RESUMEN
Clostridioides difficile (CD), a nosocomial gut pathogen, produces two major exotoxins, TcdA and TcdB, which disrupt the gut epithelial barrier and induce inflammatory/immune responses, leading to symptoms ranging from mild diarrhoea to pseudomembranous colitis and potentially to death. The expression of toxins is regulated by various transcription factors (TFs) which are induced in response to CD physiological life stages, nutritional availability, and host environment. This review summarises our current understanding on the regulation of toxin expression by TFs that interconnect with pathways of flagellar synthesis, quorum sensing, motility, biofilm formation, sporulation, and phase variation. The pleiotropic roles of some key TFs suggest that toxin production is tightly linked to other cellular processes of the CD physiology.
This review summarises the current knowledge of the transcription factors involved in regulation of toxin production, which is affected by C. difficile physiological life stages, nutritional availability, and host environment in the gut.
Asunto(s)
Toxinas Bacterianas , Clostridioides difficile , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Clostridioides difficile/genética , Clostridioides/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Cutaneous thermal injuries from burns/explosives are a major cause of morbidity and mortality and represent a monumental burden on our current health care system. Injury severity is predominantly due to potentially lethal sepsis caused by multidrug-resistant (MDR) bacteria such as Pseudomonas aeruginosa (MDR-PA). Thus, there is a critical need to develop novel and effective antimicrobials for the (i) prevention, (ii) treatment, and (iii) healing of such wounds that are complicated by MDR-P. aeruginosa and other bacterial infections. AB569 is a novel bactericidal tandem consisting of acidified NaNO2 (A-NO2-) and Na2-EDTA. Here, we first show that AB569 acts synergistically to kill all human burn wound strains of P. aeruginosa in vitro. This was found to be due, in part, to the generation of A-NO2--mediated nitric oxide (NO) formation coupled with the metal chelating properties of Na2-EDTA. Using a murine scald burn wound model of P. aeruginosa infection, an AB569-Solosite gel formulation eradicated all bacteria. Futher, we also demonstrate enhanced AB569-mediated wound healing by not only accelerating wound contraction, but also by reducing levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-1ß while increasing the levels of anti-inflammatory cytokine, IL-10, and granulocyte-colony-stimulating factor (G-CSF). We also observed better epidermal restoration in AB569-treated wounds. Taken together, we conclude that this study provides solid foundational evidence that AB569 can be used topically to treat highly problematic dermal insults, including wound, burn, blast, and likely, diabetic infections in civilian and military populations, and help relieve the economical burden that MDR organisms have on the global health care system.
Asunto(s)
Antibacterianos/farmacología , Quemaduras/tratamiento farmacológico , Ácido Edético/farmacología , Nitritos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Quemaduras/complicaciones , Quemaduras/microbiología , Colágeno/genética , Modelos Animales de Enfermedad , Ácido Edético/uso terapéutico , Geles , Óxido Nítrico/biosíntesis , Nitritos/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológicoRESUMEN
The life-threatening pandemic concerning multi-drug resistant (MDR) bacteria is an evolving problem involving increased hospitalizations, billions of dollars in medical costs and a remarkably high number of deaths. Bacterial pathogens have demonstrated the capacity for spontaneous or acquired antibiotic resistance and there is virtually no pool of organisms that have not evolved such potentially clinically catastrophic properties. Although many diseases are linked to such organisms, three include cystic fibrosis (CF), burn/blast wounds and urinary tract infections (UTIs), respectively. Thus, there is a critical need to develop novel, effective antimicrobials for the prevention and treatment of such problematic infections. One of the most formidable, naturally MDR bacterial pathogens is Pseudomonas aeruginosa (PA) that is particularly susceptible to nitric oxide (NO), a component of our innate immune response. This susceptibility sets the translational stage for the use of NO-based therapeutics during the aforementioned human infections. First, we discuss how such NO therapeutics may be able to target problematic infections in each of the aforementioned infectious scenarios. Second, we describe a recent discovery based on years of foundational information, a novel drug known as AB569. AB569 is capable of forming a "time release" of NO from S-nitrosothiols (RSNO). AB569, a bactericidal tandem consisting of acidified NaNO2 (A-NO2 -) and Na2-EDTA, is capable of killing all pathogens that are associated with the aforementioned disorders. Third, we described each disease state in brief, the known or predicted effects of AB569 on the viability of PA, its potential toxicity and highly remote possibility for resistance to develop. Finally, we conclude that AB569 can be a viable alternative or addition to conventional antibiotic regimens to treat such highly problematic MDR bacterial infections for civilian and military populations, as well as the economical burden that such organisms pose.
