Impact of Fertilizing with Raw or Anaerobically Digested Sewage Sludge on the Abundance of Antibiotic-Resistant Coliforms,Antibiotic Resistance Genes,and Pathogenic Bacteria in Soil and on Vegetables at Harvest

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.     

Safely Coupling Livestock and Crop Production Systems: How Rapidly Do Antibiotic Resistance Genes Dissipate in Soil following a Commercial Application of Swine or Dairy Manure?

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.     

Abundance of Antibiotic Resistance Genes in Bacteriophage following Soil Fertilization with Dairy Manure or Municipal Biosolids,and Evidence for Potential Transduction

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.     

Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months

Manuring of arable soils may stimulate the spread of resistance genes by introduction of resistant populations and antibiotics. We investigated effects of pig manure and sulfadiazine (SDZ) on bacterial communities in soil microcosms. A silt loam and a loamy sand were mixed with manure containing SDZ (10 or 100 mg per kilogram of soil), and compared with untreated soil and manured soil without SDZ over a 2-month period. In both soils, manure and SDZ positively affected the quotients of total and SDZ-resistant culturable bacteria [most probable number (MPN)], and transfer frequencies of plasmids conferring SDZ resistance in filter matings of soil bacteria and an Escherichia coli recipient. Detection of sulfonamide resistance genes sul1, sul2 and sul3 in community DNA by polymerase chain reaction (PCR) and hybridization revealed a high prevalence of sul1 in manure and manured soils, while sul2 was mainly found in the loamy sand treated with manure and high SDZ amounts, and sul3 was not detected. By PCR quantification of sul1 and bacterial rrn genes, a transient effect of manure alone and a long-term effect of SDZ plus manure on absolute and relative sul1 abundance in soil was shown. The dynamics in soil of class 1 integrons, which are typically associated with sul1, was analysed by amplification of the gene cassette region. Integrons introduced by manure established in both soils. Soil type and SDZ affected the composition of integrons. The synergistic effects of manure and SDZ were still detectable after 2 months. The results suggest that manure from treated pigs enhances spread of antibiotic resistances in soil bacterial communities.     

Insects Represent a Link between Food Animal Farms and the Urban Environment for Antibiotic Resistance Traits

Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.     

Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content

Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module ( rep and oriV ) of other descent. The plasmids differed in composition of the 27.0–28.3 kbp accessory region, each of which carried IS CR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.     

Association of antibiotic resistance in agricultural Escherichia coli isolates with attachment to quartz

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203 Escherichia coli isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (P < 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (P < 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms.     

Bacteriophages as antibiotic resistance genes carriers in agro-food systems

Antibiotic resistance genes (ARGs) are a global health concern. Antibiotic resistance occurs naturally, but misuse of antibiotics in humans and animals is accelerating the process of antibiotic resistance emergency, which has been aggravated by exposure to molecules of antibiotics present in clinical and agricultural settings and the engagement of many countries in water reuse especially in Middle East and North Africa region. Bacteriophages have the potential to be significant actors in ARGs transmission through the transduction process. These viruses have been detected along with ARGs in non impacted habitats and in anthropogenic impacted environments like wastewater, reclaimed water and manure amended soil as well as minimally processed food and ready to eat vegetables. The ubiquity of bacteriophages and their persistence in the environment raises concern about their involvement in ARGs transmission among different biomes and the generation of pathogenic-resistant bacteria that pose a great threat to human health. The aim of this review is to give an overview of the potential role of bacteriophages in the dissemination and the transfer of ARGs to pathogens in food production and processing and the consequent contribution to antibiotic resistance transmission through faecal oral route carrying ARGs to our dishes.     

Sulfonamide-Resistant Bacteria and Their Resistance Genes in Soils Fertilized with Manures from Jiangsu Province,Southeastern China

