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Here, we report the draft genome sequence of Gleimia europaea DSM 26657, a pathogenic gram-positive bacillus, isolated from a patient with a subcutaneous fistula in 2007 in Germany. The genome is 2.0 Mb in size with 1,813 predicted genes, having only one putative antibiotic resistance gene.
There is an urgent need for strategies to discover secondary drugs to prevent or disrupt antimicrobial resistance (AMR), which is causing >700,000 deaths annually. Here, we demonstrate that tetracycline-resistant (Tet R ) Escherichia coli undergoes global transcriptional and metabolic remodeling, including downregulation of tricarboxylic acid cycle and disruption of redox homeostasis, to support consumption of the proton motive force for tetracycline efflux. Using a pooled genome-wide library of single-gene deletion strains, at least 308 genes, including four transcriptional regulators identified by our network analysis, were confirmed as essential for restoring the fitness of Tet R E. coli during treatment with tetracycline. Targeted knockout of ArcA, identified by network analysis as a master regulator of this new compensatory physiological state, significantly compromised fitness of Tet R E. coli during tetracycline treatment. A drug, sertraline, which generated a similar metabolome profile as the arcA knockout strain, also resensitized Tet R E. coli to tetracycline. We discovered that the potentiating effect of sertraline was eliminated upon knocking out arcA, demonstrating that the mechanism of potential synergy was through action of sertraline on the tetracycline-induced ArcA network in the Tet R strain. Our findings demonstrate that therapies that target mechanistic drivers of compensatory physiological states could resensitize AMR pathogens to lost antibiotics.
Copy-out/paste-in transposition is a major bacterial DNA mobility pathway. It contributes significantly to the emergence of antibiotic resistance, often by upregulating expression of downstream genes upon integration. Unlike other transposition pathways, it requires both asymmetric and symmetric strand transfer steps. Here, we report the first structural study of a copy-out/paste-in transposase and demonstrate its ability to catalyze all pathway steps in vitro. X-ray structures of ISCth4 transposase, a member of the IS256 family of insertion sequences, bound to DNA substrates corresponding to three sequential steps in the reaction reveal an unusual asymmetric dimeric transpososome. During transposition, an array of N-terminal domains binds a single transposon end while the catalytic domain moves to accommodate the varying substrates. These conformational changes control the path of DNA flanking the transposon end and the generation of DNA-binding sites. Our results explain the asymmetric outcome of the initial strand transfer and show how DNA binding is modulated by the asymmetric transposase to allow the capture of a second transposon end and to integrate a circular intermediate.
The search for efficient antimicrobial therapies that can alleviate suffering caused by infections from resistant bacteria is more urgent than ever before. Infections caused by multi-resistant pathogens represent a significant and increasing burden to healthcare and society and researcher are investigating new classes of bioactive compounds to slow down this development. Antimicrobial peptides from the innate immune system represent one promising class that offers a potential solution to the antibiotic resistance problem due to their mode of action on the microbial membranes. However, challenges associated with pharmacokinetics, bioavailability and off-target toxicity are slowing down the advancement and use of innate defensive peptides. Improving the therapeutic properties of these peptides is a strategy for reducing the clinical limitations and synthetic mimics of antimicrobial peptides are emerging as a promising class of molecules for a variety of antimicrobial applications. These compounds can be made significantly shorter while maintaining, or even improving antimicrobial properties, and several downsized synthetic mimics are now in clinical development for a range of infectious diseases. A variety of strategies can be employed to prepare these small compounds and this review describes the different compounds developed to date by adhering to a minimum pharmacophore based on an amphiphilic balance between cationic charge and hydrophobicity. These compounds can be made as small as dipeptides, circumventing the need for large compounds with elaborate three-dimensional structures to generate simplified and potent antimicrobial mimics for a range of medical applications. This review highlights key and recent development in the field of small antimicrobial peptide mimics as a promising class of antimicrobials, illustrating just how small you can go.
Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG).