RESUMEN
There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64-labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.
Asunto(s)
Infecciones Bacterianas , Cobre/metabolismo , Fenoles , Tomografía de Emisión de Positrones/métodos , Sideróforos , Tiazoles , Animales , Bacterias/química , Bacterias/metabolismo , Infecciones Bacterianas/diagnóstico por imagen , Infecciones Bacterianas/microbiología , Modelos Animales de Enfermedad , Ecocardiografía , Femenino , Ratones , Ratones Endogámicos BALB C , Imagen Molecular , Fenoles/análisis , Fenoles/química , Fenoles/metabolismo , Sideróforos/análisis , Sideróforos/química , Sideróforos/metabolismo , Tiazoles/análisis , Tiazoles/química , Tiazoles/metabolismoRESUMEN
Multi-drug resistant (MDR) Acinetobacter baumannii (Ab) and Acinetobacter spp. present monumental global health challenges. These organisms represent model Gram-negative pathogens with known antibiotic resistance and biofilm-forming properties. Herein, a novel, nontoxic biocide, AB569, consisting of acidified nitrite (A-NO2-) and ethylenediaminetetraacetic acid (EDTA), demonstrated bactericidal activity against all Ab and Acinetobacter spp. strains, respectively. Average fractional inhibitory concentrations (FICs) of 0.25 mM EDTA plus 4 mM A-NO2- were observed across several clinical reference and multiple combat wound isolates from the Iraq/Afghanistan wars. Importantly, toxicity testing on human dermal fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2-, and thus are in the therapeutic range for effective Ab and Acinetobacter spp. treatment. Following treatment of Ab strain ATCC 19606 with AB569, quantitative PCR analysis of selected genes products to be responsive to AB569 revealed up-regulation of iron regulated genes involved in siderophore production, siderophore biosynthesis non-ribosomal peptide synthetase module (SBNRPSM), and siderophore biosynthesis protein monooxygenase (SBPM) when compared to untreated organisms. Taken together, treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical application of preventing Ab from gaining an early growth advantage during infection followed by extensive bactericidal activity upon subsequent exposures.
Asunto(s)
Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Campaña Afgana 2001- , Antibacterianos/farmacología , Desinfectantes/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Ácido Edético/farmacología , Guerra de Irak 2003-2011 , Nitritos/farmacología , Infección de Heridas/microbiología , Infecciones por Acinetobacter/epidemiología , Acinetobacter baumannii/genética , Adulto , Afganistán/epidemiología , Antibacterianos/química , Biopelículas/efectos de los fármacos , Células Cultivadas , Desinfectantes/química , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Ácido Edético/química , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Irak/epidemiología , Pruebas de Sensibilidad Microbiana , Nitritos/química , Reacción en Cadena de la Polimerasa , Piel/citología , Infección de Heridas/epidemiologíaRESUMEN
Exosomes are natural cell-derived nanovesicles of endocytic origin that enable cellular crosstalk by transferring encapsulated molecular cargos across biological barriers, thereby holding significantly complex implications in the etiology and progression of diverse disease states. Consequently, the development of exosomes-based nano-theranostic strategies has received immense consideration for advancing therapeutic interventions and disease prognosis. Their favorable biopharmaceutical properties make exosomes a unique nanoparticulate carrier for pharmaceutical drug delivery. This review provides an update on the contemporary strategies utilizing exosomes for theranostic applications in nanomedicine. In addition, we provide a synopsis of exosomal features and insights into strategic modifications that control in vivo biodistribution. We further discuss their opportunities, merits and pitfalls for cell/tissue targeted drug delivery in personalized nanotherapy.
