Keywords

1 Introduction

Nowadays, millions of tons of antibiotics are produced each year, and veterinary antibiotics are largely used to promote the growth of animals globally [1, 2]. Worldwide overuse and abuse of antibiotics in human health and livestock production over the last few decades have greatly contributed to the propagation of antibiotic resistance [3].

Antibiotics and antibiotic resistance genes (ARGs) coexist naturally in the environment. The former are universal and multifarious, while the latter can be mobile and transferable [4]. ARGs may not only be transferred via environmental media, they may also be transmitted between parents and offspring or among different species of bacteria by vertical and horizontal gene transfer (HGT), respectively. Bacteria without ARGs may acquire antibiotic resistance by taking up naked DNA released into the environment after the death of microbes carrying ARGs, as extracellular DNA can exist for a long time under the protection of soil minerals and organic colloids [5].

ARGs have been widely identified in diverse environments, including livestock production systems, wastewater and sludge, atmosphere, and soil [6,7,8,9]. Soil undoubtedly contains the most diverse and richest DNA sequences and microorganisms [10], which significantly affect the emergence and propagation of antibiotic resistance, mainly facilitated through mobile genetic elements (MGEs) and selected mutations of existing genes exerted by antibiotics and even heavy metals [11]. The prevalence of resistance determinants is highly related to antibiotic-resistant bacteria (ARB) and ARGs in various environments [12, 13]. Widespread antibiotic resistance is identified as the top of the six emerging environmental issues and global challenges humans face in this century [14].

Antibiotic resistance has been described as the quintessential “One Health” issue [15], which requires an interdisciplinary vision, coordinated study and action across three main domains: human health, animal health, and the environment. Soil is a crucial component in the One Health approach since it not only harbors a diverse natural resistome but also receives ARB and ARGs from both human waste and livestock manures which can be further transferred to humans and animals through multiple pathways, including the production and use of vegetable and animal products, the water cycle, and aerosols [1].

2 ARG Distribution in Soil

2.1 Natural ARGs in Soil

In the early twentieth century, it is confirmed that ARGs disseminated long before the antibiotic era was proposed and claimed in many studies [16, 17]. For instance, there is a high diversity of ARGs conferring resistance to β-lactams, tetracyclines, and glycopeptides, in the DNA from Late Pleistocene organisms in Arctic soil that has been frozen for 30,000 years [17]. A variety of novel β-lactamase genes and chloramphenicol resistance genes were found in frozen Alaskan soils that were largely unaffected by human activities, and the bifunctional β-lactamase gene was also first discovered in this environment [18]. Bacterial strains isolated from sediments below the land surface at the US Department of Energy’s Hanford Site in Washington state were far away from sources of anthropogenic antibiotics, but still showed a relatively high frequency of resistance [16]. Microbes obtained from the Lechuguilla Cave in New Mexico, which had been isolated for 4 million years, also showed high resistance to antibiotics in nature, with some strains even tolerating up to 14 different commercially available antibiotics [19]. Firmicutes, Arthrobacter, Bacteroidetes, γ-proteobacteria, and α-proteobacteria from nine sites of Eastern Siberia permafrost sediments buried and frozen 15,000–3,000,000 years ago showed resistance to aminoglycoside, tetracycline and chloramphenicol antibiotics [20].

Thirteen subtypes of ARGs and MGEs were identified in soil, animal waste, and deposits that were unaffected by human use of antibiotics in Tibet, China, where bacA, mexB, mexF, and mexW were dominant over other subtypes. Eight major ARG categories consisting of 73 ARGs and MGEs (integrons, transposons, plasmids, and gene cassettes) were detected at the Gondwana Research Station and the new Jang Bogo Research Station in Antarctica, less affected by humans [21]. These studies confirm that ARGs existed in soil long before antibiotics were in widespread use by humans.

2.2 ARGs in Soil with Human Activity

Although there is plenty of evidence that some ARGs occur naturally, new ARB from humans and animals can potentially enter the soil environment and become important environmental contaminants [22]. Notably, a specific subset of clinically relevant ARGs is becoming enriched in the environment [23]. Therefore, the spread of ARGs in diverse environments is mainly due to increasing selection pressure from continuous anthropogenic usage of antibiotics [24].

