Antibiotic Resistance Genes In Manure Research Articles

Assessing intracellular and extracellular distribution of antibiotic resistance genes in the commercial organic fertilizers

Compost-based organic fertilizers often contain high levels of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). Previous studies focused on quantification of total ARGs and MGEs. For a more accurate risk assessment of the dissemination risk of antibiotic resistance, it is necessary to quantify the intracellular and extracellular distribution of ARGs and MGEs. In the present study, extracellular ARGs and MGEs (eARGs and eMGEs) and intracellular ARGs and MGEs (iARGs and iMGEs) were separately analyzed in 51 commercial composts derived from different raw materials by quantitative polymerase chain reaction (qPCR) and metagenomic sequencing. Results showed that eARGs and eMGEs accounted for 11–56% and 4–45% of the total absolute abundance of ARGs and MGEs, respectively. Comparable diversity, host composition and association with MGEs were observed between eARGs and iARGs. Contents of high-risk ARGs were similar between eARGs and iARGs, with high-risk ARGs in the two forms accounting for 6.7% and 8.2% of the total abundances, respectively. Twenty-four percent of the overall ARGs were present in plasmids, while 56.7% of potentially mobile ARGs were found to be associated with plasmids. Variation partitioning analysis, null model and neutral community model indicated that the compositions of both eARGs and iARGs were largely driven by deterministic mechanisms. These results provide important insights into the cellular distribution of ARGs in manure composts that should be paid with specific attention in risk assessment and management.

Response of soil antibiotic resistance genes and bacterial communities to fresh cattle manure and organic fertilizer application

Livestock manure use in agriculture contributes to pollutants like antibiotic resistance genes (ARGs) and resistant bacteria. This practice could potentially facilitate ARGs development in soil ecosystems. Our study aimed to explore ARGs and bacterial communities in cattle manure from Ningxia beef cattle farms with varying breeding periods. We also assessed the impact of different application rates of cattle manure compost, created by mixing manure with different growing periods, on soil's physicochemical and heavy metal properties. High-throughput PCR and sequencing were used to analyze ARGs and bacterial communities. We aimed to understand ARGs dynamics in cattle manure during breeding stages and the impact of different fertilizer application rates on soil bacteria and resistance genes. We found 212 ARGs from cattle manure, spanning tetracycline, aminoglycoside, multidrug, and MLSB categories. Relative ARGs abundance was presented across breeding stages: lactation (C1), breeding (C3), pre-fattening (C4), calving (C2), and late fattening (C5). pH, total nitrogen (TN), electrical conductivity (EC), arsenic (As) and cadmium (Cd) presence significantly impacted ARGs quantity and microbial community structure in manure. Mobile genetic elements (MGEs) were the primary factor altering ARGs in manure (65.56%). Heavy metals contributed to 18.60% of ARGs changes. Manure application changed soil ARGs abundance, notably in soils with high application rates, primarily associated with aminoglycoside, multidrug and sulfonamide resistance. Soils with higher manure rates had elevated MGEs, positively correlated with most ARGs, suggesting MGEs' role in ARGs dissemination. Soil microbial community structure was influenced by fertilization, particularly with the highest application rate. Heavy metals (specifically Cd, contributing to 23.12%), microbial community changes (17.42%), and MGEs (17.38%) were the main factors affecting soil antibiotic resistance. Our study establishes a framework for understanding ARGs emergence in manure and treated soils. This informs strategies to mitigate environmental ARGs transmission and guides diverse livestock manure application and management.

Long-term impacts of conservation pasture management in manuresheds on system-level microbiome and antibiotic resistance genes.