RESUMEN
Electrogenic bacteria or exoelectrogens can transfer electrons to extracellular electron acceptors and thus have a wide range of applications to the ever-emerging fields of bioenergy, bioremediation, and biosensing. Standard state-of-the-art techniques for screening of electrogenic bacteria are inefficient, and often prevent rapid, high-throughput analyses. Herein, we created a simple, rapid, and straightforward papertronic 4- and 16-channel sensing platforms that is connected to a visual readout, allowing the naked eye to evaluate and quantify direct bacterial electrogenic capabilities. Our system integrated multiple 2-electrode sensing units into a signal amplifier circuit connected to light-emitting diode (LED) reporting units. The current generated from electrogenic bacteria in the sensing unit was amplified by the transistor and was transduced into LED illumination. The sensing units incorporated on the paper-based printed circuit boards (PCBs) absorbed bacteria-laden suspensions through capillary action, allowing for a rapid assessment (<2 min) of their electrogenic potential. Two well-known exoelectrogens, Shewanella oneidensis MR1 and Pseudomonas aeruginosa PA01, and many other mutants of the latter were selected to demonstrate the practicality of the proposed sensor. The effectiveness for on-site and portable measurements was validated by testing solid wastewater samples randomly obtained from the environment. Thus, the system described in this work highlights a novel form of a scalable, high-throughput sensing array for simple and rapid quantification of bacterial electrogenicity.
Asunto(s)
Técnicas Biosensibles , Shewanella , Electrodos , ElectronesRESUMEN
Global antibiotic resistance, driven by intensive antibiotic exposure/abuse, constitutes a serious challenge to all health care, particularly in an era when new antimicrobial development has slowed to a trickle. Recently, we published work demonstrating the discovery and partial mechanism of action of a novel bactericidal agent that is effective against both gram-positive and gram-negative multidrug-resistant bacteria. This drug, called AB569, consists of acidified nitrite (A-NO2-) and EDTA, of which there is no mechanism of resistance. Using both chemistry-, genetic-, and bioinformatics-based techniques, we first discovered that AB569 was able to generate bactericidal levels of nitric oxide (NO), while the EDTA component stabilized S-nitrosyl thiols, thereby furthering NO and downstream reactive nitrogen species production. This elegant chemistry triggered a paralytic downregulation of vital genes using RNA-seq involved in the synthesis of DNA, RNA, ATP, and protein in the representative ESKAPE pathogen, Pseudomonas aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Antibacterianos/química , Farmacorresistencia Bacteriana , Ácido Edético/química , Bacterias Gramnegativas/genética , Bacterias Grampositivas/genética , Nitritos/químicaRESUMEN
Microbial extracellular electron transfer (EET) stimulates a plethora of intellectual concepts leading to potential applications that offer environmentally sustainable advances in the fields of biofuels, wastewater treatment, bioremediation, desalination, and biosensing. Despite its vast potential and remarkable research efforts to date, bacterial electrogenicity is arguably the most underdeveloped technology used to confront the aforementioned challenges. Severe limitations are placed in the intrinsic energy and electron transfer processes of naturally occurring microorganisms. Significant boosts in this technology can be achieved with the growth of synthetic biology tools that manipulate microbial electron transfer pathways and improve their electrogenic potential. In particular, electrogenic Pseudomonas aeruginosa has been studied with the utility of its complete genome being sequenced coupled with well-established techniques for genetic manipulation. To optimize power density production, a high-throughput, rapid and highly sensitive test array for measuring the electrogenicity of hundreds of genetically engineered P. aeruginosa mutants is needed. This task is not trivial, as the accurate and parallel quantitative measurements of bacterial electrogenicity require long measurement times (~tens of days), continuous introduction of organic fuels (~tends of milliliters), architecturally complex and often inefficient devices, and labor-intensive operation. The overall objective of this work was to enable rapid (<30 min), sensitive (>100-fold improvement), and high-throughput (>96 wells) characterization of bacterial electrogenicity from a single 5 µL culture suspension. This project used paper as a substratum that inherently produces favorable conditions for easy, rapid, and sensitive control of an electrogenic microbial suspension. From 95 isogenic P. aeruginosa mutant, an hmgA mutant generated the highest power density (39 µW/cm2), which is higher than that of wild-type P. aeruginosa and even the strongly electrogenic organism, Shewanella oneidensis (25 µW/cm2). In summary, this work will serve as a springboard for the development of novel paradigms for genetic networks that will help develop mutations or over-expression and synthetic biology constructs to identify genes in P. aeruginosa and other organisms that enhance electrogenic performance in microbial fuel cells (MFCs).
