Shifts in microbial community, pathogenicity-related genes and antibiotic resistance genes during dairy manure piled up.
ABSTRACT: The uncomposted faeces of dairy cow are usually stacked on cow breeding farms, dried under natural conditions and then used as cow bedding material or they may be continuously piled up. However, no information is available to evaluate variations in the human and animal pathogen genes and antibiotic resistance during the accumulation of fresh faeces of dairy cow to manure. Here, we present the metagenomic analysis of fresh faeces and manure from a dairy farm in Ning Xia, showing a unique enrichment of human and animal pathogen genes and antibiotic resistance genes (ARGs) in manure. We found that manure accumulation could significantly increase the diversity and abundance of the pathogenic constituents. Furthermore, pathogens from manure could spread to the plant environment and enphytotic pathogens could affect the yield and quality of crops during the use of manure as a fertilizer. Levels of virulence genes and ARGs increased with the enrichment of microbes and pathogens when faeces accumulated to manure. Accumulated manure was also the transfer station of ARGs to enrich the ARGs in the environment, indicating the ubiquitous presence of environmental antibiotic resistance genes. Our results demonstrate that manure accumulation and usage without effective manure management is an unreasonable approach that could enrich pathogenic microorganisms and ARGs in the environment. The manure metagenome structure allows us to appreciate the overall influence and interaction of animal waste on water, soil and other areas impacted by faecal accumulation and the factors that influence pathogen occurrence in products from dairy cows.
Project description:Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment.
Project description:Animal manure comprises an important reservoir for antibiotic resistance genes (ARGs), but the variation in ARGs during anaerobic digestion at various temperatures and its underlying mechanism remain unclear. Thus, we performed anaerobic digestion using dairy manure at three temperature levels (moderate: 20?°C, mesophilic: 35?°C, and thermophilic: 55?°C), to analyze the dynamics of ARGs and bacterial communities by quantitative PCR and 16S rRNA gene sequencing. We found that 8/10 detected ARGs declined and 5/10 decreased more than 1.0 log during thermophilic digestion, whereas only four and five ARGs decreased during moderate and mesophilic digestion, respectively. The changes in ARGs and bacterial communities were similar under the moderate and mesophilic treatments, but distinct from those in the thermophilic system. Potential pathogens such as Bacteroidetes, Proteobacteria, and Corynebacterium were removed by thermophilic digestion but not by moderate and mesophilic digestion. The bacterial community succession was the dominant mechanism that influenced the variation in ARGs and integrons during anaerobic digestion. Thermophilic digestion decreased the amount of mesophilic bacteria (Bacteroidetes and Proteobacteria) carrying ARGs. Anaerobic digestion generally decreased the abundance of integrons by eliminating the aerobic hosts of integrons (Actinomycetales and Bacilli). Thermophilic anaerobic digestion is recommended for the treatment and reuse of animal manure.
Project description:Dairy cattle are routinely treated with antibiotics, and the resulting manure or composted manure is commonly used as a soil amendment for crop production, raising questions regarding the potential for antibiotic resistance to propagate from "farm to fork." The objective of this study was to compare the microbiota and "resistomes" (i.e., carriage of antibiotic resistance genes [ARGs]) associated with lettuce leaf and radish taproot surfaces grown in different soils amended with dairy manure, compost, or chemical fertilizer only (control). Manure was collected from antibiotic-free dairy cattle (DC) or antibiotic-treated dairy cattle (DA), with a portion composted for parallel comparison. Amendments were applied to loamy sand or silty clay loam, and lettuce and radishes were cultivated to maturity in a greenhouse. Metagenomes were profiled via shotgun Illumina sequencing. Radishes carried a distinct ARG composition compared to that of lettuce, with greater relative abundance of total ARGs. Taxonomic species richness was also greater for radishes by 1.5-fold. The resistomes of lettuce grown with DC compost were distinct from those grown with DA compost, DC manure, or fertilizer only. Further, compost applied to loamy sand resulted in twofold-greater relative abundance of total ARGs on lettuce than when applied to silty clay loam. The resistomes of radishes grown with biological amendments were distinct from the corresponding fertilizer controls, but effects of composting or antibiotic use were not measureable. Cultivation in loamy sand resulted in higher species richness for both lettuce and radishes than when grown in silty clay loam by 2.2-fold and 1.2-fold, respectively, when amended with compost.IMPORTANCE A controlled, integrated, and replicated greenhouse study, along with comprehensive metagenomic analysis, revealed that multiple preharvest factors, including antibiotic use during manure collection, composting, biological soil amendment, and soil type, influence vegetable-borne resistomes. Here, radishes, a root vegetable, carried a greater load of ARGs and species richness than lettuce, a leafy vegetable. However, the lettuce resistome was more noticeably influenced by upstream antibiotic use and composting. Network analysis indicated that cooccurring ARGs and mobile genetic elements were almost exclusively associated with conditions receiving raw manure amendments, suggesting that composting could alleviate the mobility of manure-derived resistance traits. Effects of preharvest factors on associated microbiota and resistomes of vegetables eaten raw are worthy of further examination in terms of potential influence on human microbiomes and spread of antibiotic resistance. This research takes a step toward identifying on-farm management practices that can help mitigate the spread of agricultural sources of antibiotic resistance.
