Proton-coupled transport mechanism of the efflux pump NorA.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

Membrane Permeability Dominates over Electrostatic Interactions in Dictating Drug Transport in Osmotically Shocked Escherichia coli.

To combat surging multidrug-resistant Gram-negative bacterial infections, better strategies to improve the efficacy of existing drugs are critical. Because the dual membrane cell envelope is the first line of defense for these bacteria, it is crucial to understand the permeation properties of the drugs through it. Our recent study shows that isosmotic conditions prevent drug permeation inside Gram-negative bacteria, Escherichia coli, while hypoosmotic stress enhances the process. Here, we unravel the reason behind such differential drug penetration. Specifically, we dissect the roles of electrostatic screening and low membrane permeability in the penetration failure of drugs under osmotically balanced conditions. We compare the transport of a quaternary ammonium compound malachite green in the presence of an electrolyte (NaCl) and a wide variety of commonly used organic osmolytes, e.g., sucrose, proline, glycerol, sorbitol, and urea. These osmolytes of different membrane permeability (i.e., nonpermeable sucrose and NaCl, freely permeable urea and glycerol, and partially permeable proline and sorbitol) clarify the role of osmotic stress in cell envelope permeability. The results showcase that under balanced osmotic conditions, drug molecules fail to penetrate inside E. coli cells because of low membrane permeabilities and not because of electrostatic screening imposed by the osmolytes. Contribution of the electrostatic interactions, however, cannot be completely overruled as at osmotically imbalanced conditions, drug transport across the bacterial subcellular compartments is found to be dependent on the osmolytes used.

A finely balanced order-disorder equilibrium sculpts the folding-binding landscape of an antibiotic sequestering protein.

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.

Enhancing erythromycin production in Saccharopolyspora erythraea through rational engineering and fermentation refinement: A Design-Build-Test-Learn approach.

Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66 mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.

Bioprospecting of Metabolites from Actinomycetes and their Applications.

Actinomycetes are present in various terrestrial and aquatic habitats, predominantly in the soil rhizosphere, encompassing marine and freshwater ecosystems. These microorganisms exhibit characteristics that resemble both bacteria and fungi. Numerous actinomycetes exhibit a mycelial existence and undergo significant morphological transformations. These bacteria are widely recognized as biotechnologically significant microorganisms utilized for the production of secondary metabolites. In all, over 45% of all bioactive microbial metabolites are produced by actinomycetes, which are responsible for producing around 10,000 of them. The majority of actinomycetes exhibit substantial saprophytic characteristics in their natural environment, enabling them to effectively decompose a diverse range of plant and animal waste materials during the process of decomposition. Additionally, these organisms possess a sophisticated secondary metabolic system, which enables them to synthesize almost two-thirds of all naturally occurring antibiotics. Moreover, they can create a diverse array of chemical compounds with medical or agricultural applications, including anticancer, antiparasitic, and antibacterial agents. This review aims to provide an overview of the prominent biotechnological domains in which actinobacteria and their metabolites demonstrate noteworthy applicability. The graphical abstract provides a preview of the primary sections covered in this review. This paper presents a comprehensive examination of the biotechnological applications and metabolites of actinobacteria, highlighting their potential for patent innovations.

The protective role of commensal gut microbes and their metabolites against bacterial pathogens.

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.

Antibiotic influx and efflux in Pseudomonas aeruginosa: Regulation and therapeutic implications.

Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.

Quantitative assessment, molecular docking and novel metabolic pathways reveal the interaction mechanisms between norfloxacin biodegradation and environmental implications.

Norfloxacin (NOR) is widely used in medicine and animal husbandry, but its accumulation in the environment poses a substantial threat to ecological and human health. Traditional physical, chemical, and rudimentary biological methods often fall short in mitigating NOR contamination, necessitating innovative biological approaches. This study proposes an engineered bacterial consortium found in marine sediment as a strategy to enhance NOR degradation through inter-strain co-metabolism of diverse substrates. Strategically supplementing the engineered bacterial consortium with exogenous carbon sources and metal ions boosted the activity of key degradation enzymes like laccase, manganese peroxidase, and dehydrogenase. Iron and amino acids demonstrated synergistic effects, resulting in a remarkable 70.8% reduction in NOR levels. The innovative application of molecular docking elucidated enzyme interactions with NOR, uncovering potential biodegradation mechanisms. Quantitative assessment reinforced the efficiency of NOR degradation within the engineered bacterial consortium. Four metabolic routes are herein proposed: acetylation, defluorination, ring scission, and hydroxylation. Notably, this study discloses distinctive, co-operative metabolic pathways for NOR degradation within the specific microbial community. These findings provide new ways of understanding and investigating the bioremediation potential of NOR contaminants, which may lead to the development of more sustainable and effective environmental management strategies.

