Expanding the focus of the One Health concept: links between the Earth-system processes of the planetary boundaries framework and antibiotic resistance.

The scientific community warns that our impact on planet Earth is so acute that we are crossing several of the planetary boundaries that demarcate the safe operating space for humankind. Besides, there is mounting evidence of serious effects on people's health derived from the ongoing environmental degradation. Regarding human health, the spread of antibiotic resistant bacteria is one of the most critical public health issues worldwide. Relevantly, antibiotic resistance has been claimed to be the quintessential One Health issue. The One Health concept links human, animal, and environmental health, but it is frequently only focused on the risk of zoonotic pathogens to public health or, to a lesser extent, the impact of contaminants on human health, i.e., adverse effects on human health coming from the other two One Health "compartments". It is recurrently claimed that antibiotic resistance must be approached from a One Health perspective, but such statement often only refers to the connection between the use of antibiotics in veterinary practice and the antibiotic resistance crisis, or the impact of contaminants (antibiotics, heavy metals, disinfectants, etc.) on antibiotic resistance. Nonetheless, the nine Earth-system processes considered in the planetary boundaries framework can be directly or indirectly linked to antibiotic resistance. Here, some of the main links between those processes and the dissemination of antibiotic resistance are described. The ultimate goal is to expand the focus of the One Health concept by pointing out the links between critical Earth-system processes and the One Health quintessential issue, i.e., antibiotic resistance.

Biological remediation treatments improve the health of a mixed contaminated soil before significantly reducing contaminant levels.

The remediation of mixed contaminated soil is challenging as it often requires actions to minimize metal-induced risks while degrading organic contaminants. Here, the effectiveness of different bioremediation strategies, namely, rhizoremediation with native plant species, mycoremediation with Pleurotus ostreatus spent mushroom substrate, and biostimulation with organic by-products (i.e., composted sewage sludge and spent mushroom substrate), for the recovery of a mixed contaminated soil from an abandoned gravel pit was studied. The combination of biostimulation and rhizoremediation led to the most significant increase in soil health, according to microbial indicator values. The application of composted sewage sludge led to the highest reduction in anthracene and polychlorinated biphenyls concentrations. None of the strategies managed to decrease contamination levels below regulatory limits, but they did enhance soil health. It was concluded that the biological remediation treatments improved soil functioning in a short time, before the concentration of soil contaminants was significantly reduced.

A case for the importance of following antibiotic resistant bacteria throughout the soil food web.

It is necessary to complement next-generation sequencing data on the soil resistome with theoretical knowledge provided by ecological studies regarding the spread of antibiotic resistant bacteria (ARB) in the abiotic and, especially, biotic fraction of the soil ecosystem. Particularly, when ARB enter agricultural soils as a consequence of the application of animal manure as fertilizer, from a microbial ecology perspective, it is important to know their fate along the soil food web, that is, throughout that complex network of feeding interactions among members of the soil biota that has crucial effects on species richness and ecosystem productivity and stability. It is critical to study how the ARB that enter the soil through the application of manure can reach other taxonomical groups (e.g., fungi, protists, nematodes, arthropods, earthworms), paying special attention to their presence in the gut microbiomes of mesofauna-macrofauna and to the possibilities for horizontal gene transfer of antibiotic resistant genes.

Development of a high-throughput platform to measure plasmid transfer frequency.

Antibiotic resistance represents one of the greatest threats to global health. The spread of antibiotic resistance genes among bacteria occurs mostly through horizontal gene transfer via conjugation mediated by plasmids. This process implies a direct contact between a donor and a recipient bacterium which acquires the antibiotic resistance genes encoded by the plasmid and, concomitantly, the capacity to transfer the acquired plasmid to a new recipient. Classical assays for the measurement of plasmid transfer frequency (i.e., conjugation frequency) are often characterized by a high variability and, hence, they require many biological and technical replicates to reduce such variability and the accompanying uncertainty. In addition, classical conjugation assays are commonly tedious and time-consuming because they typically involve counting colonies on a large number of plates for the quantification of donors, recipients, and transconjugants (i.e., the bacteria that have received the genetic material by conjugation). Due to the magnitude of the antibiotic resistance problem, it is critical to develop reliable and rapid methods for the quantification of plasmid transfer frequency that allow the simultaneous analysis of many samples. Here, we present the development of a high-throughput, reliable, quick, easy, and cost-effective method to simultaneously accomplish and measure multiple conjugation events in 96-well plates, in which the quantification of donors, recipients, and transconjugants is estimated from the time required to reach a specific threshold value (OD600 value) in the bacterial growth curves. Our method successfully discriminates different plasmid transfer frequencies, yielding results that are equivalent to those obtained by a classical conjugation assay.