Asunto(s)
Fuentes de Energía Bioeléctrica/microbiología , Pseudomonas aeruginosa/metabolismo , Electricidad , Transporte de Electrón , Electrones , Diseño de Equipo , Ingeniería Genética , Genoma Bacteriano , Mutación , Pseudomonas aeruginosa/genéticaRESUMEN
Antibiotic-resistant superbug bacteria represent a global health problem with no imminent solutions. Here we demonstrate that the combination (termed AB569) of acidified nitrite (A-NO2-) and Na2-EDTA (disodium ethylenediaminetetraacetic acid) inhibited all Gram-negative and Gram-positive bacteria tested. AB569 was also efficacious at killing the model organism Pseudomonas aeruginosa in biofilms and in a murine chronic lung infection model. AB569 was not toxic to human cell lines at bactericidal concentrations using a basic viability assay. RNA-Seq analyses upon treatment of P. aeruginosa with AB569 revealed a catastrophic loss of the ability to support core pathways encompassing DNA, RNA, protein, ATP biosynthesis, and iron metabolism. Electrochemical analyses elucidated that AB569 produced more stable SNO proteins, potentially explaining one mechanism of bacterial killing. Our data implicate that AB569 is a safe and effective means to kill pathogenic bacteria, suggesting that simple strategies could be applied with highly advantageous therapeutic/toxicity index ratios to pathogens associated with a myriad of periepithelial infections and related disease scenarios.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Ácido Edético/farmacología , Nitrito de Sodio/farmacología , Animales , Antibacterianos/uso terapéutico , Biopelículas/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo , Farmacorresistencia Bacteriana/efectos de los fármacos , Ácido Edético/química , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/microbiología , Redes y Vías Metabólicas , Ratones , Nitritos/química , Nitritos/farmacología , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157-194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases.
Asunto(s)
Proteínas Bacterianas/genética , Biopelículas , Pulmón/microbiología , Mutación , Nitritos/farmacología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Enfermedad Crónica , Humanos , Concentración de Iones de Hidrógeno , Plancton/metabolismo , Plancton/fisiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismoRESUMEN
The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFP-saturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.
RESUMEN
For the first time, we report a low-cost, disposable fully-papertronic screening platform for rapid screening and identification of electroactive microorganisms. This novel papertronic device is capable of simultaneous characterizing the electrogenicity of 10' s of the newly discovered, genetically engineered, bacteria. This work explored an exciting range of possibilities with the goal of fusing microbial fuel cell technology with 'papertronics,' the emerging field of paper-based electronics. Spatially distinct 64 sensing units of the array were constructed by patterning hydrophilic anodic reservoirs in paper with hydrophobic wax boundaries and utilizing 3-D multi-laminate paper structures. Full integration of a high-performance microbial sensor on paper can be achieved by improving the microbial electron exchange with the electrodes in an engineered conductive paper reservoir and reducing cathodic overpotential by using a solid electron acceptor on paper. Furthermore, the intrinsic capillary force of the paper and the increased capacity from the engineered reservoir allowed for rapid adsorption of the bacterial sample and promote immediate microbial cell attachment to the electrode, leading to instant power generation with even a small amount of the liquid.
Asunto(s)
Técnicas Biosensibles , Fenómenos Fisiológicos Bacterianos , Electrodos , Electrones , Pseudomonas aeruginosaRESUMEN
Within the past 5â¯years, tremendous advances have been made to maximize the performance of microbial fuel cells (MFCs) for both "clean" bioenergy production and bioremediation. Most research efforts have focused on parameters including (i) optimizing reactor configuration, (ii) electrode construction, (iii) addition of redox-active, electron donating mediators, (iv) biofilm acclimation and feed nutrient adjustment, as well as (v) other parameters that contribute to enhanced MFC performance. To date, tremendous advances have been made, but further improvements are needed for MFCs to be economically practical. In this review, the diversity of electrogenic microorganisms and microbial community changes in mixed cultures are discussed. More importantly, different approaches including chemical/genetic modifications and gene regulation of exoelectrogens, synthetic biology approaches and bacterial community cooperation are reviewed. Advances in recent years in metagenomics and microbiomes have allowed researchers to improve bacterial electrogenicity of robust biofilms in MFCs using novel, unconventional approaches. Taken together, this review provides some important and timely information to researchers who are examining additional means to enhance power production of MFCs.
