cleaning the surface of those earthworms with sterile water, they were placed in a clean beaker with wet filter paper and kept in a dark environment for 24 h to empty their intestinal contents.

3.2. Experimental Design and Sample Collection

After being naturally composted for 15 days, the cow manure was provided for earthworms to inhabit and undergo vermicomposting treatment. Based on the actual residual level of the heavy metal Zn in the cow manure [60,61], six groups were established and labeled as follows: CK (control group with natural composting without earthworms), CQ (vermicomposting), T1 (vermicomposting + 125 mg/kg Zn), T2 (vermicomposting + 250 mg/kg Zn), T3 (vermicomposting + 500 mg/kg Zn), and T4 (vermicomposting + 1000 mg/kg Zn). Each treatment was replicated three times.

After 15-day natural compost, the initial physicochemical properties of cow manure are shown in Table S1. ZnCl_2_ was added to different treatment groups to achieve Zn concentrations of 125, 250, 500, and 1000 mg/kg in the manure substrate. The thoroughly mixed manure was then portioned into 200 g aliquots and placed into plastic cultivation boxes (with a bottom diameter of 11 cm, top diameter of 15 cm, and height of 7.5 cm). Except for the CK group, 30 earthworms were evenly distributed onto the manure substrate in each group. The cultivation boxes were wrapped with aluminum foil to shield them from light and covered with three layers of gauze to ensure ventilation and prevent the earthworms from escaping. To maintain suitable humidity, sterile water was sprayed every two days based on weight measurements. On days 1, 7, 14, 28, 42, and 60 of the experiment, compost samples were randomly collected from three distinct points in each cultivation box. To ensure homogenization, the collected samples were freeze-dried and ground to 1 mm with a low-temperature grinder, then stored at −80 °C for subsequent analysis.

3.3. DNA Extraction

At each time point, 0.5 g of the compost sample was weighed. The Fast DNA SPIN Kit for Soil (MP Biomedicals, LLC, Santa Ana, CA, USA) was used according to the instructions for DNA extraction. Subsequently, the extracted DNA samples were temporarily stored in a −20 °C freezer for subsequent high-throughput sequencing and ARG quantification.

3.4. Quantification of ARGs and MGEs

Quantitative real-time polymerase chain reaction (qPCR) was performed on the 7500 Real-Time PCR instrument (7500, Applied Biosystems, CA, USA) using specific primers (Table S2) to quantify the existence levels of ARGs and 16S rRNA gene in different treatment groups [36,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76]. This study targeted 8 classes of ARGs, including sulfonamide resistance genes (sul-ARGs: sul1, sul2), tetracycline resistance genes (tet-ARGs: tetL, tetM, tetO, tetQ, tetX, tetW), macrolide resistance genes (erm-ARGs: ermB, ermC), quinolone resistance genes (qnr-ARGs: oqxB, qnrS, qnrB), streptomycin resistance genes (str-ARGs: strA, strB, aadA), chloramphenicol resistance genes (chl-ARGs: cfr, fexA), β-lactam resistance genes (bla-ARGs: blaampC, blaTEM-1, blaOXA-1, blaNDM), polymyxin resistance gene (mcr-ARG: mcr-1), and 2 mobile genetic elements (MGEs), including MGEs (intI1, intI2).

The qPCR reaction mixtures (20.0 μL) consisted of 10.0 μL TB Green Premix Ex Taq (Tli RNase H Plus, Takara, Kyoto, Japan), 0.4 μL of primers, 0.4 μL ROX Reference Dye II, 6.8 μL DNA-free water, and 2.0 μL DNA template [18]. The PCR amplification program consisted of an initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min (GeneAmp^®^ 9700, ABI, Foster, CA, USA). To ensure the accuracy of the qPCR results, each DNA sample was analyzed in triplicate independent qPCR run. Negative controls contained all of the components of the PCR mixture except DNA template. Positive controls were the DNA extracted from sample and verified gene sequencing [18]. Both positive and negative controls were included in every run. Standards for qPCR were prepared from positive controls, followed by linking into the pMD-18T cloning vector according to the manufacturer instructions (Takara, Japan). Plasmids were 10-fold diluted and then used to generate standard curves (R^2^ > 0.990) by monitoring the accumulation of fluorescence signals in real time on a thermal cycler. Based on the obtained cycle threshold values (C_T_), the initial copy number of ARGs was calculated using the standard curve based on the equation as follows:

