Patent Application
20230398507
In one of its aspects, the present invention relates to a system for growing a microorganism in liquid culture. In another of its aspects, the present invention relates to a method of growing a microorganism in liquid culture. In yet another of its aspects, the present invention relates to a microfluidic cartridge that may be used to grow microorganisms using the system and methods disclosed herein.
For conventional assays involving pathogenic bacteria, the step of incubating to induce bacterial growth is often rate limiting, typically taking hours to days and requiring transport to a central lab. The long lead times may have deleterious effects. For example, in antibiotic susceptibility testing, slow testing often leads to “best guess” methods to determine treatment options, which contributes to antibiotic resistance. Furthermore, traditional liquid bacteria cultures are grown in 96-well plates which requires the use of bulky and expensive plate shakers.
Accordingly, it would be desirable to have an improved system and methods for rapidly growing microorganisms in liquid culture. It would also be desirable for this improved system to be portable and more cost effective than the microorganism growth systems previously used in the art.
It is an object of the present invention to obviate or mitigate at least one of the above-mentioned disadvantages of the prior art.
It is another object of the present invention to provide a novel system, methods and apparatus for improving the rate of growth of microorganisms in liquid culture.
Accordingly, in one of its aspects, the present invention provides a system for growing a microorganism in liquid culture, comprising:
In another of its aspects, the present invention provides a system for growing a microorganism in liquid culture, comprising:
In yet another of its aspects, the present invention provides a method for growing a microorganism in a liquid culture comprising:
In yet another of its aspects, the present invention provides a microfluidic cartridge comprising:
In yet another of its aspects, the present invention provides a microfluidic cartridge used for growing a microorganism in liquid culture comprising:
Accordingly, as described herein below, the present inventors have developed a system and methods for rapid, on-site growth of microorganisms in liquid culture that is faster, less bulky and more cost efficient than traditional growth techniques.
For liquid bacterial cultures, rapid and healthy growth depends on factors including (1) sample aeration, so that bacteria samples have access to atmospheric gases (e.g., oxygen) for growth, (2) nutrient availability, where samples are thoroughly mixed to provide nutrients homogenously throughout the culture, and (3) minimization of biofilms and clumping, where shaking and agitation prevents bacteria culture from settling to the bottom of a chamber and forming biofilms or clumps that hinder reproduction.
To address the challenges of the conventional art, these principles are applied in designing a portable microorganism growth system which includes a rotatable microfluidic cartridge that is used in conjunction with an oscillation driving apparatus and an oscillation protocol optimized for mixing liquid bacterial samples.
In order to provide access to atmospheric gases (e.g., oxygen) to increase growth, the present inventors have developed a portable rotatable microfluidic cartridge that is specifically designed to increase sample aeration in several ways. First, the microfluidic cartridge contains an incubation chamber with at least one gas permeable membrane that facilitates the flow of gas into and out of the incubation chamber during the incubation process. This gas flow generates bubbles within the incubation chamber, providing more surface area for gas exchange within the sample during mixing. Second, the surface area to volume ratio of the incubation chamber, (where the surface area of the chamber is measured in the same plane as the direction of rotation of the rotating microfluidic cartridge) is configured to be larger than that of traditional 96-well plates, in order to allow for more turbulence in the incubation chamber during mixing and further to allow for better gas exchange through the gas permeable membrane. Traditional 96-well plates may for example have a surface area to volume ratio of about 19 mm−1. The microfluidic cartridges developed by the present inventors thus have a surface area to volume ratio that is larger than that of traditional 96-well plates. For example, incubation chambers of the microfluidic cartridges disclosed herein may have a surface area to volume ratio of at least 19 mm−1. Finally, the growth system is designed so that the incubation chamber is intended to be only partially filled with a liquid sample, leaving a head space of air in the sample during mixing. This headspace provides further aeration to the sample. While not wishing to be bound by any particular theory or mode of action, it is believed that the above-mentioned features of the microfluidic cartridge design facilitate optimal amounts of aeration to allow for increased microorganism growth.
