Soil is the largest reservoir of ARBs and ARGs ^[1][2][14,15]. The abundance of ARGs in soil has increased substantially since the beginning of the antibiotic era ^[3][4][16,17]. Similar to antibiotics, wastewater irrigation and manure application are two main routes for ARBs and ARGs entering the soil ^[2][5][15,18].

ARBs and ARGs have been widely detected in different types of soils around the world ^[6][19]. Taking E. coli, which has been widely studied, as an example, the existence of E. coli has been found in farmland soil, non-farmland soil, and even plant microbial communities ^[7][8][20,21]. Many E. coli strains isolated from soil carry ARGs, and most of them have multidrug resistance. Furlan et al. ^[9][22] isolated a total of 60 strains of E. coli from soil samples on Brazilian farms, of which 68.3% of them exhibited multidrug resistance profiles. Liu et al. ^[8][21] found that all soil E. coli isolated from Washington State (n = 1905) were resistant to at least four different antibiotics. Graves et al. ^[10][23] analyzed 616 strains of E. coli collected from swine manure, swine lagoon effluent, and soils that received lagoon effluent and found that these strains usually carried ARGs coding for streptomycin, spectinomycin, tetracycline, and sulfonamide.

At the same time, the relative abundance of various ARGs in soil has increased significantly in recent years, and the increase in tetracycline resistance gene levels was significantly more frequent than for other ARGs ^[11][24]. Tetracyclines ARGs are present in soils worldwide, and the relative abundance is between 10⁻⁹ and 10⁻² copies/16S rRNA gene (Table 1). Tetracyclines ARGs are also the main types of ARGs carried by phages in the soil environment ^[12][25].

]. Only competent bacteria can obtain extracellular DNA, and the competence can be naturally or artificially induced ^[38][52]. Most naturally transformable bacteria can develop into competent cells under specific circumstances (e.g., nutrient conditions, changes in bacterial density ^[39][53]). Johann et al. ^[40][54] listed 87 species of bacteria that can absorb extracellular free DNA through natural transformation, including Pseudomonas and Acinetobacter, which are commonly found in soil. The key steps of ARGs transformation are as follows: (1) bacteria actively or passively discharge ARGs into the environment; (2) extracellular ARGs become stable and ingestible in the environment; (3) extracellular ARGs are ingested into the bacterial cytoplasm; (4) exogenous ARGs integrate into bacterial chromosomes by homologous recombination or replicate autonomously as episomes ^{[37][38][39][41]}[51,52,53,55]. Chen et al. ^[42][56] found that the plasmid pK5 carrying ARGs had a strong migration ability in soil, which confirmed the widespread occurrence of the transformation process in soil.

Conjugation refers to the process by which the plasmid or chromosome carrying ARGs enters the recipient bacteria through the conjugative fimbriae produced by the donor bacteria ^[43][44][57,58]. Conjugation is considered to provide better protection from the surrounding environment and a more efficient means of genetic material entering the host cell than transformation, while often having a broader host range than bacteriophage transduction ^[32][46]. Integrative and conjugative elements (ICEs) and plasmids are the main vectors for the delivery of ARGs in conjugation ^[45][46][47][59,60,61]. Plasmids, as important mediators of conjugation, can still persist among bacterial populations without antibiotic stress and invade new strains with high frequency ^[48][49][62,63]. It can be classified into three categories according to mobility: conjugative, mobilizable, and non-mobilizable ^[45][59]. A conjugative plasmid codes for its own set of mating pair formation (MPF) genes; if it uses an MPF of another genetic element present in the cell, it is called mobilizable; other plasmids are called non-mobilizable because they are neither conjugative nor mobilizable and usually spread through transformation and transduction ^[45][47][59,61]. In addition, non-mobilizable plasmids can also be transferred by physical association with conjugative plasmids ^[50][64]. Plasmid-mediated conjugation includes multiple processes such as mating pair formation and relaxosome formation ^[47][51][61,65]. In soil, this process is susceptible to a variety of factors, such as soil bacterial population structure ^[52][66], nutrient composition ^[53][67], selective pressure of antibiotics and heavy metals ^[54][68], etc. However, the understanding of the plasmid-mediated conjugative transfer process of the complex bacterial community in soil is still limited ^[55][69]. ICEs have the properties of transposons, plasmids, and phages: both ICEs and transposons can jump on chromosomes, but transposons cannot undergo HGT; both ICEs and plasmids can transfer DNA between cells in the form of conjugation, but most ICEs cannot self-replicate as plasmids do; ICEs and phages both can detach, integrate, and replicate with host chromosomes, the difference being that ICEs transfer DNA in the form of conjugation rather than transduction ^[56][70]. The study by Gonçalves et al. ^[57][71] confirmed the role of ICEs in the soil microbial HGT process.