Animal manure improves soil fertility and organic carbon, but long-term deposition may contribute to antibiotic resistance genes (ARGs) entering the soil-water environment. Additionally, long-term impacts of applying animal manure to soil on the soil-water microbiome, a crucial factor in soil health and fertility, are not well understood. The aim of this study is to assess: (1) impacts of long-term conservation practices on the distribution of ARGs and microbial dynamics in soil, and runoff; and (2) associations between bacterial taxa, heavy metals, soil health indicators, and ARGs in manures, soils, and surface runoff in a study following 15 years of continuous management. This management strategy consists of two conventional and three conservation systems, all receiving annual poultry litter. High throughput sequencing of the 16S ribosomal RNA was carried out on samples of cattle manure, poultry litter, soil, and runoff collected from each manureshed. In addition, four representative ARGs (intl1, sul1, ermB, and blactx-m-32) were quantified from manures, soil, and runoff using quantitative PCR. Results revealed that conventional practice increased soil ARGs, and microbial diversity compared to conservation systems. Further, ARGs were strongly correlated with each other in cattle manure and soil, but not in runoff. After 15-years of conservation practices, relationships existed between heavy metals and ARGs. In the soil, Cu, Fe and Mn were positively linked to intl1, sul1, and ermB, but trends varied in runoff. These findings were further supported by network analyses that indicated complex co-occurrence patterns between bacteria taxa, ARGs, and physicochemical parameters. Overall, this study provides system-level linkages of microbial communities, ARGs, and physicochemical conditions based on long-term conservation practices at the soil-water-animal nexus.

Risk assessment and dissemination mechanism of antibiotic resistance genes in compost

In recent years, the excessive of antibiotics in livestock and poultry husbandry, stemming from extensive industry experience, has resulted in the accumulation of residual antibiotics and antibiotic resistance genes (ARGs) in livestock manure. Composting, as a crucial approach for the utilization of manure resources, has the potential to reduce the levels of antibiotics and ARGs in manure, although complete elimination is challenging. Previous studies have primarily focused on the diversity and abundance of ARGs in compost or have solely examined the correlation between ARGs and their carriers, potentially leading to a misjudgment of the actual risk associated with ARGs in compost. To address this gap, this study investigated the transfer potential of ARGs in compost and their co-occurrence with opportunistic pathogenic bacteria by extensively analyzing metagenomic sequencing data of compost worldwide. The results demonstrated that the potential risk of ARGs in compost was significantly lower than in manure, suggesting that composting effectively reduces the risk of ARGs. Further analysis showed that the microbes shifted their life history strategy in manure and compost due to antibiotic pressure and formed metabolic interactions dominated by antibiotic-resistant microbes, increasing ARG dissemination frequency. Therefore, husbandry practice without antibiotic addition was recommended to control ARG evolution, dissemination, and abatement both at the source and throughout processing.

The Antibiotics Degradation and Its Mechanisms during the Livestock Manure Anaerobic Digestion.

Antibiotics are administered to livestock at subtherapeutic levels to promote growth, and their degradation in manure is slow. High antibiotic concentrations can inhibit bacterial activity. Livestock excretes antibiotics via feces and urine, leading to their accumulation in manure. This can result in the propagation of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Anaerobic digestion (AD) manure treatment technologies are gaining popularity due to their ability to mitigate organic matter pollution and pathogens, and produce methane-rich biogas as renewable energy. AD is influenced by multiple factors, including temperature, pH, total solids (TS), substrate type, organic loading rate (OLR), hydraulic retention time (HRT), intermediate substrates, and pre-treatments. Temperature plays a critical role, and thermophilic AD has been found to be more effective in reducing ARGs in manure compared to mesophilic AD, as evidenced by numerous studies. This review paper investigates the fundamental principles of process parameters affecting the degradation of ARGs in anaerobic digestion. The management of waste to mitigate antibiotic resistance in microorganisms presents a significant challenge, highlighting the need for effective waste management technologies. As the prevalence of antibiotic resistance continues to rise, urgent implementation of effective treatment strategies is necessary.

