Antibiotic resistance gene spread due to manure application on agricultural fields

The usage of antibiotics in animal husbandry has promoted the development and abundance of antibiotic resistance in farm environments. Manure has become a reservoir of resistant bacteria and antibiotic compounds, and its application to agricultural soils is assumed to significantly increase antibiotic resistance genes and selection of resistant bacterial populations in soil. The genome location of resistance genes is likely to shift towards mobile genetic elements such as broad-host-range plasmids, integrons, and transposable elements. Horizontal transfer of these elements to bacteria adapted to soil or other habitats supports their environmental transmission independent of the original host. The human exposure to soil-borne resistance has yet to be determined, but is likely to be severely underestimated.     

Development and application of real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-streptogramin B in livestock manure and manure management systems

Erythromycin and tylosin are commonly used in animal production, and such use is perceived to contribute to the overall antimicrobial resistance (AR) reservoirs. Quantitative measurements of this type of AR reservoir in microbial communities are required to understand AR ecology (e.g., emergence, persistence, and dissemination). We report here the development, validation, and use of six real-time PCR assays for quantifying six classes of erm genes (classes A through C, F, T, and X) that encode the major mechanism of resistance to macrolides-lincosamides-streptogramin B (MLS(B)). These real-time PCR assays were validated and used in quantifying the six erm classes in five types of samples, including those from bovine manure, swine manure, compost of swine manure, swine waste lagoons, and an Ekokan upflow biofilter system treating hog house effluents. The bovine manure samples were found to contain much smaller reservoirs of each of the six erm classes than the swine manure samples. Compared to the swine manure samples, the composted swine manure samples had substantially reduced erm gene abundances (by up to 7.3 logs), whereas the lagoon or the biofilter samples had similar erm gene abundances. These preliminary results suggest that the methods of manure storage and treatment probably have a substantial impact on the persistence and decline of MLS(B) resistance originating from food animals, thus likely affecting the dissemination of such resistance genes into the environment. The abundances of these erm genes appeared to be positively correlated with those of the tet genes determined previously among these samples. These real-time PCR assays provide a rapid, quantitative, and cultivation-independent measurement of six major classes of erm genes, which should be useful for ecological studies of AR.     

Accumulation of copper and zinc from liquid manure in agricultural soils and crop plants

In Northern Italy it is a common practice to utilise slurries and other manure from intensive animal farming to fertilise agricultural soils. Due to the relatively high copper and zinc contents of these materials, this practice could lead to contamination of soils and the crops grown on them. In this study we have evaluated the extent of copper and zinc contamination of soils subjected to different levels of fertilisation with liquid manure and the copper and zinc concentrations in the edible tissues of three crops (maize, sugar beet and lucerne) grown on the same soils. There was a direct correlation between soil copper and zinc contents and the levels of application of animal wastes and copper concentrations were high enough to represent a risk, according to current European legislation. The metal contents in the edible tissues of the three crops were relatively low and variable, but showed no clear correlations with the intensity of liquid manure applications. Overall, there was no risk of contamination of the food chain, as all concentrations were well below levels considered to be toxic to animals.     

堆肥化处理对畜禽粪便中四环素类抗生素及抗性基因控制的研究进展

随着四环素类抗生素在畜禽养殖中的广泛应用,畜禽粪便已成为四环素类抗生素和抗性基因的重要富集位点,其未经处理直接施用具有潜在的生态环境和人类健康风险。堆肥化处理可有效消减畜禽粪便中的四环素类抗生素,并且对抗性基因的扩散和传播具有一定的控制效果。本综述比较了不同的堆肥化工艺对粪肥中四环素类抗生素消减的效果,并重点讨论了其微生物降解机理,总结了堆肥化处理对粪肥中四环素抗性基因消减的研究进展,进一步讨论了堆肥化处理过程中抗性基因变化的微生态机理与控制策略,最后提出了采用热水解等预处理工艺去除抗生素压力和采用厌氧堆肥化工艺增强抗性基因控制的技术建议,以及从动态的角度采用高通量的检测技术来解析抗性基因消减机制的研究策略建议。     

Transformation of Penicillium chrysogenum to sulfonamide resistance

Penicillium chrysogenum has been transformed to sulfonamide resistance by vectors containing the dihydropteroate synthetase gene from plasmid R388 controlled by the promoter and terminator sequences of the P. chrysogenum trpC gene. Transformation frequencies of four to ten transformants per microgram of vector DNA were obtained.     

