Platform and method for testing antibiotic sensitivity of a polymicrobial infection

A platform and method for testing antibiotic sensitivity of a polymicrobial infection is provided. The platform includes a body defining a plurality of sets of chambers and a plurality of wells. Each set of chambers has a plurality of chambers adapted for culturing microbes of the polymicrobial infection therein. Each well is associated with a corresponding set of chambers and has an input in fluidic communication with the outlets of the plurality of chambers in the corresponding set of chambers. Selected antibiotic therapies may be received in the wells which fluidically connect the plurality of chambers in a corresponding set of chambers such that microbes cultured in the plurality of chambers in the corresponding set of chambers are in soluble factor contact.

FIELD OF THE INVENTION

This invention relates generally to antibiotic sensitivity testing, and in particular, to a platform and method for testing antibiotic sensitivity of a polymicrobial infection by culturing multiple bacterial strains in soluble-factor communication with each other.

BACKGROUND AND SUMMARY OF THE INVENTION

Early microbiology studies are primarily based on adaptations of a one microbe, one disease hypothesis, known as Koch's postulates, introduced in the late 1800s. Koch's postulates states that a disease causing microbe should have the following four criteria: 1) the microbe must be found in all organisms affected from the disease, but not in healthy organisms; 2) the microbe must be isolatable from the disease affected organism and also grown in pure culture; 3) the pure cultured microbe should be able to cause the same disease when introduced into a healthy organism; and 4) the microbe must be able to be isolated from the infected organism and be identical to the original isolated strain. More recently, however, researchers have realized that microbes do not exist as single entities, but as complex multispecies communities in the majority of environments ranging from soil to the gastrointestinal tract of animals. These multi-microbial species interactions play a significant role in human health and pathology. For instance, the human gut alone has an estimated 500 to 1000 different microbial species. Different members within the microbial community interact locally in ways that can affect the dynamics and stability of the population as a whole. The role of these interactions in human health and pathology has recently emerged, with various studies showing that microbial communities inhabiting the gut play a critical role in human pathologies like diabetes, obesity, Parkinson's, Alzheimer's, and even cancers.

Recent advances in both throughput and cost reduction in metagenomic sequencing technologies have enabled the study of microbiomes and their participating members with unprecedented throughput and offer insight into a more systems-level understanding of the identities of microbial community members. Despite progress in these areas, much remains to be understood regarding how the individual members of a community interact with their adjacent neighbors and the physiological functions that arise from these interactions within their microenvironment. Multi-variable combinatorial screening is a universal critical step in the development of combinatorial drug treatments, stem cell differentiation, studying cell-cell/microbe interactions, among many other applications involving multi-parameter experimental optimization.

Current standard technologies for studying microbial communities include 16S ribosomal RNA (rRNA) sequencing, fluorescence in situ hybridization (FISH), macroscale (mostly pairwise) co-cultures, and computer-based modeling. 16S rRNA sequencing can provide valuable information regarding which microbial species are present within a community, but cannot elucidate what interactions occur between which members of the community. FISH can provide information regarding the spatial distribution of different microbial species within a sample, but cannot infer functional readouts, is challenging to perform and is also limited to the number of fluorescent probes and wavelengths available. Pairwise co-cultures are generally performed in conventional laboratory culture vessels like multi-well plates, tubes, and solid agar plates. Although suitable for small scale interaction screens for a handful of communities, the process is ill suited for taking on the colossal task of screening and re-assembling the vast parameter space of interactions for even small numbers of microbial species. Additionally, macroscale liquid cultures are prone to disturbance by convective flow, causing rapid mixing and dilution of localized diffusible factors. As a result, valuable local interaction phenotypes could be lost. Computer based modeling is useful for gaining understanding of how competition and cooperation within a microbial community can maintain stability, survival, and biodiversity, such as “rock-paper-scissors” interaction dynamics, and fills some of the gaps in current experimental limitations in screening multi-species interactions. However, the results obtained from modeling biological systems is highly hypothetical and often not translatable to real-world conditions.