Mitigation of antibiotic resistome in swine manure by black soldier fly larval conversion combined with composting

The increasing prevalence of antibiotic resistance genes (ARGs) in animal manure has attracted considerable attention because of their potential contribution to the development of multidrug resistance worldwide. Insect technology may be a promising alternative for the rapid attenuation of ARGs in manure; however, the underlying mechanism remains unclear. This study aimed to evaluate the effects of black soldier fly (BSF, Hermetia illucens [L.]) larvae conversion combined with composting on ARGs dynamics in swine manure and to uncover the mechanisms through metagenomic analysis. Compared to natural composting (i.e. without BSF), BSFL conversion combined with composting reduced the absolute abundance of ARGs by 93.2 % within 28 days. The rapid degradation of antibiotics and nutrient reformulation during BSFL conversion combined with composting indirectly altered manure bacterial communities, resulting in a lower abundance and richness of ARGs. The number of main antibiotic-resistant bacteria (e.g., Prevotella, Ruminococcus) decreased by 74.9 %, while their potential antagonistic bacteria (e.g., Bacillus, Pseudomonas) increased by 128.7 %. The number of antibiotic-resistant pathogenic bacteria (e.g., Selenomonas, Paenalcaligenes) decreased by 88.3 %, and the average number of ARGs carried by each human pathogenic bacterial genus declined by 55.8 %. BSF larvae gut microbiota (e.g., Clostridium butyricum, C. bornimense) could help reduce the risk of multidrug-resistant pathogens. These results provide insight into a novel approach to mitigate multidrug resistance from the animal industry in the environment by using insect technology combined with composting, in particular in light of the global “One Health” requirements.

Comparison of antibiotic resistance genes in swine manure storage pits of Iowa, USA

Antimicrobial resistance (AMR) can develop in deep-pit swine manure storage when bacteria are selectively pressured by unmetabolized antibiotics. Subsequent manure application on row crops is then a source of AMR into soil and downstream runoff water. Therefore, understanding the patterns of diverse antibiotic resistance genes (ARGs) in manure among different farms is important for both interpreting the results of the detection of these genes from previous studies and for the use of these genes as bioindicators of manure borne antibiotic resistance in the environment. Previous studies of manure-associated ARGs are based on limited samples of manures. To better understand the distribution of ARGs between manures, we characterized manures from 48 geographically independent swine farms across Iowa. The objectives of this study were to characterize the distribution of ARGs among these manures and to evaluate what factors in manure management may influence the presence of ARGs in manures. Our analysis included quantification of two commonly found ARGs in swine manure, ermB and tetM. Additionally, we characterized a broader suite of 31 ARGs which allowed for simultaneous assays of the presence or absence of multiple genes. We found the company integrator had a significant effect on both ermB (P=0.0007) and tetM gene concentrations (P=0.0425). Our broad analysis on ARG profiles found that the tet(36) gene was broadly present in swine manures, followed by the detection of tetT, tetM, erm(35), ermF, ermB, str, aadD, and intl3 in samples from 14 farms. Finally, we provide a comparison of methods to detect ARGs in manures, specifically comparing conventional and high-throughput qPCR and discuss their role in ARG environmental monitoring efforts. Results of this study provide insight into commonalities of ARG presence in manure holding pits and provide supporting evidence that company integrator decisions may impact ARG concentrations.

Insights into the impact of manure on the environmental antibiotic residues and resistance pool.