In transduction, ARGs are transferred from one bacterium to another by means of phages (bacteriovirus) and can be integrated into the chromosome of the recipient cell ^[58][72]. The phage-mediated transduction progress does not need contact between the donor and recipient, or even the simultaneous appearance of them ^[59][73]. Soil is one of the important habitats for phages and their hosts ^[60][61][74,75]. It is estimated that the number of soil virus particles (mainly phages) accounts for 10% of the total number of viruses in the world, about 4.8 × 10^{30 [62]}[76]. The special protein capsid structure of phages can effectively protect nucleic acids, and the soil is highly heterogeneous and rich in biodiversity, providing a variety of parasitic environments for phages ^[36][50]. The opaque environment of soil protects the phages from sun damage ^[63][77], which is more conducive to their long-term survival and reproduction. Therefore, compared with free ARGs and bacteria in soil, phages are more resistant to adverse environmental factors ^[64][78] and persist longer, providing a material basis for their interactions with bacteria and gene transfer. Related studies have shown that the contribution of phages to HGT was likely underestimated ^[65][66][79,80]. Olatz et al. ^[67][81] found a large number of free and replicable phages containing ARGs in farmland soil, which may lead to the production and enrichment of ARB.

Although HGT is regarded as the major pathway of ARGs spreading, there is a significant involvement of VGT. Firstly, VGT raises the possibility of spontaneous mutation of bacterial DNA ^[41][55]. Secondly, VGT promotes HGT among the bacterial community: Li et al. ^[68][4] found that VGT can significantly promote the formation of conjugants and accelerate the spread of ARGs.

2. Effects of Soil Components on the HGT of ARGs from the Micro Perspective

2.1. The Effects of Soil Components on the HGT of ARGs in Pure Bacterial System

Another part of the studies discussed the effect of soil components on HGT processes such as ARGs transformation and conjugation using the pure bacterial system (Table 2 and Table 3).

The causes, inheritance, and transmission mechanisms of antibiotic resistance are very complex and can be divided into genetic resistance and non-inherited resistance ^[26][39] (Figure 1). Non-inherited resistance refers to resistance that is not acquired through horizontal or vertical transfer of ARGs but through behaviors such as collaboration between groups ^[27][40]. Cooperative resistance, a population-based survival strategy that adapts to high antibiotic stress through the cooperation of multiple ARBs, is a typical non-inherited resistance ^[28][41]. 

Genetic resistance can be divided into intrinsic resistance and acquired resistance. Intrinsic resistance refers to the natural existence of certain genes in bacterial genomes that could generate a resistance phenotype ^[29][43]. It is an ancient, natural, and widespread environmental phenomenon that predates the selective pressures caused by modern human use of antibiotics, and multiple ARGs have been detected in Arctic permafrost unaffected by human activities ^[6][30][19,44]. Acquired resistance is a consequence of spontaneous chromosomal mutations or ARGs gained through HGT ^[31][45], which refers to the exchange of genetic material between individuals of different organisms and is the key reason for the widespread existence of ARB in clinical systems ^[32][46]. After obtaining ARGs through HGT, ARGs will achieve the amplification and continuation of these genes through reproduction between parent and child generations in VGT ^[33][47].

HGT mainly includes three pathways mediated by mobile genetic elements (MGEs), namely extracellular DNA-mediated transformation, plasmid-mediated conjugation, and phage-mediated transduction ^[32][46]. A large number of research results have shown that HGT can widely occur in the soil environment ^[34][35][36][48,49,50].