In view of the foregoing, it is evident that there exists a significant need for practical microscale co-culture tools with sufficient throughput to allow for large scale screening of microbial community interactions at the microscale. This is a critical piece of the puzzle to enable scientists to gain functional insight and understanding of how different community members interact and drive community behavior within the microenvironment. Development of microscale co-culture tools with sufficient throughput to allow for large scale screening of microbial community interactions at the microscale could lead to important insights into future clinical treatment strategies. For example, studies have shown that microbes in co-cultures can exhibit differing responses to antibiotic treatment compared to those in isolation, in which the microbial members in cohabitation can either antagonize or potentiate antibiotic susceptibility to the whole community. Hence, in the context of polymicrobial infection, interplay between two or more invading pathogens is likely clinically significant and unfortunately, unaccounted for with current methods.

Heretofore, patients found to have an infection undergo antibiotic sensitivity testing performed on their specific bacterial pathogens. This type of testing helps clinicians select antibiotic therapy in a patient-specific manner. Unfortunately, antibiotic sensitivity testing can be a poor predictor of patient outcome. For example, in the phase 3 clinical trial for cefotaxime, a “sensitive” test result, in the best case scenario, meant 93% of patients would respond. Importantly, 64% of patients who had a “resistant” test result also responded to the antibiotic therapy. Both sources of error are clinically significant. Clinicians want to treat patients with antibiotics that are effective and also provide good antimicrobial stewardship by avoiding jumping to backup/reserve antibiotics if other, more frequently used options, would also be effective.

There are a number of factors that contribute to the poor predictive value of state of the art antibiotic sensitivity tests, most of which come back to the fact that the assay is oversimplified; it is performed on pure bacterial isolates. It neglects the role of both the patient's immune system and the role other nearby pathogens may play. In the context of polymicrobial infection, interplay between two or more invading pathogens is likely clinically significant and unfortunately, unaccounted for with current methods.

Therefore, it is a primary object and feature of the present invention to provide a platform and method for testing antibiotic sensitivity of a polymicrobial infection by culturing multiple bacterial strains in soluble-factor communication with each other.

It is a further object and feature of the present invention to provide a platform and method for testing antibiotic sensitivity of a polymicrobial infection that provides a large number of combinations with high throughput within a small scale to allow for detection of diffusion-limited interaction events.

It is a still further object and feature of the present invention to provide a platform and method for testing antibiotic sensitivity of a polymicrobial infection that possesses a simple and high content data readout.

It is a still further object and feature of the present invention to provide a platform and method for testing antibiotic sensitivity of a polymicrobial infection that is scalable in terms of both number of different members in combination and cell number of each member.

It is a still further object and feature of the present invention to provide a platform and method for testing antibiotic sensitivity of a polymicrobial infection that is simple and straightforward to operate and that enables the sufficient long-term culture of specific target microbes.

In accordance with the present invention, a platform for testing antibiotic sensitivity of a polymicrobial infection is provided. The platform includes a body defining a plurality of chambers having outlets and a well having an input. Each chamber is adapted for receiving a corresponding microbe of the polymicrobial infection therein. The well is in fluidic communication with the outlets of the plurality of chambers and is well adapted for receiving a selected antibiotic therapy therein. The selected antibiotic therapy received in the well fluidically connects each of chambers such that microbes received in the plurality of chambers are in soluble factor contact.

The body further includes a permeable membrane disposed between the outlets of the plurality of chambers and the input of the well. Preferably, the permeable membrane is a porous polycarbonate membrane. The body also includes upper and lower surfaces. Each of the plurality of chambers has an opening communicating with the upper surface of the body. The well is partially defined by a closed surface within the body. The closed surface is generally parallel to the lower surface of the body. A well inlet extends between the closed surface of the well and the lower surface of the body. The well inlet allows for access to the well for loading the selected antibiotic therapy therein. An air outlet also extends between the closed surface of the well and the lower surface of the body. The air outlet allows for the purging of air from the well during the loading of the selected antibiotic therapy therein.

In accordance with a further aspect of the present invention, a platform for testing antibiotic sensitivity of a polymicrobial infection is provided. The platform includes a body defining a plurality of sets of chambers and a plurality of wells. Each set of chambers has a plurality of chambers adapted for receiving microbes of the polymicrobial infection therein. Each well is associated with a corresponding set of chambers. An input of the well is in fluidic communication with the outlets of the plurality of chambers in the corresponding set of chambers. Each well is adapted for receiving a selected antibiotic therapy wherein the selected antibiotic therapy received in the well fluidically connects the plurality of chambers in the corresponding set of chambers such that microbes received in the plurality of chambers in the corresponding set of chambers are in soluble factor contact.

