Experimental evolution of Pseudomonas aeruginosa to colistin in spatially confined microdroplets identifies evolutionary trajectories consistent with adaptation in microaerobic lung environments.

Antibiotic resistance is a continuing global health crisis. Identifying the evolutionary trajectories leading to increased antimicrobial resistance can be critical to the discovery of biomarkers for clinical diagnostics and new targets for drug discovery. While the combination of patient data and in vitro experimental evolution has been remarkably successful in extending our understanding of antimicrobial resistance, it can be difficult for in vitro methods to recapitulate the spatial structure and consequent microenvironments that characterize in vivo infection. Notably, in cystic fibrosis (CF) patients, changes to either the PmrA/PmrB or PhoP/PhoQ two-component systems have been identified as critical drivers for high levels of colistin and polymyxin resistance. When using microfluidic emulsions to provide spatially structured, low-competition environments, we found that adaptive mutations to phoQ were more successful than pmrB in increasing colistin resistance. Conversely, mutations to pmrB were readily identified using well-mixed unstructured cultures. We found that oxygen concentration gradients within the microdroplet emulsions favored adaptive changes to the PhoP/PhoQ pathway consistent with microaerobic conditions that can be found in the lungs of CF patients. We also observed mutations linked to hallmark adaptations to the CF lung environment, such as loss of motility and loss of O antigen biosynthesis (wbpL). Mutation to wbpL, in addition to causing loss of O antigen, was additionally shown to confer moderately increased colistin resistance. Taken together, our data suggest that distinct evolutionary trajectories to colistin resistance may be shaped by the microaerobic partitioning and spatial separation imposed within the CF lung.IMPORTANCEAntibiotic resistance remains one of the great challenges confronting public health in the world today. Individuals with compromised immune systems or underlying health conditions are often at an increased for bacterial infections. Patients with cystic fibrosis (CF) produce thick mucus that clogs airways and provides a very favorable environment for infection by bacteria that further decrease lung function and, ultimately, mortality. CF patients are often infected by bacteria such as Pseudomonas aeruginosa early in life and experience a series of chronic infections that, over time, become increasingly difficult to treat due to increased antibiotic resistance. Colistin is a major antibiotic used to treat CF patients. Clinical and laboratory studies have identified PmrA/PmrB and PhoP/PhoQ as responsible for increased resistance to colistin. Both have been identified in CF patient lungs, but why, in some cases, is it one and not the other? In this study, we show that distinct evolutionary trajectories to colistin resistance may be favored by the microaerobic partitioning found within the damaged CF lung.

Methyl halide transferase-based gas reporters for quantification of filamentous bacteria in microdroplet emulsions.

The application of microfluidic techniques in experimental and environmental studies is a rapidly emerging field. Water-in-oil microdroplets can serve readily as controllable micro-vessels for studies that require spatial structure. In many applications, it is useful to monitor cell growth without breaking or disrupting the microdroplets. To this end, optical reporters based on color, fluorescence, or luminescence have been developed. However, optical reporters suffer from limitations when used in microdroplets such as inaccurate readings due to strong background interference or limited sensitivity during early growth stages. In addition, optical detection is typically not amenable to filamentous or biofilm-producing organisms that have significant nonlinear changes in opacity and light scattering during growth. To overcome such limitations, we show that volatile methyl halide gases produced by reporter cells expressing a methyl halide transferase (MHT) can serve as an alternative nonoptical detection approach suitable for microdroplets. In this study, an MHT-labeled Streptomyces venezuelae reporter strain was constructed and characterized. Protocols were established for the encapsulation and incubation of S. venezuelae in microdroplets. We observed the complete life cycle for S. venezuelae including the vegetative expansion of mycelia, mycelial fragmentation, and late-stage sporulation. Methyl bromide (MeBr) production was detected by gas chromatography-mass spectrometry (GC-MS) from S. venezuelae gas reporters incubated in either liquid suspension or microdroplets and used to quantitatively estimate bacterial density. Overall, using MeBr production as a means of quantifying bacterial growth provided a 100- to 1,000-fold increase in sensitivity over optical or fluorescence measurements of a comparable reporter strain expressing fluorescent proteins. IMPORTANCE Quantitative measurement of bacterial growth in microdroplets in situ is desirable but challenging. Current optical reporter systems suffer from limitations when applied to filamentous or biofilm-producing organisms. In this study, we demonstrate that volatile methyl halide gas production can serve as a quantitative nonoptical growth assay for filamentous bacteria encapsulated in microdroplets. We constructed an S. venezuelae gas reporter strain and observed a complete life cycle for encapsulated S. venezuelae in microdroplets, establishing microdroplets as an alternative growth environment for Streptomyces spp. that can provide spatial structure. We detected MeBr production from both liquid suspension and microdroplets with a 100- to 1,000-fold increase in signal-to-noise ratio compared to optical assays. Importantly, we could reliably detect bacteria with densities down to 106 CFU/mL. The combination of quantitative gas reporting and microdroplet systems provides a valuable approach to studying fastidious organisms that require spatial structure such as those found typically in soils.