Transformation refers to the process by which competent bacteria take up DNA from outside. Unlike conjugation, transformation does not require physical contact between the donor and recipient cells, and free DNA released by cell lysis can serve as the donor for transformation ^[37][51 Existing studies on transformation not only discuss the changes of ARGs vectors (plasmids, chromosomes, etc.) and recipient bacteria in soil microcosms, EPS, sediments, or other media but also include studies on the addition of single components to simulate soil conditions ^{[70][71][72][76]}[95,96,97,101]. Relevant research (Table 2) has shown that plasmids ^[69][12] and chromosomes ^[73][98] adsorbed by soil components can still participate in transformation; Chamier et al. ^[74][99] found that the plasmid adsorbed on sand transformed significantly less efficiently than the plasmid in solution; but Dong et al. ^[76][101] considered that sediment-adsorbed plasmids had higher transformation efficiency than episomal plasmids. Montmorillonite at low concentrations (0–0.025 g/L) ^[71][96] and goethite at high concentrations (10 g/L) ^[70][95] promote transformation, while high concentrations of kaolinite (10 g/L), montmorillonite (0.025–2 g/L and 10 g/L), and biochar (2, 4, and 8 g/L) inhibit it ^[70][71][72][95,96,97]. The research on conjugation (Table 3) showed that birnessite and low concentrations of goethite (0–0.5 g/L) promoted conjugation; the effects of kaolinite and montmorillonite were irregular; goethite at high concentration (5 g/L) inhibited conjugative transfer ^[77][13]. Liu et al. ^[78][102] found that biochar can weaken the promoting effect of heavy metals on conjugation, while Zheng et al. ^[79][103] reported that pyroligneous acid and its three fractions at different temperatures had inhibitory effects on conjugative transfer. Some studies illustrated the mechanisms of soil components affecting the process of conjugation by detecting the expression of related genes ^[77][78][80][13,102,104], but most of them are speculation based on transcriptome results, and the understanding of related pathways and mechanisms is still unclear, which is worth exploring in depth.

2.2. Influence Mechanisms of Soil Components on HGT of ARGs

Although transformation, conjugation, and transduction are three independent HGT mechanisms, there are some commonalities between them when soil components are present. Soil components mostly affect the HGT process of ARGs through similar pathways: from the perspective of intracellular changes and responses, including regulation of intracellular reactive oxygen species (ROS) production, SOS response, and the expression levels of related genes, etc. ^[77][80][83][13,104,107]; from the point of view of intercellular contact and communication, it includes the influence of extracellular polymeric substances (EPS) ^[80][104] and quorum sensing ^[84][85][108,109], etc.; in addition, it also includes affecting the activity of plasmids or bacterial concentration ^[86][87][88][110,111,112].

2.2.1. Intracellular Changes and Responses

Intracellular ROS Production

ROS are generated via successive single-electron reductions, including superoxide (O₂·⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH·) ^[89][113]. Intracellular ROS generation can cause oxidative stress, which affects a series of macromolecules of bacteria (DNA, lipids, and proteins) ^[90][114]. Intracellular ROS can be scavenged by the antioxidant system, which is an intracellular defense mechanism ^[91][115]. Antioxidant enzymes (such as catalase (CAT) and superoxide dismutase (SOD)) catalyze the conversion and detoxification of corresponding oxidative groups and, finally, relieve oxidative stress ^[92][116]. Moderately generated ROS after treatment with soil components may stimulate a series of protective responses that favor the promotion of HGT. Birnessite can initiate the formation of intracellular ROS and induce oxidative stress, which is one of the important mechanisms for birnessite-promoting ARGs conjugation ^[77][13]. However, excessive production of intracellular ROS will exceed the capacity of antioxidant enzymes, resulting in severe cellular damage or death of cells, ultimately inhibiting conjugation ^[93][117].