The body further includes a permeable membrane disposed between the outlets of the plurality of chambers of the plurality of sets of chambers and the inputs of corresponding wells of the plurality of wells. The permeable membrane is a porous polycarbonate membrane. The body includes upper and lower surfaces. Each of the plurality of chambers of the plurality of sets of chambers has an opening communicating with the upper surface of the body. Each well of the plurality of wells is partially defined by a closed surface within the body. The closed surface of each well is generally parallel to the lower surface of the body.

The body further includes a plurality of well inlets and a plurality of air outlets. Each well inlet extends between the closed surface of a corresponding well and the lower surface of the body and allows access to the corresponding well for loading the selected antibiotic therapy therein. Each air outlet extends between the closed surface of the corresponding well and the lower surface of the body and allows for the purging of air from the corresponding well during the loading of the selected antibiotic therapy therein.

In accordance with a still further aspect of the present invention, a method for testing antibiotic sensitivity of a polymicrobial infection is provided. The method includes the steps of providing microbes of the polymicrobial infection in corresponding chambers of a plurality of chambers and loading a selected antibiotic therapy in a well so as to fluidically connect each of chambers such that microbes provided in the plurality of chambers are in soluble factor contact. The interaction of the selected antibiotic therapy and the microbes is then observed.

A permeable membrane may be positioned between outlets of the plurality of chambers and an input to the well. The permeable membrane is a porous polycarbonate membrane. Each of the plurality of chambers has an opening communicating with the atmosphere and the well is interconnected to the atmosphere with a well inlet. The well inlet allows for access to the well for loading the selected antibiotic therapy therein. The well may also be interconnected to the atmosphere with an air outlet. The air outlet allows for the purging of air from the well during the loading of the selected antibiotic therapy therein.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring toFIGS.1-2, a microfluidic device in accordance with the present invention is generally designated by the reference numeral10. Microfluidic device10may be formed from polystyrene (PS) or polydimethylsiloxane (PDMS), however, other materials are contemplated as being within the scope of the present invention. In the depicted embodiment, microfluidic device10includes base12having first and second ends14and16, respectively, and first and second sides18and20, respectively. First and second ends14and16, respectively, and first and second sides18and20, respectively, of base12define the outer periphery of base12. The outer periphery of base12interconnects upper and lower surfaces22and24, respectively.

As hereinafter described, microfluidic device10defines platform26for effectuating a method of multi-variable combinational screening is accordance with the present invention. It is noted that microfluidic device10may include additional platforms26provided therein for effectuating the methodology in after described, without deviation from the scope of the present invention. Referring toFIGS.2-4, in the depicted embodiment, platform26includes a vertical common well28extending along a corresponding axis27into base12from upper surface22thereof. Common well28is defined by a generally cylindrical side wall30having an upper edge32intersecting upper surface22of base12so as to define opening34. Lower edge36of side wall30defines outlet38of common well28.

Platform26further includes an array of multiplexing chambers40extending radially outward from axis27and communicating with exit38of common well28. In the depicted embodiment, six (6), generally triangular multiplexing chambers42a-42fare provided in base12. As hereinafter described, each multiplexing chamber42a-42fis identical in structure and configuration. As such, the following description of multiplexing chamber42ais understood to describe multiplexing chambers42b-42fas if fully described herein.

Multiplexing chamber42ais generally triangular in shape and is defined by first sidewall44lying in a first plane, a second sidewall46lying in a second plane and third sidewall48lying in a third plane. First, second and third sidewalls44,46and48, respectively, depend from upper, generally planar, chamber surface49and intersect lower surface24of base12at corresponding lower terminal edges44a,46aand48a, respectively. It can be appreciated that lower edges44a,46aand48aof first, second and third sidewalls44,46and48, respectively, lie in a common plane with lower surface24of base12and define opening51. First, second and third sidewall44,46and48, respectively, have a common width and, for reasons hereinafter described, first, second and third sidewall44,46and48, respectively, have a generally equal length L. Hence, multiplexing chamber42ahas an equilateral triangular configuration. The first and second planes, respectively, are provided at an angle of approximately 60 degrees to each other and intersect each other along a line, thereby defining first vertex52of the triangular configuration of multiplexing chamber42a. It can be appreciated that first vertex52is adjacent to axis27. Similarly, the first and third planes, respectively, are provided at an angle of approximately 60 degrees to each other and intersect each other along a line, thereby defining second vertex56of the triangular configuration of multiplexing chamber42a. Second and third planes, respectively, are provided at an angle of approximately 60 degrees to each other and intersect each other along a line, thereby defining third vertex60of the triangular configuration of multiplexing chamber42a.