The intensive use of antibiotics in the veterinary sector, linked to the application of manure-derived amendments in agriculture, translates into increased environmental levels of chemical residues, AR bacteria (ARB) and antibiotic resistance genes (ARG). The aim of this review was to evaluate the current evidence regarding the impact of animal farming and manure application on the antibiotic resistance pool in the environment. Several studies reported correlations between the prevalence of clinically relevant ARB and the amount and classes of antibiotics used in animal farming (high resistance rates being reported for medically important antibiotics such as penicillins, tetracyclines, sulfonamides and fluoroquinolones). However, the results are difficult to compare, due to the diversity of the used antimicrobials quantification techniques and to the different amounts and types of antibiotics, exhibiting various degradation times, given in animal feed in different countries. The soils fertilized with manure-derived products harbor a higher and chronic abundance of ARB, multiple ARG and an enriched associated mobilome, which is also sometimes seen in the crops grown on the amended soils. Different manure processing techniques have various efficiencies in the removal of antibiotic residues, ARB and ARGs, but there is only a small amount of data from commercial farms. The efficiency of sludge anaerobic digestion appears to be dependent on the microbial communities composition, the ARB/ARG and operating temperature (mesophilic vs. thermophilic conditions). Composting seems to reduce or eliminate most of antibiotics residues, enteric bacteria, ARB and different representative ARG in manure more rapidly and effectively than lagoon storage. Our review highlights that despite the body of research accumulated in the last years, there are still important knowledge gaps regarding the contribution of manure to the AMR emergence, accumulation, spread and risk of human exposure in countries with high clinical resistance rates. Land microbiome before and after manure application, efficiency of different manure treatment techniques in decreasing the AMR levels in the natural environments and along the food chain must be investigated in depth, covering different geographical regions and countries and using harmonized methodologies. The support of stakeholders is required for the development of specific best practices for prudent – cautious use of antibiotics on farm animals. The use of human reserve antibiotics in veterinary medicine and of unprescribed animal antimicrobials should be stopped and the use of antibiotics on farms must be limited. This integrated approach is needed to determine the optimal conditions for the removal of antibiotic residues, ARB and ARG, to formulate specific recommendations for livestock manure treatment, storage and handling procedures and to translate them into practical on-farm management decisions, to ultimately prevent exposure of human population.

Distribution Characteristics of Antibiotics and Antibiotic Resistance Genes in Manure and Surrounding Soil of Poultry Farm in Ningxia

Antibiotics and antibiotic resistance genes (ARGs) in livestock and poultry manure pose potential ecological risks. In order to understand the distribution characteristic of antibiotics and ARGs in manure and surrounding soils of poultry farms in Ningxia, the poultry manure and relative soil samples were collected from 12 layers of different poultry breeding farms. The compositions of antibiotics and ARGs in the samples were analyzed using UPLC-MS/MS and HT-qPCR. The results showed that:① tetracycline, aminoglycoside, and sulfonamide were the dominant antibiotics in poultry manure. The types and contents of antibiotics in poultry manure were different in different breeding periods. There were more types of antibiotics in the brooding period, the average content was high, and the initial stage showed the opposite trend. ② A small amount of antibiotics was detected in the surrounding soil only 20 m away from the poultry farm, and the poultry farm had little effect on the distribution of antibiotics in the surrounding soil. The content of quinolone in the soils with poultry manure application was significantly higher than that in the control and surrounding soil. ③ We detected 132-168 ARGs in poultry manure, and the number of aminoglycosides and tetracycline was higher. The relative abundance of ARGs in the rearing period was highest, and the initial stage showed the opposite trend. The total relative abundance of ARGs in the brooding period was highest, but the terminal period showed the opposite. There were 110 ARGs in poultry manure during all breeding periods. ④ There were 23-105 ARGs in the soils, and the number of aminoglycoside was highest, followed by multidrug ARGs. The poultry farm had a great effect on the number and relative abundance of ARGs in the surrounding soil. For example, the number and relative abundance of ARGs in the surrounding soil of poultry farms gradually decreased with the increase in the distance from the poultry farms. The number and relative abundance of ARGs in the soil with applied poultry manure were significantly increased; however, these values were lower than those in the soil 20 m away from the poultry farm. ⑤ β-lactamases, aminoglycosides, and macrolide lincosamide-streptogramin B (MLSB) ARGs were all at risk of horizontal movement in manure, and chloramphenicol ARGs were at risk of horizontal movement in soil. Correlation analysis showed that the relative abundance of aminoglycoside, tetracycline, sulfonamide, β-lactamase, and MLSB were not significantly correlated with their contents. ⑥ Different types of ARGs had related co-occurrence phenomena, such as the positive correlation between the relative abundance of ARGs in poultry manure, and aminoglycoside and β-lactamases, MGEs, multidrugs and vancomycins. The relative abundances of ARGs in soil, aminoglycoside and tetracyclines, vancomycins, sulfonamides, and MLSBs; tetracyclines and MLSBs; etc., all showed a significant positive correlation. In short, the co-occurrence among the relative abundance of ARGs in soil was significantly stronger than that in poultry manure. These results could provide the theoretical basis for the site selection of poultry farms, the selection of antibiotic types and dosages for large-scale breeding of laying hens, and the application of poultry manure.

