Fecal antibiotic resistance genes were transferred through the distribution of soil-lettuce-snail foodchain

doi:10.21203/rs.3.rs-2695627/v1

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Fecal antibiotic resistance genes were transferred through the distribution of soil-lettuce-snail foodchain

https://doi.org/10.21203/rs.3.rs-2695627/v1

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published 10 Jul, 2023

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Massive antibiotic resistance genes (ARG) were detected in the soil modified by manure, which may affect human life safety through the food chain. However, the transmission of ARGs through the soil-plant-animal food chain is still unclear. Therefore, this study used high-throughput quantitative PCR technology to explore the effects of pig manure application on ARGs and bacterial communities in soil, lettuce phyllosphere and snail excrement.The results showed that a total of 384 ARGs and 48 MEGs were detected in all samples after 75 days of incubation. The diversity of ARGs and MGEs in soil components increased significantly by 87.04%and 40%with the addition of pig manure. The absolute abundance of ARGs in the phyllosphere of lettuce was significantly higher than that of the control group, with a growth rate of 212.5%.Six common ARGs were detected between the three components of the fertilization group, indicating that there was internal transmission of fecal ARGs between the trophic levels of the food chain.Firmicutes and Proteobacteria were identified as the dominant host bacteria in the food chain system, which were more likely to be used as carriers of ARGs to promote the spread of resistance in the food chain.The results were used to evaluate the potential ecological risks of livestock and poultry manure and provide theoretical basis and scientific support for the formulation of ARGs prevention and control policies.

Pig manure

Antibiotics resistance genes

Food chain

Transfer

Mobile genetic elements

Bacterial community

For a long time, with the acceleration of intensive breeding process, antibiotics play an important role in the prevention and treatment of animal infectious diseases, promoting animal growth and improving feed conversion rate(YAJIE G et al.,2023).However, due to the inability of livestock and poultry to fully absorb these antibiotics, up to 20%~90%of antibiotics are discharged into the natural environment in the form of prototypes or metabolites(BINGZHEN J et al.,2023).The selective pressure of sensitive bacteria gradually produces resistance to it, which promotes the production of ARGs in animals induced by antibiotics, resulting in the increase of drug resistance of pathogenic bacteria, thus accelerating the production and diffusion of ARGs in the environment(HEUER H et al.,2011;QIAO M et al.,2018).As shown in Fig.1, ARGs migrate and spread in crops through plant endogenesis in the soil improved by animal manure, and plant roots obtain drug-resistant bacteria, causing serious drug-resistant pollution of farmland soil and crops(YUAN Z et al.,2022).In addition to water reuse and atmospheric deposition, the media of antibiotic resistant bacteria (ARB) and ARGs entering farmland soil are also attributed to intensive animal husbandry, agricultural practice and fertilizer application(MINJIA T et al.,2022). Farmland soil has naturally become a transfer station for ARGs transformation and expansion(HEUER H et al.,2011), and further transferred to soil plants, animals or inherent microorganisms, and even human pathogens through the food chain(HOEK A H A M V et al.,2015;Zhang.,2018).At the same time, there are a large number of mobile genetic elements (MGE) in manure, such as plasmids, integrons and transposons, which can be used as driving factors to promote the transfer and diffusion of ARGs(CASARIN P F R A et al.,2023).ARGs have become a new type of pollutant in soil environment.

More importantly, many scholars have reported that the application of manure not only leads to the enrichment of ARGs in soil, but also significantly increases the enrichment of ARGs in plants(SHIHAI L et al.,2024).Planting vegetables in manure-amended soils may lead to an increase in the abundance and diversity of some ARGs in vegetables ready for retail on the market after harvest(XIAOTING F et al.,2023;CAIXIA Z et al.,2022).A variety of tetracycline resistance genes and MGEs with high relative abundance were detected in the endophytic bacteria of three kinds of vegetable leaves harvested in the vegetable field with manure application. MGEs have a certain risk of promoting the migration and diffusion of ARGs in vegetable endophytic bacteria, which means that eating these vegetables raw may pose a threat to human health(Wang.,2021).Yin et al.detected 205 ARGs from the phyllosphere of six vegetables, among which multidrug resistance genes were the most abundant(YUE Y et al.,2022).Chen et al.showed that long-term application of chicken manure as an organic fertilizer to irrigate farmland soil would significantly increase the abundance of ARGs in the phyllosphere, with the highest enrichment factor of 2638 times(CHEN Q-L et al.,2018).Therefore, raw vegetables may be a potential way for soil ARGs to enter the human body, and its migration and driving mechanism to the lower end of the food chain through horizontal transfer need to be further explored.