$$[eqn]$$

where the base number refers to the sum of the sizes of the vector and the fragments of the target gene. The limit of detection and quantification for targeted genes were 4.85–12.34 copies/μL and 8.14–15.26 copies/μL in 20 μL of reaction mixture, respectively. The relative abundance of ARGs in the samples was assessed by normalizing their copy numbers to 16S rRNA gene with the unit of ARGs copies/16S rRNA gene copies.

3.5. Determination Methods of Various Components in Vermicompost

Based on the agricultural industry standard of China (NY 525-2012) [77], the total nitrogen (TN) of the vermicompost samples was determined through the distillation titration method. The total phosphorus (TP) was measured by spectrophotometry. The total organic carbon (TOC) was determined by the potassium dichromate oxidation-ferrous sulfate titration method. The moisture content was tested through the vacuum oven method (GB/T 8576-2010) [78]. The conductivity and pH value were tested by potentiometry (NYT 1377-2007) [79] with carbon dioxide-free water in a ratio of 0.4 g/mL.

As for the content of Zn in the vermicompost, a total of 0.20 g of freeze-dried sample was placed in a Teflon digestion tube containing 6 mL of HNO_3_, 1 mL of H_2_O_2_, and 4 mL of HF. The mixture was heated at 150 °C for 48 h, and the residue was transferred to a hot plate and boiled for 2–3 min. The temperature was maintained at 180 °C, and then 0.5 mL of HClO_4_ and 1 mL of HNO_3_ were added. The solution was heated until the residue was completely dissolved, and the filtrate was diluted to 25 mL with deionized water. Finally, the Zn concentrations in the liquid were determined using an inductively coupled plasma mass spectrometry (ICP-MS 7700, Agilent, Santa Clara, CA, USA).

3.6. Statistical Analysis

All the experiments were performed at least in triplicate. The results were plotted with Origin software 2024. Mantel test was performed using R-4.2.2 with the ggcor package [80]. The network analysis based on a correlation matrix was performed with the thresholds (significance index p ≤ 0.05 and spearman’s correlation coefficients r ≥ 0.8). Relative calculations were carried out with the psych package, and the co-occurrence networks were visualized using Gephi 0.10.1 to reveal the potential host relations [81]. Structural equation model (SEM) was conducted using plspm package to identify the potential effects of earthworm activity, Zn stress, bacterial structure, MGEs, manure physicochemical properties and nutrients on ARGs in composting systems [82]. Observed variables with loading values below 0.50 were excluded, and the significance of the estimated path coefficients was determined using a 95% confidence interval. The model performance was assessed using a goodness-of-fit (GOF) index.

4. Conclusions

This study provides novel insights into ARG prevalence in vermicomposting under Zn stress, revealing Zn’s critical role in shaping ARG dynamics. Vermicomposting achieved significantly higher total ARG removal (88.62%) from cow manure over 60 days compared to natural composting (42.01%). However, Zn stress introduced concentration-dependent trade-offs. Low Zn (125–250 mg/kg) amplified co-selective pressure from metals and antibiotics, elevating tet-, erm-, and chl-ARGs via strengthened ARG-bacteria linkages, while high Zn (500–1000 mg/kg) suppressed qnr- and mcr-ARGs by stressing intolerant bacteria, yet intensified horizontal gene transfer (HGT) through cross-resistance (e.g., plasmid–borne metal–antibiotic resistance), undermining ARG removal efficiency. These findings highlight the trade-off between Zn-induced microbial stress and ARG dissemination in vermicomposting systems, emphasizing the need to regulate Zn residues in livestock manure for sustainable composting practices.