SOS Response

SOS response is a global regulatory response to protect cells from severe DNA damage by ROS ^[94][118], which has been shown to promote the HGT of ARGs ^[83][107]. However, there are few studies on the induction of bacterial SOS responses by soil. It is speculated that the natural components in soil have limited influence on the bacteria, while the nanoscale components or other pollutants in soil may cause the excessive accumulation of ROS and induce the SOS response. For example, high concentration of nano-CeO₂ (50 mg/L) caused the up-regulation of both SOS response activation genes (lexA, recA) and DNA repair genes (umuC, umuD, uvrA, uvrB) ^[80][104], which promoted the conjugative transfer of ARGs.

Cell Membrane Permeability

Cell membrane permeability changes with the stimulation of environmental stress, and such changes are potentially related to the spread of genetic materials ^[72][97]. The increase in cell membrane permeability, which can be divided into active improvement and passive damage, may contribute to the transfer of ARGs to a certain extent ^[39][78][53,102]. On the one hand, under the action of soil components, bacteria can autonomously up-regulate the related gene expression of membrane proteins, that is, active improvement. For example, Wu et al. ^[77][13] found that birnessite up-regulated the expression level of several outer membrane protein genes (ompA, ompF, ompC), thus promoting the conjugative transfer of ARGs. On the other hand, bacteria may be physically damaged by external perturbations, resulting in the formation of pores on the cell membrane (e.g., collisions with bacteria during material mixing ^[95][96][119,120]); it is also possible that some soil components, especially nanoscale soil components (e.g., high-temperature black carbon), allow the excessive production of intracellular ROS and then damage the integrity of cell membranes; another possibility shows that the high concentration of heavy metals released from the process of interaction between soil components and bacteria indirectly promotes lipid peroxidation and induces cell membrane damage ^[97][98][121,122]; all of the above are passive damage. Both goethite ^[70][95] and montmorillonite ^[71][96] were found to promote the transformation of ARGs by causing cell membrane damage. When the integrity of the bacterial cell membrane is excessively damaged, the bacteria will die, which inhibits the conjugation of ARGs. But the ARGs released from damaged or dead bacteria are free from the soil and have the opportunity to become donors of transformation. Ma et al. ^[99][123] and Ouyang et al. ^[100][124] reported that soil minerals, such as kaolinite, goethite, and hematite, can induce bacterial death by disrupting cell membranes. In addition, bacteria can also initiate protective responses by reducing cell membrane permeability, thereby reducing the uptake of toxic substances ^[101][102][125,126], while also hindering the occurrence of HGT. For example, biochar dissolutions caused a decrease in cell membrane permeability, thus inhibiting the transformation of ARGs ^[72][97].

ATP Synthesis Capacity

The construction of conjugative transfer apparatus, replication of plasmids, and transport across cell membranes all depend on adenosine triphosphate (ATP) ^[103][127]. Soil components can affect the frequency of conjugation and transformation by regulating ATP synthesis. For example, CeO₂ caused an insufficient ATP supply, which in turn inhibited the process of conjugation of ARGs ^[80][104].

Conjugation Activity of Intracellular Plasmids

The conjugation of plasmids requires the participation of a series of conjugation-related genes and regulatory genes, such as global regulatory genes (korA, korB, etc.), DNA transfer and replication (DTR) system genes (trfAp, etc.), and MPF system genes (trbBp, etc.) ^[82][106]. Among them, the MPF system is crucial for the formation of fimbriae ^[104][128]. In Gram-negative bacteria, sexual fimbriae act as channels for DNA conjugative transfer, and both their length and flexibility affect the efficiency of bacterial contact, including collision, attachment, and detachment ^[105][129]. As to transformation, the adherence of high concentrations of mineral particles to bacteria may damage fimbriae, while its absence will greatly reduce the expression of competent genes and the formation of competent bacteria, thus affecting the transformation process of ARGs ^[70][106][95,130].

