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.

Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt.

The increasing rates of antimicrobial resistance among carbapenem-resistant Acinetobacter baumannii in the Middle East and North Africa are one of the major concerns for healthcare settings. We characterised the first A. baumannii isolate harbouring five ß-lactamases identified in Egypt. The isolate Ale25 was obtained from an ICU patient of a hospital from Alexandria. The isolate was phenotypically and genotypically screened for carbapenemase genes. The isolate was resistant to carbapenems, aminoglycosides, fluoroquinolones and cefiderocol. Whole-Genome Sequencing identified five ß-lactamase genes, blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57, together with other antibiotic resistance genes, conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol. Virulome analysis showed the presence of genes involved in adhesion and biofilm production, type II and VI secretion systems, exotoxins, etc. Multi-Locus Sequence Typing analysis identified the isolate as Sequence Types 113Pas and 2246Oxf, belonging to International Clone 7. Sequencing experiments revealed the presence of four plasmids of 2.7, 22.3, 70.4 and 240.8 Kb. All the ß-lactamase genes were located in the chromosome, except the blaPER-7, gene which was found within the plasmid of 240.8 Kb. This study highlights the threat of the emergence and dissemination of these types of isolates.

Commercial and farm fermented liquid organic amendments to improve soil quality and lettuce yield.

The anaerobic decomposition of organic wastes might lead to the formation of organic-byproducts which can then be successfully used as organic fertilizers. This study evaluated the impact of the application of two fermented liquid organic amendments (commercial vs. farm-made) at two doses of application (optimal vs. suboptimal), compared to mineral fertilization, on lettuce growth and soil quality. To this purpose, two experiments were conducted at microcosm- and field-scale, respectively. In the microcosm experiment, organically amended soils resulted in lower lettuce yield than minerally fertilized soil but, in contrast, they enhanced microbial activity and biomass, thus leading to an improvement in soil quality. The fertilization regime (organic vs. inorganic) significantly affected soil microbial composition but did not have any significant effect on structural or functional prokaryotic diversity. In the field experiment, at the optimal dose of application, organically-amended soils resulted in comparable lettuce yield to that displayed by minerally fertilized soils. The application of organic amendments did not result in an enhanced microbial activity and biomass, compared to mineral fertilization, but led to a higher soil prokaryotic diversity. Among the organically-amended plots, the optimal application dose resulted in a higher lettuce yield and soil microbial activity and biomass, but led to a decline in soil prokaryotic diversity, compared to the suboptimal application dose. Our results indicate that commercial and farm-made fermented liquid organic amendments possess the potential to ameliorate soil quality while sustaining crop yield. Given the strong influence of other factors (e.g., type of soil, dose of application) on the effects exerted by such amendments on soil quality and fertility, we recommend that an exhaustive characterization of both the amendments and the recipient soils should be carried out prior to their application, in order to better ensure their potential beneficial effects.

Coupling Proteins in Type IV Secretion.

Type IV coupling proteins (T4CPs) are essential constituents of most type IV secretion systems (T4SSs), and probably the most intriguing component in terms of their evolutionary origin and functional role. Coupling proteins have coevolved with their cognate secretion system and translocated substrates. They are present in all conjugative systems, leading to the suggestion that they play a specific role in DNA transfer. However, they are also part of many T4SSs involved in bacterial virulence, where they are required for protein translocation, with no apparent involvement in DNA secretion. Their name reflects genetic and biochemical evidence of a connecting role between the substrate and the T4SS, thus probably playing a major role in substrate recruitment. Increasing evidence supports also a role in signal transmission leading to activation of secretion. Most studies have addressed conjugative coupling proteins of the VirD4-like protein family. Their conserved features include a nucleotide-binding domain, essential for substrate translocation, a C-terminal domain involved in substrate interactions, and a transmembrane domain anchoring them to the inner membrane, which is an important regulator of protein function. Purified soluble deletion mutants display ATP hydrolysis activity and unspecific DNA binding. Elucidation of the 3D structure of the soluble deletion mutant of the conjugative coupling protein TrwB, TrwBΔN70, provided the basis for further mutagenesis studies rendering interesting insights into the structure-function of these proteins. Their key role as couplers between substrate and transporter provides biotechnological potential as targets for anti-virulence strategies, as well as for customization of substrate delivery through heterologous secretion systems.