Biofilm formation on surfaces via microbial colonization causes infections and has become a major health issue globally. The biofilm lifestyle provides resistance to environmental stresses and antimicrobial therapies. Biofilms can cause several chronic conditions, and effective treatment has become a challenge due to increased antimicrobial resistance. Antibiotics available for treating biofilm-associated infections are generally not very effective and require high doses that may cause toxicity in the host. Therefore, it is essential to study and develop efficient anti-biofilm strategies that can significantly reduce the rate of biofilm-associated healthcare problems. In this context, some effective combating strategies with potential anti-biofilm agents, including plant extracts, peptides, enzymes, lantibiotics, chelating agents, biosurfactants, polysaccharides, organic, inorganic, and metal nanoparticles, etc., have been reviewed to overcome biofilm-associated healthcare problems. From their extensive literature survey, it can be concluded that these molecules with considerable structural alterations might be applied to the treatment of biofilm-associated infections, by evaluating their significant delivery to the target site of the host. To design effective anti-biofilm molecules, it must be assured that the minimum inhibitory concentrations of these anti-biofilm compounds can eradicate biofilm-associated infections without causing toxic effects at a significant rate.
Efficacy of streptomycin sulfate for treatment of Meniere disease - ear diseases - physiological response to antibiotics
We examined the consumption of glucose from the media in which Escherichia coli ZK650 was grown. This organism, which produces the polypeptide antibiotic microcin B17 best under conditions of limiting supplies of glucose and air, was grown with a low level of glucose (0.5 mg/ml) as well as a high level (5.0 mg/ml) under both high and low aeration. Glucose consumption rates were virtually identical under both high and low aeration. Thus, glucose consumption rate is not a regulating factor in microcin B17 formation.
The E. coli AntiMicrobial Satellite(EcAMSat) mission will investigate space microgravity affects on the antibiotic resistance of E. coli, a bacterial pathogen responsible for urinary tract infection in humans and animals. EcAMSat is being developed through a partnership between NASA Ames Research Center and the Stanford University School of Medicine. Scientists believe that the results of this experiment could help design effective countermeasures to protect astronauts health during long duration human space missions.
As NASA's effort to establish a permanent residence in space continues, research on the effects of microgravity onbiological microorganisms is vital to protect or promote the health of plants and their astronaut counterparts. The purpose of this study is to determine the effects of microgravity on Pantoea agglomerans (P. agglomerans), using an analog microgravity simulator; the Rotary Cell Culture System (RCCS) developed at Johnson Space Center (JSC) in Houston, TX. P. agglomerans has been shown to be a plant growth promoter (PGPR) in ground based studies, but has also been shown to be a pathogen in both plants and immunocompromised patients. In this study, we will determine changes in the growth rate and antibiotic susceptibility of P. agglomerans when exposed to simulated microgravity.
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Abstract Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotroph that is commonly reported to exhibit diauxic population growth behavior where ferrous iron is oxidized before elemental sulfur when both are available, despite the higher energy content of sulfur. We have discovered sulfur dispersion formulations that enables sulfur oxidation before ferrous iron oxidation. The oxidation of dispersed sulfur can lower the culture pH within days below the range where aerobic ferrous iron oxidation can occur. Thus, ferric iron reduction can be observed quickly which had previously been reported over extended incubation periods with untreated sulfur. Therefore, we demonstrate that this substrate utilization pattern is strongly dependent on the cell loading in relation to sulfur concentration, sulfur surface hydrophobicity, and the pH of the culture. Our dispersed sulfur formulation, lig‐sulfur, can be used to support the rapid antibiotic selection of plasmid‐transformed cells, which is not possible in liquid cultures where ferrous iron is the main source of energy for these acidophiles. Furthermore, we find that media containing lig‐sulfur supports higher production of green fluorescent protein compared to media containing ferrous iron. The use of dispersed sulfur is a valuable new tool for the development of engineered A. ferrooxidans strains and it provides a new method to control iron and sulfur oxidation behaviors.
Abstract Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single‐carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications. To overcome this problem, we harnessed chassis‐independent recombinase‐assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum . We then integrated a non‐ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum , which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.
Abstract The objective of this study is to demonstrate that melittin, a well‐studied antimicrobial peptide (AMP), can be solubilized in an active form in bicontinuous microemulsions (BMEs) that employ biocompatible oils. The systems investigated consisted of Winsor‐III and ‐IV BME phases composed of Water/Aerosol‐OT (AOT)/Polysorbate 85/isopropyl myristate and a Winsor‐IV BME employing Polysorbate 80 and limonene. We found that melittin resided in an α‐helix‐rich configuration and was in an apolar environment for the AOT/Polysorbate 85 Winsor‐III system, suggesting that melittin interacted with the surfactant monolayer and was in an active conformation. An apolar environment was also detected for melittin in the two Winsor‐IV systems, but to a lesser extent than the Winsor‐III system. Small‐angle X‐ray scattering analysis indicated that melittin at a concentration of 1.0 g/L aq in the aqueous subphase of the Winsor‐IV systems led to the greatest impact on the BME structure (e.g., decrease of quasi‐periodic repeat distance and correlation length and induction of interfacial fluidity). The antimicrobial activity of the Polysorbate 80 Winsor‐IV system was evaluated against several bacteria prominent in chronic wounds and surgical site infections (SSIs). Melittin‐free BMEs inhibited the growth of all tested bacteria due to its oil, limonene, while the inclusion of 1.0 g/L aq of melittin in the BMEs enhanced the activity against several bacteria. A further increase of melittin concentration in the BMEs had no further enhancement. These results demonstrate the potential utility of BMEs as a delivery platform for AMPs and other hydrophilic and lipophilic drugs to inhibit antibiotic‐resistant microorganisms in chronic wounds and SSIs.