Induced development of oxytetracycline tolerance in bacterial communities from soil amended with well-aged cow manure.

The use of animal manure as organic fertilizer is a common agricultural practice that can improve soil health and crop yield. However, antibiotics and their metabolites are often present in animal manure and, hence, in manure-amended soil. The aim of this study was to assess the induced development of oxytetracycline (OTC) tolerance in soil bacterial communities as a result of the addition of OTC to soil amended with well-aged cow manure. To this purpose, soil amended with well-aged cow manure was repeatedly - three times - spiked with different OTC concentrations (0, 2, 20, 60, 150, and 500 mg OTC kg-1 dry weight soil, each time) according to a pollution-induced community tolerance (PICT) assay. The PICT detection phase was conducted in Biolog EcoPlatesTM in the presence of the following OTC concentration gradient in the wells: 0, 5, 20, 40, 60, and 100 mg L-1. For all treatments, the application of OTC in the PICT selection phase resulted in lower values of bacterial metabolic activity (i.e., lower values of average well color development) in the PICT detection phase. A significant increase in OTC tolerance was observed in soil bacterial communities that had been exposed three times to ≥ 20 mg OTC kg-1 DW soil during the PICT selection phase. In general, higher levels of OTC exposure during the PICT selection phase resulted in bacterial tolerance to higher OTC concentrations during the PICT detection phase, pointing to a dose-dependent induced tolerance. It is important to (i) rationalize the amount of antibiotics administered to livestock, and (ii) treat properly the antibiotic-containing manure before its application to agricultural soil as fertilizer.

Mycoremediation with Agaricus bisporus and Pleurotus ostreatus growth substrates versus phytoremediation with Festuca rubra and Brassica sp. for the recovery of a Pb and γ-HCH contaminated soil.

Mycoremediation with mushroom growth substrates can be used for the recovery of mixed contaminated soils due to the benefits derived from the physicochemical characteristics of the substrates, the activity of extracellular enzymes secreted by the fungi, and the presence of the fungal mycelia. The objective of this work was to assess the potential of Agaricus bisporus and Pleurotus ostreatus growth substrates (inoculated mushroom substrates vs. spent mushroom substrates) for the mycoremediation of soils co-contaminated with lead and lindane (γ-HCH). We compared the efficiency of these mycoremediation strategies with the phytoremediation with Brassica spp. Or Festuca rubra plants, in terms of both reduction in contaminant levels and enhancement of soil health. An enhanced soil health was achieved as a result of the application of mycoremediation treatments, compared to phytoremediation and control (untreated) treatments. The application of P. ostreatus inoculated substrate led to the most significant reduction in γ-HCH concentration (up to 88.9% compared to corresponding controls). In the presence of inoculated mushroom substrate, P. ostreatus fruiting bodies extracted more Pb than Brassica spp. Or F. rubra plants. Mycoremediation with P. ostreatus growth substrates appears a promising strategy for the recovery of the health of soils co-contaminated with Pb and γ-HCH.

Organic amendment treatments for antimicrobial resistance and mobile element genes risk reduction in soil-crop systems.