Levels and components of resistance to Amphorophora idaei in raspberry cultivars containing different resistance genes

Levels and components of resistance to Amphorophora idaei in raspberry cultivars containing different A. idaei resistance genes were studied under infestation tunnel, glasshouse and laboratory conditions. Each test consistently ranked raspberry cultivars, with increasing levels of resistance in the order, non-resistant (cv. Mailing Jewel), resistant cultivars containing minor genes, major gene A₁, gene A₁₀ (red raspberry) and gene A₁₀ (black raspberry) respectively. Resistance was expressed in three different ways; decreased alatae settling and feeding, decreased apterae settling and decreased aphid fecundity and rate of nymphal development. Following exposure to a large population of alatae, significantly fewer aphids settled on A₁, A₁₀ and to a lesser extent, minor gene-containing cultivars, compared to cv. Mailing Jewel. More alatae settled on the top than the bottom zone of non-resistant, minor gene resistant and two of four A₁-containing cultivars; alatae settling was low on all zones of A₁₀-containing raspberries. Aphid fecundity and nymph development patterns on different cultivars and resistance classes were similar to those found for alatae settling. After 7 days reproduction more than 30% of the nymphs developed to third or fourth instar on cv. Mailing Jewel, whilst on minor gene and major gene-containing cultivars the total number of nymphs and the proportion of later instars decreased. On resistant cultivars the nymphs were found mainly on the middle and bottom leaf zones, compared to cv. Mailing Jewel. A rapid (48 h) screening test using floating leaflets was developed and, on the basis of apterae settling, ranked cultivars reliably; it was particularly effective in distinguishing moderate and strong resistance to strain 1 A. idaei. Gene A₁-containing cultivars bred in England were much more resistant to strain 1 A. idaei than those bred in Scotland. Possible reasons for this difference are discussed, in relation to modified screening procedures and to the control of the viruses transmitted by this aphid vector.     

Traditional cattle manure application determines abundance, diversity and activity of methanogenic Archaea in arable European soil

Based on lipid analyses, 16S rRNA/rRNA gene single-strand conformation polymorphism fingerprints and methane flux measurements, influences of the fertilization regime on abundance and diversity of archaeal communities were investigated in soil samples from the long-term (103 years) field trial in Bad Lauchst?dt, Germany. The investigated plots followed a gradient of increasing fertilization beginning at no fertilization and ending at the 'cattle manure' itself. The archaeal phospholipid etherlipid (PLEL) concentration was used as an indicator for archaeal biomass and increased with the gradient of increasing fertilization, whereby the concentrations determined for organically fertilized soils were well above previously reported values. Methane emission, although at a low level, were occasionally only observed in organically fertilized soils, whereas the other treatments showed significant methane uptake. Euryarchaeotal organisms were abundant in all investigated samples but 16S rRNA analysis also demonstrated the presence of Crenarchaeota in fertilized soils. Lowest molecular archaeal diversity was found in highest fertilized treatments. Archaea phylogenetically most closely related to cultured methanogens were abundant in these fertilized soils, whereas Archaea with low relatedness to cultured microorganisms dominated in non-fertilized soils. Relatives of Methanoculleus spp. were found almost exclusively in organically fertilized soils or cattle manure. Methanosarcina-related microorganisms were detected in all soils as well as in the cattle manure, but soils with highest organic application rate were specifically dominated by a close phylogenetic relative of Methanosarcina thermophila. Our findings suggest that regular application of cattle manure increased archaeal biomass, but reduced archaeal diversity and selected for methanogenic Methanoculleus and Methanosarcina strains, leading to the circumstance that high organic fertilized soils did not function as a methane sink at the investigated site anymore.     

Positive and negative selection towards tetracycline resistance genes in manure treatment lagoons

Aims: To evaluate the role of manure treatment lagoons of swine operations in the fate of faeces‐ and feed‐borne tetracycline‐resistant genes (TRG). Methods and Results: Samples of feed, faeces, lagoon liquid and lagoon sediment in farm’s vicinity were collected at three swine operations varied on their operational practices and analysed on the presence and frequencies of incidence of sixteen TRG in upstream sources (feed, faeces) and downstream receptacles (lagoon liquid and sediments). The highest frequency of TRG incidence was observed in a farm with extensive antibiotic usage and the lowest in the antibiotic‐free farm. The study revealed a decrease in TRG richness and diversity in the downstream habitats of each farm. The observed TRG diverged into two groups, the persistent genes that were detected both upstream and downstream, and the transient genes that were detected in the upstream habitats but became nondetectable in the lagoons. Quantitative PCR analysis revealed that upstream concentrations and abundances (determined as TRG/16S rRNA gene ratios) of transient and persistent TRG were similar; however, the former were attenuated in the lagoons to the levels below the detection limit, whereas the latter were ～ 100‐1000 fold amplified in their (mostly) liquid phases and were also detected in farms’ vicinities. Conclusion: Manure lagoons of swine operations imposed both positive and negative selection towards faeces‐ and feed‐borne TRG that, respectively, caused either their proliferation or attenuation in those environments. Significance and Impact of the Study: The study reveals that discharge of antibiotic resistance genes from swine farms to the environment is linked to their positive selection (defined as an impact leading to proliferation of those genes) in manure lagoons.     

