Hyperosmotic Agents and Antibiotics Affect Dissolved Oxygen and pH Concentration Gradients in Staphylococcus aureus Biofilms.

Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 µm, P = 0.02; and 155.5 ± 27.9 µm, P = 0.016, respectively) than in untreated controls (30.1 µm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins.

A partial-thickness epidermal explant model was colonized with green fluorescent protein (GFP)-expressing Staphylococcus aureus, and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. The oxygen concentration in explants was quantified using microelectrodes. The relative effective diffusivity and porosity of the epidermis were determined using magnetic resonance imaging, while hydrogen peroxide (H2O2) concentration in explant media was measured by using microelectrodes. Secreted proteins were identified and quantified using elevated-energy mass spectrometry (MS(E)). S. aureus biofilm grows predominantly in lipid-rich areas around hair follicles and associated skin folds. Dissolved oxygen was selectively depleted (2- to 3-fold) in these locations, but the relative effective diffusivity and porosity did not change between colonized and control epidermis. Histological analysis revealed keratinocyte damage across all the layers of colonized epidermis after 4 days of culture. The colonized explants released significantly (P < 0.01) more antioxidant proteins of both epidermal and S. aureus origin, consistent with elevated H2O2 concentrations found in the media from the colonized explants (P< 0.001). Caspase-14 was also elevated significantly in the media from the colonized explants. While H2O2 induces primary keratinocyte differentiation, caspase-14 is required for terminal keratinocyte differentiation and desquamation. These results are consistent with a localized biological impact from S. aureus in response to colonization of the skin surface.

Staphylococcus aureus induces hypoxia and cellular damage in porcine dermal explants.

We developed a porcine dermal explant model to determine the extent to which Staphylococcus aureus biofilm communities deplete oxygen, change pH, and produce damage in underlying tissue. Microelectrode measurements demonstrated that dissolved oxygen (DO) in biofilm-free dermal tissue was 4.45 ± 1.17 mg/liter, while DO levels for biofilm-infected tissue declined sharply from the surface, with no measurable oxygen detectable in the underlying dermal tissue. Magnetic resonance imaging demonstrated that biofilm-free dermal tissue had a significantly lower relative effective diffusion coefficient (0.26 ± 0.09 to 0.30 ± 0.12) than biofilm-infected dermal tissue (0.40 ± 0.12 to 0.48 ± 0.12; P < 0.0001). Thus, the difference in DO level was attributable to biofilm-induced oxygen demand rather than changes in oxygen diffusivity. Microelectrode measures showed that pH within biofilm-infected explants was more alkaline than in biofilm-free explants (8.0 ± 0.17 versus 7.5 ± 0.15, respectively; P < 0.002). Cellular and nuclear details were lost in the infected explants, consistent with cell death. Quantitative label-free shotgun proteomics demonstrated that both proapoptotic programmed cell death protein 5 and antiapoptotic macrophage migration inhibitory factor accumulated in the infected-explant spent medium, compared with uninfected-explant spent media (1,351-fold and 58-fold, respectively), consistent with the cooccurrence of apoptosis and necrosis in the explants. Biofilm-origin proteins reflected an extracellular matrix-adapted lifestyle of S. aureus. S. aureus biofilms deplete oxygen, increase pH, and induce cell death, all factors that contribute to impede wound healing.

Vancomycin and maltodextrin affect structure and activity of Staphylococcus aureus biofilms.

Hyperosmotic agents such as maltodextrin negatively impact bacterial growth through osmotic stress without contributing to drug resistance. We hypothesized that a combination of maltodextrin (osmotic agent) and vancomycin (antibiotic) would be more effective against Staphylococcus aureus biofilms than either alone. To test our hypothesis, S. aureus was grown in a flat plate flow cell reactor. Confocal laser scanning microscopy images were analyzed to quantify changes in biofilm structure. We used dissolved oxygen microelectrodes to quantify how vancomycin and maltodextrin affected the respiration rate and oxygen penetration into the biofilm. We found that treatment with vancomycin or maltodextrin altered biofilm structure. The effect on the structure was significant when they were used simultaneously to treat S. aureus biofilms. In addition, vancomycin treatment increased the oxygen respiration rate, while maltodextrin treatment caused an increase and then a decrease. An increased maltodextrin concentration decreased the diffusivity of the antibiotic. Overall, we conclude that (1) an increased maltodextrin concentration decreases vancomycin diffusion but increases the osmotic effect, leading to the optimum treatment condition, and (2) the combination of vancomycin and maltodextrin is more effective against S. aureus biofilms than either alone. Vancomycin and maltodextrin act together to increase the effectiveness of treatment against S. aureus biofilm growth.