Agricultural fertilization with organic amendments of animal origin often leads to antibiotic resistance dissemination. In this study, we evaluated the effect of different treatments (anaerobic digestion, biochar application, ozonation, zerovalent iron nanoparticle application, and spent mushroom substrate addition) on the resistome in dairy cow manure-derived amendments (slurry, manure, and compost). Anaerobic digestion and biochar application resulted in the highest reduction in antibiotic resistance gene (ARG) and mobile genetic element (MGE) gene abundance. These two treatments were applied to cow manure compost, which was then used to fertilize the soil for lettuce growth. After crop harvest, ARG and MGE gene absolute and relative abundances in the soil and lettuce samples were determined by droplet digital PCR and high-throughput qPCR, respectively. Prokaryotic diversity in cow manure-amended soils was determined using 16S rRNA metabarcoding. Compared to untreated compost, anaerobic digestion led to a 38% and 83% reduction in sul2 and intl1 absolute abundances in the soil, respectively, while biochar led to a 60% reduction in intl1 absolute abundance. No differences in lettuce gene abundances were observed among treatments. We conclude that amendment treatments can minimize the risk of antibiotic resistance in agroecosystems.

Spatio-seasonal patterns of the impact of wastewater treatment plant effluents on antibiotic resistance in river sediments.

There is a growing concern about the risk of antibiotic resistance emergence and dissemination in the environment. Here, we evaluated the spatio-seasonal patterns of the impact of wastewater treatment plant (WWTP) effluents on antibiotic resistance in river sediments. To this purpose, sediment samples were collected in three river basins affected by WWTP effluents in wet (high-water period) and dry (low-water period) hydrological conditions at three locations: (i) upstream the WWTPs; (ii) WWTP effluent discharge points (effluent outfall); and (iii) downstream the WWTPs (500 m downriver from the effluent outfall). The absolute and relative abundances of 9 antibiotic resistance genes (ARGs), 3 mobile genetic element (MGE) genes, and 4 metal resistance genes (MRGs) were quantified in sediment samples, as well as a variety of physicochemical parameters, metal contents, and antibiotic concentrations in both sediment and water samples. In sediments, significantly higher relative abundances of most genes were observed in downstream vs. upstream sampling points. Seasonal changes (higher values in low-water vs. high-water period) were observed for both ARG absolute and relative abundances in sediment samples. Chemical data revealed the contribution of effluents from WWTPs as a source of antibiotic and metal contamination in river ecosystems. The observed positive correlations between ARG and MGE genes relative abundances point out to the role of horizontal gene transfer in antibiotic resistance dissemination. Monitoring plans that take into consideration spatio-temporal patterns must be implemented to properly assess the environmental fate of WWTP-related emerging contaminants in river ecosystems.

Reduction of the resistome risk from cow slurry and manure microbiomes to soil and vegetable microbiomes.

In cow farms, the interaction between animal and environmental microbiomes creates hotspots for antibiotic resistance dissemination. A shotgun metagenomic approach was used to survey the resistome risk in five dairy cow farms. To this purpose, 10 environmental compartments were sampled: 3 of them linked to productive cows (fresh slurry, stored slurry, slurry-amended pasture soil); 6 of them to non-productive heifers and dry cows (faeces, fresh manure, aged manure, aged manure-amended orchard soil, vegetables-lettuces and grazed soil); and, finally, unamended control soil. The resistome risk was assessed using MetaCompare, a computational pipeline which scores the resistome risk according to possible links between antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and human pathogens. The resistome risk decreased from slurry and manure microbiomes to soil and vegetable microbiomes. In total (sum of all the compartments), 18,157 ARGs were detected: 24% related to ansamycins, 21% to multidrugs, 14% to aminoglycosides, 12% to tetracyclines, 9% to ß-lactams, and 9% to macrolide-lincosamide-streptogramin B. All but two of the MGE-associated ARGs were only found in the animal dejections (not in soil or vegetable samples). Several ARGs with potential as resistome risk markers (based on their presence in hubs of co-occurrence networks and high dissemination potential) were identified. As a precautionary principle, improved management of livestock dejections is necessary to minimize the risk of antibiotic resistance.

Mycorrhizal-Assisted Phytoremediation and Intercropping Strategies Improved the Health of Contaminated Soil in a Peri-Urban Area.