As best seen inFIG.2, it understood that second vertex56of each multiplexing chamber42a-42fis adjacent to third vertex60of an adjacent multiplexing chamber42a-42f. In other words, second vertex56of multiplexing chamber42ais adjacent to third vertex60of adjacent multiplexing chamber42f; second vertex56of multiplexing chamber42fis adjacent to third vertex60of adjacent multiplexing chamber42e; second vertex56of multiplexing chamber42eis adjacent to third vertex60of adjacent multiplexing chamber42d; second vertex56of multiplexing chamber42dis adjacent to third vertex60of adjacent multiplexing chamber42c; second vertex56of multiplexing chamber42cis adjacent to third vertex60of adjacent multiplexing chamber42b; and second vertex56of multiplexing chamber42bis adjacent to third vertex60of adjacent multiplexing chamber42a.

Platform26further includes a plurality of variable wells64provided in base12. Variable wells64extend into base12from upper surface22thereof along corresponding axes at a location which overlaps second vertices56of each multiplexing chamber42a-42f(e.g. multiplexing chamber42a) and third vertices60of the multiplexing chamber42a-42fadjacent thereto (e.g. multiplexing chamber42f). As best seen inFIGS.3-5, each variable well64is defined by a generally cylindrical side wall66having an upper edge68intersecting upper surface22of base12so as to define opening70. Lower edge72of side wall66of each variable well64defines an outlet74having sufficient diameter to communicate with two adjacent multiplexing chambers42a-42f(e.g. multiplexing chambers42aand42f). It is contemplated to provide a semi-permeable membrane between outlets74of variable wells64and corresponding multiplexing chambers42a-42fto prevent direct liquid mixing/crosstalk between via convective flow between variable wells64and corresponding multiplexing chambers42a-42f.

In operation, it is intended for platform26to be utilized for the multiplex analysis of the interaction of three variables within a single chamber (e.g., multiplexing chambers42a-42f). By way of example, multiplexing chambers42a-42fmay be filled through opening51with a desired media, such as a solution, gel, or the like. A fixed or first variable “experimentee” is loaded in common well28so as to pass into each multiplexing chamber42a-42fthough outlet38thereof. The fixed variable “experimentee” may take the form of a microbe, a cell species, a drug or antibiotic, a soluble factor or another factor. In addition, each variable well64may be loaded with a different “experimenting” variable which passes into the two multiplexing chambers42a-42fwith which outlet74of variable well64communicates. The different “experimenting” variables may take the form of various microbes, cells, drugs, antibiotics, soluble factors or other factors. In this way, a user may simply and easily observe the interaction of the experimentee and experimenting variables within each of the multiplexing chambers42a-42f. It can be appreciated that platform26enables high-throughput screening of three interacting variables within a single multiplexing chamber42a-42fat a time with simplicity in readout.

Further, it is noted that due to configuration of the plurality of multiplexing chambers42a-42f, namely, the equilateral triangular configuration, the first, second and third vertices52,56and60, respectively, of each of the plurality of multiplexing chambers42a-42fare an equal distance apart, thereby allowing for the variables loaded into common well28and variable wells64to contribute equally in terms of interaction distance therebetween. Since the triangle is the most basic unit that can define a planar surface, the interaction network may be scaled out in both the x and y dimensions, as compared to a linear 2-way interaction network which is confined to only one dimension at a time in terms of geometrical design. Further, by utilizing a triangular configuration, a multiplexing chamber42a-42fmay be joined to an adjacent multiplexing chamber42a-42falong a single side thereof which, in turn, allows the two adjacent multiplexing chambers42a-42fto share two wells, but have a single well isolated from the adjacent multiplexing chamber. This allows for a 3-factorial but single variable comparison between any two (2) neighboring triangular multiplexing chambers. It can be appreciated that microfluidic device10may be used to screen drug combinations that exert highest potency, find transcription factor combinations that show the highest stem cell differentiation efficiency, elucidate cell-cell or microbe interactions mechanisms, optimize chemical factors for cell culture, and multi-cell/organ drug metabolism.