Microfluidic platform for spatially segregated experimental evolution studies with E. coli.

Microdroplet emulsions allow investigators to build controllable microenvironments for applications in experimental evolution and synthetic ecology. We designed a microfluidic platform that uses highly homogenous microdroplets to enable these experiments. We also present a step-by-step protocol for the rapid production of highly homogeneous microdroplets suitable for experimental evolution. We also describe protocols for the propagation and serial passage of microbial populations across a range of selection schemes and potential spatial structures. For complete details on the use and execution of this protocol, please refer to Seo et al. (2021).

Identification of Evolutionary Trajectories Associated with Antimicrobial Resistance Using Microfluidics.

In vitro experimental evolution of pathogens to antibiotics is commonly used for the identification of clinical biomarkers associated with antibiotic resistance. Microdroplet emulsions allow exquisite control of spatial structure, species complexity, and selection microenvironments for such studies. We investigated the use of monodisperse microdroplets in experimental evolution. Using Escherichia coli adaptation to doxycycline, we examined how changes in environmental conditions such as droplet size, starting lambda value, selection strength, and incubation method affected evolutionary outcomes. We also examined the extent to which emulsions could reveal potentially new evolutionary trajectories and dynamics associated with antimicrobial resistance. Interestingly, we identified both expected and unexpected evolutionary trajectories including large-scale chromosomal rearrangements and amplification that were not observed in suspension culture methods. As microdroplet emulsions are well-suited for automation and provide exceptional control of conditions, they can provide a high-throughput approach for biomarker identification as well as preclinical evaluation of lead compounds.

Polymer-Salt Aqueous Two-Phase System (ATPS) Micro-Droplets for Cell Encapsulation.

Biosample encapsulation is a critical step in a wide range of biomedical and bioengineering applications. Aqueous two-phase system (ATPS) droplets have been recently introduced and showed a great promise to the biological separation and encapsulation due to their excellent biocompatibility. This study shows for the first time the passive generation of salt-based ATPS microdroplets and their biocompatibility test. We used two ATPS including polymer/polymer (polyethylene glycol (PEG)/dextran (DEX)) and polymer/salt (PEG/Magnesium sulfate) for droplet generation in a flow-focusing geometry. Droplet morphologies and monodispersity in both systems are studied. The PEG/salt system showed an excellent capability of uniform droplet formation with a wide range of sizes (20-60 µm) which makes it a suitable candidate for encapsulation of biological samples. Therefore, we examined the potential application of the PEG/salt system for encapsulating human umbilical vein endothelial cells (HUVECs). A cell viability test was conducted on MgSO4 solutions at various concentrations and our results showed an adequate cell survival. The findings of this research suggest that the polymer/salt ATPS could be a biocompatible all-aqueous platform for cell encapsulation.

High inertial microfluidics for droplet generation in a flow-focusing geometry.