Not only plants are the transmission chain of ARGs, but also animal intestinal microorganisms are the repository of a large number of ARGs. In recent years, soil animal microbiome research has received extensive attention.Some scholars have found that long-term application of sludge or chicken manure significantly increased the abundance of ARGs in the gut of soil animal earthworms(DING J et al.,2019).The unique niche of soil animals often makes their microbial groups different from the surrounding environment. Organic fertilizer can significantly increase the abundance of ARGs in the soil collembolan microbial group. With the increase of ARGs content in the predatory collembolan microbial group, the content of ARGs in the mite microbial group of predatory collembolan also increases(DONG Z et al.,2019).Feng et al.found that the gut microbiota of plateau zokors and plateau pikas may also carry a large amount of ARGs and discharge them into the water or soil environment through feces(Feng et al.,2020).A variety of ARGs have also been found in the intestine of psyllids(QIAN X et al.,2022).These conclusions indicate that the intestinal tract of animals can act as a transport system for ARGs and pose potential risks to human health through the food chain(DONG Z et al.,2019;ZHENG F et al.,2019;Zhu et al.,2019).In our previous studies, the impact on the spread of ARGs in the food chain was limited to plants((Bao et al., 2021), and the changes in the resistance group of soil animal microorganisms in the food web have not been explored in depth.

Therefore, in this study, lettuce was planted in pig manure-improved soil, and the cultivation experiment of lettuce phyllosphere feeding snail was carried out to construct an indoor laboratory-scale conceptual model (soil-lettuce-snail as a typical structure).High-throughput quantitative PCR platform was used to explore the abundance and diversity of ARGs in each component of the food chain system. 16 SrRNA sequencing technology was used to analyze the changes of bacterial communities in each component, and the relationship between ARGs and bacterial communities was discussed. The results clarify the distribution and transfer of fecal ARGs in the food chain system, which is helpful to evaluate the ecological risk of antibiotic resistance in animal fecal sources.

2.1 Sample collection

The test soil was collected from the campus soil (0–20 cm) of Suzhou University of Science and Technology in Suzhou, and dried and sieved (2 mm) for subsequent pot experiments.Before planting lettuce, the prepared soil was divided into two parts : one part of the soil was uniformly mixed with pig manure (5%W/W, pig manure, PM group), and the other part of the soil was not applied with pig manure (control group, NPM group).After that, the management methods and growth conditions of the two groups of lettuce were exactly the same, including temperature, humidity, irrigation and light.The plants were harvested after 60 days. The plant samples were divided into underground parts (roots) and aboveground parts (leaves). Three lettuces were randomly selected from each group and the residual soil particles were washed off with deionized water.The aerial leaves and underground roots of lettuce were separated by ethanol disinfection scissors, and the lettuce phyllosphere was collected and placed in a sampling bag.On the day of lettuce harvest, lettuce was picked and fed to snails for 2 weeks, and their feces were collected in a refrigerator at -80°C.A total of 18 samples were collected (Treatment group PM:3 pig manure soil, 3 lettuce phyllosphere, 3 snail excrement;The control group NPM:3 soil without pig manure, 3 lettuce phyllosphere, 3 snail excrement).Some of the collected samples were immediately measured for relevant physical and chemical properties, some were freeze-dried for the determination of antibiotic content, and the rest were used to extract microbial DNA for the subsequent determination of ARGs and bacterial community structure diversity.The physical and chemical properties of soil and pig manure are shown in Table 1.

Table 1

Physical and chemical properties of soil and pig manure.
Sample	pH	Heavy metal concentration (mg·kg^− 1)						TCs(µg·kg^− 1)			SAs(µg·kg^− 1)			QNs(µg·kg^− 1)
Sample	pH	Cu	Zn	Pb	Cr	Ni	Cd	TC	OTC	DOX	SD	SM2	SMX	ENR	CIP	NOR
Soil	7.75	21.13	389.99	68.44	90.03	84.37	0.19	LOD	LOD	LOD	LOD	LOD	LOD	LOD	LOD	LOD
Pig manure	7.10	51.16	346.29	79.58	88.78	LOD	0.65	421.62	827.54	68.77	26.65	216.02	21.46	28.95	20.41	34.55

Note : LOD means lower than the instrument detection limit

2.2 DNA extraction

Fresh soil samples/snail excrement (0.5g) were weighed and placed in Lysing Matrix E tubes. The soil/snail excrement microbial DNA was extracted according to the experimental procedure provided by FastDNA ® Spin Kit (MP Biomedical, Santa Ana, California, USA).