Antibiotic-resistant bacteria and genes are recognized as new environmental pollutants that warrant special concern. There were few reports on veterinary antibiotic-resistant bacteria and genes in China. This work systematically analyzed the prevalence and distribution of sulfonamide resistance genes in soils from the environments around poultry and livestock farms in Jiangsu Province, Southeastern China. The results showed that the animal manure application made the spread and abundance of antibiotic resistance genes (ARGs) increasingly in the soil. The frequency of sulfonamide resistance genes was sul1 > sul2 > sul3 in pig-manured soil DNA and sul2 > sul1 > sul3 in chicken-manured soil DNA. Further analysis suggested that the frequency distribution of the sul genes in the genomic DNA and plasmids of the SR isolates from manured soil was sul2 > sul1 > sul3 overall (p<0.05). The combination of sul1 and sul2 was the most frequent, and the co-existence of sul1 and sul3 was not found either in the genomic DNA or plasmids. The sample type, animal type and sampling time can influence the prevalence and distribution pattern of sulfonamide resistance genes. The present study also indicated that Bacillus, Pseudomonas and Shigella were the most prevalent sul-positive genera in the soil, suggesting a potential human health risk. The above results could be important in the evaluation of antibiotic-resistant bacteria and genes from manure as sources of agricultural soil pollution; the results also demonstrate the necessity and urgency of the regulation and supervision of veterinary antibiotics in China.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coli-form bacteria on three occasions during 1983. Eighty percent or more of the coli-form bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherchia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 μg/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

A note on antibiotic resistance in the bacterial flora of raw sewage and sewage-polluted River Tigris in Mosul, Iraq

Polluted water samples collected from the River Tigris in the vicinity of a raw sewage outfall were examined for the incidence of antibiotic resistance among coliform bacteria on three occasions during 1983. Eighty percent or more of the coliform bacteria were resistant to one or more antibiotics. At the same time, raw sewage samples were examined for the incidence of antibiotic-resistant bacteria, and Escherichia coli, Pseudomonas spp. and Staphylococcus spp. were selected for sensitivity testing. Collectively, more than 90% of the 480 strains of the three organisms were resistant to one or more antibiotics. The minimal inhibitory concentration (MIC) of ampicillin for twenty-nine strains including coliforms, E. coli, Klebsiella sp., Serratia sp., Ps. aeruginosa, Pseudomonas sp., Micrococcus sp., Staph. aureus, Streptococcus faecalis and Bacillus sp. from raw sewage and polluted River Tigris water was determined and that for Ps. aeruginosa was 250 micrograms/ml. The high incidence of antibiotic-resistant bacteria in natural waters could be related to the widespread use of antibiotics in this locality.     

Manure Enhances Plasmid Mobilization and Survival of Pseudomonas putida Introduced into Field Soil

We report a field study on plasmid mobilization in an agricultural soil. The influence of pig manure on the mobilization of the IncQ plasmid pIE723 by indigenous plasmids or by the IncP(alpha) plasmid pGP527 into the recipient Pseudomonas putida UWC1 (Rif(supr) Nal(supr)) was studied in field soil. Six plots were prepared in duplicate, three of which were treated with manure prior to inoculation of the donor and recipient strains. As a donor strain, either Escherichia coli J53(pIE723) or E. coli 600(pIE723, pGP527) was used. Putative transconjugants obtained on a selective medium were confirmed by DNA hybridization and PCR. Plasmid mobilization by indigenous mobilizing plasmids was observed on two occasions in manured soil. Manuring of soil significantly enhanced the frequency of pIE723 mobilization by pGP527, since mobilization frequencies into P. putida UWC1 were at least 10-fold higher in manured soil than in nonmanured soil. Enhanced numbers of P. putida UWC1 transconjugant and recipient colonies could be observed in manured soil throughout the 79-day field test. Transfer of pIE723 or pG527 into indigenous soil or rhizosphere bacteria could not be detected when indigenous bacteria isolated by selective cultivation were screened for the presence of these plasmids by DNA hybridization. Furthermore, the presence of IncN-, IncP-, or IncQ-specific sequences was confirmed in total community DNA extracted directly from the manured or nonmanured soil by PCR. IncW plasmids were detectable only in manured soil, indicating entry of these plasmids into soil via manure.     

抗生素耐药基因在畜禽粪便-土壤系统中的分布、扩散及检测方法

抗生素耐药基因作为一种新型的环境污染物已引起研究者的高度关注。畜禽养殖业长期将抗生素添加到饲料中,在促进动物生长、预防和治疗动物疾病等方面起了重要作用。这些抗生素大多数不能被动物完全吸收,在动物肠道中诱导出耐抗生素细菌和抗生素耐药基因,并随着粪便排出体外。畜禽粪便作为重要的抗生素、耐抗生素细菌和抗生素耐药基因储存库,通过堆粪、施肥等农业活动进入土壤环境中,可刺激土壤中耐抗生素细菌和抗生素耐药基因的富集。耐药基因借助于基因水平转移等方式在土壤介质中进一步传播扩散,甚至进入植物中随食物链传播,对生态环境和人类健康造成极大的威胁。为了正确评估抗生素耐药基因的生态风险,本文结合国内外相关研究,系统阐述了畜禽粪便-土壤系统中抗生素耐药基因的来源、分布及扩散机制,同时探讨了细菌耐药性的主要研究方法,指出堆肥化处理仍是目前去除抗生素耐药基因的主要手段,并对今后的研究方向进行展望。     

