Electrofermentation increases concentration of poly γ-glutamic acid in Bacillus subtilis biofilms.

Fluctuations in redox conditions in bioprocesses can alter the end-products, reduce their concentration, and lengthen the process time. Electrofermentation enables rapid metabolic modulation of biosynthesis and allows control of redox imbalances in biofilm-based fermentation processes. In this study, electrofermentation is used to boost the production of the bacterial biopolymer poly-γ-glutamic acid (γ-PGA) from Bacillus subtilis ATCC 6051. When compared to control experiments (3.3 ± 0.99 g L-1 ), the application of an electrode potential E = 0.4 V versus Ag/AgCl results in a more than two-fold increase in the production of γ-PGA (9.13 ± 1.4 g L-1 ). Using an engineered B. subtilis strain, in which γ-PGA production is driven by isopropyl ß-d-1-thiogalactopyranoside, electrofermentation improves polymer concentrations from 15.4 ± 1.5 to 23.1 ± 1.6 versus g L-1 . These results confirm that electrofermentation conditions can be adopted to increase the concentration of γ-PGA and perhaps other extracellular biopolymers in industrial strains.

Polydopamine-immobilized yeast cells for portable electrochemical biosensors applied in environmental copper sensing.

The coupling of biological organisms with electrodes enables the development of sustainable, low cost, and potentially self-sustained biosensors. A critical aspect is to obtain portable bioelectrodes where the biological material is immobilized on the electrode surface to be utilized on demand. Herein, we developed an approach for the rapid entrapment and immobilization of metabolically active yeast cells in a biocompatible polydopamine layer, which does not require a separate and time-consuming synthesis. The reported approach allows obtaining the "electrical wire" of intact and active yeast cells with resulting current generation from glucose oxidation. Additionally, the electrochemical performance of the biohybrid yeast-based system has been characterized in the presence of CuSO4, a widely used pesticide, in the environmentally relevant concentration range of 20-100 µM. The system enabled the rapid preliminary monitoring of the contaminant based on variations in current generation, with a limit of detection of 12.5 µM CuSO4. The present approach for the facile preparation of portable yeast-based electrochemical biosensors paves the way for the future development of sustainable systems for environmental monitoring.

Biological and technical challenges for implementation of yeast-based biosensors.

Biosensors are low-cost and low-maintenance alternatives to conventional analytical techniques for biomedical, industrial and environmental applications. Biosensors based on whole microorganisms can be genetically engineered to attain high sensitivity and specificity for the detection of selected analytes. While bacteria-based biosensors have been extensively reported, there is a recent interest in yeast-based biosensors, combining the microbial with the eukaryotic advantages, including possession of specific receptors, stability and high robustness. Here, we describe recently reported yeast-based biosensors highlighting their biological and technical features together with their status of development, that is, laboratory or prototype. Notably, most yeast-based biosensors are still in the early developmental stage, with only a few prototypes tested for real applications. Open challenges, including systematic use of advanced molecular and biotechnological tools, bioprospecting, and implementation of yeast-based biosensors in electrochemical setup, are discussed to find possible solutions for overcoming bottlenecks and promote real-world application of yeast-based biosensors.

Biochemical and electrochemical characterization of biofilms formed on everolimus-eluting coronary stents.

Drug-eluting stents (DES) are mostly used in percutaneous coronary intervention, which is the main treatment for coronary artery occlusion. This procedure aims to restore the natural lumen, while minimizing the risk of restenosis. However, stent insertion increases the risk for infections, due to contamination of the device or insertion hub with normal skin flora. While coronary stent infection is a rare complication, it can be fatal. Currently, there is little information on biofilm formation on everolimus-eluting stents. Although everolimus is not designed as an antimicrobial agent, its antimicrobial activity should be investigated. In this study, biofilm formation on everolimus-eluting and bare metal stents (BMS) is characterized through biochemical and electrochemical methods. DES and BMS are inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, both independently and in co-culture. Biofilms formed on DES were 49.6 %, 12.9 % and 47.5 % higher than on BMS for P. aeruginosa, S. epidermidis and their co-culture, respectively. Further, the charge output for DES was 18.9 % and 59.7 % higher than BMS for P. aeruginosa and its co-culture with S. epidermidis, respectively. This observation is most likely due to higher surface roughness of DES, which favors biofilm formation. This work shows that bioelectrochemical methods can be used for rapid detection of biofilms on drug-eluting and bare metal stents, which may find application in quality assessment of stents and in characterization of stents removed after polymicrobial infections.