Phyllosphere:10 g leaves were cut into pieces and placed in a 250 mL conical flask, rinsed with sterile water for 3 times, 1 min each time, and the rinse solution was collected for later use.150 mL sterilized PB (0.01M) buffer (containing 0.1% Triton) was added, sonicated for 5 min, placed in a room temperature shaker (180 rpm), and taken out after 2 h(DURáN P et al.,2018;WAN-YING X et al.,2015). The plant tissue was filtered out with sterilized gauze, and then vacuum filtered with 0.22µm filter membrane with the previous cleaning solution. Finally, the filter membrane was cut into pieces and DNA was extracted with FastDNA ® Spin Kit kit. DNA concentration and purity were determined by NanoDrop One instrument (Thermo Fisher Scientific, USA).

2.3 High-throughput quantitative PCR analysis

The Wafergen SmartChip Real-time PCR system (Fremont, USA) was used for high throughput quantitative PCR (HT-qPCR) analysis to detect the diversity and abundance of ARGs and MGEs in soil, lettuce phyllosphere and snail feces samples. The Wafergen system has 5184 reaction holes, each reaction is a 100 nL system. In this study, 296 pairs of 384 pairs of primers were used, mainly involving 8 major ARG types (285 pairs of primers), 8 transposases, 1 class I integron, 1 clinical class I integron and 16S rRNA gene(ZHANG Y-J et al.,2019;HANG-WEI H et al.,2018).

The SmartChip was placed into Wafergen SmartChip Real-time PCR Cycler. The PCR program was set to 95°C for 10 min, followed by 40 cycles including 95°C 30 s and 60°C 30 s. Each reaction hole only retains data with a single effective melting peak and an amplification efficiency in the range of 1.7 ~ 2.3. The threshold cycle (Ct) is set to 31(JIAN-QIANG S et al.,2015). Three replicates were set for each sample, and the average of three replicates was taken for subsequent analysis.

2.4 Quantitative PCR analysis of bacterial 16S rRNA gene

The standard curve of qPCR analysis was constructed. Firstly, the bacterial 16S rRNA gene was amplified by PCR and ligated into the pGEM-T Easy vector. Then the ligation product was transformed into JM109 competent cells, and the clone containing 16S rRNA gene fragment was selected. The plasmid DNA was extracted and diluted 10 times continuously to prepare the standard. Bio-Rad CFX384 Real-Time PCR detection system (Bio-Rad, USA) was used to detect the abundance of 16S rRNA gene, and the primers and reaction procedures were the same as those of HT-qPCR.

2.5 MiSeq sequencing and data processing

In order to explore the structure and composition of bacterial community in each component of the food chain system, the primers 805R (GACTACHVGGGTATCTAATCC) and 341F (CCTACGGGNGGCWGCAG) were used to amplify the V3-V4 region of each component in the soil-lettuce-snail food chain system. Studies have shown that the V3-V4 region of the selected bacteria can reduce genomic heterogeneity and can be closer to the full-length comparison information than other variable regions(DONG-LEI S et al.,2013).The PCR amplification reaction consisted of 1 µL of 10 mM upstream and downstream primers (805 R primer with Barcode at the 5 ' end), 25 µL of Ex Taq enzyme, 1 µL of DNA template and 22 µL of ddH2O to form a 50 µL × 2 reaction system. The specific PCR amplification reaction conditions are shown in Table 4 − 1. After amplification, the products were recovered by DNA purification and recovery kit (Thermo Fisher). The concentration of the purified product was determined, and each sample was mixed with the standard of DNA 200 ng. The mixed samples were sent to Sangon Biotech (Shanghai) Co., Ltd., and 16S rRNA high-throughput sequencing of each sample was completed by llumina Hiseq 2500 platform.

2.6 Statistical analysis

The data of the treatment group were analyzed by one-way analysis of variance (ANOVA) and Duncan multiple comparison (Duncan). IBM SPSS Statistics 26 and Excel 2013 were used to analyze and test the differences in the diversity and abundance of ARGs, MGEs and the relative abundance of bacterial communities between the two groups.Origin 2021 was used to draw histograms, chords and other graphics.Heatmap was used to describe the overall distribution pattern of ARGs and MGEs.Yifan-Hu layout in Gephi (https://gephi.org/.) 0.9.2 software was used to display the common and unique ARGs and MGEs among the components of the food chain system (soil, lettuce phyllosphere, snail excrement).The correlation network was drawn by Fruchterman Reingold algorithm in Gephi software to analyze the correlation between ARGs and bacterial flora structure.