Soils of abandoned and vacant lands in the periphery of cities are frequently subjected to illegal dumping and can undergo degradation processes such as depletion of organic matter and nutrients, reduced biodiversity, and the presence of contaminants, which may exert an intense abiotic stress on biological communities. Mycorrhizal-assisted phytoremediation and intercropping strategies are highly suitable options for remediation of these sites. A two-year field experiment was conducted at a peri-urban site contaminated with petroleum hydrocarbons and polychlorinated biphenyls, to assess the effects of plant growth (spontaneous plant species, Medicago sativa, and Populus × canadensis, alone vs. intercropped) and inoculation of a commercial arbuscular mycorrhizal and ectomycorrhizal inoculum. Contaminant degradation, plant performance, and biodiversity, as well as a variety of microbial indicators of soil health (microbial biomass, activity, and diversity parameters) were determined. The rhizosphere bacterial and fungal microbiomes were assessed by measuring the structural diversity and composition via amplicon sequencing. Establishment of spontaneous vegetation led to greater plant and soil microbial diversity. Intercropping enhanced the activity of soil enzymes involved in nutrient cycling. The mycorrhizal treatment was a key contributor to the establishment of intercropping with poplar and alfalfa. Inoculated and poplar-alfalfa intercropped soils had a higher microbial abundance than soils colonized by spontaneous vegetation. Our study provided evidence of the potential of mycorrhizal-assisted phytoremediation and intercropping strategies to improve soil health in degraded peri-urban areas.

Agricultural Soils Amended With Thermally-Dried Anaerobically-Digested Sewage Sludge Showed Increased Risk of Antibiotic Resistance Dissemination.

The application of sewage sludge (SS) to agricultural soil can help meet crop nutrient requirements and enhance soil properties, while reusing an organic by-product. However, SS can be a source of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), resulting in an increased risk of antibiotic resistance dissemination. We studied the effect of the application of thermally-dried anaerobically-digested SS on (i) soil physicochemical and microbial properties, and (ii) the relative abundance of 85 ARGs and 10 MGE-genes in soil. Soil samples were taken from a variety of SS-amended agricultural fields differing in three factors: dose of application, dosage of application, and elapsed time after the last application. The relative abundance of both ARGs and MGE-genes was higher in SS-amended soils, compared to non-amended soils, particularly in those with a more recent SS application. Some physicochemical parameters (i.e., cation exchange capacity, copper concentration, phosphorus content) were positively correlated with the relative abundance of ARGs and MGE-genes. Sewage sludge application was the key factor to explain the distribution pattern of ARGs and MGE-genes. The 30 most abundant families within the soil prokaryotic community accounted for 66% of the total variation of ARG and MGE-gene relative abundances. Soil prokaryotic α-diversity was negatively correlated with the relative abundance of ARGs and MGE-genes. We concluded that agricultural soils amended with thermally-dried anaerobically-digested sewage sludge showed increased risk of antibiotic resistance dissemination.

Antibiotic Resistance in Agricultural Soil and Crops Associated to the Application of Cow Manure-Derived Amendments From Conventional and Organic Livestock Farms.

The application of organic amendments to agricultural soil can enhance crop yield, while improving the physicochemical and biological properties of the recipient soils. However, the use of manure-derived amendments as fertilizers entails environmental risks, such as the contamination of soil and crops with antibiotic residues, antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In order to delve into these risks, we applied dairy cow manure-derived amendments (slurry, fresh manure, aged manure), obtained from a conventional and an organic farm, to soil. Subsequently, lettuce and wheat plants were grown in the amended soils. After harvest, the abundance of 95 ARGs and MGE-genes from the amended soils and plants were determined by high-throughput qPCR. The structure of soil prokaryotic communities was determined by 16S rRNA amplicon sequencing and qPCR. The absolute abundance of ARGs and MGE-genes differed between treatments (amended vs. unamended), origins of amendment (conventional vs. organic), and types of amendment (slurry vs. fresh manure vs. aged manure). Regarding ARG-absolute abundances in the amendments themselves, higher values were usually found in slurry vs. fresh or aged manure. These abundances were generally higher in soil than in plant samples, and higher in wheat grain than in lettuce plants. Lettuce plants fertilized with conventional amendments showed higher absolute abundances of tetracycline resistance genes, compared to those amended with organic amendments. No single treatment could be identified as the best or worst treatment regarding the risk of antibiotic resistance in soil and plant samples. Within the same treatment, the resistome risk differed between the amendment, the amended soil and, finally, the crop. In other words, according to our data, the resistome risk in manure-amended crops cannot be directly inferred from the analysis of the amendments themselves. We concluded that, depending on the specific question under study, the analysis of the resistome risk should specifically focus on the amendment, the amended soil or the crop.