In order to ensure thorough mixing, to provide nutrients homogenously throughout the culture, and to minimize the formation of biofilms and clumping during bacterial growth, the present inventors have developed a system with an optimized mixing protocol to be used on a microfluidic cartridge. Traditional microfluidic systems have low Reynolds numbers and exhibit laminar flow regimes, which are dominated by viscous, rather than inertial forces. Thus, without turbulent mixing, microfluidic devices must rely on either passive molecular diffusion or external energy sources. Furthermore, the small, enclosed volumes characteristic of microfluidic systems restrict access of the bacterial culture to fresh oxygen and other atmospheric gases, making sample aeration difficult without bulky or complex pumps that bubble gases from an external source. By combining a rotatable microfluidic cartridge with an oscillation driving apparatus, the present inventors have developed an efficient method for mixing a bacterial sample within a microfluidic system. In this system, the oscillating driving apparatus creates a Euler force that results in chaotic advection and turbulent mixing of bacteria samples at a higher rate than in a 96-well plate or culture flask method. In an oscillating system, the Euler force (which is perpendicular to centrifugal force), may be used to generate vortical flow and/or provide uniform turbulent mixing within a microfluidic chamber of the microfluidic system. Euler forces are inertial forces that are produced when the microfluidic system (i.e., an incubation chamber) experiences cycles of unidirectional acceleration-and-deceleration rotation. Thus, mixing is influenced by chamber geometry, orientation, acceleration/deceleration rate, and angular spin. For example, as disclosed herein, the incubation chambers comprise three dimensions: length, width and depth. In certain embodiments, the length may be oriented tangentially to the direction of rotation of the cartridge. The microfluidic cartridges developed by the present inventors have been designed such that each of these dimensions allows for increased turbulent mixing within the chamber when the cartridge is rotated or oscillated. For example, the incubation chamber may be configured so that the length is greater than the width, and the length and width are each significantly greater than the depth. Further, as highlighted above, the surface area (measured in the same plane as the direction of rotation of the rotating microfluidic cartridge and calculated based on the length and width of the chamber) may be configured such that ratio of the surface area to chamber volume is larger than that of traditional 96-well plates. While not wishing to be bound by any particular theory or mode of action, it is believed that by manipulating the dimensions of the incubation chamber in this way, the microfluidic cartridge design facilitates turbulent mixing within the chamber to allow for increased microorganism growth.
Unlike traditional liquid bacteria cultures grown in 96-well plates in bulky and expensive plate shakers, the oscillation driving apparatus and microfluidic cartridge described above represent an inexpensive and portable alternative that yields faster growth of bacteria. This system may be used to either increase signal of an existing assay or to decrease assay time by achieving a measurable signal faster. In one exemplary application, the rotatable microfluidic cartridge and oscillation driving apparatus may be used in developing ultrafast, point of care antibiotic susceptibility assays which require rapid culture of bacteria with different antibiotics to determine resistance.
As illustrated through experimental data hereinbelow, the present inventors have shown that the use of a rotating microfluidic cartridge in conjunction with an oscillation driving apparatus yields superior bacterial growth rates compared with traditional shaker incubators, including 96-well plates on a shaker.
Embodiments of the present invention will be described with reference to the accompanying drawings, wherein like reference numerals denote like parts, and in which:
The present invention relates to a system for growing a microorganism in liquid culture, comprising a rotating platform on a driving apparatus; and at least one cartridge comprising a plurality of incubation chambers which rests upon said rotating platform, wherein said rotating platform provides vortical flow and/or turbulent mixing within the plurality of incubation chambers.
In another of its aspects, the present invention relates to a system for growing a microorganism in liquid culture, comprising: (a) a driving apparatus configured to house and oscillate a microfluidic cartridge; and (b) a microfluidic cartridge secured with respect to the driving apparatus, the microfluidic cartridge comprising: a body portion and at least a first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, a ratio of the first wall surface area to chamber volume ratio is at least about 19 mm−1; wherein at least a portion of at least one of the first wall and second wall is gas permeable to facilitate a flow of gas into and out of the chamber interior.
Preferred embodiments of this system may include any one or a combination of any two or more of any of the following features:
In yet another of its aspects, the present invention relates to a method for growing a microorganism in a liquid culture comprising: (a) disposing a microorganism and a suitable growth medium in a first incubation chamber, wherein the incubation chamber comprises (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein a ratio of the first wall surface area to chamber volume is at least about 19 mm−, wherein at least a portion of at least one of the first wall and second wall is gas permeable; and (b) mixing the microorganism and the growth medium by oscillating the incubation chamber back and forth along an oscillation path at a predetermined oscillation frequency.
Preferred embodiments of this method may include any one or a combination of any two or more of any of the following features:
In yet another of its aspects, the present invention relates to a microfluidic cartridge used for growing a microorganism in liquid culture comprising: (a) a body portion having a mounting portion configured to be secured with respect to a driving apparatus; and (b) at least a first incubation chamber disposed in the body portion of the first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, a ratio of the first wall surface area to chamber volume ratio is at least about 19 mm−1; wherein at least a portion of at least one of the first wall and second wall is gas permeable.
Preferred embodiments of this apparatus may include any one or a combination of any two or more of any of the following features:
As used herein, certain terms may have the following defined meanings.
