Patent Application
20190291052
The present disclosure relates to bacteria removal. More particularly, it relates to deployable microtraps to sequester motile bacteria.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the description of example embodiments, serve to explain the principles and implementations of the disclosure.
In a first aspect of the disclosure, a microtrap is described, the microtrap: an outer surface separating an inner volume of the microtrap from an outer volume; and at least one opening allowing entry of motile bacteria from the outer volume into the inner volume.
The development of strategies to reduce the load of unwanted bacteria is a fundamental challenge in industrial processing, environmental sciences and medical applications. The present disclosure describes methods to sequester motile bacteria from a liquid, based on passive, deployable micro-traps that confine bacteria. The microtraps use micro-funnels that open into trapping chambers. Even in low concentrations, micro-traps afford a 70% reduction in the amount of bacteria in a liquid sample, with a potential to reach a reduction greater than 90%, as shown by modelling improved geometries. The present disclosure describes a new approach to contain the growth of bacteria without chemical means, an advantage of particular importance given the alarming growth of pan-drug-resistant bacteria.
Existing approaches to restrict the presence of bacteria in an environment are known to have important limitations. For example, antibacterial agents, or antibiotics, interfere with bacteria's biochemical or genetic processes, but their excessive use poses serious concerns related to the emergence of pan-drug resistant strains. Other methods to limit bacterial growth are based on the application of high temperatures, or high energy irradiation. For example, pasteurization is an effective, widespread method, but creates undesired free radicals and thermolytic byproducts. Gamma ray irradiation treatments require expensive equipment and a source of radiation. Microfiltration and sonication require considerable external energy. As these approaches are often invasive and expensive, the development of alternative or complementary strategies to reduce bacterial loads will be extremely beneficial in a broad field of applications.
The present disclosure describes how to exploit the bacteria's dynamic behavior to control the load of bacteria in liquid environments, by leveraging their motility and interaction with surfaces to trap them into microscale engineered particles. Key human pathogens, such as Salmonella enterica, Helicobacter pylori, Vibrio cholerae, Vibrio vulnificus, Pseudomonas aeruginosa, and virulent strains of Escherichia coli are motile: they use their flagella to reach specific niches in the host. For example, Salmonella—which is found in contaminated food and water and represents the causative agent of gastroenteritis—swims towards and adheres to gastro-intestinal villi before entering the intestinal cells. Enterobacteriaceae in general, live in close association with surfaces. These surfaces can generate attractive hydrodynamic forces on swimming bacteria (swimmers), resulting in their accumulation in the proximity of boundaries. The attractive forces between bacteria and surfaces provide specific tropism to the target site on the intestinal cell's surface, thus permitting the pre-docking phase at the onset of infection.
Understanding of how bacteria move and interact with surfaces has, over the last decade, been significantly furthered by the advances in microfabrication techniques. For example, the effect of surfaces in redirecting bacteria has been exploited in the design of two-dimensional (2D) funnel walls. These structures favor bacteria crossing funnels in one direction over the other, based on the surface interaction of individual swimmers. The funnels have been used to rectify the random motility of bacteria and thereby concentrate them. However, funnel walls have only been realized on surfaces, and have therefore not been deployed in liquid samples to date. The present disclosure leverages recent advances in the resolution of three-dimensional (3D) printing to develop three-dimensional micro-traps that rectify the motility of bacteria, and trap them into deployable, microscale particles.
Funnel apertures connect inner chambers of the micro-traps with the outside liquid.
The surface-attached micro-traps were tested by imaging the accumulation of bacteria within the traps over time, for four different geometries as illustrated in
Experiments were conducted with two species of bacteria: the enteric bacterium Escherichia coli, which represents the classic model for bacterial motility, and the marine pathogen Vibrio coralhilyticus, which swims rapidly with a strategy that significantly differs from E. coli's. The concentration of bacteria inside the micro-traps was quantified by image analysis and compared with the concentration of bacteria in the external suspension, determined with the same approach. In all micro-traps, an accumulation of bacteria within the structure was detected, for both E. coli and V. coralhilyticus, demonstrating the ability of the micro-traps to trap swimming bacteria.