Application of in situ bioremediation strategies in soils amended with sewage sludges.

Increasing soil loss and the scarcity of useful land requires new reusing strategies. Thus, recovery of polluted soils recovery offers a chance for economic and social regeneration. With this objective, different soil cleaning technologies have been developed during the last few decades. On one hand, classical physical and/or chemical technologies can be found which are efficient, but have high costs and impacts upon ecosystems. On the other hand, biological methods (such as phytoremediation, bioremediation and vermiremediation) are relatively cost effective and eco-friendly, but also more time-consuming. These biological methods and their yields have been widely studied but little is known about the interaction between different soil cleaning methods. The combination of different biological strategies could lead to an improvement in remediation performance. Hence, in the present work, different micro-, vermi- and phyto-remediation combinations are applied in a sewage sludge polluted landfill in Gernika-Lumo (Basque Country) which was used as a disposal point for decades, in search of the treatment (single) or combination (dual or triple) of treatments with best remediation yields. Eight experimental groups were applied (n=3) placing earthworms (E), bacteria (B), plants (P), bacteria+earthworms (B+E), bacteria+plants (B+P), plants+earthworms (P+E) plants+bacteria+earthworms (P+B+E) and a non-treated (N.T.) group in the experimental plot (Landfill 17), for 12 months. In order to assess the efficiency of each treatment, a complete characterization (chemical and ecotoxicological) was carried out before and after remediation. Results showed high removal rates for dieldrin (between 50% and 78%) in all the experimental groups. In contrast, removal rates around 20-25% were achieved for heavy metals (Cd 15%-35%; Ni 24%-37%; Pb 15%-33%; Cr 7%-39%) and benzo(a)pyrene (19.5%-28%). The highest reductions were observed in dual (P+E, B+E) and triple (P+B+E) treatments. The best elimination yields were obtained after P+B+E treatment, as highlighted by the battery of ecotoxicological tests and bioassays performed with earthworms, plants and bacteria.

Successful remediation of soils with mixed contamination of chromium and lindane: Integration of biological and physico-chemical strategies.

Soils contaminated by organic and inorganic pollutants like Cr(VI) and lindane, is currently a main environmental challenge. Biological strategies, such as biostimulation, bioaugmentation, phytoremediation and vermiremediation, and nanoremediation with nanoscale zero-valent iron (nZVI) are promising approaches for polluted soil health recovery. The combination of different remediation strategies might be key to address this problem. For this reason, a greenhouse experiment was performed using soil without or with an organic amendment. Both soils were contaminated with lindane (15 mg kg-1) and Cr(VI) (100 or 300 mg kg-1). After one month of aging, the following treatments were applied: (i) combination of bioaugmentation (actinobacteria), phytoremediation (Brassica napus), and vermiremediation (Eisenia fetida), or (ii) nanoremediation with nZVI, or (iii) combination of biological treatments and nanoremediation. After 60 days, the wellness of plants and earthworms was assessed, also, soil health was evaluated through physico-chemical parameters and biological indicators. Cr(VI) was more toxic and decreased soil health, however, it was reduced to Cr(III) by the amendment and nZVI and, to a lesser extent, by the biological treatment. Lindane was more effectively degraded through bioremediation. In non-polluted soils, nZVI had strong deleterious effects on soil biota when combined with the organic matter, but this effect was reverted in soils with a high concentration of Cr(VI). Therefore, under our experimental conditions bioremediation might be the best for soils with a moderate concentration of Cr(VI) and organic matter. The application of nZVI in soils with a high content of organic matter should be avoided except for soils with very high concentrations of Cr(VI). According to our study, among the treatments tested, the combination of an organic amendment, biological treatment, and nZVI was shown to be the strategy of choice in soils with high concentrations of Cr(VI) and lindane, while for moderate levels of chromium, the organic amendment plus biological treatment is the most profitable treatment.