As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof
As used in the specification and claims, the term “RiboGrow™” refers to the use of a rotating platform system, as described herein, for increasing growth of a cell, such as a microorganism, in a liquid culture. For instance, a RiboGrow™ method for increasing growth of a microorganism in a liquid culture may be based on placing a cell culture medium comprising at least one microorganism in at least one chamber of a cartridge comprising a plurality of incubation chambers, the liquid within the plurality of incubation chambers of said cartridge being sealed within the chambers by a breathable membrane; rotating the cartridge to generate vortical flow and/or turbulent mixing within the plurality of incubation chambers; and incubating the rotating cartridge at a temperature optimized to induce growth of the microorganism.
As used herein, the term “cell culture media,” refers to a media where a microorganism is capable of rapid growth.
As used herein, the term “breathable membrane” refers to a membrane that is pervious to gases and impervious to liquids as well as microorganisms. In some embodiments, a breathable membrane is a bio-compatible polymer film.
Disclosed herein are systems for growing a microorganism in liquid culture. Systems for growing a microorganism in liquid culture may comprise (a) a rotating platform on a driving apparatus; and (b) at least one cartridge comprising a plurality of incubation chambers which rests upon said rotating platform, wherein said rotating platform provides turbulent mixing within the plurality of incubation chambers.
As further disclosed herein, systems for growing a microorganism in liquid culture may comprise (a) a driving apparatus configured to house and oscillate a microfluidic cartridge; and (b) a microfluidic cartridge secured with respect to the driving apparatus, the microfluidic cartridge comprising: a body portion and at least a first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein a ratio of the first wall surface area to chamber volume is at least about 19 mm−1; wherein at least a portion of at least one of the first wall and second wall is gas permeable to facilitate a flow of gas into and out of the chamber interior.
In one of its aspects, the present invention provides a microfluidic cartridge for growing a microorganism in liquid culture. The microfluidic cartridge may comprise (a) a body portion having a mounting portion configured to be secured with respect to a driving apparatus; and (b) at least a first incubation chamber disposed in the body portion of the first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein a ratio of the first wall surface area to chamber volume is at least about 19 mm−1; wherein at least a portion of at least one of the first wall and second wall is gas permeable.
In certain preferred embodiments, the mounting portion of body of the microfluidic cartridge may be configured to allow the cartridge to remain secured to the driving apparatus when the cartridge oscillates at a predetermined angular acceleration with a predetermined oscillation angle.
In certain preferred embodiments, the incubation chamber of the microfluidic cartridge may be configured so that when the microfluidic cartridge is in use and engaged by a driving apparatus, the first incubation chamber is translated back and forth along an oscillation path at a predetermined oscillation frequency, creating turbulent mixing within the first incubation chamber. In certain embodiments, turbulent mixing within the first incubation chamber may be accomplished as a result of the design of the incubation chamber. For example, the incubation chamber may be designed such that the ratio of the first wall surface area to chamber volume is at least greater than that of traditional 96-well plates. By way of non-limiting example, traditional 96-well plates may have a surface area to volume ratio of about 19 mm−1. By using an incubation chamber designed to include a surface area to volume ratio of at least 19 mm−1, the present invention may facilitate greater mixing capabilities than achievable by traditional 96-well plates and thus may facilitate higher growth rates of microorganisms in said incubation chambers than on 96-well plates. In certain preferred embodiments, the ratio of the first wall surface area to chamber volume may be at least greater than about 20 mm−1 or greater than about 25 mm−1 or greater than about 30 mm−1 or greater than about 35 mm−1 or greater than about 40 or greater than about 45 mm−1 or greater than about 50 mm−1.
As further shown in
In some embodiments, the cartridge may have a diameter in the range of about 30 mm or about 40 mm or about 50 mm or about 60 mm or about 70 mm or about 80 mm or about 90 mm or about 100 mm or about 110 mm to about 120 mm or about 130 mm or about 140 mm or about 150 mm or about 160 mm or about 170 mm or about 180 mm or about 190 mm or about 200 mm. In some embodiments, the cartridge may have a diameter sufficient to be portable and/or easy to handle. For example, the cartridge may be as small as 30 mm and still be easy to hold and as large as 200 mm and still be portable. When the cartridge diameter is smaller than 30 mm, the cartridge may be difficult to handle. When the cartridge diameter is larger than 200 mm, the cartridge may be difficult to transport. In certain preferred embodiments, the cartridge may have a diameter of approximately 120 mm.
As further shown in
In some embodiments, the body portion 102 of the microfluidic cartridge 100 may comprise a polymer. Examples of polymers that make up the body portion 102 may include but are not limited to: poly(methyl methacrylate) (PMMA), polycarbonate, polyethylene, polypropylene, polystyrene, polyesters, polyvinyl chloride (PVC), cyclic olefin polymer (COP), cyclic olefin copolymer (COC) and nylon.