Distribution Characteristics of Antibiotics and Antibiotic Resistance Genes in Manure and Surrounding Soil of Cattle Farms in Ningxia

Livestock breeding intensively uses veterinary antibiotics in concentrated feeding operations to improve growth and control disease. Consequently, livestock and poultry manure is an important repository of antibiotics and antibiotic resistance genes (ARGs). To understanding the distribution of antibiotics and ARGs in manure and surrounding soils of cattle farms in Ningxia, cattle manure from five breeding periods (lactation, calving, growing, pre-fattening, and post-fattening periods) and comparative soil samples were collected from the largest beef-breeding area in Ningxia. The compositions of ARGs in the samples were analyzed by UPLC-MS/MS and HT-qPCR. The results showed that:① Tetracycline, quinolone, and sulfonamide were the dominant antibiotics in cattle manure. The content of antibiotics in the manure varied greatly between different breeding periods. High amounts of antibiotics were detected during the pre-fattening and lactation periods, and the lowest amounts were detected during the calving period. ② Quinolone and tetracycline were the dominant antibiotics in the soils, and the detection rate and content of quinolone were highest. The cattle farms did not affect the distribution of antibiotics in the surrounding soils. The content of quinolone and tetracycline in the soils with cattle manure application were significantly higher than control and surrounding soils. ③ We detected 79-142 ARGs in cattle manure, with aminoglycosides the most common form. The number and relative abundance of ARGs were highest during the growing period and lowest during the calving period. The dominant ARGs were tetQ, ermF, and tetO-01 across all the breeding periods. ④ There were 35-79 ARGs in the tested soils, and multidrug and aminoglycoside ARGs were dominant. The cattle farms did not affect the number and relative abundance of ARGs in the surrounding soils; however, manure application significantly affected the number and relative abundance of ARGs. ⑤ Sulfonamide and chloramphenicol ARGs are at risk of mobilization and horizontal transport. A correlation analysis showed that the relative abundances of aminoglycoside and tetracycline in cattle manure were significantly positively correlated with their contents. ⑥ Aminoglycoside, sulfonamide, chloramphenicol, and mobile genetic elements (MGEs) in manure were significantly or extremely significantly positively correlated with aminoglycosides and sulfonamides in the soils, whereas macrolides were negatively correlated with vancomycin. These results provide baseline data to inform controls on the variety and dosages of feed and veterinary drugs in cattle farms and the application of organic fertilizers in agriculture.

Using the heat generated from electrically conductive concrete slabs to reduce antibiotic resistance in beef cattle manure

Proper treatment is necessary to reduce antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in livestock manure before land application. Conventional stockpiling suffers unreliable removal efficiency, while composting can be complicated and expensive. The objective of this study was to test the feasibility of a novel heat-based technology, i.e., stockpiling manure on conductive concrete slabs, to inactivate ARB and ARGs in beef cattle manure. In this study, two independent bench-scale trials were conducted. In both trials, samples were taken from manure piles on conductive concrete slabs and regular slabs (i.e., heated and unheated piles). In the heated pile of the first trial, 25.9% and 83.5% of the pile volume met the EPA Class A and Class B biosolids standards, respectively. For the heated pile of the second trial, the two values were 43.9% and 74.2%. In both trials, nearly all forms of the total and resistant Escherichia coli and enterococci were significantly lower in the heated piles than in the unheated piles. Besides, significant reduction of ARGs in heated piles was observed in the first trial. Through this proof-of-concept study, the new technology based on conductive concrete slabs offers an alternative manure storage method to conventional stockpiling and composting with respect to reduce ARB and ARGs in manure.

A comparison of antibiotics, antibiotic resistance genes, and bacterial community in broiler and layer manure following composting.

