38 research outputs found
Selectively Permeable Microbial Nanoculture System as Artificial Microniches
Microfluidics-based approaches for isolating microbes, replicating their microenvironments and growth conditions can provide new platforms to elucidate many fundamental biological questions. For instance, the spatial confinement of microorganisms in microcompartments can spawn new insights on the inherent advantages of cell clustering and biofilm formation in microbial survival and virulence mechanisms. As the worldwide antibiotic crisis persists, there is an urgent need for emerging technologies that enable the scrutiny of the evolution of multidrug resistance, and facilitate the study of cooperative or antagonistic microbial interactions. Here we describe a method to generate artificial multifunctional microniches in order to encapsulate and interrogate microbes. Using a flow-focusing microfluidic device, microorganisms including Pseudomonas aeruginosa and Escherichia coli (Gram-negative bacteria), Streptococcus mutans (Gram-positive bacterium) and Candida albicans (fungus) were grown in a polydimethylsiloxane (PDMS)-based nanoculture system. The selective permeability of the nanocultures to small molecules including antibiotics, signaling molecules and fluorescent dyes allows assessing essential cell functionalities (e.g. growth kinetics, cell vitality, pH change), as well as characterize inter-species or inter-kingdom interactions, and microbial dynamics in response to antibiotic attacks. The multifunctional nanoculture system has broad potential for studying microbiome dynamics, and identifying new bioactive molecules such as antimicrobials from these defined microcosms
Electrochemical Control of Bacterial Persister Cells
The emergence of antibiotic-resistant bacteria has presented an increasing challenge to infection control. Conventional methods of antibacterial treatment involving high dose of antibiotics or surgical intervention have proven insufficient for eradicating persistent infections, such as those associated with medical implants. It is well recognized that bacterial populations commonly contain a small percentage of phenotypic variants, known as persister cells, which are metabolically inactive and extremely tolerant to antibiotics. When the antibiotic treatment is stopped, surviving persister cells can regenerate the bacterial population with a similar percentage of persister cells. Thus, persistence presents a great challenge to curing chronic infections.
In this study, we introduced a novel method for controlling bacterial persistence based on a phenomenon we named electrochemical control of persister cells (ECCP). We demonstrate that bacterial persister cells can be effectively eliminated by low-level direct currents (DCs); e.g. treatment with 70 μA/cm2 DC for 1 h using stainless steel (SS) 304 reduced the number of viable planktonic persister cells of Pseudomonas aeruginosa PAO1 by 98% compared to the untreated control. In addition to persister killing by applying DC alone, synergistic effects were observed when treating persister cells with 70 μA/cm2 DC and 1.5 μg/mL tobramycin together using SS 304 electrodes. The same level of DC was also found to be cidal to biofilm associated persister cells of P. aeruginosa PAO1. Based on this discovery, the electrophysiological properties of P. aeruginosa PAO1 cells treated with 70 μA/cm2 DC using carbon and SS 304 electrodes were characterized both at the cellular and genetic levels to understand the mechanism of ECCP. We found that DC treatments affected surface charge and membrane integrity of P. aeruginosa, leading to increase intracellular concentration of metal cations as observed via scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) analysis. Moreover, electrochemical treatments mediated via carbon electrodes provoked the permeabilization of the cells to extracellular materials, and increased their susceptibility to antibiotics, which led to complete eradication of the persisters. These findings are corroborated by DNA microarray analysis, which revealed that DC treatments have profound effects on the physiology of persister cells, altering the regulation of genes involved in antibiotic resistance, pyocin-related functions, and SOS response. Comparative transmission electron microscopy (TEM) studies of the stationary phase P. aeruginosa PAO1 cells confirmed that DC treatments resulted in the compartmentalization of intracellular contents, release of outer membrane vesicles, or cell lysis.