From phytoremediation of soil contaminants to phytomanagement of ecosystem services in metal contaminated sites.

Since the emergence of phytoremediation, much research has focused on its development for (i) the removal of metals from soil and/or (ii) the reduction of metal bioavailability, mobility, and ecotoxicity in soil. Here, we review the lights and shades of the two main strategies (i.e., phytoextraction and phytostabilization) currently used for the phytoremediation of metal contaminated soils, irrespective of the level of such contamination. Both strategies face limitations to become successful at commercial scale and, then, often generate skepticism regarding their usefulness. Recent innovative approaches and paradigms are gradually establishing these phytoremediation strategies as suitable options for the management of metal contaminated soils. The combination of these phytotechnologies with a sustainable and profitable site use (a strategy called phytomanagement) grants value to the many benefits that can be obtained during the phytoremediation of metal contaminated sites, such as, for instance, the restoration of important ecosystem services, e.g. nutrient cycling, carbon storage, water flow regulation, erosion control, water purification, fertility maintenance, etc.

Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

Subcellular location of the coupling protein TrwB and the role of its transmembrane domain.

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

Early gene expression in Pseudomonas fluorescens exposed to a polymetallic solution.

The molecular response of Pseudomonas fluorescens cells exposed to a mixture of heavy metals remains largely unknown. Here, we studied the temporal changes in the early gene expression of P. fluorescens cells exposed to three doses of a polymetallic solution over two exposure times, through the application of a customized cDNA microarray. At the lowest metal dose (MD/4), we observed a repression of the Hsp70 chaperone system, MATE and MFS transporters, TonB membrane transporter and histidine kinases, together with an overexpression of metal transport (ChaC, CopC), chemotaxis and glutamine synthetase genes. At the intermediate metal dose (MD), several amino acid transporters, a response regulator (CheY), a TonB-dependent receptor and the mutT DNA repair gene were repressed; by contrast, an overexpression of genes associated with the antioxidative stress system and the transport of chelates and sulfur was observed. Finally, at the highest metal dose (4MD), a repression of genes encoding metal ion transporters, drug resistance and alginate biosynthesis was found, together with an overexpression of genes encoding antioxidative proteins, membrane transporters, ribosomal proteins, chaperones and proteases. It was concluded that P. fluorescens cells showed, over exposure time, a highly complex molecular response when exposed to a polymetallic solution, involving mechanisms related with chemotaxis, signal transmission, membrane transport, cellular redox state, and the regulation of transcription and ribosomal activity.

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells.

BACKGROUND: The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. METHODS: These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent. RESULTS: This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in ß2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound. CONCLUSIONS: All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties. GENERAL SIGNIFICANCE: Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function.

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.

Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB.

TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein. We have prepared a mutant, TrwBΔN50 that lacks one of the two α-helices in the TMD. The mutant has been studied both in detergent suspension and reconstituted in lipid vesicles. Deletion of a single helix from the TMD is enough to increase markedly the affinity of TrwB for ATP. The deletion changes the secondary structure of the cytosolic domain, whose infrared spectroscopy (IR) spectra become similar to those of the mutant TrwBΔN70 lacking the whole TMD. Interestingly, when TrwBΔN50 is reconstituted into lipid membranes, the cytosolic domain orients itself towards the vesicle interior, opposite to what happens for wild-type TrwB. In addition, we analyze the secondary structure of the TMD and TMD-lacking mutant TrwBΔN70, and found that the sum IR spectrum of the two protein fragments is different from that of the native protein, indicating the irreversibility of changes caused in TrwB by deletion of the TMD.

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.

Molecular characterization of multidrug resistant Acinetobacter baumannii clinical isolates from Alexandria, Egypt.