Electrochemical Characteristics of Shewanella loihica PV-4 on Reticulated Vitreous Carbon (RVC) with Different Potentials Applied.

The current output of an anodic bioelectrochemical system (BES) depends upon the extracellular electron transfer (EET) rate from electricigens to the electrodes. Thus, investigation of EET mechanisms between electricigens and solid electrodes is essential. Here, reticulated vitreous carbon (RVC) electrodes are used to increase the surface available for biofilm formation of the known electricigen Shewanella loihica PV-4, which is limited in conventional flat electrodes. S. loihica PV-4 utilizes flavin-mediated EET at potential lower than the outer membrane cytochromes (OMC), while at higher potential, both direct electron transfer (DET) and mediated electron transfer (MET) contribute to the current output. Results show that high electrode potential favors cell attachment on RVC, which enhances the current output. DET is the prevailing mechanism in early biofilm, while the contribution of MET to current output increased as the biofilm matured. Electrochemical analysis under starvation shows that the mediators could be confined in the biofilm. The morphology of biofilm shows bacteria distributed on the top layer of honeycomb structures, preferentially on the flat areas. This study provides insights into the EET pathways of S. loihica PV-4 on porous RVC electrodes at different biofilm ages and different set potential, which is important for the design of real-world BES.

Biofilm Detection by a Fiber-Tip Ball Resonator Optical Fiber Sensor.

Bacterial biofilms are one of the most important challenges that modern medicine faces due to the difficulties of diagnosis, antibiotic resistance, and protective mechanisms against aggressive environments. For these reasons, methods that ensure the inexpensive and rapid or real-time detection of biofilm formation on medical devices are needed. This study examines the possibilities of using optical- and fiber-based biosensors to detect and analyze early bacterial biofilms. In this study, the biofilm-forming model organism Pseudomonas aeruginosa was inoculated on the surface of the optical sensor and allowed to attach for 2 h. The biosensors were made by a fiber-tip ball resonator, fabricated through a CO2 laser splicer on a single-mode fiber, forming a weak reflective spectrum. An optical backscatter reflectometer was used to measure the refractive index detected by the sensors during different growth periods. The early biofilm concentration was determined by crystal violet (CV) binding assay; however, such a concentration was lower than the detection limit of this assay. This work presents a new approach of biofilm sensing in the early attachment stage with a low limit of detection up to 10-4 RIU (refractive index units) or 35 ± 20 × 103 CFU/mL (colony formed units).

Electroactivity of weak electricigen Bacillus subtilis biofilms in solution containing deep eutectic solvent components.

Bacillus subtilis is a Gram-positive, spore-forming bacterium with a versatile and adaptable metabolism, which makes it a viable cell factory for microbial production. Electroactivity has recently been identified as a cellular characteristic linked with the metabolic activity of B. subtilis. The enhancement of B. subtilis electroactivity can positively enhance bioproduction of high-added value metabolites under electrofermentative conditions. Here, we explored the use of deep eutectic solvents (DESs) and DES components as biocompatible nutrient additives for enhancing electroactivity of B. subtilis. The strongest electroactivity was obtained in an aqueous choline chloride: glycerol (1:2 mol mol-1) eutectic mixture. At low concentration (50-500 mM), this mixture induced a pseudo-diauxic increase in planktonic growth and increased biofilm formation, likely due to a nutritional and osmoprotectant effect. Similarities in electroactivity enhancements of choline chloride-based eutectic mixtures and quinone redox metabolism in B. subtilis were detected using high performance liquid chromatography and differential pulse voltammetry. Results show that choline chloride-based aqueous eutectic mixtures can enhance biomass and productivity in biofilm-based electrofermentation. However, the specific mechanism needs to be fully elucidated.

Assessment of physiological and electrochemical effects of a repurposed zinc dithiocarbamate complex on Acinetobacter baumannii biofilms.