3.1 Abundance and diversity of ARGs and MGEs in each component of food chain system

A total of 384 ARGs and 48 MEGs were detected by HT-qPCR.According to their potential resistance to antibiotics, they can be classified into nine main types of resistance:Aminoglycoside, Beta-Lactamase,FCA,MLSB,Multidrug,Sulfonamide,Tetracycline,Vancomycin and others.As shown in Fig. 2,the overall number of snail excrement components was the highest,and the highest number of detections could be as high as 214. Due to its unique dietary structure and living environment, snails have become a reservoir of a large number of ARGs(STEFANIE M et al.,2014).The lowest number was detected in lettuce phyllosphere, ranging from 54 to 82.The number of ARGs and MGEs in soil, lettuce phyllosphere and snail excrement increased with the application of pig manure, which were 40%~87.04%, 9.57%~30.47% and 12.66%~13.96%, respectively. Among them, the number of ARGs and MGEs detected in soil components was the most significant, and the growth rate of sulfonamide resistance genes was the highest, which was 506.06%.Since vegetables are rooted in the soil and take in nutrients or pollutants, the soil is an important reservoir for ARGs intrinsic resistance and acquired resistance(XUEXIA Y, et al.,2022;YUN Z et al.,2022), and has the characteristics of easy accumulation and enrichment of ARGs.Therefore, the introduction of ARGs from manure is likely to increase the diversity of ARGs in the trophic components of the food chain system(PU Q et al.,2020), and promote the diffusion of ARGs from soil to the next trophic level of vegetables and food chain(PU C et al.,2019).

The absolute abundance of ARGs in soil, lettuce phyllosphere and snail excrement ranged from 1.86E + 09 ~ 4.41E + 09,4.78E + 11 ~ 1.50E + 12,6.16E + 10 ~ 8.39E + 10 (copies/g), respectively (Fig. 3). The absolute abundances of ARGs and MGEs in soil, lettuce phyllosphere and snail excrement were 1.36 ~ 20.54 times, 2.43 ~ 3.13 times and 1.36 ~ 1.85 times higher than those in the control, respectively. The absolute abundance of ARGs showed lettuce phyllosphere > snail excrement > soil. The absolute abundance of ARGs in the phyllosphere of lettuce was significantly higher than that of the control group, with a growth rate of 212.5%.On the one hand, manure was applied to input exogenous ARGs into the soil, and exogenous microorganisms can directly enter the plant for colonization through the absorption and migration of plant rhizosphere(XUEXIA Y et al.,2022;ISHI K et al.,2022).On the other hand, the application of manure also promoted the proliferation of indigenous drug-resistant bacteria. The drug-resistant genes carried by drug-resistant bacteria spread in the soil environment. In the process of colonization through the rhizosphere of plants to the interior of plants, the resistance obtained by exogenous soil microorganisms was transferred into the interior of plants, which also led to an increase in the abundance of drug-resistant bacteria in the endophytic system of plants.The absolute abundance of MGEs in the soil components was 20.54 times higher than that of the control group. The abundance of MGEs had a strong positive correlation with multiple ARGs (Fig. 13), which meant that the change trend of MGEs in the soil components was the same as that of ARGs and significantly higher than that of the control soil(ZHU B et al.,2017;CERQUEIRA F et al.,2020).The absolute abundance of ARGs and MGEs in snail excrement did not change significantly compared with other components, only increased by 36.20%~84.62%. The effects of different pollutants on the intestinal microorganisms of soil animals were different. Fecal ARGs may be difficult to migrate and colonize the snail intestine through the food chain in a short time. More studies are needed to reveal the mechanism of resistance group changes.

Figure 4 lists the absolute abundance of ARGs in different types of ARGs in each component of the food chain. The abundance of Tetracycline was the highest in the soil, lettuce phyllosphere and snail excrement, which was 1–3 orders of magnitude higher than other types of ARGs.Sulfonamide had the highest growth rate in soil components and lettuce phyllosphere components, which was about 2 ~ 3 orders of magnitude higher than the control group. Vancomycin increased most significantly in snail excrement, which was 346.67%.The high concentration of tetracycline antibiotics in the environmental media of animal husbandry has a direct selection effect on the bacteria carrying TRGs (tetracycline resistance genes, TRGs), making livestock and poultry manure a huge reservoir of TRGs(SUI Q et al.,2018;ZHU Y-G et al.,2019), which leads to the wide spread of TRGs.In this study, the concentrations of TC, OTC and DOX antibiotics in pig manure were the highest, which also confirmed this conclusion.