Project description:Animal manures and municipal biosolids recycled onto crop production land carry antibiotic-resistant bacteria that can influence the antibiotic resistome of agricultural soils, but little is known about the contribution of bacteriophage to the dissemination of antibiotic resistance genes (ARGs) in this context. In this work, we quantified a set of ARGs in the bacterial and bacteriophage fractions of agricultural soil by quantitative PCR. All tested ARGs were present in both the bacterial and phage fractions. We demonstrate that fertilization of soil with dairy manure or human biosolids increases ARG abundance in the bacterial fraction but not the bacteriophage fraction and further show that pretreatment of dairy manure can impact ARG abundance in the bacterial fraction. Finally, we show that purified bacteriophage can confer increased antibiotic resistance to soil bacteria when combined with selective pressure. The results indicate that soilborne bacteriophage represents a substantial reservoir of antibiotic resistance and that bacteriophage could play a significant role in the horizontal transfer of resistance genes in the context of an agricultural soil microbiome. Overall, our work reinforces the advisability of composting or digesting fecal material prior to field application and suggests that application of some antibiotics at subclinical concentrations can promote bacteriophage-mediated horizontal transfer of ARGs in agricultural soil microbiomes.
Project description:Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.
Project description:The production of extended-spectrum β-lactamases (ESBLs) conferring resistance to new derivatives of β-lactams is a major public health threat if present in pathogenic Gram-negative bacteria. The objective of this study was to characterize ceftiofur (TIO)- or cefotaxime (FOX)-resistant Escherichia coli isolated from dairy cow manure. Twenty-four manure samples were collected from four farms and incubated under anaerobic conditions for 20 weeks at 4 °C or at 25 °C. A total of 37 TIO- or FOX-resistant E. coli were isolated from two of the four farms to determine their susceptibility to 14 antibiotics. Among the 37 resistant E. coli, 10 different serotypes were identified, with O8:H1 being the predominant serotype (n = 17). Five isolates belonged to each of serotypes O9:NM and O153:H42, respectively. All 37 cephalosporin resistant isolates were multi-resistant with the most prevalent resistance spectrum being amoxicillin-clavulanic acid-ampicillin-cefoxitin-ceftiofur-ceftriaxone-chloramphenicol-streptomycin-sulfisoxazole-tetracycline-trimethoprim-sulfamethoxazole. The genomes of 18 selected isolates were then sequenced and compared to 14 selected human pathogenic E. coli reference genomes obtained from public repositories using different bioinformatics approaches. As expected, all 18 sequenced isolates carried at least one β-lactamase bla gene: TEM-1, TEM-81, CTX-M115, CTX-M15, OXA-1, or CMY-2. Several other antibiotic resistance genes (ARGs) and virulence determinants were detected in the sequenced isolates and all of them harbored antimicrobial resistance plasmids belonging to classic Inc groups. Our results confirm the presence of diverse ESBL producing E. coli isolates in dairy cow manure stored for a short period of time. Such manure might constitute a reservoir of resistance and virulence genes for other bacteria that share the same environment.
Project description:The antibiotic monensin is fed to dairy cows to increase milk production efficiency. A fraction of this monensin is excreted into the cow manure. Previous studies have found that cow manure containing monensin can negatively impact the performance of anaerobic digesters, especially upon first introduction. Few studies have examined whether the anaerobic digester microbiome can adapt to monensin during the operating time. Here, we conducted a long-term time series study of four lab-scale anaerobic digesters fed with cow manure. We examined changes in both the microbiome composition and function of the anaerobic digesters when subjected to the dairy antibiotic monensin. In our digesters, monensin was not rapidly degraded under anaerobic conditions. The two anaerobic digesters that were subjected to manure from monensin feed-dosed cows exhibited relatively small changes in microbiome composition and function due to relatively low monensin concentrations. At higher concentrations of monensin, which we dosed directly to control manure (from dairy cows without monensin), we observed major changes in the microbiome composition and function of two anaerobic digesters. A rapid introduction of monensin to one of these anaerobic digesters led to the impairment of methane production. Conversely, more gradual additions of the same concentrations of monensin to the other anaerobic digester led to the adaptation of the anaerobic digester microbiomes to the relatively high monensin concentrations. A member of the candidate OP11 (Microgenomates) phylum arose in this anaerobic digester and appeared to be redundant with certain Bacteroidetes phylum members, which previously were dominating.IMPORTANCE Monensin is a common antibiotic given to dairy cows in the United States and is partly excreted with dairy manure. An improved understanding of how monensin affects the anaerobic digester microbiome composition and function is important to prevent process failure for farm-based anaerobic digesters. This time series study demonstrates how anaerobic digester microbiomes are inert to low monensin concentrations and can adapt to relatively high monensin concentrations by redundancy in an already existing population. Therefore, our work provides further insight into the importance of microbiome redundancy in maintaining the stability of anaerobic digesters.