2.2.2. Cell-Cell Contact and Quorum Sensing

The EPS consists of exopolysaccharides, nucleic acids, proteins, lipids, and other biomolecules, which determine the surface properties of bacteria (e.g., surface charge) and are critical for inter-bacterial adhesion and communication ^[107][131]. It was concluded by Tsuneda et al. ^[108][132] that, if the EPS amount is relatively small, cell adhesion onto solid surfaces is inhibited by electrostatic interaction, and cell adhesion is enhanced by polymeric interaction when it is relatively large. This process may affect the contact behavior between bacteria (e.g., conjugation) and may also enrich the plasmid or block the contact between the plasmid and bacteria, thereby affecting transformation. However, few studies have paid attention to the effects of soil components on bacterial EPS production and the transfer of ARGs, while relatively many studies have focused on soil pollutants. For example, Yu et al. ^[80][104] found that CeO₂, a typical nanoparticle pollutant in soil, weakened inter-bacterial contact by inhibiting the synthesis of polysaccharides in EPS. EPS can also act as a permeability barrier to limit the increase in cell membrane permeability and hinder the transformation of ARGs ^[109][133]. Wang et al. ^[75][100] found that the transformation ability of free ARGs was higher than that of activated sludge EPS when calculated by per ng DNA, and lower when calculated by per g volatile suspended solids (VSS). This phenomenon proved that although activated sludge EPS had a certain inhibitory effect on gene transfer. Due to the large amount of ARGs contained in EPS, it has a significant enrichment effect on ARGs, and may be an important environmental source of extracellular ARGs for bacteria. Bacterial quorum sensing is a form of bacterial cell-to-cell communication that enables bacteria to sense the presence and number of other bacteria within their surrounding environment and to rapidly respond to changes in population density ^[110][134]. Autoinducers such as acyl-homoserine lactones (AHLs) are common signaling molecules for quorum sensing ^[111][135]. Zhang et al. ^[85][109] found that six AHLs could promote the conjugation frequency to varying degrees between the same bacteria genera during the advanced treatment of drinking water using biologically activated carbon.

2.2.3. Bacterial Uptake of Extracellular ARGs

The Competent State of Bacteria

Bacterial cells must first develop a regulated physiological state of competence for natural transformation, which allows the occurrence of stable uptake, integration, and functional expression of extracellular DNA ^[39][53]. Taking Bacillus subtilis (B. subtilis) as an example, the formation of its competence requires the competency stimulating factor (CSF) ^[112][136], while the strong adsorption of CSF by kaolinite and montmorillonite reduced the transformation of ARGs ^[70][95]. In addition, the development of a competent state is also affected by various environmental stresses, such as population density, starvation, and DNA damage ^{[113][114][115]}[137,138,139]. Soil bacterial communities normally live under conditions of starvation ^[116][140]. Inaoka et al. ^[113][137] noticed that the competent genes of B. subtilis 168 were up-regulated under these conditions and tended to be competent. Transformation is entirely directed by the recipient cell, and all required proteins are encoded in the core genome ^[117][141], so we should pay more attention to the gene expression of the recipient cell. Mineral-cell adhesion may influence the expression of competent genes in bacteria, thereby interfering with the development of a competent state ^[70][95].

Availability of Extracellular ARGs

As early as around 2000, the adsorption of DNA by soil components and the transformation activity of the adsorbed DNA have been thoroughly studied, not only for plasmids but also for chromosomes ^[74][99]. Some important components in soil can protect DNA from being degraded through the adsorption of nucleases so that it can be retained in the environment for a long time ^[69][12], and the adsorbed DNA still has transformation activity ^[73][98]. The interface that adsorbs DNA and the ion species or concentrations in the surrounding environment will affect the desorption and configuration of DNA ^[118][142]. Hu et al. ^[71][96] believed that the adsorption and desorption processes of ARGs by montmorillonite would cause a locally high concentration of ARGs around the montmorillonite particles, which was beneficial to the uptake of free ARGs by competent bacteria.

2.2.4. Bacterial Concentration

The HGT process and the proliferation of ARB and ARGs in soil are intrinsically dependent on bacterial growth and concentration ^[87][88][111,112], especially for conjugation ^[119][143], but there are few studies on soil components that affect the concentration of bacteria and then influence the HGT of ARGs. The production of conjugants will be inhibited when the donor-to-recipient concentration ratio (R_D/R) is too high ^{[119][120][121]}[143,144,145]. Dahlberg et al. ^[120][144] found that the lowest concentration of donor bacteria created the highest conjugation frequency of plasmids.