Enhanced degradation of enrofloxacin in mariculture wastewater based on marine bacteria and microbial carrier.

This study aimed to isolate marine bacteria to investigate their stress response, inhibition mechanisms, and degradation processes under high-load conditions of salinity and enrofloxacin (ENR). The results demonstrated that marine bacteria exhibited efficient pollutant removal efficiency even under high ENR stress (up to 10 mg/L), with chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN) and ENR removal efficiencies reaching approximately 88%, 83%, 61%, and 73%, respectively. The predominant families of marine bacteria were Bacillaceae (50.46%), Alcanivoracaceae (32.30%), and Rhodobacteraceae (13.36%). They responded to ENR removal by altering cell membrane properties, stimulating the activity of xenobiotic-metabolizing enzymes and antioxidant systems, and mitigating ENR stress through the secretion of extracellular polymeric substance (EPS). The marine bacteria exhibited robust adaptability to environmental factors and effective detoxification of ENR, simultaneously removing carbon, nitrogen, phosphorus, and antibiotics from the wastewater. The attapulgite carrier enhanced the bacteria's resistance to the environment. When treating actual mariculture wastewater, the removal efficiencies of COD and TN exceeded 80%, TP removal efficiency exceeded 90%, and ENR removal efficiency approached 100%, significantly higher than reported values in similar salinity reactors. Combining the constructed physical and mathematical models of tolerant bacterial, this study will promote the practical implementation of marine bacterial-based biotechnologies in high-loading saline wastewater treatment.

Actinomycetes are a natural resource for sustainable pest control and safeguarding agriculture.

Actinomycetes, a diverse group of bacteria with filamentous growth characteristics, have long captivated researchers and biochemists for their prolific production of secondary metabolites. Among the myriad roles played by actinomycete secondary metabolites, their historical significance in the field of biocontrol stands out prominently. The fascinating journey begins with the discovery of antibiotics, where renowned compounds like streptomycin, tetracycline, and erythromycin revolutionized medicine and agriculture. The history of biocontrol traces its roots back to the early twentieth century, when scientists recognized the potential of naturally occurring agents to combat pests and diseases. The emergence of synthetic pesticides in the mid-twentieth century temporarily overshadowed interest in biocontrol. However, with growing environmental concerns and the realization of the negative ecological impacts of chemical pesticides, the pendulum swung back towards exploring sustainable alternatives. Beyond their historical role as antibiotics, actinomycete-produced secondary metabolites encompass a rich repertoire with biopesticide potential. The classification of these compounds based on chemical structure and mode of action is highlighted, demonstrating their versatility against both plant pathogens and insect pests. Additionally, this review provides in-depth insights into how endophytic actinomycete strains play a pivotal role in biocontrol strategies. Case studies elucidate their effectiveness in inhibiting Spodoptera spp. and nematodes through the production of bioactive compounds. By unraveling the multifunctional roles of endophytic actinomycetes, this review contributes compelling narrative knowledge to the field of sustainable agriculture, emphasizing the potential of these microbial allies in crafting effective, environmentally friendly biocontrol strategies for combating agricultural pests.

Validation of aminodeoxychorismate synthase and anthranilate synthase as novel targets for bispecific antibiotics inhibiting conserved protein-protein interactions.

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.

In vitro Antibacterial Activity and Secondary Metabolite Profiling of Endolichenic Fungi Isolated from Genus Parmotrema.