In one of its aspects, the present invention provides a microfluidic cartridge for growing a microorganism in liquid culture wherein the microfluidic cartridge may comprise (a) a body portion having a mounting portion configured to be secured with respect to a driving apparatus; and (b) at least a first incubation chamber disposed in the body portion of the first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein at least a portion of at least one of the first wall and second wall is gas permeable. By way of non-limiting example, in certain preferred embodiments, either the first wall of the incubation chamber, the second wall of incubation chamber or both may be gas permeable to permit a flow of gas into and out of the incubation chamber. This gas permeability may be accomplished by sealing the incubation chamber on the first wall, second wall, or both with a breathable membrane.
In some embodiments, the membrane may be a clear, gas permeable biaxially-oriented polyethylene terephthalate film attached using an adhesive. In some embodiments, the breathable membrane may be attached only on one side of the body portion of the microfluidic cartridge. In other embodiments, the breathable membrane may be attached to both sides of the body of the microfluidic cartridge. In certain preferred embodiments, the membrane may be a flexible membrane. In other embodiments, the membrane may be a non-flexible membrane.
The addition of a breathable, gas-permeable membrane allows for sample aeration so that bacteria samples have access to atmospheric gases (e.g., oxygen) for growth. Moreover, the breathable sealing membranes also allow respiration, cell viability and cell growth to be maintained in leak-proof incubation chambers since the membrane does not peel and is impervious to liquids. In fact, many cellular-based assays depend upon continuing respiration for accuracy and reproducibility of the assays, and an extended period of ongoing cellular metabolism may be required for cells held in such plates. Membranes of the present disclosure assure uniformity of gas exchange and thus cellular respiration from chamber-to-chamber and sample-to-sample across the cartridge. This uniformity is important for experimental accuracy and valid comparisons among different cell samples held in different chambers within a cartridge.
In some embodiments, when a microfluidic cartridge comprises a gas permeable thermopolymer from which the body of the cartridge is molded, the microfluidic cartridge itself may function as a suitable breathable membrane.
In certain preferred embodiments, the permeable membranes may be of a thickness such that they are impervious to microorganisms and allow for sufficient oxygen permeability through the membrane. Consequently, when applied and adhered to an incubation chamber as described herein, microbial contaminants are likewise excluded from the sample chambers of the cartridge. The amount of gas permeability necessary depends on experimental design.
In one of its aspects, the present invention provides a system for growing a microorganism in liquid culture comprising (a) a driving apparatus configured to house and oscillate a microfluidic cartridge; and (b) a microfluidic cartridge secured with respect to the driving apparatus, the microfluidic cartridge comprising: a body portion and at least a first incubation chamber comprising (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein a ratio of the first wall surface area to chamber volume is at least about 19 mm−1; wherein at least a portion of at least one of the first wall and second wall is gas permeable to facilitate a flow of gas into and out of the chamber interior.
In certain preferred embodiments, the driving apparatus may comprise a direct current (DC) motor. In some embodiments, the DC motor is brushless, while in other embodiments, the DC motor may be brush motor. Examples of DC motors may include but are not limited to stepper motors or servo motors.
In certain preferred embodiments, the motor may be configured such that the driving apparatus oscillates the incubation chamber back an forth at a predetermined frequency. By way of non-limiting example, the predetermined oscillation frequency may be between about 1 and 5 Hz. In certain preferred embodiments, the oscillation frequency may be about 4 Hz. In other preferred embodiments, the oscillation frequency may be about 2 Hz.
In some embodiments, the motor may be configured such that the driving apparatus oscillates the incubation chamber with an oscillation angle in a range of from 30 degrees and 330 degrees. In some embodiments, the motor may be configured to oscillate with an oscillation angle in a range of from about 30 degrees or about 40 degrees or about 50 degrees or about 60 degrees or about 70 degrees or about 80 degrees or about 90 degrees or about 100 degrees or about 110 degrees or about 120 degrees or about 130 degrees or about 140 degrees or about 150 degrees or about 160 degrees or about 170 degrees to about 180 degrees or about 190 degrees or about 200 degrees or about 210 degrees or about 220 degrees or about 230 degrees or about 240 degrees or about 250 degrees or about 260 degrees or about 270 degrees or about 280 degrees or about 290 degrees or about 300 degrees or about 310 degrees or about 320 degrees or about 330 degrees.