Economic and environmental assessment of bacterial poly(3-hydroxybutyrate) production from the organic fraction of municipal solid waste.

The management of municipal solid waste is a major logistic and environmental problem worldwide. Nonetheless, the organic fraction of municipal solid waste (OFMSW) is a valuable source of nutrients which can be used for a variety of purposes, according to the Circular Economy paradigm. Among the possible applications, the bioproduction of a biodegradable polyester, poly(3-hydroxybutyrate) [P(3HB)], using OFMSW as carbon platform is a promising strategy. Here, an economic and environmental assessment of bacterial P(3HB) production from OFMSW is presented based on previously published results. The SuperPro Designer® software was used to simulate P(3HB) production under our experimental parameters. Two scenarios were proposed depending on the fermentation medium: (1) enzymatic hydrolysate of OFMSW supplemented with glucose and plum waste juice; and (2) basal medium supplemented with glucose and plum waste juice. According to our results, both scenarios are not economically feasible under our experimental parameters. In Scenario 1, the low fermentation yield, the cost of the enzymes, the labour cost and the energy consumption are the factors that most contribute to that result. In Scenario 2, the cost of the extraction solvent and the low fermentation yield are the most limiting factors. The possibility of using process waste as raw material for the generation of other products must be investigated to enhance economic feasibility. From an environmental viewpoint, the photochemical oxidation potential (derived from the use of anisole as extraction solvent) and the generation of acid rain and global warming effect (caused by the burning of fuels for power generation) are the most relevant impacts associated to P(3HB) production under our experimental parameters.

Keep and promote biodiversity at polluted sites under phytomanagement.

The phytomanagement concept combines a sustainable reduction of pollutant linkages at risk-assessed contaminated sites with the generation of both valuable biomass for the (bio)economy and ecosystem services. One of the potential benefits of phytomanagement is the possibility to increase biodiversity in polluted sites. However, the unique biodiversity present in some polluted sites can be severely impacted by the implementation of phytomanagement practices, even resulting in the local extinction of endemic ecotypes or species of great conservation value. Here, we highlight the importance of promoting measures to minimise the potential adverse impact of phytomanagement on biodiversity at polluted sites, as well as recommend practices to increase biodiversity at phytomanaged sites without compromising its effectiveness in terms of reduction of pollutant linkages and the generation of valuable biomass and ecosystem services.

Gentle remediation options for soil with mixed chromium (VI) and lindane pollution: biostimulation, bioaugmentation, phytoremediation and vermiremediation.

Gentle Remediation Options (GROs), such as biostimulation, bioaugmentation, phytoremediation and vermiremediation, are cost-effective and environmentally-friendly solutions for soils simultaneously polluted with organic and inorganic compounds. This study assessed the individual and combined effectiveness of GROs in recovering the health of a soil artificially polluted with hexavalent chromium [Cr(VI)] and lindane. A greenhouse experiment was performed using organically-amended vs. non-amended mixed polluted soils. All soils received the following treatments: (i) no treatment; (ii) bioaugmentation with an actinobacteria consortium; (iii) vermiremediation with Eisenia fetida; (iv) phytoremediation with Brassica napus; (v) bioaugmentation + vermiremediation; (vi) bioaugmentation + phytoremediation; and (vii) bioaugmentation + vermiremediation + phytoremediation. Soil health recovery was determined based on Cr(VI) and lindane concentrations, microbial properties and toxicity bioassays with plants and worms. Cr(VI) pollution caused high toxicity, but some GROs were able to partly recover soil health: (i) the organic amendment decreased Cr(VI) concentrations, alleviating toxicity; (ii) the actinobacteria consortium was effective at removing both Cr(VI) and lindane; (iii) B. napus and E. fetida had a positive effect on the removal of pollutants and improved microbial properties. The combination of the organic amendment, B. napus, E. fetida and the actinobacteria consortium was the most effective strategy.