The endolichenic fungi are an unexplored group of organisms for the production of bioactive secondary metabolites. The aim of the present study is to determine the antibacterial potential of endolichenic fungi isolated from genus Parmotrema. The study is continuation of our previous work, wherein a total of 73 endolichenic fungi were isolated from the lichenized fungi, which resulted in 47 species under 23 genera. All the isolated endolichenic fungi were screened for preliminary antibacterial activity. Five endolichenic fungi-Daldinia eschscholtzii, Nemania diffusa, Preussia sp., Trichoderma sp. and Xylaria feejeensis, were selected for further antibacterial activity by disc diffusion method. The zone of inhibition ranged from 14.3 ± 0.1 to 23.2 ± 0.1. The chemical composition of the selected endolichenic fungi was analysed through GC-MS, which yielded a total of 108 compounds from all the selected five endolichenic fungi. Diethyl phthalate, 1-hexadecanol, dibutyl phthalate, n-tetracosanol-1, 1-nonadecene, pyrrol[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methyl) and tetratetracontane were found to be common compounds among one or the other endolichenic fungi, which possibly were responsible for antibacterial activity. GC-MS data were further analysed through Principal Component Analysis which showed D. eschscholtzii to be with unique pattern of expression of metabolites. Compound confirmation test revealed coumaric acid to be responsible for antibacterial activity in D. eschscholtzii. So, the study proves that endolichenic fungi that inhabit lichenized fungal thalli could be a source of potential antibacterial compounds.

Evaluation of the Effect of Lactobacillus acidophilus ATCC 4356 Bacteriocin against Staphylococcus aureus.

Background: Lactobacillus acidophilus is lactic acid bacteria that produce bacteriocins. Bacteriocins are antimicrobial peptides or proteins that exhibit activity against closely related bacteria. The aim of this study was to determine the effect of L. acidophilus ATCC 4356 bacteriocin against Staphylococcus aureus. Material and Methods. We used four different phenotypic methods for antimicrobial activities against two standard strains: methicillin-resistant S. aureus (MRSA) ATCC 33591 and methicillin-susceptible S. aureus (MSSA) ATCC 25923. The methods were (1) agar well diffusion, (2) overlay soft agar, (3) paper disk, and (4) modification of punch hole. The ammonium sulfate method was used to concentrate crude bacteriocin, and ultrafiltration and dialysis tubes were used to remove ammonium sulfate from the bacteriocins. Each method was repeated in triplicate. Result: L. acidophilus ATCC 4356 showed antimicrobial activity against both MRSA and MSSA standard strains only by the overlay soft agar method and not by the agar well diffusion, punch hole modification, and paper disk methods. No antimicrobial effects were observed in crude bacteriocins concentrated. Conclusion: The growth inhibition of S. aureus in overlay soft agar method may be due to the production of bacteriocin-like substances. The overlay soft agar method is a qualitative test, so there is a need for further study to optimize the conditions for the production of bacteriocin-like substances in the culture supernatant and precise comparison between the inhibitory activity and pheromone secretion of different strains.

Microalgae enhanced co-metabolism of sulfamethoxazole using aquacultural feedstuff components: Co-metabolic pathways and enzymatic mechanisms.

The application of antibiotics in freshwater aquaculture leads to increased contamination of aquatic environments. However, limited information is available on the co-metabolic biodegradation of antibiotics by microalgae in aquaculture. Feedstuffs provide multiple organic substrates for microalgae-mediated co-metabolism. Herein, we investigated the co-metabolism of sulfamethoxazole (SMX) by Chlorella pyrenoidosa when adding main components of feedstuff (glucose and lysine). Results showed that lysine had an approximately 1.5-fold stronger enhancement on microalgae-mediated co-metabolism of SMX than glucose, with the highest removal rate (68.77% ± 0.50%) observed in the 9-mM-Lys co-metabolic system. Furthermore, we incorporated reactive sites predicted by density functional theory calculations, 14 co-metabolites identified by mass spectrometry, and the roles of 18 significantly activated enzymes to reveal the catalytic reaction mechanisms underlying the microalgae-mediated co-metabolism of SMX. In lysine- and glucose-treated groups, five similar co-metabolic pathways were proposed, including bond breaking on the nucleophilic sulfur atom, ring cleavage and hydroxylation at multiple free radical reaction sites, together with acylation and glutamyl conjugation on electrophilic nitrogen atoms. Cytochrome P450, serine hydrolase, and peroxidase play crucial roles in catalyzing hydroxylation, bond breaking, and ring cleavage of SMX. These findings provide theoretical support for better utilization of microalgae-driven co-metabolism to reduce sulfonamide antibiotic residues in aquaculture.

Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

Gyrase and Topoisomerase IV: Recycling Old Targets for New Antibacterials to Combat Fluoroquinolone Resistance.