Carbapenem resistant Acinetobacter baumannii is a major global concern, especially in countries of the Middle East and North Africa, where the antibiotic resistance rates are on the rise. The aim of this study was to study the genomic characteristics and antimicrobial susceptibility profile of thirty-six multidrug resistant A. baumannii clinical isolates obtained in hospitals from Alexandria, Egypt. Antibiotic resistance rates were estimated by determination of Minimum Inhibitory Concentrations. Carbapenemase genes, other antibiotic resistance genes and virulence factors were then screened by the use of Whole Genome Sequencing. Isolates were also subjected to Multi Locus Sequence Typing (MLST) using the Pasteur Scheme and to core genome MLST to study their clonal relatedness. In addition, plasmid analysis was performed by the use of a commercial kit and S1- Pulsed Field Gel Electrophoresis, and Hybridization experiments with DIG-labeled DNA probes for bla NDM-1, blaPER-7 and bla GES-like were performed to locate these genes. The majority of isolates were resistant to ß-lactams (including carbapenems), fluoroquinolones, aminoglycosides and trimethoprim; and some showed resistance to cefiderocol and minocycline. We identified 8 different bla OXA-51-like variants including bla OXA-51, bla OXA-64, bla OXA-65, bla OXA-66, bla OXA-68, bla OXA-91, bla OXA-94 and bla OXA-336; bla OXA-23, bla NDM-1, bla PER-7, bla GES-like and bla ADC-like and other antibiotic resistance genes, some of these genes were within transposons or class 1 integrons. Multiple virulence factors responsible for adherence, biofilm production, type II and type VI secretion systems, exotoxins, exoenzymes, immune modulation and iron uptake were observed and 34 out of 36 isolates showed motility. Thirty-five out of 36 isolates clustered with International Clones 2, 4, 5, 7, 8 and 9; and 9 STs were identified including ST570, ST2, ST600, ST15, ST113, ST613, ST85, ST158, ST164. Plasmids ranging in size from 1.7 to 70 kb were found; bla NDM-1 and blaPER-7 genes were located in the chromosome and bla GES-like genes were simultaneously located in the chromosome and in a plasmid of 70kb. In conclusion, this study revealed a wide spectrum of antibiotic resistance genes and a variety of lineages among A. baumannii isolated in hospitals from Alexandria, and highlights the importance of investigating the molecular epidemiology to control the spread of multi-drug resistant isolates.

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.

Plasmid content of carbapenem resistant Acinetobacter baumannii isolates belonging to five International Clones collected from hospitals of Alexandria, Egypt.

Multidrug resistant Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. During the last decades it has become a major threat for healthcare settings due to the high antibiotic resistance rates among these isolates. Many resistance determinants are coded by conjugative or mobilizable plasmids, facilitating their dissemination. The majority of plasmids harbored by Acinetobacter species are less than 20 Kb, however, high molecular weight elements are the most clinically relevant since they usually contain antibiotic resistance genes. The aim of this work was to describe, classify and determine the genetic content of plasmids harbored by carbapem resistant A. baumannii isolates belonging to predominant clonal lineages circulating in hospitals from Alexandria, Egypt. The isolates were subjected to S1-Pulsed Field Gel Electrophoresis experiments to identify high molecular weight plasmids. To further analyze the plasmid content and the genetic localization of the antibiotic resistance genes, isolates were sequenced by Illumina Miseq and MinION Mk1C and a hybrid assembly was performed using Unicycler v0.5.0. Plasmids were detected with MOBsuite 3.0.3 and Copla.py v.1.0 and mapped using the online software Proksee.ca. Replicase genes were further analyzed through a BLAST against the Acinetobacter Plasmid Typing database. Eleven plasmids ranging in size from 4.9 to 205.6 Kb were characterized and mapped. All isolates contained plasmids, and, in many cases, more than two elements were identified. Antimicrobial resistance genes such as bla OXA-23, bla GES-like, aph(3')-VI and qacEΔ1 were found in likely conjugative large plasmids; while virulence determinants such as septicolysin or TonB-dependent receptors were identified in plasmids of small size. Some of these resistance determinants were, in turn, located within transposons and class 1 integrons. Among the identified plasmids, the majority encoded proteins belonging to the Rep_3 family, but replicases of the RepPriCT_1 (Aci6) family were also identified. Plasmids are of high interest as antibiotic resistance control tools, since they may be used as genetic markers for antibiotic resistance and virulence, and also as targets for the development of compounds that can inhibit transfer processes.