By way of example, it can be understood that device10is amenable to solid culture and liquid culture or a combination of both. More specifically, for solid culture of prokaryotic cells (such as bacteria), variable wells64and multiplexing chambers42a-42fmay be filled with a warm agar solution including culture media (for example, Luria Broth (LB), Tryptic Soy Broth (TSB), Mueller Hinton Broth (MHB), or the like). The warm agar solution is allowed to solidify at room temperature or lower, e.g. 4° C. Device10filled with the agar solution can be stored for an extended period of time before use. Thereafter, bacteria may be inoculated by pipetting a bacteria solution onto the solid agar surface at opening51of a corresponding multiplexing chamber42a-42fand thereafter cultured therein with device10facing either up or down. The experimenting variables such as drugs, antibiotics, other soluble factors or cells are added to device10via pipetting the solution on the solid agar surface at openings70of variable wells64and allowing the solution to absorb/diffuse therein.

For solid culture of eukaryotic cells (such as mammalian cells), variable wells64and multiplexing chambers42a-42fcan be filled as heretofore described with a hydrogel solution (such as collagen, matrigel, polyethylene glycol (PEG) gels, or the like) including culture media (such as Dulbecco's Modified Eagle's medium (DMEM), Roswell Park Memorial Institute medium (RPMI), or the like) and allowed to solidify to form a gel matrix. Cells may be premixed with the gel and loaded together into device10or seeded on top of the solidified gel matrix at opening51of a corresponding multiplexing chamber42a-42fand/or openings70of variable wells64.

For a mixed culture of eukaryotic cells and prokaryotic cells, a first portion of variable wells64may be loaded with eukaryotic cell-compatible gels such as collagen, matrigel, PEG gels, or the like, while a second portion of variable wells64may be loaded with prokaryotic cell-compatible gels such as agar. It is noted that for this “mixed gel” type of culture, one gel has to be fully solidified prior to adding the other gel to prevent the mixing thereof.

For a mixed solid and liquid co-culture system, either variable wells64or multiplexing chambers42a-42fmay be preloaded as heretofore described with a solid gel matrix prior to loading a liquid media in order to prevent direct liquid convection between variable wells64. In other words, either variable wells64or multiplexing chambers42a-42fcan receive the solid gel matrix, but the solid gel matrix has to be loaded first and allowed to solidify prior to loading the liquid media. For example, for a solid prokaryotic cell co-culture with eukaryotic cells in liquid, variable wells64are first loaded with a solid agar gel, followed by adding liquid eukaryotic cell culture media into multiplexing chambers42a-42f. The eukaryotic cells are then seeded into multiplexing chambers42a-42fincluding liquid cell culture media, while the prokaryotic cells are seeded on top of the solid agar matrix at openings70in variable wells64and allowed to adhere.

It is noted that the experimenting variables such as drugs, antibiotics, other soluble factors can be added to variable wells64in a variety of ways. As noted above, if variable wells64are preloaded with a solid culture gel such as agar or collagen, then the experimenting variable can be added on top of the solid culture gel at opening70of a corresponding variable well64and allowed to absorb or diffuse into the solid culture gel. Alternatively, an experimenting variable or variables may be mixed with the liquid gel solution prior to loading in a corresponding variable well64. Thereafter, the mixture may be loaded into the corresponding variable well64. The experimenting variable-infused gels can be stored for an extended period of time without cross-contamination/mixing as long as the corresponding multiplexing chambers42a-42fare left empty and not filled with liquid or solid media. In this manner, diffusion of the experimenting variable into the corresponding multiplexing chamber42a-42fis only initiated upon the adding of liquid/solid media into the corresponding multiplexing chamber42a-42f.