A numerical study of aqueous droplet generation in a high Reynolds (Re) number air flow was performed in a microfluidic flow-focusing geometry. Droplet breakup mechanisms, flow regime mapping, droplet morphology, and droplet generation frequency were studied in a high initial air flow under various flow conditions. Several flow regimes were identified including dripping, unstable dripping, plugging, stratified flow, multi-satellite droplet formation, and unstable jetting. Unstable dripping, multi-satellite droplet formation, and unstable jetting have been observed as new flow regimes in our study. We found that the high inertial air flow remarkably induces the formation of these new flow regimes by retaining unique droplet generation mechanisms and morphology. In particular, the polydisperse spray of tiny droplets is formed at the junction in the multi-satellite droplet formation regime, while at the end of a jet in the unstable jetting regime. On the other hand, stable droplet generation occurs in the dripping and plugging regimes, while generated droplets in the unstable dripping, unstable jetting, and multi-satellite droplet formation regimes are unstable. The maximum generation frequency of ~ 1900 Hz was obtained under the unstable dripping regime. It was found that increasing Re number results in droplet size reduction, while higher capillary (Ca) number leads to bigger droplets.

Catalytic activity of nickel nanoparticles stabilized by adsorbing polymers for enhanced carbon sequestration.

This work shows the potential of nickel (Ni) nanoparticles (NPs) stabilized by polymers for accelerating carbon dioxide (CO2) dissolution into saline aquifers. The catalytic characteristics of Ni NPs were investigated by monitoring changes in diameter of CO2 microbubbles. An increase in ionic strength considerably reduces an electrostatic repulsive force in pristine Ni NPs, thereby decreasing their catalytic potential. This study shows how cationic dextran (DEX), nonionic poly(vinyl pyrrolidone) (PVP), and anionic carboxy methylcellulose (CMC) polymers, the dispersive behaviors of Ni NPs can be used to overcome the negative impact of salinity on CO2 dissolution. The cationic polymer, DEX was less adsorbed onto NPs surfaces, thereby limiting the Ni NPs' catalytic activity. This behavior is due to a competition for Ni NPs' surface sites between the cation and DEX under high salinity. On the other hand, the non/anionic polymers, PVP and CMC could be relatively easily adsorbed onto anchoring sites of Ni NPs by the monovalent cation, Na+. Considerable dispersion of Ni NPs by an optimal concentration of the anionic polymers improved their catalytic capabilities even under unfavorable conditions for CO2 dissolution. This study has implications for enhancing geologic sequestration into deep saline aquifers for the purposes of mitigating atmospheric CO2 levels.

High-Throughput Aqueous Two-Phase System Droplet Generation by Oil-Free Passive Microfluidics.

Aqueous two-phase system (ATPS) droplet generation has significant potential in biological and medical applications because of its excellent biocompatibility. However, the ultralow interfacial tension of ATPS makes droplet generation extremely challenging when compared with the conventional water-in-oil (W/O) system. In this paper, we passively produced ATPS droplets with a wide range of droplet size and high production rate without the involvement of an oil phase and external forces. For the first time, we reported important information of the flow rate and capillary (Ca) number for passive, oil-free ATPS droplet generation. It was found that the range of Ca numbers of the continuous phase under the jetting flow regime is 0.3-1.7, as compared to less than 0.1 in the W/O system, indicating the ultralow interfacial tension in ATPS. In addition, we successfully generated ATPS droplets with a radius as small as 7 µm at the maximum frequency up to 300 Hz, which has not been achieved in previous studies. The size and generation frequency of ATPS droplets can be controlled independently by adjusting the inlet pressures and corresponding flow rates. We found that the droplet size is correlated with the pressure and flow rate ratios with the power-law exponents of 0.8 and 0.2, respectively.

Microbubbles Loaded with Nickel Nanoparticles: A Perspective for Carbon Sequestration.