Trade-offs between microbiome diversity and productivity in a stratified microbial mat.

Productivity is a major determinant of ecosystem diversity. Microbial ecosystems are the most diverse on the planet yet very few relationships between diversity and productivity have been reported as compared with macro-ecological studies. Here we evaluated the spatial relationships of productivity and microbiome diversity in a laboratory-cultivated photosynthetic mat. The goal was to determine how spatial diversification of microorganisms drives localized carbon and energy acquisition rates. We measured sub-millimeter depth profiles of net primary productivity and gross oxygenic photosynthesis in the context of the localized microenvironment and community structure, and observed negative correlations between species richness and productivity within the energy-replete, photic zone. Variations between localized community structures were associated with distinct taxa as well as environmental profiles describing a continuum of biological niches. Spatial regions in the photic zone corresponding to high primary productivity and photosynthesis rates had relatively low-species richness and high evenness. Hence, this system exhibited negative species-productivity and species-energy relationships. These negative relationships may be indicative of stratified, light-driven microbial ecosystems that are able to be the most productive with a relatively smaller, even distributions of species that specialize within photic zones.

Microbiosensor for the detection of acetate in electrode-respiring biofilms.

The goal of this work was to develop a microbiosensor to measure acetate concentration profiles inside biofilms in situ. The working principle of the microbiosensor was based on the correlation between the acetate concentration and the current generated during acetate oxidation by Geobacter sulfurreducens. The microbiosensor consisted of a 30-µm carbon microelectrode with an open tip as a working electrode, with G. sulfurreducens biofilm on the tip and a pseudo Ag/AgCl reference electrode, all enclosed in a glass outer case with a 30-µm tip diameter. The microbiosensor showed a linear response in the 0-1.6mM acetate concentration range with a 79±8µM limit of detection (S/N=2). We quantified the stirring effect and found it negligible. However, the interfering effect of alternative electron donors (lactate, formate, pyruvate, or hydrogen) was found to be significant. The usefulness of the acetate microbiosensor was demonstrated by measuring acetate concentration depth profiles within a G. sulfurreducens biofilm. The acetate concentration remained at bulk values throughout the biofilm when no current was passed, but it decreased from the bulk values to below the detection limit within 200µm when current was allowed to pass. The zero acetate concentration at the bottom of the biofilm showed that the biofilm was acetate-limited.

Regulation of electron transfer processes affects phototrophic mat structure and activity.

Phototrophic microbial mats are among the most diverse ecosystems in nature. These systems undergo daily cycles in redox potential caused by variations in light energy input and metabolic interactions among the microbial species. In this work, solid electrodes with controlled potentials were placed under mats to study the electron transfer processes between the electrode and the microbial mat. The phototrophic microbial mat was harvested from Hot Lake, a hypersaline, epsomitic lake located near Oroville (Washington, USA). We operated two reactors: graphite electrodes were polarized at potentials of -700 mVAg/AgCl [cathodic (CAT) mat system] and +300 mVAg/AgCl [anodic (AN) mat system] and the electron transfer rates between the electrode and mat were monitored. We observed a diel cycle of electron transfer rates for both AN and CAT mat systems. Interestingly, the CAT mats generated the highest reducing current at the same time points that the AN mats showed the highest oxidizing current. To characterize the physicochemical factors influencing electron transfer processes, we measured depth profiles of dissolved oxygen (DO) and sulfide in the mats using microelectrodes. We further demonstrated that the mat-to-electrode and electrode-to-mat electron transfer rates were light- and temperature-dependent. Using nuclear magnetic resonance (NMR) imaging, we determined that the electrode potential regulated the diffusivity and porosity of the microbial mats. Both porosity and diffusivity were higher in the CAT mats than in the AN mats. We also used NMR spectroscopy for high-resolution quantitative metabolite analysis and found that the CAT mats had significantly higher concentrations of osmoprotectants such as betaine and trehalose. Subsequently, we performed amplicon sequencing across the V4 region of the 16S rRNA gene of incubated mats to understand the impact of electrode potential on microbial community structure. These data suggested that variation in the electrochemical conditions under which mats were generated significantly impacted the relative abundances of mat members and mat metabolism.