Intensive animal husbandry has greatly increased the use of antibiotics and stimulated the development and spread of ARGs in agroecosystems [25]. Livestock production practices using antibiotics and the subsequent application of organic fertilizers to land can induce ARG expression in bacteria and/or mutation to produce new ARGs [26]. All major classes of antibiotics except vancomycin were found in manure-amended farm soils from three Chinese provinces (Beijing, Zhejiang, Fujian) after the use of in-feed and therapeutic antibiotics in swine production [25]. Compared with antibiotic-free manure or soil controls, the top 63 ARGs of 149 unique resistance genes, detected in these large-scale swine farm samples via high-capacity quantitative PCR arrays, are enriched up to 28,000-fold [25]. In addition, leachate from sewage and landfills increases the abundance and diversity of ARGs and bacteria in the soil [27]. One study found that as the composting time in landfill increased, the abundance of target genes sul1 and tetO in solid waste decreased, whereas the abundance of target genes in leachate increased [27]. Therefore, antibiotic residues and ARGs in landfills represent a potential risk to the environment.

In general, the external input has a profound effect on ARG pollution. Moreover, the soil bacteria commonly vary in different locations, and they respond to the environment differently; this is closely related to effects of human activities in those different regions [13]. Table 1 lists the types and abundance of ARB and ARGs in soils from different countries and regions.

Table 1 Distribution of ARGs and ARB in soils and sediments
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3 Anthropogenic Sources of ARGs in Soil

3.1 ARGs from Manure

3.1.1 Manure Production and Antibiotic Use in Animal Production

Livestock feedlots from industrial farms (concentrated animal feeding operations, CAFOs) are the major source of animal manure worldwide, especially in developed countries [37]. In the USA, nearly 500 million pounds of animal feces are produced per year, with industrial farms generating approximately 300 million pounds, 90% of which is periodically disposed of by application as organic fertilizer for agriculture [38]. In China, over 80% of the manures produced in industrial farms are used on agricultural fields [39]. Household farms serve as another crucial source of manure. About 23% and 50% of the manures from integrated livestock farms and individual household farms, respectively, were first composted before field application in China [39]. The proportion of the manure from industrial farms is likely to increase as livestock husbandry transits to more integrated systems, giving rise to greater utilization of antibiotics in livestock production for disease prevention and animal growth enhancement because the administration of antibiotic usage lags behind [40]. Commensal microbiota and pathogenic microorganisms can exchange genes and spread in high-density farms during sub-therapeutic administration of antimicrobials. It is urgent to eliminate and reduce the use of nontherapeutic and sub-therapeutic administration of antimicrobials in poultry production [41]. Information on antibiotic utilization is important for evaluating their effects on the environment. Therefore, it is necessary to carry out market surveys to estimate the production of antibiotics and their utilization in animal and human medicines.

3.1.2 Effect of Manure Application on Soil Resistome

The overuse of antibiotics in livestock increases the risk of transmission of ARGs and MGEs from manures to soil environments [42, 43]. Highly diverse ARGs conferring resistance to tetracyclines, β-lactams, aminoglycosides, macrolides, chloramphenicol, sulfonamides, and multiple drugs exist in manures and manure composts [44]. The highest relative abundance of ARGs (relative to 16S rRNA gene) measured in manures ranged from 10−3 to 10−1 and 10−2 to 10−1 in Chinese and Finnish farms, respectively [2]. The transmission of ARGs from manure into soil might also be facilitated when ARGs co-occur with MGEs [45]. Many ARGs in manures are carried on plasmids or integrons [46, 47], implying a high risk of transmission from the manure into the soil ecosystem.

