Resistance Genes In Soil Research Articles

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.

Dynamics of oxytetracycline and resistance genes in soil under long-term intensive compost fertilization in Northern China.

In the present study, we explored the dynamics of antibiotics (ciprofloxacin, norfloxacin, enrofloxacin, and oxytetracycline), tetracycline resistance genes (TRGs), and bacterial communities over 2013-2015 in soils fertilized conventionally or with two levels (82.5 and 165t/ha) of compost for 12years. In the soil receiving 165t/ha of compost, only oxytetracycline was 46% higher than that in the conventionally fertilized soil. Transient enrichment of both tetM (20% to 9-fold) and tetK (25% to 67-fold) was observed in multiple instances immediately after the application of compost. The majority of genera which positively correlated with tetM or tetK were affiliated to Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The structural equation model analysis indicated that fertilization regimes directly affected the bacterial composition and antibiotics and had an indirect effect on the abundance of tetK and tetM via these antibiotics. In summary, this study shed light into the complex interactions between fertilization, antibiotics, and antibiotic resistance pollution in greenhouse soil.

Does nano silver promote the selection of antibiotic resistance genes in soil and plant?

Growing evidences have demonstrated that heavy metal contamination can promote the proliferation of antibiotic resistance genes (ARGs) via co-selection. However, effects of nano-metal-materials on the occurrence and level of ARGs in the soil and plant, have not been fully explored. To gain insights into this impact, we conducted a pot experiment by adding nano‑silver particles (AgNPs) as a stimuli and Ag ion (AgNO3) and tetracycline as a comparison. By using high throughput quantitative PCR, our results indicated that application of AgNPs (~20 nm and ~50 nm) at a concentration of 100 ppm resulted in no significant changes in the abundance of ARGs in either soil or phyllosphere (P > 0.05). Nevertheless, the overall pattern of resistome, especially in soil, was shifted following AgNPs application, with a significance increase in the relative abundance of efflux pumps genes, which is an important mechanism for co-selection of ARGs by heavy metals. By comparison, Ag ion at an equivalent Ag mass of AgNPs markedly increased ARGs abundance and shifted ARGs profile in soil, indicating that free Ag ion had a stronger impact on ARGs than AgNPs. These findings provide new insights in assessing the risks of manufactured nanomaterials accumulated in the environment.

Factors that affect the occurrence and distribution of antibiotic resistance genes in soils from livestock and poultry farms

Livestock manure is generally dumped directly onto open soil or used to enhance the soil fertility. However, there are growing concerns regarding the impact of these practices on the development and dissemination of antibiotic resistance genes (ARGs) in soil. In this study, we sampled soils treated with manure from 10 large-scale farms (pig, beef cattle, and chicken farms) and those from farmland without manure. The results showed that the abundance of ARGs was more than 2.62 times higher in the soil samples treated with livestock manure than the farmland soil without manure. The abundances of ARGs and intI1 in all samples were in the following order: pig farms > chicken farms > beef cattle farms. tetX, sul1, sul2, and tetG were the dominant ARGs in farm soil. The concentrations of tetracycline antibiotics and sulfonamide antibiotics were 0.15-4.76 mg/kg and 0-2.62 mg/kg, respectively, in the soils treated with manure, which were higher than those in farmland soils without manure. Redundancy analysis (P < 0.05) and network analysis (P < 0.01, R > 0.80) demonstrated that copper, zinc, actinomycetes, and tetracycline antibiotics were the main factors that affected the distribution of ARGs in soils treated with livestock manure.

Discovery of Novel Antibiotic Resistance Determinants in Forest and Grassland Soil Metagenomes.