To design novel systems to effectively control infections associated with biofilms and persister cells, the safety and the efficacy of ECCP were evaluated in co-culture models with human epithelial cells and P. aeruginosa PAO1. In addition, a pilot animal study was conducted to investigate the effects of electrochemical currents using a rabbit model of sinus infections. P. aeruginosa PAO1 was used to induce rhinosinusitis in rabbits, which were then treated with antibiotics, or antibiotics with electrical current, and compared with the untreated controls. The results of this study validated the effectiveness of electrochemical treatment in reducing both biofilms and planktonic cells in vivo. Overall, these findings improved the understanding of the electrophysiology of bacterial persister cells, and provided new insight for designing novel systems to effectively control infections associated with biofilms and persister cells
Topological defects at smectic interfaces as a potential tool for the biosensing of living microorganisms
Characterizing the anchoring properties of smectic liquid crystals (LCs) in
contact with bacterial solutions is crucial for developing biosensing
platforms. In this study, we investigate the anchoring properties of a smectic
LC when exposed to Bacillus Subtilis and Escherichia coli bacterial solutions
using interfaces with known anchoring properties. By monitoring the optical
response of the smectic film, we successfully distinguish different types of
bacteria, leveraging the distinct changes in the LC's response. Through a
comprehensive analysis of the interactions between bacterial proteins and the
smectic interface, we elucidate the potential underlying mechanisms responsible
for these optical changes. Additionally, we introduce the utilization of
topological defects; the focal conic domains (FCDs), at the smectic interface
as an indicative measure of the bacterial concentration. Our findings
demonstrate the significant potential of smectic LCs and their defects for
biosensing applications and contribute to our understanding of bacteria- LC
interactions, paving the way for advancements in pathogen detection and
protein-based sensing
Differential Gene Expression to Investigate the Effects of Low-level Electrochemical Currents on Bacillus subtilis
With the emergence and spread of multidrug resistant bacteria, effective methods to eliminate both planktonic bacteria and those embedded in surface-attached biofilms are needed. Electric currents at μA-mA/cm2 range are known to reduce the viability of bacteria. However, the mechanism of such effects is still not well understood. In this study, Bacillus subtilis was used as the model Gram-positive species to systematically investigate the effects of electrochemical currents on bacteria including the morphology, viability, and gene expression of planktonic cells, and viability of biofilm cells. The data suggest that weak electrochemical currents can effectively eliminate B. subtilis both as planktonic cells and in biofilms. DNA microarray results indicate that the genes associated with oxidative stress response, nutrient starvation, and membrane functions were induced by electrochemical currents. These findings suggest that ions and oxidative species generated by electrochemical reactions might be important for the killing effects of these currents
Microbial Nanoculture as an Artificial Microniche
Microbes self-organize in microcolonies while transitioning to a sessile form within a protective biofilm matrix. To enable the detailed study of microbial dynamics within these microcolonies, new sessile culture systems are needed that sequester cells and mimic their complex growth conditions and interactions. We present a new nanoliter-scale sessile culture system that is easily implemented via microfluidics-enabled fabrication. Hundreds of thousands of these nanocultures can be easily generated and imaged using conventional or confocal microscopy. Each nanoculture begins as a several nanoliter droplet of suspended cells, encapsulated by a polydimethylsiloxane (PDMS) membrane. The PDMS shell provides long-lasting mechanical support, enabling long term study, and is selectively permeable to small molecules including antibiotics, signaling molecules and functional fluorescent probes. Thus, as microcolonies mature within the nanocultures, they can be stressed or interrogated using selected probes to characterize cell physiological properties, antibiotic susceptibilities, and antagonistic interactions. We demonstrate this platform by investigating broad ranges of microcolony dynamics, including direct and indirect bacterial-fungal interactions. This versatile new tool has broad potential for addressing biological questions associated with drug resistance, chronic infections, microbiome dynamics, and antibiotic discovery
\u3cem\u3eCandida Albicans\u3c/em\u3e Stimulates \u3cem\u3eStreptococcus Mutans\u3c/em\u3e Microcolony Development via Cross-Kingdom Biofilm-Derived Metabolites