3.2 Enrichment of ARGs in soil-lettuce-snail food chain system

In this study, the Heatmap showed the enrichment of ARGs in the soil-lettuce-snail food chain system under different treatments. ARGs were significantly stratified among the components of the food chain (Fig. 5), and the ARGs and MGEs dominated by different components and control areas were significantly different.Aminoglycoside, Tetracycline, and MLSB accounted for 8.19%,9.34%, and 6.65% of the total gene abundance in the soil components of the manure application, and only 1.59%, 0.11%, and 0.65% of the total gene abundance in the control area.ANT(6), APH3-III, ANT(6) -Ia, AAC (3)-Via (Aminoglycoside), tetM, tetW, tetR, tetQ (Tetracycline), ErmF, ErmQ, ErmB (MLSB), IS6100, intI1_337old, IS613, ISCR1, IS1247 (MGEs) were the dominant ARGs and MGEs in the soil components of manure application.Studies have shown that the easy enrichment of erm genes is due to the easy acquisition of mobile genetic elements such as plasmids or transposons, which can migrate and spread between different host bacteria(FAN M et al.,2023).Aminoglycoside, Multidrug and Tetracycline were the dominant resistance gene types in lettuce phyllosphere of manure group. Among them, AAC(3) -IV, APH(6) -Ia, aacA43, AAC(3) -Via (Aminoglycoside), czcA, sugE, mel_1, pbrT (Multidrug), tetA (P), tetM, tetR (Tetracycline), ISEcp1, IncI _repI1, IS3, IS1247, ISCR1 (MGEs) were the dominant ARGs and MGEs, which were about 1 ~ 6 orders of magnitude higher than those in the control area.The dominant ARGs in snail excrement of fertilization group were Multidrug, Aminoglycoside and Tetracycline. QacF_H, ttgA, qacH_351, copA (Multidrug), aadA2, APH(6)-Id, aadA21, AAC(3)-Via (Aminoglycoside), tetR, tetA, tetM, tetQ (Tetracycline), IS200-2, IS200-1, IS1247, intI1_337 old, ISCR1 (MGEs) were the dominant ARGs and MGEs. Among them, Multidrug, Aminoglycoside and Tetracycline accounted for 3.61%, 2.27%and 15.55%of the total gene abundance, respectively, while Multidrug, Aminoglycoside and Tetracycline in the control area accounted for 3.16%, 3.12% and 14.09% of the total gene abundance.

Therefore, Aminoglycoside and Tetracycline are the gene types dominated by each component, while AAC(3)-Via (Aminoglycoside), tetM, tetR (Tetracycline), ISCR1 and IS1247 (MGEs) are the co-dominant ARGs and MGEs. Through network analysis (Fig. 14), AAC(3)-Via, tetM, tetR were significantly correlated with 10 ARGs in the food chain system, such as IS26, IS200-2, IS200-1, etc. ARGs can be transferred horizontally with MGEs, so that ARGs can migrate and spread among different species of microorganisms, thereby increasing their risk of transmission in the food chain. The detection of high content of MGEs is helpful to promote the horizontal transfer of ARGs(ZEWEN W et al.,2024), including the integron intI1_337old detected in soil and snail feces. Integron as MGEs is considered to be one of the important reasons for the horizontal transfer of ARGs(CHEN B et al.,2015).It has been reported that integrons can also coexist with tetracycline in binding plasmids, such as tetM and tetR, which can be easily transferred to receptors together(Ye et al.,2022).

3.3 Common and specific ARGs in each component of soil-lettuce-snail food chain system

It is considered to be an effective method to study the transfer and diffusion of ARGs by analyzing the common and unique ARGs contained in different sample types(QING-LIN C et al.,2017).In this study, we used the ' Yifan Hu ' algorithm 's Bipartite network analysis to explore the types of ARGs that may migrate to the lettuce phyllosphere and snails after applying pig manure to the soil.

As shown in Fig. 6, we explored the sources of ARGs in fertilized soil and non-fertilized soil through a two-sided network diagram, and cluster III showed the unique ARGs and MGEs types in pig manure. Then, the two-sided network diagram was drawn with pig manure, lettuce phyllosphere of fertilization group and snail manure of fertilization group as the research objects, revealing that the resistance group was mainly divided into 7 clusters (Fig. 7).Cluster I indicated that there were 7 ARGs (6 types) in the three components, which were tetM, tnpA-2, mel_1, aacA43, sul1, sul2 and ermF, respectively. Cluster III was the common ARGs in the phyllosphere of pig manure and fertilized lettuce, which were vatB and vanRB.The detection of common ARGs in the resistant group means that the application of pig manure may spread ARGs to the lettuce phyllosphere and snail feces samples through the transfer of the food chain. However, common ARGs are also detected in the lettuce phyllosphere and snail feces of the control group (Fig. 8). Therefore, it is necessary to explore the changes in the abundance of ARGs.