Effect of farmyard manure on the trace element content of soils and of plants grown thereon

Farmyard manure, added to six soils at intervals over a two-year period until the equivalent of 240 tons of fresh manure per acre had been applied, significantly increased pH values, organic matter and nitrogen contents, water-soluble boron levels and total amounts of copper, zinc and molybdenum. The uptake of boron and manganese by ladino clover was less, and that of molybdenum was greater, on the manured than on the unmanured soils. It is suggested that the influence of manure on trace-element availability may be an indirect one, due to its effect on soil reaction rather than to any increased amount of these elements that may be added in the manure.Contribution No. 377, Chemistry Division, Science Service.     

Short-term effects of amoxicillin on bacterial communities in manured soil

Antibiotic-resistant bacteria, nutrients and antibiotics that enter the soil by means of manure may enhance the proportion of bacteria displaying antibiotic resistance among soil bacteria and may affect bacterial community structure and function. To investigate the effect of manure and amoxicillin added to manure on soil bacterial communities, microcosm experiments were performed with two soil types and the following treatments: (1) nontreated, (2) manure-treated, (3) treated with manure supplemented with 10 mg amoxicillin kg(-1) soil and (4) treated with manure supplemented with 100 mg amoxicillin kg(-1) soil, with four replicates per treatment. Manure significantly increased the total CFU count and the amoxicillin-resistant CFU count of both soil types. However, only the soil with a history of manure treatment showed a significant increase in the relative number of amoxicillin-resistant bacteria as a result of amoxicillin amendment. The majority of plasmids exogenously isolated from soil originated from soil treated with amoxicillin-supplemented manure. All 16 characterized plasmids carried the bla-TEM gene, and 10 of them belonged to the IncN group. The bla-TEM gene was detected in DNA directly extracted from soil by dot-blot hybridization of PCR amplicons and showed an increased abundance in soil samples treated with manure. Molecular fingerprint analysis of 16S rRNA gene fragments amplified from soil DNA revealed significant effects of manure and amoxicillin on the bacterial community of both soils.     

Veterinary antibiotics in the aquatic and terrestrial environment

The fate of antibiotics in the environment, and especially antibiotics used in animal husbandry, is subject to recent studies and the issue of this review. The assumed quantity of antibiotics excreted by animal husbandry adds up to thousands of tonnes per year. Administered medicines, their metabolites or degradation products reach the terrestrial and aquatic environment by the application of manure or slurry to areas used agriculturally, or by pasture-reared animals excreting directly on the land, followed by surface run-off, driftage or leaching in deeper layers of the earth. The scientific interest in antimicrobially active compounds in manure and soil, but also in surface and ground water, has increased during the last decade. On the one side, scientific interest has focused on the behaviour of antibiotics and their fate in the environment, on the other hand, their impact on environmental and other bacteria has become an issue of research. Analytical methods have now been developed appropriately and studies using these new techniques provide accurate data on concentrations of antimicrobial compounds and their residues in different organic matters. Some antibiotics seem to persist a long time in the environment, especially in soil, while others degrade very fast. Not only the fate of these pharmaceuticals but their origin as well is an object of scientific interest. Besides human input via wastewater and other effluents, livestock production has been recognised as a source of contamination. One main concern with regard to the excessive use of antibiotics in livestock production is the potential promotion of resistance and the resulting disadvantages in the therapeutic use of antimicrobials. Since the beginning of antibiotic therapy, more and more resistant bacterial strains have been isolated from environmental sources showing one or multiple resistance. There have been several attempts to use antibiotic resistance patterns in different bacteria as indicators for various sources of faecal pollution. This review gives an overview of the available data on the present use of veterinary antibiotics in agriculture, on the occurrence of antibiotic compounds and resistant bacteria in soil and water and demonstrates the need for further studies.     