The utility of bicontinuous microemulsions (BMEs) as carriers of the antimicrobial peptide (AMP) gramicidin D and antiseptic chlorhexidine was investigated for possible topical delivery to chronic wounds. The two water-insoluble solutes dissolved in pre-formed one-phase BMEs of Water/Polysorbate 80/Limonene/Ethanol/Glycerol and Water/Aerosol-OT (AOT)/Polysorbate 85/Isopropyl Myristate and an AOT/Polysorbate 85 Winsor-III system, achieving gramicidin and chlorhexidine concentrations of 1.0 wt% and 0.5% individually and 0.5% and 0.3% in mixtures at 22°C, respectively. Small-angle neutron scattering measurements demonstrated that both solutes decreased surfactant interfacial activity and increased interfacial fluidity for the Polysorbate 80 system. For the AOT/Polysorbate 85 systems, ellipsoidal aggregates consisting of gramicidin and likely adsorbed surfactant and oil formed, while chlorhexidine enhanced the surface activity of surfactants. According to bioassays performed on artificial skin, the incorporation of melittin, gramicidin, and chlorhexidine in general enhanced the bioactivity of Polysorbate 80 BMEs for 24 h treatment against relevant antibiotic-resistant bacteria found on skin relative to controls. Yet, BME treatments were less effective than aqueous melittin control, in contrast to well diffusion bioassays performed previously. The results reflect the strong impact of AMPs and antiseptics on BME structure and dynamics and the complexity of formulating BMEs for optimal antimicrobial activity.
Staphylococcus epidermidis and Staphylococcus aureus are highly problematic bacteria in hospital settings. A major challenge is their ability to form biofilms on abiotic or biotic surfaces. Biofilms are well-organized, multicellular bacterial aggregates that resist antibiotic treatment and often lead to recurrent infections. Bacterial cell wall-anchored (CWA) proteins are important players in biofilm formation and infection. Many have putative stalk-like regions or regions of low complexity near the cell wall-anchoring motif. Recent work demonstrated the strong propensity of the stalk region of S. epidermidis accumulation-associated protein (Aap) to remain highly extended under solution conditions that typically induce compaction. This behavior is consistent with the expected function of a stalk-like region that is covalently attached to the cell wall peptidoglycan and projects the adhesive domains of Aap away from the cell surface. In this study, we evaluate whether the ability to resist compaction is a common theme among stalk regions from various staphylococcal CWA proteins. Circular dichroism spectroscopy was used to examine secondary structure changes as a function of temperature and cosolvents along with sedimentation velocity analytical ultracentrifugation, size-exclusion chromatography, and SAXS to characterize structural characteristics in solution. All stalk regions tested are intrinsically disordered, lacking secondary structure beyond random coil and polyproline type II helix, and they all sample highly extended conformations. Remarkably, the Ser-Asp dipeptide repeat region of SdrC exhibited nearly identical behavior in solution when compared to the Aap Pro/Gly-rich region, despite highly divergent sequence patterns, indicating conservation of function by various distinct staphylococcal CWA protein stalk regions.
Abstract Membrane‐embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell factory strains to hydrophobic bioproducts. Here, we report on the identification of small multidrug resistance (SMR) transporters in early‐branching anaerobic fungi (Neocallimastigomycetes). The SMR class of transporters is commonly found in bacteria but has not previously been reported in eukaryotes. In this study, we show that SMR transporters from anaerobic fungi can be produced heterologously in the model yeast Saccharomyces cerevisiae , demonstrating the potential of these proteins as targets for further characterization. The discovery of these novel anaerobic fungal SMR transporters offers a promising path forward to enhance bioproduction from engineered microbial strains.