First of all, from the soil to the lettuce food chain system, we can find that aacA43, mel_1, and vanRB are detected in a small amount in the lettuce phyllosphere without manure application. The reason may be that the particles in the air provide attachment sites for ARGs, and ARGs are generated by aerosol attachment to the lettuce leaf surface(GAO M et al.,2017).However, with the application of pig manure, its abundance increased significantly by 233.94%, 312.00% and 228.23%, respectively, among which mel_1 had the strongest proliferation ability. The remaining ARGs such as tetM, tnpA-2, sul1, sul2, ermF and vatB were not detected in the lettuce phyllosphere of the NPM group, which clearly indicated that tetM, tnpA-2, sul1, sul2, ermF and vatB were derived from pig manure and transferred to the roots and leaves of lettuce through horizontal gene transfer (HGT). Plants, especially agricultural products, can be used as potential reservoirs and transmission tools for ARGs(An et al.,2022).Wang et al.also detected many ARGs similar to manure-improved soil in lettuce and lettuce endophytes(WANG F-H et al.,2015).Zhang et al.used chicken manure as fertilizer to add to the soil planted with bitter chrysanthemum, and common ARGs were detected between endophytes and soil flora(Zhang et al.,2021).The above studies have shown that the application of manure may lead to the transfer of ARGs to vegetables.

In the food chain system from lettuce to snail, mel_1 in lettuce components was the only source of mel_1 in snail excrement in the fertilization group. Therefore, manure application successfully promoted the colonization of mel_1 in snail intestine through the gene level of soil-lettuce-snail food chain.In addition to mel_1, tetM, tnpA-2, aacA43, sul1, sul2 and ermF were also detected in the snail excrement of the control group. Because the soil animal microbiome may contain a variety of resistant groups, its intestinal microorganisms are the repository of ARGs(FLORENT M et al.,2023).However, according to the abundance change of ARGs in Fig. 9, the abundance of tetM, tnpA-2, aacA43, sul1 and sul2 in snail excrement increased by 78.95%, 4.39%, 17.96%, 20% and 21.15% respectively with the application of pig manure, and tetM had the strongest proliferation ability. In addition, the abundance of ermF in snail excrement decreased with the application of pig manure, and the application of manure had little effect on ermF.

Therefore, the proliferation of aacA43, mel_1 and vanRB in lettuce and the proliferation of tetM, tnpA-2, aacA43, sul1 and sul2 in snail fecal components can be explained as two reasons. First, MGEs play a key role in the transfer of ARGs level genes. Studies have shown that the transfer of ARGs from exogenous ARB to local microorganisms through MGEs is an important mechanism affecting the dynamics of ARGs in soil(YAN H et al.,2023). MGEs can promote fecal ARGs to pass through the soil-lettuce-snail food chain from the internal path through HGT to root and leaf endophytes. When snails eat lettuce, ARGs are transferred into the snail intestine, resulting in an increase in the abundance of ARGs in lettuce and snail excrement. Among them, tetM has an extremely significant correlation with MGEs such as IS6100, IS1247, and IS26 (P < 0.001) (Fig. 13), and its growth is so significant that it may be the promotion of MGEs.The second reason is that manure application not only inputs exogenous ARGs into the soil, but also promotes the proliferation of indigenous drug-resistant bacteria, resulting in the enrichment of ARGs in the soil(PENG S et al.,2015;JECHALKE S et al.,2014). The selective pressure of ARGs in pig manure induces the increase of the proportion of endogenous drug-resistant bacteria in lettuce, which leads to the increase of ARGs abundance in lettuce endogenous system and snail excrement.

3.4 Changes of bacterial community in soil-lettuce-snail food chain system

3.4.1 Analysis of α diversity of bacterial community in each component of food chain system

Shannon, Simpson, Chao1, OTU and other diversity indexes were used to characterize the richness and diversity of bacterial communities in each component of the food chain system (Fig. 10). The four α diversity indexes in this study showed that the application of pig manure increased the bacterial diversity and richness in soil, lettuce phyllosphere and snail manure components. Among them, the bacterial community in soil components had the most diversity and richness, and the bacterial community in lettuce phyllosphere components had the least diversity and richness.

3.4.2 Comparison of bacterial phylum level in each component of food chain system

Compared with the control treatment, the application of pig manure significantly changed the composition and distribution of bacterial communities in soil, lettuce phyllosphere and snail excrement. The application of manure transformed Actinobacteria and Firmicutes, which were previously the main dominant phylum in the soil, into Bacteroidetes and Firmicutes.Under two different treatments, although Firmicutes (27%~40%) was the most dominant phylum, the application of pig manure still caused changes in bacterial diversity in lettuce phyllosphere.Among the snail excrement components, the dominant phylum changed from Bacteroidetes (47%) in the blank group to Proteobacteria (63%) (Fig. 12).