Organic fertilization changes the response of mycelium of arbuscular mycorrhizal fungi and their sporulation to mineral NPK supply

The synergetic effect of organic (cow manure) and mineral fertilization on the development arbuscular mycorrhizal (AM) fungi was demonstateded. The length of AM mycelium and sporulation were used as sensitive markers of the physiological state of soil AM fungal population. In manured treatments, both parameters increased in proportion with increasing mineral fertilization. In unmanured soil, the opposite trend was observed for the length of AM hyphae, which decreased with increasing mineral fertilization. Correlation analysis showed the dependence of length of AM hyphae and sporulation on soil available phosphorus. The correlation was negative in soil with no mineral fertilization and positive in soil supplied with luxury doses of mineral fertilizer.     

Occurrence and patterns of antibiotic resistance in vertebrates off the Northeastern United States coast

The prevalence of antibiotic-resistant bacteria in the marine environment is a growing concern, but the degree to which marine mammals, seabirds and fish harbor these organisms is not well documented. This project sought to identify the occurrence and patterns of antibiotic resistance in bacteria isolated from vertebrates of coastal waters in the northeastern United States. Four hundred and seventy-two isolates of clinical interest were tested for resistance to a suite of 16 antibiotics. Fifty-eight percent were resistant to at least one antibiotic, while 43% were resistant to multiple antibiotics. A multiple antibiotic resistance index value ≥0.2 was observed in 38% of the resistant pathogens, suggesting exposure of the animals to bacteria from significantly contaminated sites. Groups of antibiotics were identified for which bacterial resistance commonly co-occurred. Antibiotic resistance was more widespread in bacteria isolated from seabirds than marine mammals, and was more widespread in stranded or bycaught marine mammals than live marine mammals. Structuring of resistance patterns based on sample type (live/stranded/bycaught) but not animal group (mammal/bird/fish) was observed. These data indicate that antibiotic resistance is widespread in marine vertebrates, and they may be important reservoirs of antibiotic-resistant bacteria in the marine environment.     

临床分离4544株革兰阴性杆菌的耐药分布与变迁

目的了解玉溪市革兰阴性杆菌的耐药分布与变迁,为临床用药提供依据。方法对玉溪市人民医院1999—2004年临床各科送检的各类标本中培养分离出的4544株革兰阴性杆菌作回顾性分析。结果9种（属）细菌对25种药物的药敏结果耐药率〉50％者72种次（40．9％）,〈20％者44种次（25．0％）。总的耐药情况为不动杆菌和阴沟肠杆菌最高,铜绿假单胞菌和大肠埃希菌次之,甲型副伤寒沙门菌和福氏志贺菌最低。亚胺培南对多种细菌有较高的敏感覆盖率。有4种细菌对10种抗菌药物耐药率的上升具有临床意义（P〈0.01或P〈0．05）,其中以甲型副伤寒沙门菌对氟喹诺酮类上升最为显著。结论临床分离革兰阴性杆菌的耐药形势十分严峻,应定期监测区域内细菌的耐药变化．指导临床合坪用药。     

The MAR1 transporter is an opportunistic entry point for antibiotics

The vast quantities of antibiotics used in modern agriculture contaminate the environment and threaten human health. Recent studies have shown that crop plants grown in soil fertilized with manure from antibiotic-treated animals can accumulate antibiotic within the plant body, thus making them an additional antibiotic exposure route for consumers. Until recently, mechanisms of antibiotic entry and subcellular partitioning within plant cells were virtually unknown. We have uncovered and characterized a transporter gene in Arabidopsis thaliana, MAR1, which appears to control antibiotic entry into the chloroplast. Antibiotic resistance via MAR1 is specific to the aminoglycoside class, and is conferred by loss-of-function mutations, which is rather unusual, since most transporter-based antibiotic resistance is conferred by overexpression or gain-of-function mutations in efflux pumps with poor substrate specificity. Since MAR1 overexpression lines exhibit various iron starvation phenotypes, we propose that MAR1 transports an iron chelation molecule that is mimicked specifically by aminoglycoside antibiotics, and this facilitates their entry into the chloroplast. Knowledge about MAR1 enhances our understanding of how antibiotics might enter the plant cell, which may aid in the production of crop plants that are incapable of antibiotic accumulation, as well as further the development of new plant-based antibiotic resistance markers.Key words: antibiotic, contamination, transport, import, chloroplast, membrane, iron, chelation, nicotianamine