Utilization of manures or manure composts may increase the types and abundance of ARGs in soil via import of manure-borne ARGs and antibiotic residues [25]. After application of manure or compost, the relative abundance of some ARGs may decrease directly in soil, whereas the relative abundance of others may initially be enhanced followed by gradual attenuation [36]. This initial period of increasing relative abundance enhances the risk of transmission of manure-borne ARGs to microorganisms in soil niches, especially in the case of repeated and long-term applications of manures and composts into agricultural soil [2]. Manure-introduced bacteria hosting ARGs might be outcompeted by native soil microorganisms and gradually disappear from the soil microbiome [48]. Nevertheless, some manure-borne ARGs may still persist in the soil via gene transfer to indigenous microorganisms [43], and these ARGs may be further enriched due to the growth enhancement effects of introduced nutrients from the fertilizer [49, 50]. Therefore, applications of manure or compost may affect soil ARGs and MGEs by direct transfer of manure-borne ARGs and selection by manure-introduced antibiotics [2]. It is thus essential to distinguish manure-introduced ARGs and MGEs from the intrinsic soil resistome and indigenous soil ARGs and MGEs enhanced by manure or compost applications, as well as to identify the ARG hosts selected by antibiotic residues and other co-selection pressures.

3.2 ARGs from Wastewater

It was estimated that global production of wastewater reached 135 and 275 km3 in the domestic and manufacturing sectors in 2010, respectively [51]. Approximately 167 km3 of global manufacturing wastewater comes from Russia, China, the USA, India, Indonesia, Brazil, and Japan [52]. According to the AQUASTAT data, only 60% of municipal wastewater is treated effectively [52].

In the past 20 years, the concentration of antibiotics has increased in untreated wastewater, which is rich in nutrients and bacteria [53]. The wastewater treatment system is thus considered to be a high-risk environment for the selection and transfer of microbial genetic material and is also one of the most important sources of ARGs in other water environments. Studies in irrigated soil in the Pearl River Delta Region in Southern China have suggested that wastewater irrigation can significantly increase the concentration of tetracycline and sulfamethazine and the relative abundance of ARGs associated with those drugs [54]. Compared with nonirrigated soil, the absolute abundance of ARGs other than tetQ, the aadA, intI1, qacE+qacE△1, and IncP-1 plasmids (linked to multi-antibiotic resistance) increased by two orders of magnitude in soils irrigated with untreated wastewater for 100 years in Mexican soil [55]. In 12 urban park soils with reclaimed irrigation water in Victoria, Australia, a total of 40 unique ARGs were identified, with β-lactam resistance genes being most prevalent by high-throughput qPCR and terminal restriction fragment length polymorphism techniques. Compared with domestic wastewater-irrigated soils, there are higher concentrations of antibiotics and abundance of ARGs in soils irrigated by fishpond water, indicating that different types of wastewater exert different impacts on ARGs in the soil [54]. Generally, the increase of ARGs may be attributed to the accumulation rather than the in situ enrichment of ARGs in soil, and short-term wastewater irrigation cannot attenuate the accumulation of ARGs [55].

3.3 ARGs from Sludge

In the European Union, a total of 10 million tons (dry matter) of sewage sludge was produced in 2010 [56, 57], with 76% from Germany, the UK, Italy, France, and others. In the USA, over 8 million tons of solids is produced annually and 55% is applied to arable land [57]. In China, 6.25 million tons of dry solids were produced in 2013, with an average annual increase of 13% [58].

Antibiotics and ARGs might be absorbed to and enriched in sludge during sewage treatment. The abundance of ARGs significantly increased by an average of 947-fold in activated sludge compared to influent samples, which might be due to the increase in the total number of bacteria [59]. Various optimization measures have been tried to reduce the abundance of ARGs in treated sludge/biosolids, such as the addition of natural zeolite to the sludge [60] and bio-drying [61]. Regardless of the disposal method of sludge, the process of treatment, or the use of recycled products, ARGs and ARB can be easily transferred into the soil. Bacteria can share genetic information through HGT with MGEs allowing ARGs to transfer from microorganisms in the sludge to indigenous microbes in the soil [62]. Furthermore, it indicates that sludge serves as a hot spot for the enrichment of ARGs and MGEs among bacteria, and its application may increase the HGT activities of ARGs in soil regardless of the difference in bacterial populations between sludge and soil [60].

Land application of sewage sludge is the key management approach of sludge disposal and an important channel for ARG spread to farmland [63], leading to the dissemination of ARGs into the soil [64]. Compared with ARGs in unamended soil, there was a short-term increase within 20 days in the number of ARGs in amended soils, but there was no significant difference in the relative abundance of ARGs [65]. The sludge can be mixed with straw or bark to produce organic fertilizer through microbial fermentation. The changing composition of the bacterial community is responsible for the reduction of ARGs upon composting, mainly because the high temperature produced by composting is conducive to the suppression or reduction of resistant bacteria and hence their resistance genes [66]. This was found to be driven by several key effects: (1) most of the pathogenic microorganisms were killed when temperatures exceeded 50°C for at least 5 days during composting; (2) high temperatures affect microbial enzyme activity, changing the rate of enzymatic reactions and ultimately affecting cell synthesis; and (3) the extracellular DNA in the sludge contains some ARGs whose rate of hydrolysis and biodegradation increases with increasing temperature.