In some embodiments, the motor may be configured such that the driving apparatus oscillates the incubation chamber with an oscillation angle in a range of from 150 degrees and 210 degrees. In some embodiments, the motor may be configured such that the driving apparatus oscillates the incubation chamber with an oscillation angle in a range of from 30 to 330 degrees, or from 100 degrees to 260 degrees.
In some embodiments, the motor is configured such that the driving apparatus oscillates the incubation chamber at an angular acceleration in a range of about 100 rad/s2 or about 120 rad/s2 or about 140 rad/s2 or about 160 rad/s2 or about 180 rad/s2 or about 200 rad/s2 or about 220 rad/s2 or about 240 rad/s2 or about 260 rad/s2 or about 280 rad/s2 to about 300 rad/s2 or about 320 rad/s2 or about 340 rad/s2 or about 360 rad/s2 or about 380 rad/s2 or about 400 rad/s2 or about 420 rad/s2 or about 440 rad/s2 or about such that the driving apparatus oscillates the incubation chamber at an angular acceleration in a range of 100 to 500 rad/s2. In some embodiments, the motor is configured such that the driving apparatus oscillates the incubation chamber at an angular acceleration in a range of 200 to 300 rad/s2.
In certain preferred embodiments, the system and methods for growing a microorganism in liquid culture described herein may further comprise an incubator configured to incubate a microorganism in a microfluidic cartridge. By way of non-limiting example, in certain preferred embodiments the incubator may comprise a heating element. The heating element may comprise metal heating elements (i.e. iron/chromium/aluminum (FeCrAl) wires, nickel/chrome (Ni/Cr) 80/20 wires, copper/nickel (Cu/Ni) wires). In some embodiments, the heating element may comprise ceramic heating elements (i.e. MoSi2, PTC ceramics). In some embodiments, the heating element may comprise polymer PTC heating elements (i.e. PTC rubber material). In some embodiments, the heating element may comprise composite heating elements.
In yet another of its aspects, the present invention provides methods of growing a microorganism in liquid culture. Methods of growing a microorganism in liquid culture may comprise: (a) disposing a microorganism and a suitable growth medium in a first incubation chamber, wherein the incubation chamber comprises (i) a first wall, (ii) a second wall opposed to the first wall, and (iii) at least one sidewall interconnecting the first wall and the second wall to define a chamber interior having a chamber volume and configured to contain a liquid, wherein a ratio of the first wall surface area to chamber volume is at least about 19 mm−1, wherein at least a portion of at least one of the first wall and second wall is gas permeable; and (b) mixing the microorganism and the growth medium by oscillating the incubation chamber back and forth along an oscillation path at a predetermined oscillation frequency.
In certain preferred embodiments, the methods disclosed herein for growing a microorganism in liquid culture may further comprise disposing a microorganism and a suitable growth medium in at least one additional incubation chamber, wherein the growth medium in the first incubation chamber comprises an anti-microbial agent free cell culture medium, and the growth medium in the at least one additional incubation chamber comprises comprising at least one anti-microbial agent.
In some embodiments, the anti-microbial agent is an antibiotic. Examples of antibiotics may include but are not limited to, a bactericidal antibiotic, a bacteriostatic antibiotic, a beta-lactam antibiotic, an aminoglycoside antibiotic, an ansamycin antibiotic, a macrolide antibiotic, a sulfonamide antibiotic, a quinolone antibiotic, an oxazolidinone antibiotic, a glycopeptide antibiotic, an anthraquinone antibiotic, an azole antibiotic, a nucleoside antibiotic, a peptide antibiotic, a polyene antibiotic, a polyether antibiotic, a steroid antibiotic, a tetracycline antibiotic, a dicarboxylic acid antibiotic, a metal or a metal ion antibiotic, a silver compound antibiotic, an oxidizing antibiotic or an antibiotic that releases free radicals or active oxygen, or a cationic antimicrobial agent.
In some embodiments, the methods disclosed herein comprise growing a microorganism in cell culture media. In some embodiments, the microorganism may be selected from the group of prokaryotic cells and eukaryotic cells. In some embodiments, the prokaryotic cells are Gram-negative bacteria. In some embodiments, the Gram-negative bacteria is selected from the group of Escherichia coli, Salmonella, Shigella, Enterobaceriaceae, Pseudomonas, Moraxella, Helicobacter, Strenotrophomonas, Bdellovibrio, and Legionella. In some embodiments, the prokaryotic cells are Gram-positive bacteria. In some embodiments, the Gram-positive bacteria is selected from the group of Enterococcus, Staphylococcus, Streptococcus, Actinomyces, Bacillus, Clostridium, Corynebacterium, Listeria, and Lactobacillus. In some embodiments, the eukaryotic cells are fungal cells. In some embodiments, the fungal cells are yeast. In some embodiments, the yeast is Candida.