Acinetobacter baumannii is an infectious agent of global proportion and concern, partly due to its proficiency in development of antibiotic resistance phenotypes and biofilm formation. Dithiocarbamates (DTC) have been identified as possible alternatives to the current antimicrobials. We report here the evaluation of several DTC-metal complexes against A. baumannii planktonic cells and biofilms. Among the DTC-metal complexes and DTCs tested, ZnL1 (N-methyl-1-phenyldithiocarbamato-S,S' Zn(II)), originally designed as an antitumor agent, is effective against biofilm forming A. baumannii. A MIC value of 12.5 µM, comparable to that of Gentamicin (5 µM) was measured for planktonic cells in tryptic soy broth. Spectroscopy, microscopy and biochemical analyses reveal cell membrane degradation and leakage after treatment with ZnL1. Bioelectrochemical analyses show that ZnL1 reduces biofilm formation and decreases extracellular respiration of pre-formed biofilms, as corroborated by microscopic analyses. Due to the affinity of Zn to cells and the metal chelating nature of L1 ligand, we hypothesize ZnL1 could alter metalloprotein functions in the membranes of A. baumannii cells, leading to altered redox balance. Results indicate that the DTC-Zn metal complex is an effective antimicrobial agent against early A. baumannii biofilms under laboratory conditions.

Nanotechnologies for control of pathogenic microbial biofilms.

Biofilms are formed at interfaces by microorganisms, which congregate in microstructured communities embedded in a self-produced extracellular polymeric substance (EPS). Biofilm-related infections are problematic due to the high resistance towards most clinically used antimicrobials, which is associated with high mortality and morbidity, combined with increased hospital stays and overall treatment costs. Several new nanotechnology-based approaches have recently been proposed for targeting resistant bacteria and microbial biofilms. Here we discuss the impacts of biofilms on healthcare, food processing and packaging, and water filtration and distribution systems, and summarize the emerging nanotechnological strategies that are being developed for biofilm prevention, control and eradication. Combination of novel nanomaterials with conventional antimicrobial therapies has shown great potential in producing more effective platforms for controlling biofilms. Recent developments include antimicrobial nanocarriers with enzyme surface functionality that allow passive infection site targeting, degradation of the EPS and delivery of high concentrations of antimicrobials to the residing cells. Several stimuli-responsive antimicrobial formulation strategies have taken advantage of the biofilm microenvironment to enhance interaction and passive delivery into the biofilm sites. Nanoparticles of ultralow size have also been recently employed in formulations to improve the EPS penetration, enhance the carrier efficiency, and improve the cell wall permeability to antimicrobials. We also discuss antimicrobial metal and metal oxide nanoparticle formulations which provide additional mechanical factors through externally induced actuation and generate reactive oxygen species (ROS) within the biofilms. The review helps to bridge microbiology with materials science and nanotechnology, enabling a more comprehensive interdisciplinary approach towards the development of novel antimicrobial treatments and biofilm control strategies.

Biosynthesis of Gold Nanoparticles by Vascular Cells in vitro.

Biosynthesis of gold nanoparticles (AuNPs) for antimicrobial and chemotherapeutic applications is a well-established process in microbial hosts such as bacterial, fungi, and plants. However, reports on AuNPs biosynthesis in mammalian cells are scarce. In this study, bovine aortic endothelial cells (BAECs) and bovine aortic smooth muscle cells (BASMCs) were examined for their ability to synthesize AuNPs in vitro. Cell culture conditions such as buffer selection, serum concentration, and HAuCl4 concentration were optimized before the biosynthesized AuNPs were characterized through visible spectrometry, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. BAECs and BASMC produced small, spherical AuNPs that are semi-crystalline with a similar diameter (23 ± 2 nm and 23 ± 4 nm). Hydrogen peroxide pretreatment increased AuNPs synthesis, suggesting that antioxidant enzymes may reduce Au3+ ions as seen in microbial cells. However, buthionine sulfoximine inhibition of glutathione synthesis, a key regulator of oxidative stress, failed to affect AuNPs generation. Taken together, these results show that under the right synthesis conditions, non-tumor cell lines can produce detectable concentrations of AuNPs in vitro.

Evaluation of extracellular electron transfer in Pseudomonas aeruginosa by co-expression of intermediate genes in NAD synthetase production pathway.