Project description:Antibiotic resistance is a public health problem of growing concern. Animal manure application to soil is considered to be a main cause of the propagation and dissemination of antibiotic residues, antibiotic-resistant bacteria (ARB), and antibiotic resistance genes (ARGs) in the soil-water system. In recent decades, studies on the impact of antibiotic-contaminated manure on soil microbiomes have increased exponentially, in particular for taxonomical diversity and ARGs' diffusion. Antibiotic resistance genes are often located on mobile genetic elements (MGEs). Horizontal transfer of MGEs toward a broad range of bacteria (pathogens and human commensals included) has been identified as the main cause for their persistence and dissemination. Chemical and bio-sanitizing treatments reduce the antibiotic load and ARB. Nevertheless, effects of these treatments on the persistence of resistance genes must be carefully considered. This review analyzed the most recent research on antibiotic and ARG environmental dissemination conveyed by livestock waste. Strategies to control ARG dissemination and antibiotic persistence were reviewed with the aim to identify methods for monitoring DNA transferability and environmental conditions promoting such diffusion.
Project description:Metagenomic investigations have the potential to provide unprecedented insights into microbial ecologies, such as those relating to antimicrobial resistance (AMR). We characterized the microbial resistome in livestock operations raising cattle conventionally (CONV) or without antibiotic exposures (RWA) using shotgun metagenomics. Samples of feces, wastewater from catchment basins, and soil where wastewater was applied were collected from CONV and RWA feedlot and dairy farms. After DNA extraction and sequencing, shotgun metagenomic reads were aligned to reference databases for identification of bacteria (Kraken) and antibiotic resistance genes (ARGs) accessions (MEGARes). Differences in microbial resistomes were found across farms with different production practices (CONV vs. RWA), types of cattle (beef vs. dairy), and types of sample (feces vs. wastewater vs. soil). Feces had the greatest number of ARGs per sample (mean = 118 and 79 in CONV and RWA, respectively), with tetracycline efflux pumps, macrolide phosphotransferases, and aminoglycoside nucleotidyltransferases mechanisms of resistance more abundant in CONV than in RWA feces. Tetracycline and macrolide-lincosamide-streptogramin classes of resistance were more abundant in feedlot cattle than in dairy cow feces, whereas the ?-lactam class was more abundant in dairy cow feces. Lack of congruence between ARGs and microbial communities (procrustes analysis) suggested that other factors (e.g., location of farms, cattle source, management practices, diet, horizontal ARGs transfer, and co-selection of resistance), in addition to antimicrobial use, could have impacted resistome profiles. For that reason, we could not establish a cause-effect relationship between antimicrobial use and AMR, although ARGs in feces and effluents were associated with drug classes used to treat animals according to farms' records (tetracyclines and macrolides in feedlots, ?-lactams in dairies), whereas ARGs in soil were dominated by multidrug resistance. Characterization of the "resistance potential" of animal-derived and environmental samples is the first step toward incorporating metagenomic approaches into AMR surveillance in agricultural systems. Further research is needed to assess the public-health risk associated with different microbial resistomes.
Project description:Antibiotics are widely added to swine forage and are the main reason for the environmental accumulation of antibiotic resistance genes (ARGs) in swine manure-dwelling microorganisms. Aerobic composting (AC) and anaerobic digestion (AD) are efficient methods for converting swine manure to bio-fertilizer while degrading residual antibiotics. However, the influence of these methods on ARG accumulation and the difference in their efficiency have rarely been investigated. In this study, we explored the variations in four tetracycline antibiotics (TCs) and their associated ARGs and in microbial communities after AC and AD treatment. After full-scale manure AC and AD, the four TCs were removed effectively. AD had a higher TC removal efficiency than AC and a slower rate of TC-associated ARG accumulation. In addition, the community structure was more stable in the AC and AD manures than in untreated manure, and the relationship among microbial species also evolved into competition from mutualism after both AC and AD treatment. It was also speculated that the genera Acholeplasma and Arthrobacter were the possible hosts of tetO, tetW, and tetQ; the shift in the prokaryotic community composition and the alleviation of selective pressure by TC degradation led to decreased relative abundance of ARGs in AD- and AC-treated manure.