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.

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 Pseudomonas aeruginosa 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.

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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.

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Recently, we reported synergistic effects between 70 μA/cm(2) direct current and tobramycin in killing Pseudomonas aeruginosa PAO1 persister cells, a phenomenon we named electrochemical control of persister cells (ECCP; Niepa et al. Biomaterials 33: 7356-7365, 2012). To understand the mechanism of ECCP, the effects of electrochemical treatments mediated via stainless steel 304 and carbon electrodes on P. aeruginosa PAO1 were systematically compared using complementary approaches in this study. Electron microscopic analysis revealed that μA/cm(2) level direct current (DC) caused substantial changes in the structure and membrane integrity of P. aeruginosa PAO1 cells. DC treatments using SS304 electrodes induced cell lysis, while the same level of DC generated using carbon electrodes led to aggregation of intracellular proteins and increased permeabilization of P. aeruginosa PAO1 cells to antibiotics. The profound effects of DC on the physiology of persister cells were corroborated with DNA microarray analysis, which revealed the induction of genes associated with pyocin production and SOS response in DC-treated persister cells. Interestingly, sequential treatment using DC mediated with carbon electrodes followed by tobramycin was found more effective than concurrent treatment; and total eradication of persister cells was achieved.

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Bacteria at fluid interfaces endure physical and chemical stresses unique to these highly asymmetric environments. The responses of Pseudomonas aeruginosa PAO1 and PA14 to a hexadecane-water interface are compared. PAO1 cells form elastic films of bacteria, excreted polysaccharides and proteins, whereas PA14 cells move actively without forming an elastic film. Studies of PAO1 mutants show that, unlike solid-supported biofilms, elastic interfacial film formation occurs in the absence of flagella, pili, or certain polysaccharides. Highly induced genes identified in transcriptional profiling include those for putative enzymes and a carbohydrate metabolism enzyme, alkB2; this latter gene is not upregulated in PA14 cells. Notably, PAO1 mutants lacking the alkB2 gene fail to form an elastic layer. Rather, they form an active film like that formed by PA14. These findings demonstrate that genetic expression is altered by interfacial confinement, and suggest that the ability to metabolize alkanes may play a role in elastic film formation at oil-water interfaces.