3.4 ARGs from the Atmosphere

ARGs in soil can also be exchanged and transmitted through air [67]. Especially in heavily polluted air, the increased concentration of airborne particles is conducive to microbial transport as it provides more adhesion sites [6]. Studies have indicated that farms produce a large number of microorganisms, such as ARB, pathogenic bacteria, and opportunistic pathogens, which are easily aerosolized during animal production activities and fecal disposal, hence becoming a potential repository and carrier of ARGs [68].

ARGs can enter soil through either dry deposition or wet deposition. Dry deposition is caused by turbulent diffusion, gravity sedimentation, molecular diffusion, etc. and can transport aerosol particles carrying ARGs to the surface of the earth, where molecular forces enable them to adhere to soils [69]. Wet deposition refers to the process in which aerosol particles, participating in the formation of cloud droplets, are removed from the atmosphere by rainfall and snowfall. Cloud removal makes the aerosol particles with ARGs themselves become part of the cloud droplets as a result of condensation nuclei, and subcloud removal refers to the formation of a droplet of rain, snow, ice, etc., which can adsorb and contain aerosol particles with ARGs [70]. The ARGs in air may disseminate across different regions, as the pollutants can be transported and settled to soil in remote areas [13]. Therefore, the ARGs coming from the air also increase in the soil [71]. In return, a series of soil pollutants and microbes may also be aerosolized and further exacerbate ARG spread via air.

4 The Persistence of ARGs in Soil

Although there are a number of reports focused on transmission of ARGs from soil to plants, and the effects of anthropogenic activities and environmental factors on transmission of ARGs, the persistence of ARGs in soil is still unclear. The potential factors that influence the dynamics and persistence of ARGs in soil are thus attracting more attention.

In a field study, the relative abundance of sul1, sul2, and ermF increased rapidly in soil after slurry application and dissipation rate of ermF in slurry-treated soils was higher than sul1 and sul2 in dry-stack-amended soils [72]. The relative abundance of ARGs increased rapidly at day 1 after application of manure, followed by a decrease to the background levels by day 60, whereas a slight increase of the relative abundance of ARGs, followed by a decrease to the background levels by day 32 after application of compost [73]. The half-lives of ermB, sul1, tetA, tetW, and tetX in soil range from 13 days to 81 days after the application of different ratios of the biosolids from a wastewater treatment plant [74]. Half-lives of ARGs (0.40–3.87 d) and specific genes (ermF, 1.42–3.51 d; tetG, 0.43–2.86 d; tetX, 1.35–8.79 d) differed in the treated soils according to the addition of different sludge composts [75]. However, long-term application of manure compost in a red soil did not greatly increase the relative abundance of ARGs and MGEs in dryland soil or paddy soil, compared to the soil without any fertilization over 26 years [9]. Therefore, the persistence of ARGs varies considerably depending on fertilizer treatments and a series of factors, such as cropping systems, soil oxygen status, the introduction of nutrients, and other factors related to microbial community structure. Therefore, the removal rates of ARGs in soil are highly related to several factors: transport of extracellular DNA containing ARGs and cells carrying ARGs [76], binding of ARGs to soil or organic matter [77], decay of extracellular ARGs [78], and death of host bacteria [79].

5 The Transmission of ARGs in Soil to Groundwater and Underground Water

There is an increasing concern that the resistant bacteria generated in farm activities may migrate into soil and groundwater with the land application of manure. Antibiotic resistance contaminants in soil (especially large animal facilities) may be carried by runoff and erosion to secondary reception systems (surface water and groundwater) or leach directly to groundwater [80]. Several ARGs directly impacted by animal agriculture were characterized in natural groundwater [81]. Furthermore, seven tetracycline resistance genes (tetM, tetO, tetQ, tetW, tetC, tetH, and tetZ) in lagoons and groundwater adjacent to swine production facilities were monitored and source tracked from 2000 through 2003 [82]. Results showed that gene sequences in the impacted groundwater are highly identical to those in the lagoon. Additionally, novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, swine manure seriously affected the ARGs in groundwater, as well as part of the indigenous gene pool [82].