If variable wells64are filled with a liquid media/reagent such as phosphate-buffered saline (PBS), LB or DMEM, the experimenting variables may be added to a corresponding variable well64by pipetting the experimenting variables into the liquid media. However, such liquid media are less amenable to long term storage and transportation when received with device10. In order to overcome this limitation, the liquid media and/or the experimenting variables may be dried by desiccation or lyophilization inside device10after loading. After drying, the dried liquid media inside variable wells64can be stored for an extended period of time without cross-contamination/mixing therebetween as long as corresponding multiplexing chambers42a-42fare left empty and not filled with a liquid or a solid media. To “re-activate” the dried liquid media, water or other liquid/solid media solutions may be added to a corresponding variable well64to re-dissolve the liquid media therein. As described above, diffusion into multiplexing chambers42a-42fis only initiated upon the filing of multiplexing chambers42a-42fwith a liquid/solid media.

It is contemplated to affix a removable membrane to upper surface22of base12which overlaps openings70to variable wells64to isolate the media inside variable wells64from the external embodiment during storage. Similarly, a removable membrane may be affixed to lower surface24of base12which overlaps openings51to multiplexing chambers42a-42fto further isolate the dried liquid media inside variable wells64from the external embodiment during storage.

Referring toFIGS.2a-2b, using the design principles of the microfluidic device10described above, it can be appreciated that the scale of platform26may be expanded to include a higher number of variable wells64and multiplexing chambers, generally designated by the reference numeral42. By way of example, platform26may be expanded to provide for a scaled-out symmetrical hexagonal design,FIG.2aor scaled-out in a single direction,FIG.2b. It can be appreciated that multiplexing chambers42are identical in structure to microfluidic chambers42a-42f, and as such, the prior description of microfluidic chamber42ais understood describe multiplexing chamber42as if fully described herein. The choice of the configuration depends on the scale of the experiment (number of combinations required) and the distance of interactions in question.

Referring toFIG.7, an alternate embodiment of a microfluidic device in accordance with the present invention is generally designated by the reference numeral100. As hereinafter described, microfluidic device100incorporates multiple platforms101for testing the antibiotic sensitivity of a polymicrobial infection. Microfluidic device100includes first and second layers102and104, respectively,FIG.7. Referring toFIGS.7-9, first layer102is formed from a polymeric material (e.g., polystyrene) and includes upper and lower surfaces106and108, respectively, interconnected by first and second ends110and112, respectively, and first and second sides114and118, respectively. A plurality of wells120are provided in upper surface106. In the depicted embodiment, the plurality of wells120are arranged in two rows and seven columns. However, the number and arrangement of the plurality of wells120in upper surface106of first layer102may be varied without deviating from the scope of the present invention.

Each of the plurality of wells120includes an opening122communicating with upper surface106of first layer102and is defined by a generally planer lower surface124spaced from upper surface106of first layer102by a sidewalls126a-126dand generally parallel to lower surface108of first layer102. In the depicted embodiment, sidewalls126a-126dhave identical depths D and identical widths W. However, the depths and widths of sidewalls126a-126dmay be varied without deviating from the scope of the present invention. In addition, sidewall126aand sidewall126care generally parallel to each other and perpendicular to sidewalls126band126d. Similarly, sidewall126band sidewall126dare generally parallel to each other and perpendicular to sidewalls126aand126c. As described, well120has a generally square configuration in cross-section. A media inlet128extends between lower surface124of each of the plurality of wells120and lower surface108of first layer102at a location adjacent the intersection of sidewalls126aand126b. In addition, an air outlet130extends between lower surface124of the plurality of wells120and lower surface108of first layer102at a location adjacent the intersection of sidewalls126aand126b.

Referring toFIG.9a, an alternate construction of the plurality of wells in first layer102is generally designated by the reference numeral120a. Each of the plurality of wells120aincludes an opening122aextending through first layer102. Opening122ahas a first end123communicating with upper surface106of first layer102and a second end125communicating with lower surface108of first layer102. Sidewalls127a-127ddefine opening122aand are generally perpendicular to upper and lower surfaces106and108, respectively, of first layer102. It is contemplated for sidewalls127a-127dhave identical depths D and identical widths W. However, the depths and widths of sidewalls127a-127dmay be varied without deviating from the scope of the present invention. In addition, sidewall127aand sidewall127care generally parallel to each other and perpendicular to sidewalls127band127d. Similarly, sidewall127band sidewall127dare generally parallel to each other and perpendicular to sidewalls127aand127c. As described, each of the plurality of wells120ahas a generally square configuration in cross-section.