Soil represents a significant reservoir of antibiotic resistance genes (ARGs), which can potentially spread across distinct ecosystems and be acquired by pathogens threatening human as well as animal health. Currently, information on the identity and diversity of these genes, enabling anticipation of possible future resistance development in clinical environments and the livestock sector, is lacking. In this study, we applied functional metagenomics to discover novel sulfonamide as well as tetracycline resistance genes in soils derived from forest and grassland. Screening of soil metagenomic libraries revealed a total of eight so far unknown ARGs. The recovered genes originate from phylogenetically diverse soil bacteria (e.g., Actinobacteria, Chloroflexi, or Proteobacteria) and encode proteins with a minimum identity of 46% to other antibiotic resistance determinants. In particular forest soil ecosystems have so far been neglected in studies focusing on antibiotic resistance. Here, we detected for the first time non-mobile dihydropteroate synthase (DHPS) genes conferring resistance to sulfonamides in forest soil with no history of exposure to these synthetic drugs. In total, three sulfonamide resistant DHPSs, differing in taxonomic origin, were discovered in beech or pine forest soil. This indicates that sulfonamide resistance naturally occurs in forest-resident soil bacterial communities. Besides forest soil-derived sulfonamide resistance proteins, we also identified a DHPS affiliated to Chloroflexi in grassland soil. This enzyme and the other recovered DHPSs confer reduced susceptibility toward sulfamethazine, which is widely used in food animal production. With respect to tetracycline resistance, four efflux proteins affiliated to the major facilitator superfamily (MFS) were identified. Noteworthy, one of these proteins also conferred reduced susceptibility toward lincomycin.

RefSoil+: a Reference Database for Genes and Traits of Soil Plasmids.

Plasmids harbor transferable genes that contribute to the functional repertoire of microbial communities, yet their contributions to metagenomes are often overlooked. Environmental plasmids have the potential to spread antibiotic resistance to clinical microbial strains. In soils, high microbiome diversity and high variability in plasmid characteristics present a challenge for studying plasmids. To improve the understanding of soil plasmids, we present RefSoil+, a database containing plasmid sequences from 922 soil microorganisms. Soil plasmids were larger than other described plasmids, which is a trait associated with plasmid mobility. There was a weak relationship between chromosome size and plasmid size and no relationship between chromosome size and plasmid number, suggesting that these genomic traits are independent in soil. We used RefSoil+ to inform the distributions of antibiotic resistance genes among soil microorganisms compared to those among nonsoil microorganisms. Soil-associated plasmids, but not chromosomes, had fewer antibiotic resistance genes than other microorganisms. These data suggest that soils may offer limited opportunity for plasmid-mediated transfer of described antibiotic resistance genes. RefSoil+ can serve as a reference for the diversity, composition, and host associations of plasmid-borne functional genes in soil, a utility that will be enhanced as the database expands. Our study improves the understanding of soil plasmids and provides a resource for assessing the dynamics of the genes that they carry, especially genes conferring antibiotic resistances. IMPORTANCE Soil-associated plasmids have the potential to transfer antibiotic resistance genes from environmental to clinical microbial strains, which is a public health concern. A specific resource is needed to aggregate the knowledge of soil plasmid characteristics so that the content, host associations, and dynamics of antibiotic resistance genes can be assessed and then tracked between the environment and the clinic. Here, we present RefSoil+, a database of soil-associated plasmids. RefSoil+ presents a contemporary snapshot of antibiotic resistance genes in soil that can serve as a reference as novel plasmids and transferred antibiotic resistances are discovered. Our study broadens our understanding of plasmids in soil and provides a community resource of important plasmid-associated genes, including antibiotic resistance genes.

Distribution of antibiotic resistance genes in soils and crops. A field study in legume plants (Vicia faba L.) grown under different watering regimes

Social concern has raised during the last years due to the development of antibiotic resistance hotspots in different environmental compartments, including the edible parts of crops. To assess the influence of the water quality used for watering, we collected samples from soil, roots, leaves and beans from the legume plant Vicia faba (broad beans) in three agricultural peri-urban plots (Barcelona, NE Spain), irrigated with either groundwater, river water, or reclaimed water. Antibiotic resistance genes (ARGs) sul1, tetM, qnrS1, blaCTX-M-32,blaOXA-58, mecA, and blaTEM were quantified by real-time PCR, along with 16S rDNA and intl1 sequences, as proxies for bacterial abundance and integron prevalence, respectively. Microbiome composition of all samples were analyzed by high-throughput DNA sequencing. Results show a gradient of bacterial species diversity and of ARG prevalence from highly diverse soil samples to microbially-poor beans and leaves, in which Rhizobiales essentially displaced all other groups, and that presented very small loads of ARGs and integron sequences. The data suggest that the microbiome and the associated resistome were likely influenced by agricultural practices and water quality, and that future irrigation water legal standards should consider the specific Physiology of the different crop plants.