In certain embodiments, methods of growing a microorganism in liquid culture may further comprise the step of incubating the microorganism by placing the incubation chamber in an incubator for a predetermined incubation period optimized to induce growth of the microorganism.
In some embodiments, incubating the microorganism may be conducted in a bacterial growth broth solution. By way of non-liming example, the bacterial growth broth solution may be a cation-adjusted broth solution, such as Mueller Hinton broth, lysogeny broth, super optimal broth, super optimal broth with catabolite repression, terrific broth, or M9 minimal broth.
In some embodiments, incubating the microorganism is conducted at a temperature in the range of 20° C. to 60° C. In some embodiments, incubating the microorganism is conducted at a temperature in the range of 30° C. to 50° C. In some embodiments, the microorganism may be incubated for at least 15, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360, 420, or 480 or more minutes. In some embodiments, incubating the microorganism is conducted at a temperature in the range of about 20° C., or about 21° C., or about 22° C., or about 23° C., or about 24° C., or about 25° C., or about 26° C., or about 27° C., or about 28° C., or about 29° C., or about 30° C. or about 31° C. or about 32° C. or about 33° C. or about 34° C. or about 35° C. or about 36° C. or about 37° C. or about 38° C. or about 39° C. to about 40° C. or about 41° C. or about 42° C. or about 43° C. or about 44° C. or about 45° C. or about 46° C. or about 47° C. or about 48° C. or about 49° C. or about or about 51° C., or about 52° C., or about 53, ° C., or about 54° C., or about 55° C., or about 56° C., or about 57° C., or about 58° C., or about 59° C. or about 60° C.
In certain preferred embodiments, incubating the microorganism may be conducted at a temperature in the range of 33° C. to 47° C., or more preferably at a temperature in the range of 36° C. to 44° C.
In some embodiments, the microorganism may be incubated at room temperature e.g., about 25° C. In some embodiments, incubating the microorganism may be conducted at a temperature of about 37° C.
In some embodiments, the methods disclosed herein comprise a RiboGrow™ method. In some embodiments, the RiboGrow™ method is followed by lysis of the microorganism and release of a ribonucleic acid (RNA) molecule from the cells. In some embodiments, the cell lysate comprises an ribosomal RNA molecule. In some embodiments, the ribosomal RNA molecule is from a prokaryotic organism, or a fungal organism.
In some embodiments, the methods disclosed herein may further comprise lysing the microorganism to form a lysate. In certain preferred embodiments, lysis may include (a) subjecting a sample to mechanical lysis to cause disruption of a cellular membrane in the cellular material; (b) contacting the sample with an alkaline material to produce a lysate composition comprising the target chemical compound; and (c) recovering the lysate composition from the sample. Methods for lysing include those disclosed in International Patent Application No. PCT/US2018/045211, filed on Aug. 3, 2018, which is herein incorporated by reference in its entirety.
In some embodiments, the methods disclosed herein further comprise detecting the quantity of a nucleic acid molecule from a microorganism in a sample. In some embodiments, the methods disclosed herein comprise comparing the quantity of a nucleic acid molecule in the antimicrobial agent-free inoculate to the quantity of a nucleic acid molecule in the antimicrobial agent inoculate.
In some embodiments, determining the quantity of a nucleic acid molecule in a plurality of inoculates comprises a sandwich assay. In some embodiments, determining the quantity of a nucleic acid molecule in a plurality of inoculates comprises using an electrochemical sensor platform.
In some embodiments, the buffer solution used to neutralize a cell lysate comprises a detector probe. In some embodiments a detector probe is added separately after a cell lysate is neutralized. In some embodiments, the detector probe comprises one or more nucleic acids. In some embodiments, the nucleic acids comprise one or more modified oligonucleotides. In some embodiments, the detector probe comprises a plurality of nucleic acids. In some embodiments, the detector probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acids. In some embodiments, the detector probe comprises at least one deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or any combination thereof. In some embodiments, the detector probe comprises one or more DNA. In some embodiments, the detector probe comprises a plurality of DNA. In some embodiments, the detector probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more DNA. In some embodiments, the detector probe comprises one or more PNAs. In some embodiments, the detector probe comprises a plurality of PNAs. In some embodiments, the detector probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more PNAs. In some embodiments, the detector probe comprises one or more LNAs. In some embodiments, the detector probe comprises a plurality of LNAs. In some embodiments, the detector probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more LNAs.