The highest average accumulation (4-fold higher concentration of bacteria within the micro-traps than outside) was observed in the 3-layer micro-traps. Furthermore, the accumulation increased with the number of layers in the micro-trap: the 3-layer traps had approximately double the accumulation compared to the 1-layer box, demonstrating that the multi-layer design is effective in enhancing trapping. The increase in accumulation with increasing number of layers is also in agreement with earlier experiments with 2D arrays of funnels, described by Galajda, P., Keymer, J., Chaikin, P. & Austin, R., A wall of funnels concentrates swimming bacteria, J. Bacteriol. 189, 8704-7 (2007), the disclosure of which is incorporated herein by reference in its entirety.
The trapping mechanism is based on the interaction of motile bacteria with 3D funnel-like apertures, and relies on rectification and confinement. Upon approaching the micro-traps by random motility, bacteria preferentially swim along the surface of the funnels and—due to the funnel's shape—are directed through the funnel's aperture and into the inner chamber. The asymmetric shape of the funnels makes it less likelihood for a bacterium to swim out through the funnel in the reverse direction, resulting in an accumulation of cells inside the micro-trap. This effect is enhanced by the presence of multiple layers of funnels, which guide bacteria further into the interior of the micro-traps, and decreases the outward flux of bacteria. The funnels, therefore, establish an asymmetry in the random motion of bacteria, resulting in a preferential overall flow from outside the microtrap to within the microtrap.
The influence of micro-trap geometry on trapping efficiency was further investigated through a mathematical model. The model simulated 105 bacterial trajectories for each geometry, and quantified the percentage of trapped bacteria at its steady state. It was found that funnel-like apertures accumulate 35% more bacteria than cylindrical apertures. Cylindrical apertures that form an acute angle with the micro-traps' internal walls, also trap bacteria due to their asymmetry toward the inside of the micro-traps. The predicted accumulation increased up to approximately 3-fold for the 3-layer micro-trap. These results show a good comparison with the experimental observations, and confirm that the mechanism of bacterial accumulation in the micro-traps is the rectification of bacterial motility due to surface interactions, a fundamental process likely applicable to all motile bacteria. Therefore, the microtraps mechanism of sequestering bacteria will be applicable to a wide range of microbial swimmers.
As further testing, micro-traps in a bacterial suspension to determine their ability to lower the bacterial load. The deployable micro-traps were designed by stacking a single dome on top of a 1-layer box, and mirroring this stack to obtain an egg-shaped particle. Thus the deployable structures are a combination of the building blocks tested in
It can be seen from
The depletion efficiency can be affected by both funnel geometry and number of micro-traps. Comparing the bacterial depletion (measured after 180 min and normalized by the no-trap control) effected by 300 micro-traps with either asymmetric cylindrical apertures or funnel-like apertures, showed that the latter were 22% more effective in depleting bacteria from the solution. This result indicates that funnel geometry is an important design factor that can be optimized, possibly in a species-specific manner, to achieve highest trapping efficiency in different applications. Doubling the number of micro-traps, from 300 to 600, resulted in a 15% increase in the bacterial depletion after 180 min, from 60% to 75%. This finding is in line with the theoretical limit obtained considering micro-traps acting independently: given that 300 micro-traps captured 60% of the bacteria, the additional 300 micro-traps were expected to capture 60% of the remaining 40% of bacteria, i.e. an additional 24% (still independent of the absolute number of bacteria).
The accumulation of bacteria cmt inside the micro-traps was also estimated imposing the concentration of bacteria in the absence of micro-traps (control experiments) to be 1 everywhere in the volume. When introducing the micro-traps, the concentration inside them increases. Consequently, the concentration in the outside medium will be less than 1. It is possible to describe the higher concentration in the micro-traps as cmt=Vmt−1(1−cs)+cs, where Vmt is the internal volume fraction of the micro-traps with respect to the total volume and cs is the concentration of bacteria in the suspension outside the micro-traps. This calculation revealed an 18-fold accumulation of bacteria (at time 180 min) within the micro-traps, with respect to the concentration in the suspension. This value is larger than that measured in surface-immobilized micro-traps, probably due to the free motion of the deployed micro-traps in the suspension.