A rationale for the high limits of quantification of antibiotic resistance genes in soil

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR.The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Investigating the environmental risks from the use of spray-dried cephalosporin mycelial dreg (CMD) as a soil amendment

Cephalosporin mycelial dreg (CMD) is a by-product of the pharmaceutical industry. Spray-drying is widely used for the dewatering process prior to the application of CMD as a soil amendment. However, the potential environmental behaviors and risks of spray-dried CMD amendment remain unclear. Here, a lab-scale incubation experiment was conducted to investigate the salinity, phytotoxicity, introduced antibiotics, heavy metals and the potential impacts of resistance genes in CMD-amended soil. Spray-dried CMD amendment generally increased soil salinity and only high dosed soils showed phytotoxic effects at the end of the incubation period, implying the physiological damage to plant growth. The introduced antibiotics quickly degraded over time, indicating a relatively low environmental persistence. Heavy metal slightly increased in soil receiving spray-dried CMD, and regulations should be developed to avoid metal accumulation. A decreased diversity and distinct patterns of β-lactam resistance genes as well as a dose-effect of their enrichment were observed in CMD-amended soil, which might be partially explained by the specific metals and introduced antibiotics. Antibiotic resistance genes in soil may be a valuable tool for evaluating the environmental risk associated with use of CMD as a soil amendment.

Tetracycline and Sulfonamide Antibiotic Resistance Genes in Soils From Nebraska Organic Farming Operations.

There is widespread agreement that agricultural antibiotic resistance should be reduced, however, it is unclear from the available literature what an appropriate target for reduction would be. Organic farms provide a unique opportunity to disentangle questions of agricultural antibiotic drug use from questions of antibiotic resistance in the soil. In this study, soil was collected from 12 certified organic farms in Nebraska, evaluated for the presence of tetracycline and sulfonamide resistance genes (n = 15 targets), and correlated to soil physical, chemical, and biological parameters. Tetracycline and sulfonamide antibiotic resistance genes (ARGs) were found in soils from all 12 farms, and 182 of the 196 soil samples (93%). The most frequently detected gene was tetG (55% of samples), followed by tet(Q) (49%), tet(S) (46%), tet(X) (30%), and tetA(P) (29%). Soil was collected from two depths. No differences in ARGs were observed based on soil depth. Positive correlations were noted between ARG presence and soil electrical conductivity, and concentrations of Ca, Na, and Mehlich-3 phosphorus. Data from this study point to possible relationships between selected soil properties and individual tetracycline resistance genes, including tet(O) which is a common target for environmental samples. We compared organic farm results to previously published data from prairie soils and found significant differences in detection frequency for 12 genes, eight of which were more commonly detected in prairie soils. Of interest, when tetracycline ARG results were sorted by gene mechanism, the efflux genes were generally present in higher frequency in the prairie soils, while the ribosomal protection and enzymatic genes were more frequently detected in organic farm soils, suggesting a possible ecological role for specific tetracycline resistance mechanisms. By comparing soil from organic farms with prairie soils, we can start to determine baseline effects of low-chemical input agricultural production practices on multiple measures of resistance.

Application of biosolids drives the diversity of antibiotic resistance genes in soil and lettuce at harvest

High-throughput quantitative polymerase chain reaction (PCR) of antibiotic resistance genes (ARGs) was used to profile the composition and diversity of ARGs in biosolid-amended soil and in lettuce roots and shoots. Biosolid application significantly increased the ARGs in soil and influenced the soil antibiotics resistome mainly through exogenous introduction. Four dominant ARGs occurred in sterilized and water-washed leaves, namely catB8, php, vanB and str, which accumulated to >50% of total abundance. Nine subclasses of ARGs were determined in both sterilized and water-washed leaves, and they were shared ARGs among soil, roots and shoots. This suggests that the ARGs in ready-to-eat lettuce were mainly located in endophytes and the internal route may contribute more to the ARGs in routinely consumed raw lettuce. Moreover, a previously unsuspected shift in multridrug ARGs to root and shoot compartments due to biosolid application was discovered. These results reveal that biosolid application drives the evolution and dissemination of ARGs in the soil-plant system.