Asunto(s)
Fuentes de Energía Bioeléctrica , Biopelículas , Consorcios Microbianos , Oxidación-Reducción , Biología SintéticaRESUMEN
Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are chronic pulmonary diseases that affect ~70,000 and 251 million individuals worldwide, respectively. Although these two diseases have distinctly different pathophysiologies, both cause chronic respiratory insufficiency that erodes quality of life and causes significant morbidity and eventually death. In both CF and COPD, the respiratory microbiome plays a major contributing role in disease progression and morbidity. Pulmonary pathogens can differ dramatically during various stages of each disease and frequently cause acute worsening of lung function due to disease exacerbation. Despite some similarities, outcome and timing/type of exacerbation can also be quite different between CF and COPD. Given these clinical distinctions, both patients and physicians should be aware of emerging therapeutic options currently being offered or in development for the treatment of lung infections in individuals with CF and COPD. Although interventions are available that prolong life and mitigate morbidity, neither disorder is curable. Both acute and chronic pulmonary infections contribute to an inexorable downward course and may trigger exacerbations, culminating in loss of lung function or respiratory failure. Knowledge of the pulmonary pathogens causing these infections, their clinical presentation, consequences, and management are, therefore, critical. In this review, we compare and contrast CF and COPD, including underlying causes, general outcomes, features of the lung microbiome, and potential treatment strategies.
RESUMEN
Pseudomonas aeruginosa is a metabolically voracious bacterium that is easily manipulated genetically. We have previously shown that the organism is also highly electrogenic in microbial fuel cells (MFCs). Polarization studies were performed in MFCs with wild-type strain PAO1 and three mutant strains (pilT, bdlA and pilT bdlA). The pilT mutant was hyperpiliated, while the bdlA mutant was suppressed in biofilm dispersion chemotaxis. The double pilT bdlA mutant was expected to have properties of both mutations. Polarization data indicate that the pilT mutant showed 5.0- and 3.2-fold increases in peak power compared to the wild type and the pilT bdlA mutant, respectively. The performance of the bdlA mutant was surprisingly the lowest, while the pilT bdlA electrogenic performance fell between the pilT mutant and wild-type bacteria. Measurements of biofilm thickness and bacterial viability showed equal viability among the different strains. The thickness of the bdlA mutant, however, was twice that of wild-type strain PAO1. This observation implicates the presence of dead or dormant bacteria in the bdlA mutant MFCs, which increases biofilm internal resistance as confirmed by electrochemical measurements.
Asunto(s)
Fuentes de Energía Bioeléctrica , Biopelículas , Pseudomonas aeruginosa/genética , MutaciónRESUMEN
The LysR member of bacterial transactivators, OxyR, governs transcription of genes involved in the response to H2O2 and organic (alkyl) hydroperoxides (AHP) in the Gram-negative pathogen, Pseudomonas aeruginosa. We have previously shown that organisms lacking OxyR are rapidly killed by <2 or 500 mM H2O2 in planktonic and biofilm bacteria, respectively. In this study, we first employed a bioinformatic approach to elucidate the potential regulatory breadth of OxyR by scanning the entire P. aeruginosa PAO1 genome for canonical OxyR promoter recognition sequences (ATAG-N7-CTAT-N7-ATAG-N7-CTAT). Of >100 potential OxyR-controlled genes, 40 were strategically selected that were not predicted to be involved in the direct response to oxidative stress (e.g., catalase, peroxidase, etc.) and screened such genes by RT-PCR analysis for potentially positive or negative control by OxyR. Differences were found in 7 of 40 genes when comparing an oxyR mutant vs. PAO1 expression that was confirmed by ß-galactosidase reporter assays. Among these, phnW, encoding 2-aminoethylphosphonate:pyruvate aminotransferase, exhibited reduced expression in the oxyR mutant compared to wild-type bacteria. Electrophoretic mobility shift assays indicated binding of OxyR to the phnW promoter and DNase I footprinting analysis also revealed the sequences to which OxyR bound. Interestingly, a phnW mutant was more susceptible to t-butyl-hydroperoxide (t-BOOH) treatment than wild-type bacteria. Although we were unable to define the direct mechanism underlying this phenomenon, we believe that this may be due to a reduced efficiency for this strain to degrade t-BOOH relative to wild-type organisms because of modulation of AHP gene transcription in the phnW mutant.