In some embodiments, the detector probe comprises a detectable label. In some embodiments, the detectable label is selected from a radionuclide, an enzymatic label, a chemiluminescent label, a hapten, and a fluorescent label. In some embodiments, the detectable label is a fluorescent molecule. In some embodiments, the fluorescent molecule is selected from a fluorophore, a cyanine dye, and a near infrared (NIR) dye. In some embodiments, the fluorescent molecule is fluorescein. In some embodiments, the fluorescent molecule is fluorescein isothiocyanate (FITC). In some embodiments, the detectable label is a hapten. In some embodiments, the hapten is selected from DCC, biotin, nitropyrazole, thiazolesulfonamide, benzofurazan, and 2-hydroxyquinoxaline. In some embodiments, the detectable label is biotin.
In some embodiments, the methods disclosed herein comprise contacting the neutralized cell lysate with a capture solution comprising a capture probe. In some embodiments, the capture probe comprises a capture sequence comprising a plurality of nucleic acids. In some embodiments, the nucleic acids comprise one or more modified oligonucleotides. In some embodiments, the capture probe comprises a plurality of nucleic acids. In some embodiments, the capture probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleic acids. In some embodiments, the capture probe comprises at least one of deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or any combination thereof. In some embodiments, the capture probe comprises DNA. In some embodiments, the capture probe comprises a plurality of DNA. In some embodiments, the capture probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more DNA. In some embodiments, the capture probe comprises one or more PNAs. In some embodiments, the capture probe comprises a plurality of PNAs. In some embodiments, the capture probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more PNAs. In some embodiments, the capture probe comprises one or more LNAs. In some embodiments, the capture probe comprises a plurality of LNAs. In some embodiments, the capture probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more LNAs. In some embodiments, at least a portion of the capture sequence is complementary to at least a portion of a nucleic acid molecule from the microorganism. In some embodiments, the capture probe further comprises a bead. In some embodiments, the bead is attached to the capture sequence. In some embodiments, the bead is a magnetic bead.
In some embodiments, the methods disclosed herein comprise contacting the neutralized cell lysate with a solution comprising streptavidin.
In some embodiments, the methods disclosed herein comprise detecting the quantity of a nucleic acid molecule from a microorganism in a sample. In some embodiments, the methods disclosed herein comprise comparing the quantity of a nucleic acid molecule in the antimicrobial agent-free inoculate to the quantity of a nucleic acid molecule in the antimicrobial agent inoculate. In some embodiments, the nucleic acid molecule is a deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or a combination thereof.
In some embodiments, the methods disclosed herein further comprise a RiboResponse™ method. In some embodiments, the RiboResponse™ method comprises determining the quantity of an RNA molecule from the microorganism. In some embodiments, the RNA is a mature RNA. In some embodiments, the RNA is a precursor RNA. In some embodiments, the RNA is a ribosomal RNA (rRNA). In some embodiments, the rRNA is a 16S RNA or 23 S RNA. In some embodiments, the microorganism is a prokaryote. In some embodiments, the prokaryote is a Gram-negative bacterium. In some embodiments, the prokaryote is a Gram-positive bacterium. In some embodiments, the microorganism is fungal (e.g., candida).
The RiboResponse™ platform is quantitative in that more bacteria would result in more ribosomes and, hence, ribosomal RNA, resulting in a higher detection signal when ribosomal RNA is detected.
Methods for determining the quantity of an RNA molecule from the microorganism include those disclosed in International Patent Application No. PCT/US2018/047075, filed on Aug. 20, 2018, which is herein incorporated by reference in its entirety.
In some embodiments, when the methods disclosed herein comprise detecting the quantity of a nucleic acid molecule from a microorganism in a sample, the method can be completed in less than 4 hours or less, 3 hours or less, 2.5 hours or less, 2 hours or less, 90 minutes or less, 60 minutes or less, 45 minutes or less, or 30 minutes or less.
In some embodiments, the methods disclosed herein further comprise determining the susceptibility of a microorganism to an antimicrobial agent.
In some embodiments, in the methods and systems disclosed herein, at least one of the plurality of incubation chambers comprises at least one antimicrobial agent inoculate that comprises a microorganism in a cell culture media that contains an antimicrobial agent. In some embodiments, the plurality of inoculates comprises (a) at least one antimicrobial agent-free inoculate that comprises a microorganism in a cell culture media that does not contain an antimicrobial agent; (b) at least one antimicrobial agent inoculate that comprises a microorganism in a cell culture media that contains an antimicrobial agent; and (c) at least one antimicrobial agent inoculate that comprises a microorganism in a cell culture media that contains two antimicrobial agents. In some embodiments, the plurality of inoculates comprises (a) at least one antimicrobial agent-free inoculate that comprises a microorganism in a cell culture media that does not contain an antimicrobial agent; (b) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more antimicrobial agent inoculates that each comprise a microorganism in a cell culture media that contains an antimicrobial agent; and (c) at least one antimicrobial agent inoculate that comprises a microorganism in a cell culture media that contains two antimicrobial agents. In some embodiments, the plurality of inoculates comprises (a) at least one antimicrobial agent-free inoculate that comprises a microorganism in a cell culture media that does not contain an antimicrobial agent; (b) at least one antimicrobial agent inoculate that comprises a microorganism in a cell culture media that contains an antimicrobial agent; and (c) 1, 2, 3, 4, or 5 or more antimicrobial agent inoculates that each comprise a microorganism in a cell culture media that contains two antimicrobial agents. In some embodiments, the cell culture media for at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more antimicrobial agent inoculates contain different antimicrobial agents. In some embodiments, the cell culture media for at least 2, 3, 4, or 5 or more antimicrobial agent inoculates contain different combinations of antimicrobial agents.