Referring toFIGS.7and10-11, second layer104is formed from a polymeric material (e.g., polystyrene) and includes upper and lower surfaces132and134, respectively, interconnected by first and second ends136and138, respectively, and first and second sides140and142, respectively. A plurality of sets144of chambers146a-146dextend through second layer104between the upper and lower surfaces132and134, respectively, thereof. It is in intended for the number of the plurality of sets144of chambers146a-146din second layer104to correspond to the number of the plurality of wells120in upper surface106of first layer102. As such, in the depicted embodiment, the plurality of sets144of chambers146a-146dare arranged in two rows and seven columns.

In the depicted embodiment, each set144of chambers146a-146dincludes four chambers146a-146dof identical configuration and proportion arranged in two rows and two columns. However, the number and configuration of chambers146a-146dmay be varied, as desired. Further, in view of the foregoing, it can be understood that the description of chamber146ahereinafter provided describes chambers146b-146das if fully described herein. Chamber146aincludes an upper opening148communicating with upper surface132of second layer104and a lower opening150communication with lower surface134of second layer104. Sidewalls154a-154dextending between upper surface132and lower surface134of second layer104so as to define chamber146a. Sidewalls154a-154dhave identical depths D1and identical widths W1. In addition, sidewall154aand sidewall154care generally parallel to each other and perpendicular to sidewalls154band154d. Similarly, sidewall154band sidewall154dare generally parallel to each other and perpendicular to sidewalls154aand154c.

In order to construct platform101of microfluidic device100, first and second layers102and104, respectively, are positioned such that lower surface134of second layer104is directed at upper surface106of first layer102,FIG.7. Permeable membrane160is positioned between lower surface134of second layer104is directed at upper surface106of first layer102. By way of example, permeable membrane160may take the form of a 0.2 micrometer (μm) porous polycarbonate membrane. Thereafter, first and second layers102and104, respectively, are bonded together in any conventional manner such that first and second ends136and138, respectively, and first and second sides140and142, respectively, of second layer104are aligned with first and second ends110and112, respectively, and first and second sides114and118, respectively, of first layer102, thereby capturing permeable member160therebetween.

With first and second layers102and104, respectively, bonded together as heretofore described, each set144of chambers146a-146dis aligned with a corresponding one of the plurality of wells120,FIGS.6and12a-12d, or alternatively, with one of the plurality of wells120a,FIGS.8aand9a. With each set144of chambers146a-146daligned with a corresponding one of the plurality of wells120, sidewalls154aof chambers146aand146bof each set144of chambers146a-146dare generally co-planar with sidewall126aof a corresponding well120of each of the plurality of wells120; sidewalls154bof chambers146band146cof each set144of chambers146a-146dare generally co-planar with sidewall126bof a corresponding well120of each of the plurality of wells120; sidewalls154cof chambers146cand146dof each set144of chambers146a-146dare generally co-planar with sidewall126cof a corresponding well120of each of the plurality of wells120; and sidewalls154dof chambers146dand146aof each set144of chambers146a-146dare generally co-planar with sidewall126dof a corresponding well120of each of the plurality of wells120. Permeable member160separates each set144of chambers146a-146dfor a corresponding well120of each of the plurality of wells120.

Alternatively, with each set144of chambers146a-146dis aligned with a corresponding one of the plurality of wells120a, sidewalls154aof chambers146aand146bof each set144of chambers146a-146dare generally co-planar with sidewall127aof a corresponding well120aof each of the plurality of wells120a; sidewalls154bof chambers146band146cof each set144of chambers146a-146dare generally co-planar with sidewall127bof a corresponding well120aof each of the plurality of wells120a; sidewalls154cof chambers146cand146dof each set144of chambers146a-146dare generally co-planar with sidewall127cof a corresponding well120aof each of the plurality of wells120a; and sidewalls154dof chambers146dand146aof each set144of chambers146a-146dare generally co-planar with sidewall127dof a corresponding well120aof each of the plurality of wells120a. Permeable member160separates each set144of chambers146a-146dfor a corresponding well120aof each of the plurality of wells12a.