Two dimensional simulations of the deployable micro-traps were carried out to assess the effect of geometry on the depletion efficiency. In agreement with experiments, simulations showed that (i) the accumulation of bacteria in the micro-traps increases with time up to a plateau, and (ii) the accumulation was ˜22% higher for micro-traps with funnel apertures compared to cylindrical apertures. Absolute concentrations of trapped bacteria were higher in simulations than in experiments, likely due to the 2D nature of the simulations.
Simulations allowed to predict the effect of future increases in 3D printing resolution, which will permit further increases in the geometrical parameter most influential for trapping efficiency: the number of layers of the micro-traps. The plateau depletion values obtained from simulations have been normalized by the experimental depletion values of the cylindrical aperture micro-traps. The bacterial accumulation in micro-traps was compared with one, three and five internal layers and different aperture geometries. It was found that the depletion of bacteria in the solution increases with the number of layers, from 60% for 1 layer to 75% for 3 layers and 95% for 5 layers. The reason is that, with more layers, bacteria are ‘stashed away’ further into the micro-trap and the flux of bacteria out of the micro-trap by random motility decreases. While the precise numbers will be different in 3D compared to 2D, it can be expected that the increase of the number of layers will also contribute very significantly to the efficiency of 3D micro-traps, and that the systematic optimization of the traps' geometry will lead to yet more effective and faster accumulation. As the resolution of 3D printing improves, so will the possibilities for engineered microstructures that interact with microorganisms in controllable and potentially beneficial ways.
The present disclosure describes how deployable micro-traps can be fabricated in high-throughput and can considerably reduce the load of bacteria from a liquid suspension within tens of minutes. This approach uses a completely passive mechanism that does not require heating, chemical additions or large amounts of energy. The intrinsic selection process favoring the trapping of the most motile (hence, often, most virulent) bacteria is a considerable advantage of this method. The design of these structures can be guided by the extensive recent research focused on understanding microbial swimming and the interaction between microorganisms and surfaces, enabling the optimization of deployable microstructures and making their design species- and application-specific. This approach, in combination with continuing improvements in 3D micro-manufacturing, can reduce the number of micro-traps required to achieve the desired reduction in bacterial load, by optimizing multiple elements of micro-trap design, including funnel geometry and number of layers.
Micro-traps can represent an appealing alternative to the use of pharmacological agents, such as antibiotics, whose extensive use has created a well-known red-queen effect by driving the emergence of resistant strains. Micro-traps can also be used in synergy with antibiotics. For example, micro-traps could be loaded with antibiotics at resulting concentrations much lower than usually given in bulk—as the killing action will be localized inside the particles—and noxious effects of the antibiotics on the host are avoided. In this approach, rather than dosing antibiotics homogeneously everywhere, bacteria would swim into antibiotic-laden traps. The antibiotic traps can be further made more effective as well as potentially species-specific, by augmenting the antibiotic with chemo-attractants. These loading approaches can require the use of low-diffusivity compounds, or compounds partly trapped into a solid or gel matrix, to avoid diffusion severely limiting the time scale of micro-trap operation. In other words, the compounds should be reasonably confined to within the trap, for if they were to diffuse quickly to outside the trap, their efficacy would drop.
After use, the micro-traps can be removed from the liquid using large-pore filters (e.g., pore size of ˜100 μm), a cheap and fast filtering procedure. Therefore, micro-traps can be deployed in the gut of animals and patients, for example, for sampling. In this manner, 3D micro-technologies may open the road to a new “pharmacology”, not based on chemistry, but on the possibility to interfere mechanically with the dynamic properties of pathogens and other cells.
In some embodiments, the microtraps can be fabricated on substrates, using a semitransparent, negative tone photoresist as the building material. The polymerized resist is biocompatible, has a low density and a Young's module of 5 GPa. In some embodiments, the deployable micro-traps were 150×220 μm in size and had funnels with 45 μm and 10 μm diameter apertures, over a total length of 25 To remove them from the substrate, 20 μL of distilled water was cast on top of the produced arrays, and the micro-traps were gently scratched with a sterile steel inoculation loop, allowing them to float. The micro-traps were then collected and freed of possible production residues by washing them in 0.2 mL tubes containing 100 μL of ultrapure water. The water-filled tubes with micro-traps were exposed to 50 mBar vacuum for 5 minutes and then spun for few seconds. The procedure was repeated until all micro-traps precipitated. The supernatant was collected, and the micro-traps were dehydrated under vacuum (50 mBar) for 1 h. The micro-traps were then sterilized by exposing them to UV light for 30 minutes.