6 The Transmission of ARGs from Soil to Plant

The enhanced water fluxes and nutrient input in the rhizosphere can stimulate bacterial metabolic activities and the transfer of conjugative plasmids in their inhabitants [83]. These conditions establish “hot spots” for microorganism gene transfer activity in the phytosphere, including both the rhizosphere and the phyllosphere [50]. ARGs in manure-amended soil can be potentially transferred to vegetables, including those that are eaten raw or with minimal processing [31]. It has been shown that organic lettuce may be apt to carry more diverse ARGs. For example, one recent study reported up to eightfold higher absolute abundance of 134 ARGs in organic lettuce than conventionally produced lettuce [84]. Furthermore, long-term exposure of plants to antibiotics and ARGs in soil has increased the risk that ARGs carried by pathogens can enter the food chain via contaminated crops, indicating that consumption of raw leafy vegetables may potentially increase direct human exposure to ARGs [85, 86].

More and more studies show that endophytic bacteria (root endophytes, leaf endophytes, and phyllosphere microorganisms) can acquire antibiotic resistance from manure-amended soil, and sulfonamide and tetracycline resistance genes, such as sul1, sul2, tetC, and tetG, have been detected in harvested vegetable samples [87]. Yang et al. [88] showed that nine subclasses of ARGs were shared among soil, roots, and shoots of lettuce and that ARGs were mainly located in endophytes within the lettuce [88]. These endophytic bacteria are the main carriers and disseminators of drug-resistant genomes and mobile genetic elements, and they can acquire drug resistance through various ways [89]. Such bacteria may be directly transmitted through the chain of soil vegetables from animal feces and act as an important driver of the change of multi-antibiotic-resistant bacteria in vegetables [90]. Therefore, microorganisms and secretions (sugars, organic acids, and amino acids) in the rhizosphere may affect the migration and attenuation of ARGs in soil [91, 92].

Additionally, the potentially transferable gene pool in the phytosphere was found to be highly mobile and directly correlated with host fitness. The conditions in these hot spots fluctuate temporally as they are heterogeneous and dynamic environments [50]. Therefore, it is difficult to evaluate the extent to which these factors affect HGT processes of ARGs under different conditions [93].

7 Effect of Anthropogenic Activities and Natural Factors on Transmission of ARGs

The development of bacterial resistance to antibiotics poses a major threat to human health [94]. The application of antibiotics in human medicines and livestock production and the use of manures and municipal wastes in agriculture are regarded as pivotal selective pressures on the dissemination of ARGs [25, 95]. In addition, resistance of bacteria to antibiotics co-occurs with other environmental factors, such as lack of nutrition, extreme temperature stress, and oxidative conditions [94, 96]. The current state of knowledge on the dissemination of ARGs induced by anthropological activities and natural factors can be described as follows.

7.1 Agricultural Regulation

7.1.1 Soil Management

Common soil management practices like cropping systems, irrigation, and fertilization are known to influence both the introduced and intrinsic soil resistome. Manure is rich in available carbon for bacterial growth and may also carry co-selectants such as antibiotics and metals [25]. Additionally, all these fertilizers also contain organic and inorganic contaminants, which have been related to a series of negative environmental impacts on the dissemination of antibiotic resistance [97]. Irrigation with treated wastewater may increase ARG levels in soil bacteria, potentially adding to the global dissemination of clinically relevant antibiotic resistance. It is also evident that ARG and ARB risks could be impacted differently under different cropping systems, e.g., dryland vs. paddy soils. Wang et al. [9] showed that the relative abundance of tetPA, oprJ, mexF, and acrA_5 increased, whereas other genes decreased in paddy soil compared to the dryland soil, indicating that the overall pattern of ARGs in the soil varies according to cropping system and may have been driven by the differences between aerobic and anaerobic conditions [9]. Therefore, the origin, dynamics, and propagation of ARGs in soils are closely related to different agricultural practices and the physicochemical properties of soils. It is important to understand comprehensively the dynamics of intrinsic and introduced ARGs in soil and therefore develop strategies for risk mitigation.