Pseudomonas aeruginosa (PA) is an electrogenic bacterium, in which extracellular electron transfer (EET) is mediated by microbially-produced phenazines, especially pyocyanin. Increasing EET rate in electrogenic bacteria is key for the development of biosensors and bioelectrofermentation processes. In this work, the production of pyocyanin, Nicotinamide Adenine Dinucleotide (NAD) and NAD synthetase by the electrogenic strain PA-A4 is determined using a Microbial Fuel Cell (MFC). Effects of metabolic inhibition and enhancement of pyocyanin and NAD synthetase on NAD/NADH levels and electrogenicity was demonstrated by short chronoamperometry measurements (0-48 h). Combined overexpression of two intermediate NAD synthetase production genes-nicotinic acid mononucleotide adenyltransferase (nadD) and quinolic acid phosphoribosyltransferase (nadC) genes, which are distant on the PA genomic map, enabled co-transcription and increased NAD synthetase activity. The resulting PA-A4 nadD + nadC shows increases in pyocyanin concentration, NAD synthetase activity, NAD/NADH levels, and MFC potential, all significantly higher than its wild type. Extracellular respiratory mechanisms in PA are linked with NAD metabolism, and targeted increased yield of NAD could directly lead to enhanced EET. A previous attempt at enhancing NAD synthetase for electrogenicity by targeting the terminal NAD synthetase gene (nadE) in standard P. aeruginosa PA01 had earlier been reported. Our work however, poses another route to electrogenicity enhancement in PA using; a combination of nadD and nadC. Further experiments are needed to understand specific intracellular mechanisms governing how over-expression of nadD and nadC induced activity of NadE protein. These findings significantly advance the knowledge of the versatility of NAD biosynthetic genes in PA electrogenicity.

Optimizing Silanization to Functionalize Stainless Steel Wire: Towards Breast Cancer Stem Cell Isolation.

Chemically modified metal surfaces have been used to recognize and capture specific cell types and biomolecules. In this work, stainless steel wires were functionalized with aptamers against breast cancer stem cell markers. Stainless steel wires were first electropolished and silanized via electrodeposition. Aptamers were then attached to the silanized surface through a cross-linker. The functionalized wires were able to capture the target cells in an in vitro test. During surface modification steps, wires were analyzed by atomic force microscopy, cyclic voltammetry, scanning electron and fluorescence microscopy to determine their surface composition and morphology. Optimized conditions of silanization (applied potential, solution pH, heat treatment temperature) for obtaining an aptamer-functionalized wire were determined in this work together with the use of several surface characterization techniques suitable for small-sized and circular wires. These modified wires have potential applications for the in vivo capture of target cells in blood flow, since their small size allows their insertion as standard guidewires in biomedical devices.

Dinuclear silver(i) complexes with a pyridine-based macrocyclic type of ligand as antimicrobial agents against clinically relevant species: the influence of the counteranion on the structure diversification of the complexes.

New dinuclear silver(i) complexes with N,N',N'',N'''-tetrakis(2-pyridylmethyl)-1,4,8,11-tetraazacyclotetradecane (tpmc), [Ag2(NO3)(tpmc)]NO3·1.7H2O (1), [Ag2(CF3SO3)2(tpmc)] (2), and [Ag2(tpmc)](BF4)2 (3) were synthesized and characterized by NMR (1H and 13C), IR and UV-Vis spectroscopy, cyclic voltammetry and molar conductivity measurements. The molecular structures of the complexes were determined by single-crystal X-ray diffraction analysis. The spectroscopic and crystallographic data showed that the structure of the complexes strongly depends on the nature of the counteranion of silver(i) salt used for their synthesis. The antimicrobial activity of complexes 1-3 was examined against Gram-positive and Gram-negative bacteria and different species of unicellular fungus Candida spp. The ability of these complexes to inhibit the formation of Candida biofilms and to eradicate the already formed biofilms was tested in the standard microtiter plate-based assay. In addition, a bioelectrochemical testing of the antimicrobial activity of complex 1 against early biofilm was also performed. The obtained results indicated that complexes 1-3 showed increased activity toward Gram-negative bacteria and Candida spp. and could inhibit the formation of biofilms. In most cases, these complexes had positive selectivity indices and showed similar or even better activity with respect to the clinically used silver(i) sulfadiazine (AgSD). The values of the binding constants for complexes 1-3 to bovine serum albumin (BSA) were found to be high enough to indicate their binding to this biomolecule, but not so high as to prevent their release upon arrival at the target site. Moreover, the positive values of partition coefficients for these complexes indicated their ability to be transported through the cell membrane. Once inside the cell, complexes 1-3 could induce the formation of the reactive oxygen species (ROS) in C. albicans cells and/or interact with DNA. Taken together, silver(i) complexes with the tpmc ligand could be considered as novel antimicrobial compounds with favourable pharmacological properties, being safer than AgSD.