Candida albicans is frequently detected with heavy infection of Streptococcus mutans in plaque-biofilms from children affected with early-childhood caries, a prevalent and costly oral disease. The presence of C. albicans enhances S. mutans growth within biofilms, yet the chemical interactions associated with bacterial accumulation remain unclear. Thus, this study was conducted to investigate how microbial products from this cross-kingdom association modulate S. mutans build-up in biofilms. Our data revealed that bacterial-fungal derived conditioned medium (BF-CM) significantly increased the growth of S. mutans and altered biofilm 3D-architecture in a dose-dependent manner, resulting in enlarged and densely packed bacterial cell-clusters (microcolonies). Intriguingly, BF-CM induced S. mutans gtfBC expression (responsible for Gtf exoenzymes production), enhancing Gtf activity essential for microcolony development. Using a recently developed nanoculture system, the data demonstrated simultaneous microcolony growth and gtfB activation in situ by BF-CM. Further metabolites/chromatographic analyses of BF-CM revealed elevated amounts of formate and the presence of Candida-derived farnesol, which is commonly known to exhibit antibacterial activity. Unexpectedly, at the levels detected (25–50 μM), farnesol enhanced S. mutans-biofilm cell growth, microcolony development, and Gtf activity akin to BF-CM bioactivity. Altogether, the data provide new insights on how extracellular microbial products from cross-kingdom interactions stimulate the accumulation of a bacterial pathogen within biofilms
One-Step Generation of Cell-Encapsulating Compartments via Polyelectrolyte Complexation in an Aqueous Two Phase System
Diverse
fields including drug and gene delivery and live cell encapsulation
require biologically compatible encapsulation systems. One widely
adopted means of forming capsules exploits cargo-filled microdroplets
in an external, immiscible liquid phase that are encapsulated by a
membrane that forms by trapping of molecules or particles at the drop
surface, facilitated by the interfacial tension. To eliminate the
potentially deleterious oil phase often present in such processes,
we exploit the aqueous two phase system of poly(ethylene glycol) (PEG)
and dextran. We form capsules by placing dextran-rich microdroplets
in an external PEG-rich phase. Strong polyelectrolytes present in
either phase form complexes at the drop interface, thereby forming
a membrane encapsulating the fluid interior. This process requires
considerable finesse as both polyelectrolytes are soluble in either
the drop or external phase, and the extremely low interfacial tension
is too weak to provide a strong adsorption site for these molecules.
The key to obtaining microcapsules is to tune the relative fluxes
of the two polyelectrolytes so that they meet and complex at the interface.
We identify conditions for which complexation can occur inside or
outside of the drop phase, resulting in microparticles or poor encapsulation,
respectively, or when properly balanced, at the interface, resulting
in microcapsules. The resulting microcapsules respond to the stimuli
of added salts or changes in osmotic pressure, allowing perturbation
of capsule permeability or triggered release of capsule contents.
We demonstrate that living cells can be sequestered and interrogated
by encapsulating <i>Pseudomonas aeruginosa</i> PAO1 and
using a Live/Dead assay to assess their viability. This method paves
the way to the formation of a broad variety of versatile functional
membranes around all aqueous capsules; by tuning the fluxes of complexing
species to interact at the interface, membranes comprising other complexing
functional moieties can be formed
Design of a Well-Defined Poly(Dimethylsiloxane)-Based Microbial Nanoculture System
Organosilanes contain hydrocarbon-like backbones, allowing them to react with silicone-based agents in the presence of a catalyst and polymerize into membranes with tunable transport and mechanical properties. Owing to their high hydrophobicity, Poly(dimethylsiloxane) (PDMS) membranes, and more particularly, Sylgard® 184, have been used for applications including drug delivery, gas separation, and microfluidics fabrication. However, the undefined composition of the material and its ability to leach out uncured oligomers make its functionalization and usage challenging for many biological applications. This article presents the design of a novel culture system generated using PDMS-based membranes to study microbial dynamics. The microbial culture system that is referred to as “nanoculture” serves to encapsulate and grow microbes in semipermeable membranes. The mechanical properties of the membranes are reinforced through osmotic annealing, which enable the nanocultures to withstand high shear stress similar to environmental conditions while maintaining transport properties essential to microbial communication and growth. The present study lays the foundation for a novel microbial culture system that would enable the cultivation of microorganisms in environments other than laboratory conditions.</p