The distribution of main bacterial phylum in each component of the food chain system is quite different. As shown in Fig. 13, the relative abundances of Bacteroidetes and Firmicutes in soil samples were higher, ranging from 0.05%~32.74% and 21.06%~30.69%. Except for Bacteroidetes and Verrucomicrobia, the other bacteria showed a decreasing trend with the application of pig manure.The dominant bacteria in lettuce tissues were Firmicutes, and their relative abundance in lettuce phyllosphere was 2.08%~2.74%. Except that the relative abundance of Proteobacteria in NPM group was 4.17%, the relative abundance of other bacteria was less than 2%. Bacteroidetes and Firmicutes showed an upward trend due to the application of pig manure.Proteobacteria and Bacteroidetes were the main bacteria at the phylum level in snail excrement samples. The relative abundance of these two types of bacteria was the highest at 31.29%~54.30% and 26.33%~41.01%, respectively, except for Proteobacteria. Bacteroidetes, Actinobacteria, Verrucomicrobia and Firmicutes decreased by 55.75%, 2.38%, 151.03% and 55.48% respectively due to the application of pig manure.Overall, the application of pig manure changed the composition and distribution of bacterial communities in the soil-lettuce-snail system, and the relative abundance of some bacterial phylum in the phyllosphere of soil and lettuce changed significantly.

3.5 Co-occurrence of ARGs and MGEs in food chain system

Based on all paired Pearson correlations, we used a network diagram to visualize the co-occurrence relationship between ARGs with abundance top30 and MGEs with abundance top10 in the food chain system. The network diagram of the soil-lettuce-snail food chain system consists of 38 nodes (including 28 ARGs and 10 MGEs) and 215 connections (Fig. 13).There were 10 kinds of MGEs in the food chain system, which were IS26, IS200-2, IS200-1, IS6100, EAE_05855, IS1247, IncN_korA, ISEcp1, TN5403 and ISCR1, respectively. There was a significant correlation with various types of ARGs. Among them, IS6100, IS1247 and IS26 had a very significant correlation with 26 ARGs (P < 0.001). IS6100 and qacH_351, IS1247 and aadA2, IS26 and tetM had the strongest correlation.TN5403 and IS200-1 were significantly correlated with 22 ARGs (P < 0.001), among which TN5403 was most significantly correlated with pcoA and IS200-1 was most significantly correlated with ttgB.IS200-2, ISCR1 and ISEcp1 were significantly correlated with 21 ARGs (P < 0.001), and ttgB and ttgA resistance genes were significantly correlated with the above three MGEs, indicating that the transmission of ttgB and ttgA genes can be promoted by IS200-2, ISCR1 and ISEcp1, and IS200-2, ISCR1 and ISEcp1 are the main transmissible carriers.In addition, we also found that although the abundance of sulfonamide is not prominent in the components of the food chain, sul1 and sul2 have a significant correlation with IS26, IS6100, TN5403 and other MGEs due to the prominent growth rate of manure application in each component.The sul gene can generally be transferred and transmitted by integron, mobile plasmid and transposon(SARAH H et al.,2023;CHAO L et al.,2023;BOMBAYWALA S et al.,2021).The sul2 gene is usually located on a smaller non-conjugating plasmid or a larger mobile multi-resistant plasmid(RUPERT B et al.,2022;YING M et al.,2022). The plasmid as one of the mobile elements is an important way for ARGs horizontal gene transfer.In summary, IS6100, IS1247 and IS26 are most closely related to ARGs in the food chain system. MGEs play an important role in the lateral transfer of ARGs in the food chain system as a driving factor. Therefore, the presence of a large number of MGEs in manure will further promote the horizontal transmission of fecal ARGs between the soil-lettuce-snail food chain system.