7.1.2 Biochar Amendment

Biochar, a soil amendment produced via pyrolysis/carbonization of plant- and animal-based biomass, is widely used in agriculture to increase the soil water holding capacity and improve soil fertility [98]. π-π interactions play an important role between the aromatic rings on antibiotics and biochar [99, 100], probably driving selection or co-selection of ARGs among soil microorganisms.

Biochar amendment may significantly decrease the abundance of ARGs in non-planted soil [101]. The underlying mechanisms could be attributed to two factors: (1) the mobility of antibiotics and heavy metals being decreased through adsorption and (2) the bacterial community structure being influenced by the addition of biochar, with corresponding effects on the resistome [102, 103]. In another study, mushroom and rice straw biochar were both produced at 500°C for 4 h under oxygen-limited conditions using a muffle furnace. Whereas the application of mushroom biochar effectively removed ARGs and pathogenic bacteria, this is not the case with the rice straw biochar [104]. Therefore, further systematic and integrated study is needed to unveil the relationship between biochar physicochemical properties and their influence on the persistence and/or propagation of ARGs in soil.

7.2 Soil Properties

Soil physical, chemical, and biological parameters have been identified as environmental stresses that induce or maintain antibiotic resistance evolution and transmission [77]. Pearson correlation analysis indicated that there is a correlation between the abundance of ARGs and antibiotics or soil properties (pH and soil organic matter). The concentrations of tetracyclines and abundance of ARGs decreased with increasing soil depth [54, 105]. Relative studies showed that the abundance of ARGs in soil was increased after the long-term application of sewage sludge [11], whereas another study found inconsistent levels of ARGs in different sites of amended soil [106].

Soil microbial phylogenetic structure is commonly regarded as the dominant biotic determinant of ARG compositions [107], as soil microbes are not only the producers of many antibiotic compounds [4] but also hosts to various ARGs. Many studies have shown that soil physicochemical properties, plant species, and climate, as well as chemical substances, can significantly influence the diversity, structure, and function of microbial communities in soil [108, 109]. Moreover, it has been demonstrated that microbial community structures revealed great differences among the paddy soil samples, which were consistent with differences in pH values [110]. In addition, the total tet gene copies were highly related to soil organic matter near swine feedlots [111]. All the results verified that soil type and physicochemical properties exert an impact on the microbial community, which was correlated with the fate of ARGs.

7.3 Environment Pollutants

7.3.1 Heavy Metals

It is a common practice to add certain heavy metals into animal feeds to promote growth and control disease, and these metals are subsequently accumulated by soils after the application of manure and sewage sludge [112, 113]. There is a link between the use of Zn in feed additives and the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in animal production [114]. Positive correlations exist between some ARGs and heavy metals in manures and manure-amended soils [115]. The positive correlation between ARGs and heavy metals is conducive to the survival of bacteria in a polluted environment, which promotes the spread of ARGs in soil to some extent [116].

It is vital to recognize that metals can co-select antibiotic resistance and drive strong selection of bacterial assemblages in various environments [117]. The types, abundance, and mobility potential of ARGs were significantly influenced by long-term heavy metal contamination and result in the unpredicted risk of the ARG dissemination in environments contaminated with heavy metals. Furthermore, the co-selection for antibiotic resistance induced by heavy metals has been highlighted, particularly when the same gene confers resistance to multiple types of metals and antibiotics and when diverse genes encoding metal and antibiotic resistance coexist on the same MGE.

7.3.2 Nanomaterials

Recently, it was estimated that the global market requirement for metal oxide nanoparticles will increase from 270,041 tons in 2012 to 1,663,168 tons by 2020 [118]. Although nanoparticles exist in various environments, such as wastewater effluent and sludge, knowledge about their influence on ARB and ARGs is lacking. The application of nanoalumina significantly promoted the HGT of multidrug resistance genes encoded by the plasmids of RP4, RK2, and pCF10 [119]. It is also reported that AgNP exposure reduced the occurrence of ARGs in collembolan gut microbiota [120]. The conjugative transfer of the RP4 plasmid from E. coli to Salmonella spp. treated with nanoalumina was enhanced 200-fold, indicating that the environmental and health risks from nanomaterials could include promoting sensitive bacteria to obtain antibiotic resistance [119].