Shifts in microbial community,pathogenicity-related genes and antibiotic resistance genes during dairy manure piled up

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.     

Durable resistance to stripe rust is due to three specific resistance genes in French bread wheat cultivar Apache

Quantitative resistance is postulated to be more durable than qualitative (R-gene mediated) resistance, which is usually quickly overcome by the pathogen population. Despite its wide use for nearly 10?years in France, the French bread wheat cultivar Apache remains resistant to stripe rust. Here, we investigated the genetic architecture of cv. Apache resistance to examine whether its durability could be explained by quantitative characteristics. We identified quantitative trait loci (QTL) by composite interval mapping of disease progress data recorded throughout 4?years of field assays. These assays included inoculation with three different pathotypes on a segregating population originating from a cross between cv. Apache and cv. Taldor, a French cultivar susceptible to stripe rust. Three QTLs derived from Apache, QYr.inra-2AS, QYr.inra-2BL and QYr.inra-4B, were detected. Each of these QTLs contributed between approximately 15 and 69?% of the phenotypic variance and corresponds to a race-specific resistance gene. We showed that QYr.inra-2AS and QYr.inra-2BS map to the positions of Yr17 and Yr7, respectively, whereas QYr.inra-4B corresponds to an adult plant resistance gene. Our results demonstrate that a combination of two or more race-specific resistance genes can confer durable resistance provided that it is properly managed at a continental level. Race-specific resistance genes should not be removed from breeding programs provided that they are properly managed.     

Effect of long-term application of animal manure on physical properties of three soils

Surface soil samples to 15 cm depth were taken from replicated plots in an ongoing long-term field experiment involving application of animal manure on three soils in Virginia. The sampled plots had received either no manure or the equivalent of 289,000 kg ha^–1 of manure as dry weight. The manure was applied annually at the beginning of each spring for 15 years from 1978 through 1992. The plots were cropped similarly since 1978. Soil textures were a fine sandy loam at Holland in the Atlantic Coastal Plain region, a silt loam at Blacksburg in the Appalachian region, and a clay loam at Orange in the Piedmont region of Virginia. The following measurements were made on subsamples: liquid and plastic limits, wet aggregate stability, aggregate size distribution, dispersible clay percentage, water retention at 0. 03, 0.1, 0.3, 0.5, 1.0, and 1.5 MPa tension, and modulus of rupture of moulded briquettes at a water content corresponding to 0.1 MPa tension. Organic matter content by the Walkley-Black method was significantly higher in the manure-treated soils at all three locations. Increases were 3% for the sandy loam and 25% for the silt loam and clay loam. From these values it was estimated that at least 95% of the total applied manure had been degraded over the 15 years. Results showed that the liquid and plastic limits for all three soils were higher (p<0.05) for the manure-treated samples. However, the differences in the limits were only 2 to 3%. The modulus of rupture values were lowered by addition of the animal manure. Decreases (p<0.05) occurred for the silt loam and clay loam samples. The wet aggregate stability increased and the dispersible clay decreased in the manure-treated soils. Increases (p<0.05) in wet aggregate stability occurred for the sandy loam and silt loam samples. Decreases (p<0.05) in dispersible clay were measured for the sandy loam and clay loam samples. Water retention was consistently, but only slightly, increased by manure addition. The increases, in the order of sample texture, were clay loam > sandy loam silt loam. Increases tended to be higher at the lower values of tension. Manure addition consistently increased the weight percentages of aggregates passing a given mesh size. Increases, in order of sample texture, were silt loam > clay loam > sandy loam. In their entirety, these results show that the manure produced measurable changes in the soil physical properties. The magnitude of the changes, in most cases, were small and depended on the soil texture. Given the high total amount of manure applied, the results indicate that manure-induced physical changes in the soil were small and evidently did not accumulate over time. Rapid microbial degradation of the manure could be responsible for the lack of marked changes in the soil physical properties.     