This work reports a microfluidic study investigating the feasibility of accelerating gaseous carbon dioxide (CO2) dissolution into a continuous aqueous phase with the use of metallic nickel (Ni) nanoparticles (NPs) under conditions specific to carbon sequestration in saline aquifers. The dissolution of CO2 bubbles at different pH levels and salinities was studied to understand the effects that the intrinsic characteristics of brine in real reservoir conditions would have on CO2 solubility. Results showed that an increased shrinkage of CO2 bubbles occurred with higher basicity, while an increased expansion of CO2 bubbles was observed with a proportional increase in salinity. To achieve acceleration of CO2 dissolution in acidic brine containing high salinity content, the catalytic effect of Ni NPs was investigated by monitoring change in CO2 bubble size at various Ni NPs concentrations. The optimal concentration for the Ni NPs suspension was determined to be 30 mg L-1; increasing the concentration up to 30 mg L-1 showed a significant increase in the dissolution of CO2 bubbles, but increasing from 30 to 50 mg L-1 displayed a decrease in catalytic potential, due to the decreased translational diffusion coefficient that occurs at higher concentrations. The optimal additive concentration of Ni NPs was tested with variations of solution at acidic and basic conditions and different levels of salinity to reveal how effectively the Ni NPs behave under real reservoir conditions. At the acidic level, Ni NPs proved to be more effective in catalyzing CO2 dissolution and can sufficiently alleviate the negative impact of salinity in brine.

Effects of titanium dioxide nanoparticles derived from consumer products on the marine diatom Thalassiosira pseudonana.

Increased manufacture of TiO2 nanoproducts has caused concern about the potential toxicity of these products to the environment and in public health. Identification and confirmation of the presence of TiO2 nanoparticles derived from consumer products as opposed to industrial TiO2 NPs warrant examination in exploring the significance of their release and resultant impacts on the environment. To this end, we examined the significance of the release of these particles and their toxic effect on the marine diatom algae Thalassiosira pseudonana. Our results indicate that nano-TiO2 sunscreen and toothpaste exhibit more toxicity in comparison to industrial TiO2 and inhibited the growth of the marine diatom T. pseudonana. This inhibition was proportional to the exposure time and concentrations of nano-TiO2. Our findings indicate a significant effect, and therefore, further research is warranted in evaluation and assessment of the toxicity of modified nano-TiO2 derived from consumer products and their physicochemical properties.

Influence of siloxane on the transport of ZnO nanoparticles from different release pathways in saturated sand.

The production of nanomaterials (NMs) is expected to grow continuously, yet their transformation, transport, release mechanisms, and interactions with contaminants under environmental conditions remain poorly understood. Few studies have investigated the effects of contaminants on fate and transport of NMs, especially siloxanes that are widely found in products. It is hypothesized that the model contaminant, siloxane (e.g., 1,1,3,3-tetramethyldisiloxane (TMDS)) may influence the mechanisms and transport kinetics of NMs under different release pathways. Sand column experiments were carried out under two different scenarios: the release from a mixed TMDS and nano-ZnO suspension (A) and the release of nano-ZnO from sand contaminated with TMDS (B). Results show that interparticle reactions are dominant in (A) and particle-porous interactions are responsible for blocking effects governing in (B). Insights, especially the kinetics of nano-ZnO from co-transport by a contaminant and from porous media preloaded with a contaminant, and environmental factors affecting the release and retention of nano-ZnO in saturated sand are unveiled. These two dominant transport mechanisms (e.g., interparticle reactions and blocking effects) were derived. This study indicates that the release of ZnO NPs is influenced by the presence of TMDS; the extent of mobility and their transport pathways depend on the pre-existence of TMDS in porous media.

The enhancement of ammonium removal from ethanolamine wastewater using air-cathode microbial fuel cells coupled to ferric reduction.

A microbial fuel cell (MFC) with biological Fe(III) reduction was implemented for simultaneous ethanolamine (ETA) degradation and electrical energy generation. In the feasibility experiment using acetate as a substrate in a single-chamber MFC with goethite and ammonium at a ratio of 3.0(mol/mol), up to 96.1% of the ammonium was removed through the novel process related to Fe(III). In addition, the highest voltage output (0.53V) and maximum power density (0.49Wm(-2)) were obtained. However, the ammonium removal and electrical performance decreased as acetate was replaced with ETA. In the long-term experiment, the electrical performance markedly decreased where the voltage loss increased due to Fe deposition on the membranes.