In operation, different microbes are provided in each chamber146a-146dof each set144of chambers146a-146d. The microbes may take the form of bacteria, viruses, fungi, yeasts, parasites, antibiotics or a combination thereof. By way of example, different bacterial strains (e.g., bacterial strains162and164inFIGS.12a-12d) may be provided or cultured in media166in each chamber146a-146dof each set144of chambers146a-146d. For example, a small volume (10 μL) of four samples of bacterial strains from the same patient may be provided or cultured individually in each chamber146a-146d. Permeable membrane160is specifically chosen so that media166from chambers146a-146dof each set144of chambers146a-146ddoes not flow through permeable membrane160into a corresponding well120or120awhen the corresponding well120or120ais empty (in other words, filled with air),FIGS.12aand12b. When utilizing the plurality of wells120, each of the plurality of wells120may be filled through media inlets128with different medias, e.g., different antibiotic medias, collectively designated by the reference numeral168,FIG.12c. It can be appreciated that microfluidic device100may be flipped upside down to facilitate filling of the plurality of wells120given that the surface tension of media166in chambers146a-146dof each set144of chambers146a-146dretains media166therein. Alternatively, wherein utilizing the plurality of wells120a, each of the plurality of wells120amay be filled through second end125of opening122awith different medias, e.g., different antibiotic medias, as heretofore described. It can be appreciated that microfluidic device100may be flipped upside down to facilitate filling of the plurality of wells120agiven that the surface tension of media166in chambers146a-146dof each set144of chambers146a-146dretains media166therein.

Once the plurality of wells120or120awith different medias168(e.g., different antibiotic therapies), each of the plurality of wells120or120aserves as a liquid pool that fluidically connects each of chambers146a-146dof a corresponding set144of chambers146a-146dthrough permeable membrane160. For example, with well120filled with media168, bacteria162and164in chambers146a-146dof the corresponding set144of chambers146a-146dwill be in soluble factor contact through diffusion,FIG.12d. The structure of permeable membrane160is intended to be efficient in preventing bacteria migration, while providing sufficiently fast diffusion. By analyzing the different media168in each of the plurality of wells120or120a, the susceptibility of the combination of specific bacterial pathogens cultured in chambers146a-146dto various antibiotic therapies provided in the plurality of wells120or120amay be simply and easily assessed.

As described, microfluidic device100allows for the simultaneous testing of various antibiotic therapies to be performed on a combination of specific microbes provided in sets144of chambers146a-146d. It can be understood that the number of chambers in each set144of chambers146a-146dmay be increased or decreased to correspond to the number of microbes in a desired combination. Further, it can be appreciated the open-microfluidic nature enables unique advantages in accessibility, allowing the microbes, e.g. bacteria162and164, to be easily recollected for traditional antibiotic sensitivity or antibiotic susceptibility measures or biofilm assessment.

Further, it is contemplated to pre-load the plurality of wells120or120awith a selected antibiotic so as to provide microfluidic device100as a pre-packaged kit to test different microbes, e.g., gram negative and positive bacteria, thereagainst. For example, the plurality of wells120or120amay be preloaded with: penicillins, including amoxicillin +/− clavulanate, ampicillin +/− sulbactam, and piperacillin +/− tazobactam; cephalosporins, including cefepime, cefoxitin, cefazolin, and ceftriaxone; carbapenems, including meropenem and ertapenem; monobactams, including aztreonam; fluoroquinolones, including ciprofloxacin; aminoglycosides, including gentamicin; macrolides, including azithromycin; and others, including vancomycin, clindamycin, rifampin, trimethoprim +/− sulfamethoxazole and tetracycline. A removable membrane may be affixed lower surface108of first layer102of which overlaps openings122aof the plurality of wells120ato isolate the media within the plurality of wells120afrom the external embodiment during storage. Similarly, a removable membrane may be affixed to upper surface132of second layer104which overlaps openings148of chambers146a-146dto further isolate the media inside the plurality of wells120or120afrom the external embodiment during storage. When using the microfluidic device100, any removable membranes affixed thereto may be removed thereby allowing a user to load different microbes in each chamber146a-146dof each set144of chambers146a-146d, as heretofore described, to test the gram negative and positive bacteria against the pre-loaded antibiotic.

Various modes of carrying out the invention are contemplated as being within the scope of the following claims particularly pointing out and distinctly claiming the subject matter, which is regarded as the invention.