不同施肥处理对土壤水稳定性团聚体及有机碳分布的影响

以国家褐潮土16 a的长期肥料试验为平台(北京昌平),研究长期不同施肥对耕层土壤水稳定性团聚体及其有机碳的影响。主要研究结果:与耕种农田土壤相比,长期撂荒(CK0)可以提高水稳定性大团聚体的含量及其有机碳含量和储量。而农田耕作后,破坏了水稳性大团聚体,相应地增加水稳性微团聚体的含量。与长期不施肥种植作物(CK)相比,长期施氮磷钾肥(NPK)、氮磷钾配施有机肥(NPKM)和氮磷钾秸秆还田(NPKS)处理对水稳性团聚体数量分布和平均重量直径(MWD)有显著影响,其中对2mm和0.25 2mm水稳性大团聚体的促进作用最明显,说明施肥处理增加的新碳主要向0.25 2mm和2mm团聚体富集。在不同水平水稳性团聚体中,2mm和0.25 2mm两个级别的水稳性大团聚体有机碳的含量显著高于0.0530.25mm和0.053mm水稳性微团聚体。化肥与有机肥配施(NPKM)处理可提高水稳性大团聚体含量,改善土壤团聚体的结构。长期小麦-玉米→小麦-大豆复种轮作并施氮磷钾化肥的处理(NPKF)各级团聚体中有机碳的含量高于长期小麦-玉米轮作并施氮磷钾化肥的处理(NPK)。     

Loss of sulfonamide resistance in Neisseria meningitidis

Four strains of Neisseria meningitidis were studied during serial passage. From two strains which originally were sulfonamide resistant, variants developed that had altered susceptibility to sulfonamides. One of the variants became relatively highly sulfonamide-sensitive, the other exhibited merely reduced sulfonamide resistance. There was a difference in the resistance pattern for two sulfonamides (sulfaisodimidine and sulfamethoxazole), and the effect of inoculum size and growth conditions in three different media could be demonstrated. Although the patterns of susceptibility to other antibacterial agents were different for the strains studied, no further susceptibility alterations occurred in parallel to the sulfonamide sensitivity changes. The variants also lost their ability to liberate free endotoxin.     

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils

Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.     

Accumulation of carbon and nitrogen by old arable land reverting to woodland

The accumulation of carbon (C) and nitrogen (N) was measured on two sites on Rothamsted Farm that had been fenced off some 120 years ago and allowed to revert naturally to woodland. The sites had previously been arable for centuries. One had been chalked and was still calcareous; the other had never been chalked and the pH fell from 7.1 in 1883 to 4.4 in 1999. The acidic site (Geescroft wilderness) is now a deciduous wood, dominated by oak (Quercus robor); the calcareous site (Broadbalk wilderness) is now dominated by ash (Fraxinus excelsior), with sycamore (Acer pseudoplatanus) and hawthorn (Craetagus monogyna) as major contributors. The acidic site gained 2.00 t C ha^?1 yr^?1 over the 118‐year period (0.38 t in litter and soil to a depth of 69 cm, plus an estimated 1.62 t in trees and their roots); the corresponding gains of N were 22.2 kg N ha^?1 year^?1 (15.2 kg in the soil, plus 6.9 kg in trees and their roots). The calcareous site gained 3.39 t C ha^?1 year^?1 over the 120‐year period (0.54 t in the soil, plus an estimated 2.85 t in trees and roots); for N the gains were 49.6 kg ha^?1 yr^?1 (36.8 kg in the soil, plus 12.8 kg in trees and roots). Trees have not been allowed to grow on an adjacent part of the calcareous site. There is now a little more C and N in the soil from this part than in the corresponding soil under woodland. We argue from our results that N was the primary factor limiting plant growth and hence accumulation of C during the early stages of regeneration in these woodlands. As soil organic N accumulates and the sites move towards N saturation, other factors become limiting. Per unit area of woodland, narrow strips; that is, wide hedges with trees, are the most efficient way of sequestering C – provided that they are not short of N.     

Higher diversity of Rhizobium leguminosarum biovar viciae populations in arable soils than in grass soils

The bacterial genetic diversity after long-term arable cultivation was compared with that under permanent grassland using replicated paired contrasts. Pea-nodulating Rhizobium leguminosarum populations were sampled from pairs of arable and grass sites at four locations in Yorkshire, United Kingdom. Isolates were characterized using both chromosomal (16S-23S ribosomal DNA internal transcribed spacer PCR-restriction fragment length polymorphism) and plasmid (group-specific repC PCR amplification) markers. The diversities of chromosomal types, repC profiles, and combined genotypes were calculated using richness in types (adjusted to equal sample sizes by rarefaction), Shannon-Wiener index, and Simpson's index. The relative differences in diversity within each pair of sites were similar for all three diversity measures. Chromosomal types, repC profiles, and combined genotypes were each more diverse in arable soils than in grass soils at two of the four locations. The other comparisons showed no significant differences. We conclude that rhizobial diversity can be affected by differences between these two management regimens. Multiple regression analyses indicated that lower diversity was associated with high potential nitrogen and phosphate levels or with acidity.     

Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites

1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.