7.3.3 Microplastics

Microplastics in aquatic environments may adsorb antibiotics on their surfaces, resulting in their long-range dissemination and entry into the food chain [121]. Sophorolipid can stimulate ARG dispersion mediated by bacteria/phage in soil co-contaminated with microplastic and tetracycline [122]. Compared to free-living bacteria, the transfer rate of plasmid DNA in phylogenetically diverse bacteria was increased, and this was associated with microplastics [123]. These reports indicate that microplastics could be the hot spot for HGT between phylogenetically distinct pathogens in the environment.

8 Direct Relationships to Human Health

Most clinically relevant ARGs may originate from environmental bacteria [124]. ARGs carried by soil bacteria that are also found in human clinical pathogens [125] and novel ones conferring high-level resistance against sulfonamides, aminoglycosides, and a broad range of β-lactams have been discovered [126]. Functional metagenomic analysis of soil-inhabiting bacteria revealed a high nucleotide identity (>99%) when comparing the multidrug-resistant resistome of soil bacteria with those in clinical human pathogens, inferring possible HGT among bacteria from these different environments [127]. It was also revealed that the contiguous nature of the intI1 with the ARGs carried by the soil bacteria and clinical pathogens facilitated the HGT process [127]. The intI1, belonging to class 1 integrons, is closely related to the integration of multiple ARGs at the same genetic site. In natural and anthropogenically influenced environments, integrons have been acknowledged to be the prevalent carriers of multiple ARGs [46]. A substantially increased abundance of intI1 was reported when manure was applied to archived soils, inferring the transfers of multiple resistances from soil environments to clinical activities. This resistance could be intensified by the applications of excessive antibiotic-contaminated manures to land.

To date, several limitations for confirming ARG transfer from environmental microbiomes to human pathogens have been identified although credible evidence of this process is yet to be well or fully documented [128, 129]. In any case many ARG hosts exist on the surfaces of leafy vegetables (arugula, cilantro) grown in manure-amended soil that are eaten raw [93, 130]. This represents a direct food chain link between the environment and humans. After incubation of lettuce or cilantro leaves, the total DNA of the bacterial community was obtained from the enrichment, and multiple resistance plasmids were found in E. coli isolates, indicating that bacteria colonizing leafy vegetables are the original source of transposons and integrons [130, 131]. As multiple ARGs in the bacterial genomes are being found in the plasmids, it is obvious that antibiotic resistance pathogens or “superbugs” may propagate on a global scale. This is supported by the occurrence of plasmid-carrying mcr-1 resistant to colistin (polymyxin E) in various pathogens worldwide [132]. Colistin has been revived as a last resort drug to tackle the threat of multidrug-resistant bacterial outbreaks [133]. The plasmid-carrying mcr-1 was first reported in animal and human sources in Shanghai, China. Nowadays, mcr-1 has been identified in samples from various sources including human pathogens, swine, poultry, pork, and environmental sources worldwide [132]. The consumption of pork has also been reported to significantly promote the spread of the ARGs in Dutch travelers [134].

9 Technology for Reducing the Introduction of ARGs into Soil

Much effort is being spent to mitigate the threat of antibiotic resistance, namely, developing new or alternative antibiotics and reducing antibiotic usage [135, 136]. An alternative and complementary stewardship initiative consists of monitoring and removing ARGs as environmental pollutants [29]. Various treatment processes, such as aerobic composting, anaerobic digestion, and disinfection, have been applied and tested for their efficacy in removing ARGs and antibiotics (Table 2). The intention is to reduce the selection pressure and introduction of ARGs into soil through manure and compost application and wastewater irrigation.