For the experiments on bacterial accumulation with dome-shaped and multiple-layer box-shaped micro-traps, a polydimethylsiloxane (PDMS) gasket was built around the bottom coverslip with the micro-traps attached. We added 50 μL of a bacterial suspension, and placed the samples in the testing apparatus. Since the volume of the bacterial suspension was much larger than the inner volume of each micro-trap, it was possible to quantify the accumulation of bacteria inside individual micro-traps while neglecting the depletion of bacteria in the outer medium, i.e., the micro-traps are considered immersed in an infinite bacterial suspension. To measure the accumulation of bacteria inside the microtraps, the bacteria was counted in a volume corresponding to an area of 40×40 μm and a height of 10 to 40 μm. The obtained value was compared with the number of bacteria in the same volume outside the structures at the same height from the bottom coverslip.
For the experiments on bacterial depletion with deployable micro-traps, nine μL of LB medium were added to the tubes containing the micro-traps. To make sure that the medium had penetrated inside the micro-traps, a 50 mBar vacuum was applied again for 5 minutes. Control samples followed the same procedure. In each tube, 1 μL of E. coli was inoculated, grown in 5 mL of LB at 37° C. up to a density of ˜0.7 OD600. The tubes containing bacteria and micro-traps were mounted parallel to the surface on a vertically rotating wheel at ˜1.4 rpm at room temperature to avoid precipitation. At different time points (depending on the experiment), 2 μL of the supernatant from the respective samples were collected and the bacteria counted by optical microscopy in a micro-chamber slide. Five different areas of the micro-chamber for each sample were photographed and analyzed by software to determine the number of bacteria present. The counting method was validated with a separate experiment, where optical density (OD) readings were used as a reference. The correlation between the two measurement was linear with R2=0.93.
For the time course experiments, independent sets of micro-traps were used for each time point, and the corresponding control samples. Starting from a single culture of E. coli, bacteria were divided into 8 Eppendorf tubes, 4 containing Luria Broth (LB) medium, and 300 micro-traps each and 4 containing only LB medium. Each experiment was performed (together with its control) starting from an independent culture.
For the numerical simulations, a Langevin model was employed, previously validated also in the presence of flow, which captures the effect of the boundary on the swimming direction of bacteria close to the surface. To treat the interactions between bacteria and the surfaces of the micro-traps, when a bacterium arrives within 1 μm distance from a surface its incident angle was constrained to the surface to be 2.5 degrees, which was found to be the most stable angle for E. coli swimming near a surface. The swimming speed was set to 15 μm/s and the rotational diffusivity to 0.4 s−1, typical values for E. coli. The model was validated for the case of bacteria swimming between two parallel surfaces, by comparing the predicted accumulation of bacteria near the surfaces with the prior observations for E. coli.
Microtraps were tested with Vibrio coralliilyticus strain YB2 dsRed, grown in Marine Broth 2216, and Escherichia coli strains AW405 and JM109, grown in Luria Broth (LB) medium. The deployable micro-traps and boxes were fabricated using a commercial 3D direct-laser-lithography system. To carry out numerical modeling, the equations of motion were integrated numerically for 105 simulated bacteria using a fourth-order Runge-Kutta scheme.
In some embodiments, the microtrap is made of a material impermeable to the liquid in which the microtrap is immersed, and impenetrable by the motile bacteria. In some embodiments, the microtrap comprises stacked chambers, as visible in
The examples set forth above are provided to those of ordinary skill in the art as a complete disclosure and description of how to make and use the embodiments of the disclosure, and are not intended to limit the scope of what the inventor/inventors regard as their disclosure.
Modifications of the above-described modes for carrying out the methods and systems herein disclosed that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
It is to be understood that the disclosure is not limited to particular methods or systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
The present application claims priority to U.S. Provisional Patent Application No. 62/645,628, filed on Mar. 20, 2018, the disclosure of which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62645628 | Mar 2018 | US |