Tracking antibiotic resistance genes in soil irrigated with dairy wastewater

The application of dairy wastewater to agricultural soils is a widely used practice to irrigate crops and recycle nutrients. In this study, small-scale field plots were irrigated monthly (6 times) with dairy wastewater (100%), wastewater diluted to 50% with irrigation (canal) water, and diluted wastewater spiked with copper sulfate (50 mg Cu L−1), while control plots were irrigated with canal water. In addition, half of all plots were either planted with wheat or were left as bare soil. Biweekly soil samples were collected during this period and processed to determine the occurrence and abundance of antibiotic resistance genes [blaCTX-M-1, erm(B), sul1, tet(B), tet(M), and tet(X)] and a class 1 integron-integrase gene (intI1) via quantitative real-time PCR (qPCR). Only sul1 and tet(X) were detected in soil (3 out of 32 samples) before the wastewater treatments were applied. However, the occurrence and relative abundance (normalized to 16S rRNA gene copies) of most genes [erm(B), intI1, sul1, and tet(M)] increased dramatically after wastewater irrigation and levels were maintained during the entire study period. blaCTX-M-1 was the only gene not detected in wastewater-treated soils, which is likely related to its absence in the dairy wastewater. Relative gene levels in soil were found to be statistically similar among the treatments in most cases, regardless of the wastewater percentage applied and presence or absence of plants. The key result from this study is that dairy wastewater irrigation significantly enlarges the reservoir of ARGs and intI1 in soils, while detection of these genes rarely occurred in soil irrigated only with canal water. In addition, elevated levels of Cu in the wastewater and treated soil did not produce a concomitant increase of the ARG levels.

Changes in bacterial community structure and antibiotic resistance genes in soil in the vicinity of a pharmaceutical factory

China is the largest global producer of antibiotics. With the demand for antibiotics increasing every year, it is necessary to assess potential environmental risks and the spread of antibiotic resistance genes (ARGs) associated with antibiotic production. Here, we investigated the occurrence and distribution of ARGs in soil in the vicinity of a pharmaceutical factory. The results showed that antibiotic concentrations were under the detection limit; however, ARGs were present in soil and tended to be enriched near the factory. A significant correlation between the relative abundance of intI-1 and tetracycline ARGs implied that horizontal gene transfer might play an important role in the spread of ARGs. The occurrence of these ARGs could be the results of previous antibiotic contamination. However, the soil bacterial community structure seemed to be more affected by nutrients or other factors than by antibiotics. Overall, this study supports the viewpoint that long-term pharmaceutical activity might have a negative effect on environmental health, thus, underscoring the need to regulate antibiotic production and management.

Enrichment of antibiotic resistance genes in soil receiving composts derived from swine manure, yard wastes, or food wastes, and evidence for multiyear persistence of swine Clostridium spp.

The impact of amendment with swine manure compost (SMC), yard waste compost (YWC), or food waste compost (FWC) on the abundance of antibiotic resistance genes in soil was evaluated. Following a commercial-scale application of the composts in a field experiment, soils were sampled periodically for a decade, and archived air-dried. Soil DNA was extracted and gene targets quantified by qPCR. Compared with untreated control soil, all 3 amendment types increased the abundance of gene targets for up to 4 years postapplication. The abundance of several gene targets was much higher in soil amended with SMC than in soil receiving either YWC or FWC. The gene target ermB remained higher in the SMC treatment for a decade postapplication. Clostridia were significantly more abundant in the SMC-amended soil throughout the decade following application. Eight percent of Clostridium spp. isolates from the SMC treatment carried ermB. Overall, addition of organic amendments to soils has the potential to increase the abundance of antibiotic resistance genes. Amendments of fecal origin, such as SMC, will in addition entrain bacteria carrying antibiotic resistance genes. Environmentally recalcitrant clostridia, and the antibiotic resistance genes that they carry, will persist for many years under field conditions following the application of SMC.