Table 2 Technologies tested to reduce the introduction of ARGs into soil
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9.1 Aerobic Compost

Aerobic composting is widely used to treat organic wastes and manure. The effects of aerobic composting on the variation of ARGs are based on different processing technologies. For example, continuous thermophilic composting can significantly reduce ARGs and integrons in animal manure [140]. Superabsorbent polymers are also considered suitable amendments for reducing ARGs in swine aerobic compost [143]. Conversely, several types of ARGs cannot be effectively removed by aerobic composting, with compost products, thus remaining an important reservoir of ARGs [49]. Temperature was viewed as an important factor to influence the fate of ARGs in aerobic digested sludge where the shift in temperature would largely change the bacterial community [137]. The ARGs and MGEs from sewage sludge were significantly reduced by hyperthermophilic composting (temperatures up to 90°C), as were other indicators during the composting process (mushroom biochar, rhamnolipid, Tween 80 and other surfactants, etc.) [144, 145]. Additionally, the temperatures under elevated thermophilic composting up to 70–80°C without exogenous heating and additives (zeolite, superphosphate, or zeolite and ferrous sulfate) on the removal rate of ARGs during chicken manure composting were investigated. The removal of ARGs in manure was 86.5%, 68.6%, and 72.2% in zeolite, superphosphate, and zeolite and ferrous sulfate, respectively, which is higher than the control [146].

9.2 Anaerobic Digestion

Anaerobic digestion of manure from livestock and poultry can produce methane, while the digested residue can be applied as organic fertilizer [147]. Additionally, there is some evidence that anaerobic digestion can reduce populations of ARB in manure and biosolids, as well as genes coding for resistance to antibiotics, and may thus be an effective approach for recycling agricultural waste and removing ARGs [148]. Several treatment strategies including pretreatment, thermophilic digestion, two-stage digestion, additives, and solid-state digestion were employed to compare the removal of ARGs by anaerobic digestion [149]. Operating parameters played crucial roles in the reduction efficiency of ARGs. For example, longer solids retention time showed a higher level of ARG removal rate in anaerobic digestion of residual sludge [66]. Therefore, it is necessary to critically evaluate existing anaerobic treatment processes for their potential in mitigating ARGs and their propagation in the environment.

9.3 Disinfection

Various techniques are used in wastewater treatment, such as UV, chlorine, and ozone disinfection. To induce bacterial damage, these techniques employ different mechanisms. Comparative studies of disinfection methods on ARG removal are widely reported in the literature [141, 150]. Disinfection process of ARB cells is significantly related to the disinfectants’ relative activities and consumption by important cell constituents, including amino acids, saccharides, lipids, and nucleic acids [151]. The frequency of the transfers of ARGs was determined under different rates of UV or chlorine, indicating that UV disinfection and chlorination display different influences on conjugative transfer and that the dose of both UV and chlorine would be important factor in removing ARGs [152]. Additionally, when the ozone dose was 2 mg/L, the abundance of ARGs had higher removal efficiency, but there was no significant correlation between the concentration of ozone and ARG removal efficiency. Although inactivation of ARB via destroying bacterial DNA or the cellular structure could occur during disinfection processes, ARGs may still exist in the cell debris, constituting a potential downstream threat to human health.

10 Conclusions

Although the relevance of ARB and ARGs as environmental contaminants is widely recognized, there is currently limited evidence to assess the related human health risks quantitatively and objectively. Therefore, it is difficult to define threshold values for the maximum admissible levels of ARB and ARGs in treated wastewater and in sludge and manure used in agriculture. Increasing water scarcity and soil degradation will drive increased need for water reuse, and beneficial reuse of municipal sludge and manures, likely increasing inputs of ARGs and ARB into soil. Thus far, several primary strategies have been proposed to mitigate the propagation of antibiotic resistance, namely, the restriction of antibiotic use in clinical activities and veterinary applications, and to promote new drug design.

Given the urgency of the problem, secondary or follow-on strategies are also needed to help preserve the effectiveness of existing antibiotics. These include establishment of a global systematic and publicly available surveillance network, including regular, consecutive measurement of antibiotic application and the diversity of antibiotic resistance from agriculture and clinical activities. Continuous surveillance of antibiotic resistance is conductive to disease diagnosis, effective antimicrobial stewardship, and policy setting, thus the overriding importance of undertaking surveillance across the One Health framework. Governments should pay more attention to ARG pollution with investment in both basic and applied research to provide a strong scientific basis for formulating effective mitigation practices and a standardized assessment system to document risk reduction of soil and water contaminated by antibiotics and ARGs. This will provide reference data for prevention, reduction, and removal of these important environmental pollutants.