3.6 Co-occurrence of ARGs and bacterial community in food chain system

In order to study the correlation between bacterial communities and ARGs in the food chain system and find potential host bacteria of ARGs, this study selected the bacterial community with abundance of top30 genus level and the ARGs with abundance of top10, and explored the relationship between ARGs and bacterial community in the food chain system through network analysis.The network diagram of ARGs and dominant bacterial genera in the soil-lettuce-snail food chain system consists of 20 nodes (including 10 bacterial genera and 10 ARGs) and 85 connections (Fig. 14).The results showed that there was an interaction between ARGs and bacterial communities.All bacterial genera belong to four bacterial phyla : Bacteroidetes, Firmicutes, Verrucomicrobia and Proteobacteria.Among them, Firmicutes and Proteobacteria are the dominant host phyla in the soil-lettuce-snail food chain system, containing 7 and 5 bacterial genera, respectively.Therefore, it is easier to act as a carrier of ARGs and promote the spread of resistance in the food chain.AadA2 has the most potential host bacteria, 10.Secondly, tetM, tetD, tetG, sul1, sul2 and qacF_H all have 9 identical potential host bacteria, Aeromonas, Shewanella, Mucilaginibacter, Pseudomonas, Comamonas, Lactococcus, Algoriphagus, Luteolibacter, Paenibacillus. Among them, sul2 was significantly correlated with 8 bacterial genera except Pseudomonas (P < 0.001).Both tetA and tetR were carried by Aeromonas, Mucilaginibacter, Pseudomonas, Comamonas, Shewanella, Lactococcus and Algoriphagus. Different from tetA and tetR, the potential host bacteria of tetX were Algoriphagus, Lactococcus, Luteolibacter, Paenibacillus, Shewanella, Flavobacterium and Aeromonas.The large number of potential host bacteria of ARGs indicates that the evolution of bacterial community structure may be an important mechanism affecting the distribution of ARGs in the manure-amended soil-lettuce-snail food chain system.Chen 's results also suggest that soil microbes may be transferred to the leaf surface by some way, and the change of ARGs in the phyllosphere can be achieved by changing the phyllosphere microorganisms(CHEN Q-L et al.,2018).The composition and distribution of bacterial communities at different interfaces such as soil, river and sludge are important factors affecting the dynamic changes of ARGs spectrum(TELMA F, et al.,2019).These results suggest that bacterial community structure as another driving factor plays an important role in the spread of ARGs(CHENG J et al.,2021).By studying the changes in the distribution of bacterial community composition and the host relationship with ARGs, the risk of ARGs transmission in the food chain can be further clarified.

In summary, the application of manure will introduce ARGs into the trophic levels of the food chain, increase the abundance and diversity of ARGs and MGEs in each component, and change the composition and distribution of bacterial communities. There is a significant correlation between ARGs and MGEs in the food chain system. As a driving factor, MGEs will aggravate the spread and diffusion of ARGs in the food chain.Firmicutes and Proteobacteria, as the dominant host bacteria in the food chain system, are highly correlated with the enrichment of ARGs, and are more likely to be used as carriers of ARGs to promote the transfer of resistance in the food chain.Therefore, fecal ARGs can be transmitted between the soil-vegetable-animal environment system, and may also be transferred to human microorganisms and pathogens. The treatment and utilization of animal manure should be more effectively guided and controlled.

Acknowledgements

We are grateful to the Youth Program of the National Natural Science Foundation of China (41701564),Suzhou Science and Technology Plan Program (SNG201613),and the Natural Science Foundation of the Higher Education institutions of Jiangsu Province, China (17KJB610010) for providing financial support for this study. We would like to thank Editage (www. edita ge. cn) for English language editing.

Statements&Declarations

Ethics Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Author Contribution

Conceptualization, Yuan Zhang; methodology, Yuan Zhang; software, Jiayi Zhao; validation, Minglong Chen, Xinyue Tang, Yijia WANG and Yun Zou;formal analysis, Jiayi Zhao; investigation, Jiayi Zhao and Minglong Chen; resources, Minglong Chen; data curation, Jiayi Zhao; writing—original draft preparation, Jiayi Zhao; writing—review and editing, Yuan Zhang; visualization,Jiayi Zhao; supervision, Yuan Zhang; project administration, Yuan Zhang; funding acquisition, Yuan Zhang. All the authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Youth Program of the National Natural Science Foundation of China(41701564),Suzhou Science and Technology Plan Program(SNG201613),and Natural Science Foundation of the Higher Education Institutions of Jiangsu Province, China (17KJB610010).

Conflict of Interest

The authors declare no competing interests.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Fecal antibiotic resistance genes were transferred through the distribution of soil-lettuce-snail foodchain

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Abstract

Figures

1 Introduction

2 Materials And Methods

2.1 Sample collection

2.2 DNA extraction

2.3 High-throughput quantitative PCR analysis

2.4 Quantitative PCR analysis of bacterial 16S rRNA gene

2.5 MiSeq sequencing and data processing

2.6 Statistical analysis

3 Results And Discussion

3.1 Abundance and diversity of ARGs and MGEs in each component of food chain system

3.2 Enrichment of ARGs in soil-lettuce-snail food chain system

3.3 Common and specific ARGs in each component of soil-lettuce-snail food chain system

3.4 Changes of bacterial community in soil-lettuce-snail food chain system

3.4.1 Analysis of α diversity of bacterial community in each component of food chain system

3.4.2 Comparison of bacterial phylum level in each component of food chain system

3.5 Co-occurrence of ARGs and MGEs in food chain system

3.6 Co-occurrence of ARGs and bacterial community in food chain system

4 Conclusion

Declarations

References

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