In some embodiments, the microorganism is susceptible to the antimicrobial agent if the quantity of nucleic acid molecules of the microorganism in the antimicrobial agent-free inoculate is more than the quantity of nucleic acid molecules of the microorganism in an inoculate comprising the microorganism and the antimicrobial agent. In some embodiments, the microorganism is not susceptible to the antimicrobial agent if the quantity of nucleic acid molecules of the microorganism in the antimicrobial agent-free inoculate is nearly equal, equal, or less than the quantity of nucleic acid molecules of the microorganism in an inoculate comprising the microorganism and the antimicrobial agent.
In certain embodiments, the methods and systems disclosed herein may further comprise generating one or more reports. In some embodiments, the methods disclosed herein further comprise transmitting one or more reports. In some embodiments, the report includes information on the susceptibility of a microorganism to one or more antimicrobial agents or combinations of antimicrobial agents. In some embodiments, the report provides recommendations on a therapeutic regimen. In some embodiments, the report provides recommendations on the dosage of an antimicrobial agent.
Embodiments of the present invention will now be illustrated with reference to the following examples which should not be used to construe or limit the scope of the present invention.
A Cook Medical MINC Benchtop incubator was modified to house a brushless DC motor and spinchuck. The motor was programmed to oscillate at an angular acceleration of 240 rad/s2 and with an oscillation angle of 180 degrees. Microfluidic cartridges were laser cut from poly(methyl methacrylate) (PMMA) using a Trotec® Speedy 360 laser engraver. According to one example, the incubator and cartridge design are illustrated in
Bacteria were cultured overnight by diluting 5 μL of stock E. coli glycerol with 5 mL of cation-adjusted Mueller Hinton (MH2) broth, diluted, recultured and rediluted to obtain a desired final concentration of 5×105 colony-forming units per milliliter (CFU/mL). Two hundred microliters of the diluted bacteria-MH2 broth solution was added into each incubation chamber of the incubation cartridge or to a 96-well plate and immediately sealed with the half-breathable membrane.
The cultures were placed in either the modified incubator of
Analysis was conducted on 150 μL of sample using a Luminex MagPix assay instrument with custom capture probes designed to hybridize with oligos on Luminex MagPlex-TAG microspheres. The total number of rRNA copies in the sample was determined at 0, 60, and 90 minute time intervals.
In this Example, using the relevant materials and methodology described in Example 1, bacterial were grown on two separate incubation cartridges in an incubator spinstand, and compared to a 96-well plate on an spin-stand incubator. The first incubation cartridge did not include an air permeable membrane while the second incubation cartridge did include an air permeable membrane.
In this Example, using the relevant materials and methodology described in Example 1, bacteria were grown on an incubation cartridge in an incubator spinstand or in 96-well plate in a tabletop shaking incubator. Both the incubation cartridge and the 96-well plate included either liquid or dried down antibiotic agents (“Abx”) (e.g., ampicillin (“A”), cefazolin (“C”), ciprofloxacin (“Q”), or ceftriaxone (“X”)) in some chambers, in addition to some agent-free chambers.
The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.
While this invention has been described with reference to illustrative embodiments and examples, the description is not intended to be construed in a limiting sense. Thus, various modifications of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to this description. It is therefore contemplated that the appended claims will cover any such modifications or embodiments.
All publications, patents and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
This application is a continuation of U.S. application Ser. No. 16/641,793, filed on Feb. 25, 2020, which claims the benefit of PCT Application No. 2018/048906, filed Aug. 30, 2018, which claims the benefit of priority of U.S. provisional patent application Ser. No. 62/552,332, filed Aug. 30, 2017; which are entitled Device for Optimization of Microorganism Growth in Liquid Culture. The contents of these applications being incorporated herein in their entirety by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62552332 | Aug 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16641793 | Feb 2020 | US |
| Child | 18234171 | US |