Membrane insertion stabilizes the structure of TrwB, the R388 conjugative plasmid coupling protein.

TrwB is an integral membrane protein that plays a crucial role in the conjugative process of plasmid R388. We have recently shown [Vecino et al., Biochim. Biophys. Acta 1798(11), 2160-2169 (2010)] that TrwB can be reconstituted into liposomes, and that bilayer incorporation increases its affinity for nucleotides and its specificity for ATP. In the present contribution we examine the structural effects of membrane insertion on TrwB, by comparing the protein in reconstituted form and in the form of protein/lipid/detergent mixed micelles. TrwB was reconstituted in PE:PG:CL (76.3:19.6:4.1mol ratio) with a final 99:1 lipid:protein mol ratio. This lipid mixture is intended to mimic the bacterial inner membrane composition, and allows a more efficient reconstitution than other lipid mixtures tested. The studies have been carried out mainly using infrared spectroscopy, because this technique provides simultaneously information on both the lipid and protein membrane components. Membrane reconstitution of TrwB is accompanied by a decrease in ß-sheet contents and an increase in ß-strand structures, probably related to protein-protein contacts in the bilayer. The predominant α-helical component remains unchanged. The bilayer-embedded protein becomes thermally more stable, and also more resistant to trypsin digestion. The properties of the bilayer lipids are also modified in the presence of TrwB, the phospholipid acyl chains are slightly ordered, and the phosphate groups at the interface become more accessible to water. In addition, we observe that the protein thermal denaturation affects the lipid thermal transition profile.

Reconstitution in liposome bilayers enhances nucleotide binding affinity and ATP-specificity of TrwB conjugative coupling protein.

Bacterial conjugative systems code for an essential membrane protein that couples the relaxosome to the DNA transport apparatus, called type IV coupling protein (T4CP). TrwB is the T4CP of the conjugative plasmid R388. In earlier work we found that this protein, purified in the presence of detergents, binds preferentially purine nucleotides trisphosphate. In contrast a soluble truncated mutant TrwBΔN70 binds uniformly all nucleotides tested. In this work, TrwB has been successfully reconstituted into liposomes. The non-membranous portion of the protein is almost exclusively oriented towards the outside of the vesicles. Functional analysis of TrwB proteoliposomes demonstrates that when the protein is inserted into the lipid bilayer the affinity for adenine and guanine nucleotides is enhanced as compared to that of the protein purified in detergent or to the soluble deletion mutant, TrwBΔN70. The protein specificity for adenine nucleotides is also increased. No ATPase activity has been found in TrwB reconstituted in proteoliposomes. This result suggests that the N-terminal transmembrane segment of this T4CP interferes with its ATPase activity and can be taken to imply that the TrwB transmembrane domain plays a regulatory role in its biological activity.

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.

Incorporation of Antibiotics into Solid Lipid Nanoparticles: A Promising Approach to Reduce Antibiotic Resistance Emergence.

Antimicrobial resistance is one of the biggest threats to global health as current antibiotics are becoming useless against resistant infectious pathogens. Consequently, new antimicrobial strategies are urgently required. Drug delivery systems represent a potential solution to improve current antibiotic properties and reverse resistance mechanisms. Among different drug delivery systems, solid lipid nanoparticles represent a highly interesting option as they offer many advantages for nontoxic targeted drug delivery. Several publications have demonstrated the capacity of SLNs to significantly improve antibiotic characteristics increasing treatment efficiency. In this review article, antibiotic-loaded solid lipid nanoparticle-related works are analyzed to summarize all information associated with applying these new formulations to tackle the antibiotic resistance problem. The main antimicrobial resistance mechanisms and relevant solid lipid nanoparticle characteristics are presented to later discuss the potential of these nanoparticles to improve current antibiotic treatment characteristics and overcome antimicrobial resistance mechanisms. Moreover, solid lipid nanoparticles also offer new possibilities for other antimicrobial agents that cannot be administrated as free drugs. The advantages and disadvantages of these new formulations are also discussed in this review. Finally, given the progress of the studies carried out to date, future directions are discussed.