Electrochemical scaffold generates localized, low concentration of hydrogen peroxide that inhibits bacterial pathogens and biofilms.

We hypothesized that low concentrations of H2O2 could be generated through the electrochemical conversion of oxygen by applying an electric potential to a conductive scaffold and produce a low, but constant, concentration of H2O2 that would be sufficient to destroy biofilms. To test our hypothesis we used a multidrug-resistant Acinetobacter baumannii strain, because this species is often implicated in difficult-to-treat biofilm infections. We used conductive carbon fabric as the scaffold material ("e-scaffold"). In vitro experiments demonstrated the production of a maximum constant concentration of ~25 µM H2O2 near the e-scaffold surface. An e-scaffold was overlaid onto an existing A. baumannii biofilm, and within 24 h there was a ~4-log reduction in viable bacteria with an ~80% decrease in biofilm surface coverage. A similar procedure was used to overlay an e-scaffold onto an existing A. baumannii biofilm that was grown on a porcine explant. After 24 h, there was a ~3-log reduction in viable bacteria from the infected porcine explants with no observable damage to the underlying mammalian tissue based on a viability assay and histology. This research establishes a novel foundation for an alternative antibiotic-free wound dressing to eliminate biofilms.

Multiple cathodic reaction mechanisms in seawater cathodic biofilms operating in sediment microbial fuel cells.

In this study, multiple reaction mechanisms in cathodes of sediment microbial fuel cells (SMFCs) were characterized by using cyclic voltammetry and microelectrode measurements of dissolved oxygen and pH. The cathodes were acclimated in SMFCs with sediment and seawater from San Diego Bay. Two limiting current regions were observed with onset potentials of approximately +400 mVAg/AgCl for limiting current I and -120 mVAg/AgCl for limiting current II. The appearance of two catalytic waves suggests that multiple cathodic reaction mechanisms influence cathodic performance. Microscale oxygen concentration measurements showed a zero surface concentration at the electrode surface for limiting current II but not for limiting current I, which allowed us to distinguish limiting current II as the conventional oxygen reduction reaction and limiting current I as a currently unidentified cathodic reaction mechanism. Microscale pH measurements further confirmed these results.

Localized electron transfer rates and microelectrode-based enrichment of microbial communities within a phototrophic microbial mat.

Phototrophic microbial mats frequently exhibit sharp, light-dependent redox gradients that regulate microbial respiration on specific electron acceptors as a function of depth. In this work, a benthic phototrophic microbial mat from Hot Lake, a hypersaline, epsomitic lake located near Oroville in north-central Washington, was used to develop a microscale electrochemical method to study local electron transfer processes within the mat. To characterize the physicochemical variables influencing electron transfer, we initially quantified redox potential, pH, and dissolved oxygen gradients by depth in the mat under photic and aphotic conditions. We further demonstrated that power output of a mat fuel cell was light-dependent. To study local electron transfer processes, we deployed a microscale electrode (microelectrode) with tip size ~20 µm. To enrich a subset of microorganisms capable of interacting with the microelectrode, we anodically polarized the microelectrode at depth in the mat. Subsequently, to characterize the microelectrode-associated community and compare it to the neighboring mat community, we performed amplicon sequencing of the V1-V3 region of the 16S gene. Differences in Bray-Curtis beta diversity, illustrated by large changes in relative abundance at the phylum level, suggested successful enrichment of specific mat community members on the microelectrode surface. The microelectrode-associated community exhibited substantially reduced alpha diversity and elevated relative abundances of Prosthecochloris, Loktanella, Catellibacterium, other unclassified members of Rhodobacteraceae, Thiomicrospira, and Limnobacter, compared with the community at an equivalent depth in the mat. Our results suggest that local electron transfer to an anodically polarized microelectrode selected for a specific microbial population, with substantially more abundance and diversity of sulfur-oxidizing phylotypes compared with the neighboring mat community.