Type IV Coupling Proteins as Potential Targets to Control the Dissemination of Antibiotic Resistance.

The increase of infections caused by multidrug-resistant bacteria, together with the loss of effectiveness of currently available antibiotics, represents one of the most serious threats to public health worldwide. The loss of human lives and the economic costs associated to the problem of the dissemination of antibiotic resistance require immediate action. Bacteria, known by their great genetic plasticity, are capable not only of mutating their genes to adapt to disturbances and environmental changes but also of acquiring new genes that allow them to survive in hostile environments, such as in the presence of antibiotics. One of the major mechanisms responsible for the horizontal acquisition of new genes (e.g., antibiotic resistance genes) is bacterial conjugation, a process mediated by mobile genetic elements such as conjugative plasmids and integrative conjugative elements. Conjugative plasmids harboring antibiotic resistance genes can be transferred from a donor to a recipient bacterium in a process that requires physical contact. After conjugation, the recipient bacterium not only harbors the antibiotic resistance genes but it can also transfer the acquired plasmid to other bacteria, thus contributing to the spread of antibiotic resistance. Conjugative plasmids have genes that encode all the proteins necessary for the conjugation to take place, such as the type IV coupling proteins (T4CPs) present in all conjugative plasmids. Type VI coupling proteins constitute a heterogeneous family of hexameric ATPases that use energy from the ATP hydrolysis for plasmid transfer. Taking into account their essential role in bacterial conjugation, T4CPs are attractive targets for the inhibition of bacterial conjugation and, concomitantly, the limitation of antibiotic resistance dissemination. This review aims to compile present knowledge on T4CPs as a starting point for delving into their molecular structure and functioning in future studies. Likewise, the scientific literature on bacterial conjugation inhibitors has been reviewed here, in an attempt to elucidate the possibility of designing T4CP-inhibitors as a potential solution to the dissemination of multidrug-resistant bacteria.

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location.

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwBR388, the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwBR388. To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJpKM101 and the cytosolic domain of TrwBR388 has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobBCloDF13 of mobilizable plasmid CloDF13, together with its TMD-less mutant MobBΔTMD have been studied. Mating studies showed that the chimeric protein is functional in vivo and that it exerted negative dominance against the native proteins TrwBR388 and TraJpKM101. Also, it was observed that the TMD of MobBCloDF13 is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobBCloDF13 does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwBR388, the TMD is not an essential element for the polar location of MobBCloDF13. The main conclusion is that the characteristics described for the paradigmatic TrwBR388 T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobBCloDF13 presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.

Effects of the application of an organic amendment and nanoscale zero-valent iron particles on soil Cr(VI) remediation.

Chromium is considered an environmental pollutant of much concern whose toxicity depends, to a great extent, on its valence state, with Cr(VI) being more soluble, bioavailable, and toxic, compared to Cr(III). Nanoremediation is a promising strategy for the remediation of metal pollutants by changing their valence state. However, among other aspects, its effectiveness for soil remediation is seriously hampered by the interaction of nanoparticles with soil organic matter. In this study, soil was (i) amended with two doses of a municipal solid organic waste and (ii) artificially polluted with 300 mg Cr(VI) kg-1 DW soil. After a period of aging, a nanoremediation treatment with nanoscale zero-valent iron particles (1 g nZVI kg-1 DW soil) was applied. The efficiency of the remediation treatment was assessed in terms of Cr(VI) immobilization and recovery of soil health. The presence of the organic amendment caused (i) a decrease of redox potential, (ii) Cr(VI) immobilization via its reduction to Cr(III), (iii) a stimulation of soil microbial communities, and (iv) an improvement of soil health, compared to unamended soil. By contrast, nZVI did not have any impact on Cr(VI) immobilization nor on soil health. It was concluded that, unlike the presence of the organic amendment, nanoremediation with nZVI was not a valid option for soils polluted with Cr(VI) under our experimental conditions.