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , terc-Butilhidroperóxido/farmacología , Huella de ADN , Ensayo de Cambio de Movilidad Electroforética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Electrogenicity, or bacterial electron transfer capacity, is an important application which offers environmentally sustainable advances in the fields of biofuels, wastewater treatment, bioremediation, desalination, and biosensing. Significant boosts in this technology can be achieved with the growth of synthetic biology that manipulates microbial electron transfer pathways, thereby potentially significantly improving their electrogenic potential. There is currently a need for a high-throughput, rapid, and highly sensitive test array to evaluate the electrogenic properties of newly discovered and/or genetically engineered bacterial species. In this work, we report a single-sheet, paper-based electrofluidic (incorporating both electronic and fluidic structure) screening platform for rapid, sensitive, and potentially high-throughput characterization of bacterial electrogenicity. This novel screening array uses (i) a commercially available wax printer for hydrophobic wax patterning on a single sheet of paper and (ii) water-dispersed electrically conducting polymer mixture, poly(3,4-ethylenedioxythiophene):polystyrene sulfonate, for full integration of electronic and fluidic components into the paper substrate. The engineered 3-D, microporous, hydrophilic, and conductive paper structure provides a large surface area for efficient electron transfer. This results in rapid and sensitive power assessment of electrogenic bacteria from a microliter sample volume. We validated the effectiveness of the sensor array using hypothesis-driven genetically modified Pseudomonas aeruginosa mutant strains. Within 20 min, we observed that the sensor platform successfully measured the electricity-generating capacities of five isogenic mutants of P. aeruginosa while distinguishing their differences from genetically unmodified bacteria.
RESUMEN
Bacterial biofilms may form on indwelling medical devices such as prosthetic joints, heart valves and catheters, causing challenging-to-treat infections. We have previously described the 'electricidal effect', in which bacterial biofilms are decreased following exposure to direct electrical current. Herein, we sought to determine if the decreased bacterial quantities are due to detachment of biofilms or cell death and to investigate the role that reactive oxygen species (ROS) play in the observed effect. Using confocal and electron microscopy and flow cytometry, we found that direct current (DC) leads to cell death and changes in the architecture of biofilms formed by Gram-positive and Gram-negative bacteria. Reactive oxygen species (ROS) appear to play a role in DC-associated cell death, as there was an increase in ROS-production by Staphylococcus aureus and Staphylococcus epidermidis biofilms following exposure to DC. An increase in the production of ROS response enzymes catalase and superoxide dismutase (SOD) was observed for S. aureus, S. epidermidis and Pseudomonas aeruginosa biofilms following exposure to DC. Additionally, biofilms were protected from cell death when supplemented with antioxidants and oxidant scavengers, including catalase, mannitol and Tempol. Knocking out SOD (sodAB) in P. aeruginosa led to an enhanced DC effect. Microarray analysis of P. aeruginosa PAO1 showed transcriptional changes in genes related to the stress response and cell death. In conclusion, the electricidal effect results in death of bacteria in biofilms, mediated, at least in part, by production of ROS.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Electricidad , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Silenciamiento del Gen , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
In this work, PEGylated D-amino acid K122-4 peptide (D-K122-4-PEG), derived from the type IV pilin of Pseudomonas aeruginosa, coated on 304 stainless steel was investigated for its corrosion resistant properties in a sodium chloride solution by various electrochemical measurements, surface characterization and molecular dynamics simulation. As a comparison, stainless steel electrodes coated with non-PEGylated D-amino acid retroinverso peptide (RI-K122-4) and D-amino acid K122-4 peptide (D-K122-4) were used as control variables during electrochemical tests. It was found that the D-K122-4-PEG coating is able to protect the stainless steel from corrosion in the solution. The RI-K122-4 coating shows corrosion resistant property and should be investigated further, while the D-K122-4 peptide coating, in contrast, shows little to no effect on corrosion. The morphological characterizations support the corrosion resistance of D-K122-4-PEG on stainless steel. The adsorption of D-K122-4 molecules occurs preferentially on Fe2O3, rather than Cr2O3, present on the stainless steel surface.