Animal manure is an important source of antibiotics and antibiotic resistance genes (ARGs) in the environment. However, the difference of antibiotic residues and ARG profiles in layer and broiler manure as well as their compost remains unexplored. In this study, we investigated the profiles of twelve antibiotics, seventeen ARGs, and class 1 integrase gene (intI1) in layer and broiler manure, and the corresponding compost at large-scale. Compared with layer manure, broiler manure exhibited approximately six times more residual tetracyclines, especially chlortetracycline. The relative abundances of qnrS and ermA genes in broiler manure were significantly higher than those in layer manure. The concentration of tetracyclines not only had a significantly positive correlation with tetracycline resistance genes (tetA and tetC) but was also positively correlated with quinolone resistance (qepA, qnrB, and qnrS) and macrolide resistance (ermA and ermT). Most ARGs in manure were reduced after composting. However, the relative abundance of sulfonamide resistance gene sul1 increased up to 2.41% after composting, which was significantly higher than that of broiler (0.41%) and layer (0.62%) manure. The associated bacterial community was characterized by high-throughput 16S rRNA gene sequencing. The relative abundances of thermophilic bacteria had significant positive correlations with the abundance of sul1 in compost. The composting has a significant impact on the ARG-associated gut microbes in poultry manure. Variation partitioning analysis indicated that the change of bacterial community compositions and antibiotics contributed partially to the shift in ARG profiles. The results indicate that at industry-scale production broiler manure had more antibiotics and ARGs than layer manure did, and composting decreased most ARG abundances in poultry manure except for sulfonamide resistance genes.

The insufficient extraction of DNA from swine manures may underestimate the abundance of antibiotic resistance genes as well as ignore their potential hosts

Swine manure is considered as an extensive reservoir of antibiotic resistance genes (ARGs) spreading in the natural environment after application in soils. To understand whether ARGs abundance in swine manure is underestimated and the hosts are ignored, this study successively extracted DNA from swine manure six times and determined the abundance of several ARGs, class I integron (intI1), and 16S rRNA as well as the microbial communities. It is found that successive six DNA extraction of swine manures elevated the yield of DNA and strongly increased the abundance of ARGs, intI1, and 16S rRNA. Compared with single DNA extraction, the most dominant bacterial phylum in swine manures shifted from Proteobacteria to Firmicutes, and the dominant bacterial genera changed from Acinetobacter Clostridium after six DNA extraction. The ignored abundance of bacterial phylum and genus emphasized the possible hosts carrying these genes should be paid more attention. It is suggested that the successive DNA extraction of manures is required in the future study to improve the knowledge of estimating the risk and hosts of ARGs in manures entering the environment.

The removal of moisture and antibiotic resistance genes in dairy manure by microwave treatment.

To characterize the drying of dairy manure during microwave (MW) heating, and to determine the impacts of microwave radiation on reductions of antibiotic resistance genes (ARGs), this study investigated on understanding the effects of microwave heating on solid streams of flushed dairy manure. A series of experiments were performed to determine the rate of drying, moisture removal percentages, change in solids, and the level of ARGs. Manure was exposed to microwave radiation for 30-300 s at a frequency of 2450 MHz. The results showed moisture removal rate (U) up to 0.63-g water per minute per gram of dairy manure. During MW treatment, volatile solid (VS) content was relatively stable. The MW treatment resulted in reduction of ARGs such as sulII, intI1, and tnpA. The ARG concentrations (sulII, intI1, and tnpA) were reduced by 2 orders of magnitude in less than 1 min of microwave heating. The preliminary results of this study showed that MW treatment can be a viable option for drying of dairy manure and reducing ARGs in manure.

Variation of heavy metal speciation, antibiotic degradation, and potential horizontal gene transfer during pig manure composting under different chlortetracycline concentration