Occurrence of antibiotics and antibiotic resistance genes in soils from wastewater irrigation areas in the Pearl River Delta region, southern China

The occurrence and distribution of tetracycline (TC) and sulfamethazine (SMZ), and the corresponding antibiotic resistance genes (ARGs) were investigated in six agricultural sites in the Pearl River Delta (PRD) region in southern China. Irrigation water and irrigated soils at two different depths (0–10 and 10–20cm) were analyzed. The total concentrations of TC and SMZ in irrigation water ranged from 69.3 to 234ng/L and from 4.00 to 58.2ng/L, respectively, while the total concentrations of TC and SMZ in irrigated soils ranged from 5.00 to 21.9μg/kg and from 1.30 to 4.20μg/kg, respectively. After long-term irrigation with domestic and fishpond wastewater in the field, the residual TC and SMZ and their corresponding ARGs in soils were significantly higher in fishpond-irrigated soils (Dongguan and Shenzhen) than in domestic wastewater-irrigated soils (Foshan, Guangzhou, Huizhou and Zhongshan). The concentrations of antibiotics and their ARGs were significantly higher in irrigation water than in irrigated soils, which indicated that wastewater was the primary source of antibiotics in the soil environments. The domestic and fishpond wastewater were important repositories of antibiotics and their ARGs, which require effective treatment before their discharge into the environment. Other factors such as soil physicochemical properties, manure application, irrigation water sources and cropping patterns also affect the antibiotic concentrations and ARG abundances. The residual antibiotic concentrations statistically correlated with the corresponding ARGs in irrigation water and irrigated soils, both of which decreased with increasing soil depth, indicating that the concentration of antibiotics in the environment exerted a selection pressure on the microorganisms in the environment.

Changes to tetracyclines and tetracycline resistance genes in arable soils after single and multiple applications of manure containing tetracyclines.

The influence of manure containing tetracyclines (TCs) on the prevalence of antibiotic resistance genes in soils remains poorly understood. Here, three different TCs (oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC)) were mixed respectively with unpolluted manure to fertilize arable soil. The soil received either a single application of 0μgkg-1, 300μgkg-1 (TC and CTC), or 700μgkg-1 (OTC) or multiple applications every 14days for 140days. Four tetracycline resistance genes (TRGs), including tet(A), tet(L), tet(M), and tet(Q), were monitored. Although the abundances of the four TRGs in the single application treatment initially increased rapidly, they decreased over time and were significantly lower than those of the repeated treatments after day 112. All additions of TCs stopped on day 140, but we continued to assess the long-term accumulation of TRGs. Most of the TRGs were detected even after the TC-containing manures had not been applied for more than 15months. The abundance of the TRGs after ceasing fertilization with the TC-containing manures was higher in the repeated application treatments than in the single application treatments. Therefore, more attention should be paid to repeated applications of antibiotic-containing manure to arable soils.

Water flow paths are hotspots for the dissemination of antibiotic resistance in soil

Antibiotic resistance genes in soil pose a potential risk for human health. They can enter the soil by irrigation with untreated or insufficiently treated waste water. We hypothesized that water flow paths trigger the formation of antibiotic resistance, since they transport antibiotics, multi-resistant bacteria and free resistance genes through the soil. To test this, we irrigated soil cores once or twice with waste water only, or with waste water added with sulfamethoxazole (SMX) and ciprofloxacin (CIP). The treatments also contained a dye to stain the water flow paths and allowed to sample these separately from unstained bulk soil. The fate of SMX and CIP was assessed by sorption experiments, leachate analyses and the quantification of total and extractable SMX and CIP in soil. The abundance of resistance genes to SMX (sul1 and sul2) and to CIP (qnrB and qnrS) was quantified by qPCR. The sorption of CIP was larger than the dye and SMX. Ciprofloxacin accumulated exclusively in the water flow paths but the resistance genes qnrB and qnrS were not detectable. The SMX concentration in the water flow paths doubled the concentration of the bulk soil, as did the abundance of sul genes, particularly sul1 gene. These results suggest that flow paths do function as hotspots for the accumulation of antibiotics and trigger the formation of resistance genes in soil. Their dissemination also depends on the mobility of the antibiotic, which was much larger for SMX than for CIP.