Beyond their requisite functions in many critical DNA processes, the bacterial type II topoisomerases, gyrase and topoisomerase IV, are the targets of fluoroquinolone antibacterials. These drugs act by stabilizing gyrase/topoisomerase IV-generated DNA strand breaks and by robbing the cell of the catalytic activities of these essential enzymes. Since their clinical approval in the mid-1980s, fluoroquinolones have been used to treat a broad spectrum of infectious diseases and are listed among the five "highest priority" critically important antimicrobial classes by the World Health Organization. Unfortunately, the widespread use of fluoroquinolones has been accompanied by a rise in target-mediated resistance caused by specific mutations in gyrase and topoisomerase IV, which has curtailed the medical efficacy of this drug class. As a result, efforts are underway to identify novel antibacterials that target the bacterial type II topoisomerases. Several new classes of gyrase/topoisomerase IV-targeted antibacterials have emerged, including novel bacterial topoisomerase inhibitors, Mycobacterium tuberculosis gyrase inhibitors, triazaacenaphthylenes, spiropyrimidinetriones, and thiophenes. Phase III clinical trials that utilized two members of these classes, gepotidacin (triazaacenaphthylene) and zoliflodacin (spiropyrimidinetrione), have been completed with positive outcomes, underscoring the potential of these compounds to become the first new classes of antibacterials introduced into the clinic in decades. Because gyrase and topoisomerase IV are validated targets for established and emerging antibacterials, this review will describe the catalytic mechanism and cellular activities of the bacterial type II topoisomerases, their interactions with fluoroquinolones, the mechanism of target-mediated fluoroquinolone resistance, and the actions of novel antibacterials against wild-type and fluoroquinolone-resistant gyrase and topoisomerase IV.

Biodegradation of ciprofloxacin using machine learning tools: Kinetics and modelling.

Recently, the rampant administration of antibiotics and their synthetic organic constitutes have exacerbated adverse effects on ecosystems, affecting the health of animals, plants, and humans by promoting the emergence of extreme multidrug-resistant bacteria (XDR), antibiotic resistance bacterial variants (ARB), and genes (ARGs). The constraints, such as high costs, by-product formation, etc., associated with the physico-chemical treatment process limit their efficacy in achieving efficient wastewater remediation. Biodegradation is a cost-effective, energy-saving, sustainable alternative for removing emerging organic pollutants from environmental matrices. In view of the same, the current study aims to explore the biodegradation of ciprofloxacin using microbial consortia via metabolic pathways. The optimal parameters for biodegradation were assessed by employing machine learning tools, viz. Artificial Neural Network (ANN) and statistical optimization tool (Response Surface Methodology, RSM) using the Box-Behnken design (BBD). Under optimal culture conditions, the designed bacterial consortia degraded ciprofloxacin with 95.5% efficiency, aligning with model prediction results, i.e., 95.20% (RSM) and 94.53% (ANN), respectively. Thus, befitting amendments to the biodegradation process can augment efficiency and lead to a greener solution for antibiotic degradation from aqueous media.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Relationship of Cytotoxic and Antimicrobial Effects of Triphenylphosphonium Conjugates with Various Quinone Derivatives.

Quinone derivatives of triphenylphosphonium have proven themselves to be effective geroprotectors and antioxidants that prevent oxidation of cell components with participation of active free radicals - peroxide (RO2·), alkoxy (RO·), and alkyl (R·) radicals, as well as reactive oxygen species (superoxide anion, singlet oxygen). Their most studied representatives are derivatives of plastoquinone (SkQ1) and ubiquinone (MitoQ), which in addition to antioxidant properties also have a strong antibacterial effect. In this study, we investigated antibacterial properties of other quinone derivatives based on decyltriphenylphosphonium (SkQ3, SkQT, and SkQThy). We have shown that they, just like SkQ1, inhibit growth of various Gram-positive bacteria at micromolar concentrations, while being less effective against Gram-negative bacteria, which is associated with recognition of the triphenylphosphonium derivatives by the main multidrug resistance (MDR) pump of Gram-negative bacteria, AcrAB-TolC. Antibacterial action of SkQ1 itself was found to be dependent on the number of bacterial cells. It is important to note that the cytotoxic effect of SkQ1 on mammalian cells was observed at higher concentrations than the antibacterial action, which can be explained by (i) the presence of a large number of membrane organelles, (ii) lower membrane potential, (iii) spatial separation of the processes of energy generation and transport, and (iv) differences in the composition of MDR pumps. Differences in the cytotoxic effects on different types of eukaryotic cells may be associated with the degree of membrane organelle development, energy status of the cell, and level of the MDR pump expression.