Overuse of heavy metal and antibiotics in livestock husbandry has led to the accumulation of heavy metal resistance genes (HMRGs) and antibiotic resistance genes (ARGs) in environment. This research aims to reveal the variation of heavy metal speciation and potential horizontal gene transfer (HGT) of HMRGs and ARGs in manure composting under different initial chlortetracycline (CTC) concentrations. Treatments spiked with 20 mg/kg CTC (treatment P1), 100 mg/kg CTC (treatment P2), and the control (treatment CK) were operated. Results showed that CTC could be completely removed in the thermophilic phase of all the treatments despite of the initial concentrations. Bioavailable Cu in treatments CK, P1, and P2 declined by 14.5%, 27.1%, and 26.7% and bioavailable Zn declined by 15.3%, 29.5%, and 12.1%, respectively, after the composting, respectively. Relative abundance of HMRGs decreased by 6.49 log, 8.88 log, and 5.77 log, respectively, in treatments CK, P1, and P2. Relative abundance of ARGs decreased by 3.37 log, 4.86 log, and 3.32 log, respectively, in treatments CK, P1, and P2. Composting could effectively reduce genes pcoD, pcoA, zntA, tetQ, and tetA, which might locate on the same plasmid. CTC of 100 mg/kg promoted the co-selection of ARGs and HMRGs and increased the potential HGT of gene cusA.

Acidic conditions enhance the removal of sulfonamide antibiotics and antibiotic resistance determinants in swine manure

Manure pH may vary depending on its inherent composition or additive contents. However, the effect of pH on the fate of antibiotics and antibiotic resistance determinants in manure remains unclear. This work demonstrated that pH adjustment promoted the removal of different sulfonamide antibiotics (SAs) within swine manure under incubation conditions, which increased from 26–60.8% to 75.0–86.0% by adjusting the initial pH from neutral (7.4) to acidic (5.4–4.8). Acidification was also demonstrated to inhibit the accumulation of antibiotic resistance genes in manure during incubation. Acidified manure contained both lower absolute and relative abundances of sul1 and sul2 than those at a neutral pH like 7.4. Further investigation indicated that acidification promoted the reduction of sul genes in manure by restricting sulfonamide-resistant bacteria (SRB) proliferation and inhibiting IntI1 accumulation. Furthermore, pH adjustment significantly influenced the composition of the manure bacterial community after incubation, which increased Firmicutes and decreased Proteobacteria. Close relationships were observed between pH-induced enrichment of the Firmicutes bacterial phylum, enhanced SAs degradation, and the fates of antibiotic resistance determinants. Overall, lowering the pH of manure promotes the degradation of SAs, decreases sul genes and SRB, and inhibits horizontal sul gene transfer, which could be a simple yet highly-effective manure management option to reduce antibiotic resistance.

Effect of Co-composting of Chicken Manure with Chinese Medicinal Herbal Residues on Antibiotic Resistance Genes

Livestock manure is an important pathway by which antibiotic resistance genes (ARGs) enter the environment. To reduce the occurrence of antibiotic resistance genes in manures, we studied the variations of ARGs and mobile gene elements (MGEs) during the 46-day co-composting of chicken manure with Chinese medicinal herbal residues. The abundance of 100 ARGs and 5 MGEs were measured by Real-Time Quantitative PCR with 0, 3, 7, 14, 28, and 46 d co-composting. We detected 21 ARGs, 2 integrase genes (intI1 and intI2), and 3 transposase genes (tnpA-01, tnpA-02, and tnpA-03). The abundance of 5 MGEs significantly declined with co-composting time, particularly tnpA-01 and tnpA-02, which were reduced by two orders of magnitude. The abundance of aacA/aphD and aadE were significantly reduced (P<0.05) in aminoglycoside resistance genes. In β-lactam resistance genes, the strongest relationships were demonstrated between blaOXA1 and compost days (P=0.016), and the removal rate was 78.63%. The average removal rate was 90.39% for amide resistance genes, which decreased significantly with composting time. The removal ratios were different among tetracycline resistance genes. For example, the removal ratios of tetG and tetR were 99.77% and 31.72%, respectively. The highest removal rate of qnrD was 99.89%. The removal rate of sulⅢ was as high as 99.88%, while sulⅠ showed an increasing trend. Correlations between ARGs and MGEs were significant correlation for tnpA-01 and ARGs (P<0.05). The trend of ARGs with composting time indicates that the composting of Chinese medicine residues and chicken manure can significantly reduce the abundance of ARGs, thus reducing the risk of ARGs being distributed via livestock manure application.