Prevalence of antibiotic resistance genes in soils after continually applied with different manure for 30 years

A 30year field experiment with a wheat-soybean cropping system were performed to compare the long-term anthropogenic influence on soil ARGs. Compared with chemical fertilization, the occurrence of 38 ARGs and the abundance of seven ARGs (tetL, tetB(P), tetO, tetW, sul1, ermB, and ermF) were significantly increased by long term exposure of pig manure. However, application of wheat straw and cow manure not substantially affected the abundance of ARGs except cow manure increased the abundance of tetM and tetW to a detectable level. Relative abundance of Firmicutes, Gammaproteobacteria and Bacteroidetes and ARGs observed in the soil were significantly correlated. Integrase gene I1 (intI1) is commonly linked to genes conferring resistance to antibiotics, it was significantly increased in pig manure treated soils and showed a high positive correlation with the abundance of ARGs which were significantly affected by pig manure. Concentrations of Cu, Zn and Pb were also increased in manure treated soil and positive correlated with the relative abundance of intI1 and most of the ARGs. These results indicated that long-term animal manure application to soils has polluted the soil, especially for pig manure, and it should be scrutinized as part of future stewardship programs.

Detection of sulfonamide resistant bacteria and resistance genes in soils

Manure application could accelerate the environmental dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in soils. In this study, the prevalence of sulfonamide resistant bacteria and resistance genes was investigated in agricultural soils to which organic manures had been applied in Tianjin, China. Anti-sulfonamide bacteria were found in the range of 3.29 × 104 to 1.70 × 105 CFU/g dry soil, occupying 1.5% to 2.2% of total viable counts. And sulI and sulII genes were detected in all sampling sites, with relative abundances of 5.69 × 10−5 to 6.95 × 10−4 and 4.28 × 10−4 to 1.25 × 10−3 respectively. No significant correlations between cultivable sulfonamide resistant bacteria and sul genes were found in this study. While sulI showed significant positive correlation with soil organic matter. Overall, the results highlight that soil plays an important role in resistance genes capture as the environmental reservoir.

Impact of pre-application treatment on municipal sludge composition, soil dynamics of antibiotic resistance genes, and abundance of antibiotic-resistance genes on vegetables at harvest

In many jurisdictions sludge recovered from the sewage treatment process is a valued fertilizer for crop production. Pre-treatment of sewage sludge prior to land application offers the potential to abate enteric microorganisms that carry genes conferring resistance to antibiotics. Pre-treatment practices that accomplish this should have the desirable effect of reducing the risk of contamination of crops or adjacent water with antibiotic resistance genes carried in these materials. In the present study, we obtained municipal sludge that had been subjected to one of five treatments. There were, anaerobic-digestion or aerobic-digestion, in both instances with and without dewatering; and heat-treatment and pelletization. Each of the five types of biosolids was applied to an agricultural field at commercial rates, following which lettuce, carrots and radishes were planted. Based on qPCR, the estimated antibiotic gene loading rates were comparable with each of the five biosolids. However, the gene abundance in soil following application of the pelletized biosolids was anomalously lower than expected. Following application, the abundance of antibiotic resistance genes decreased in a generally coherent fashion, except sul1 which increased in abundance during the growing season in the soil fertilized with pelletized biosolids. Based on qPCR and high throughput sequencing evidence for transfer of antibiotic resistance genes from the biosolids to the vegetables at harvest was weak. Clostridia were more abundant in soils receiving any of the biosolids except the pelletized. Overall, the behavior of antibiotic resistance genes in soils receiving aerobically or anaerobically-digested biosolids was consistent and coherent with previous studies. However, dynamics of antibiotic resistance genes in soils receiving the heat treated pelletized biosolids were very different, and the underlying mechanisms merit investigation.