Influence of High Intensity Focused Ultrasound on the Microstructure and c-di-GMP Signaling of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are typically more tolerant to antimicrobials compared to bacteria in the planktonic phase and therefore require alternative treatment approaches. Mechanical biofilm disruption from ultrasound may be such an alternative by circumventing rapid biofilm adaptation to antimicrobial agents. Although ultrasound facilitates biofilm dispersal and may enhance the effectiveness of antimicrobial agents, the resulting biological response of bacteria within the biofilms remains poorly understood. To address this question, we investigated the microstructural effects of Pseudomonas aeruginosa biofilms exposed to high intensity focused ultrasound (HIFU) at different acoustic pressures and the subsequent biological response. Confocal microscopy images indicated a clear microstructural response at peak negative pressures equal to or greater than 3.5 MPa. In this pressure amplitude range, HIFU partially reduced the biomass of cells and eroded exopolysaccharides from the biofilm. These pressures also elicited a biological response; we observed an increase in a biomarker for biofilm development (cyclic-di-GMP) proportional to ultrasound induced biofilm removal. Cyclic-di-GMP overproducing mutant strains were also more resilient to disruption from HIFU at these pressures. The biological response was further evidenced by an increase in the relative abundance of cyclic-di-GMP overproducing variants present in the biofilm after exposure to HIFU. Our results, therefore, suggest that both physical and biological effects of ultrasound on bacterial biofilms must be considered in future studies.

Extracellular Electron Transfer Powers Enterococcus faecalis Biofilm Metabolism.

Enterococci are important human commensals and significant opportunistic pathogens. Biofilm-related enterococcal infections, such as endocarditis, urinary tract infections, wound and surgical site infections, and medical device-associated infections, often become chronic upon the formation of biofilm. The biofilm matrix establishes properties that distinguish this state from free-living bacterial cells and increase tolerance to antimicrobial interventions. The metabolic versatility of the enterococci is reflected in the diversity and complexity of environments and communities in which they thrive. Understanding metabolic factors governing colonization and persistence in different host niches can reveal factors influencing the transition to biofilm pathogenicity. Here, we report a form of iron-dependent metabolism for Enterococcus faecalis where, in the absence of heme, extracellular electron transfer (EET) and increased ATP production augment biofilm growth. We observe alterations in biofilm matrix depth and composition during iron-augmented biofilm growth. We show that the ldh gene encoding l-lactate dehydrogenase is required for iron-augmented energy production and biofilm formation and promotes EET.IMPORTANCE Bacterial metabolic versatility can often influence the outcome of host-pathogen interactions, yet causes of metabolic shifts are difficult to resolve. The bacterial biofilm matrix provides the structural and functional support that distinguishes this state from free-living bacterial cells. Here, we show that the biofilm matrix can immobilize iron, providing access to this growth-promoting resource which is otherwise inaccessible in the planktonic state. Our data show that in the absence of heme, Enterococcus faecalis l-lactate dehydrogenase promotes EET and uses matrix-associated iron to carry out EET. Therefore, the presence of iron within the biofilm matrix leads to enhanced biofilm growth.

Draft Genome Sequence of Enterobacter sp. Strain EA-1, an Electrochemically Active Microorganism Isolated from Tropical Sediment.

Enterobacter sp. strain EA-1 is an electrochemically active bacterium isolated from tropical sediment in Singapore. Here, the annotated draft genome assembly of the bacterium is reported. Whole-genome comparison indicates that Enterobacter sp. EA-1, along with a previously sequenced Enterobacter isolate from East Asia, forms a distinct clade within the Enterobacter genus.

Weak electricigens: A new avenue for bioelectrochemical research.

Electroactivity appears to be a phylogenetically diverse trait independent of cell wall classification, with both Gram-negative and Gram-positive electricigens reported. While numerous electricigens have been observed, the majority of research focuses on a select group of highly electroactive species. Under favorable conditions, many microorganisms can be considered electroactive, either through their own mechanisms or exogenously-added mediators, producing a weak current. Such microbes should not be dismissed based on their modest electroactivity. Rather, they may be key to understanding what drives extracellular electron transfer in response to transient limitations of electron acceptor or donor, with implications for the study of pathogens and industrial bioprocesses. Due to their low electroactivity, such populations are difficult to grow in bioelectrochemical systems and characterise with electrochemistry. Here, a critical review of recent research on weak electricigens is provided, with a focus on the methodology and the overall relevance to microbial ecology and bioelectrochemical systems.