Composting increased persistence of manure-borne antibiotic resistance genes in soils with different fertilization history

Different long-term fertilization regimes may change indigenous microorganism diversity in the arable soil and thus might influence the persistence and transmission of manure-born antibiotic resistance genes (ARGs). Different manure origins and composting techniques might affect the fate of introduced ARGs in farmland. A four-month microcosm experiment was performed using two soils, which originated from the same field and applied with the same chemical fertilizer or swine manure for 26 years, to investigate the dynamics of ARGs in soil amended with manure or compost from the farm and an agro-technology company. High throughput qPCR and sequencing were applied to quantify ARGs using 144 primer sets and microorganism in soil. Fertilization history had little effect on dynamics of manure-borne ARGs in soil regardless of manure origin or composting. Very different half-lives of ARGs and mobile genetic elements from farm manure and commercial manure were observed in both soils. Composting decreased abundance of most ARGs in manure, but increased the persistence of manure-introduced ARGs in soil irrespective of fertilization history, especially for those from farm manure. These findings help understanding the fate of ARGs in manured soil and may inform techniques to mitigate ARGs transmission.

Variation pattern of antibiotic resistance genes and microbial community succession during swine manure composting under different aeration strategies

AbstractBACKGROUNDAnimal manure is one of the significant sources of antibiotic resistance genes (ARGs) and an emerging pollutant that poses a threat to human beings. Composting is a common method of treating manure. The effect of composting on the ARGs in manure has become an active research area in recent years. In this study, three common aeration strategies were used to study the removal of ARGs and to clarify their microbial mechanism during composting, i.e. forced aeration (with three ventilation rates), turn windrow and static pile.RESULTSResults showed that the variation pattern of ARGs was highly dependent on the succession of the microbial community, and environmental factors accounted for 44% of the variation. Most ARGs (tetW/tetO/tetH, qnrS, ermB and blaTEM) could be significantly reduced after composting for 77 days, but sulfonamide resistance genes (sulI/sulII) and the integrase gene (intI) were highly enriched, particularly in forced aeration treatment. These enrichments were closely correlated with an increase in the genera belonging to Actinobacteria, Proteobacteria and Bacteroidetes and a decrease in the genera affiliated with Firmicutes.CONCLUSIONSThe temporal variations in ARGs were consistent under different aeration treatments. Further study should focus on optimizing the composting conditions to reduce recalcitrant ARGs. © 2019 Society of Chemical Industry

Compost-bulking agents reduce the reservoir of antibiotics and antibiotic resistance genes in manures by modifying bacterial microbiota

Sawdust, rice husk, and mushroom residues are commonly used as bulking agents during manure composting; this work evaluated their potentiality for enhancing the removal of antibiotics and antibiotic resistance genes (ARGs) under a temperature-consistent condition. Results indicated that the addition of all the compost-bulking agents increased antibiotic removal in swine manure with increasing rates of 14.9%–33.4%; however they showed less effect on the reduction of residual antibiotics in chicken manure where fluoroquinolone (FQs) antibiotics are the dominant antibiotics, partly owing to the weak promoting effects of bulking agents on FQs degradation. The addition of bulking agents somehow hindered the reduction of ARGs within swine manure, whereas there were obvious reductions in the total relative abundance of ARGs in chicken manure with bulking agents added. Among the three bulking agents, sawdust was the most efficient enhancer for antibiotic removal in both manures, and rice husk exhibited the best performance on ARGs reduction in chicken manure. The relationship between antibiotics, ARGs and bacteria communities was subsequently delineated. Proteobacteria was proposed to play key roles on the effect of bulking agent addition on antibiotics and ARGs in swine manure. Particularly, the increased Xanthomonadaceae contributed much to the promoted antibiotic degradation as well as the high level of ARGs in swine manure with sawdust added. By contrast, the changes in dominant bacterial families by the addition of bulking agents into chicken manure were not strong enough to effectively enhance antibiotic removal, but largely influenced the ARGs abundance. The large reductions of Paenibacillaceae and Staphylococcaceae by rice husk addition were closely related to the reduced ARGs in chicken manure with rice husk added.