New bioactive test structure for tracking single cells using gelling agents

Abstract

The present invention relates to a novel bioactive test structure for single cell tracking using a gelling agent and a bioactive test system including the test structure. The present invention also relates to bioactive tests, drug sensitivity tests, antibiotic screening and diagnostic methods using the test structure. The bioactive test structure of the present invention realizes very fast and simple drug sensitivity tests, drug screening and bacterial diagnosis of bacteria, especially Mycobacterium tuberculosis. In particular, the use of the test structure allows DST and bacterial diagnosis to be performed only by pretreatment, without the need to concentrate human sputum samples, without considering the inoculation effect, ensuring faster, more accurate and simpler testing compared to conventional tuberculosis diagnosis or DST systems. In addition, the test structure of the present invention is capable of performing tuberculosis diagnosis and drug sensitivity tests at the same time. Therefore, the present invention provides an effective alternative to the prior art.

Description

Technical Field

[0001] The present invention relates to a novel bioactivity test structure for tracking single cells using a gelling agent and a bioactivity test system including the test structure.

[0002] In addition, the present invention also relates to biological activity testing, drug sensitivity testing, antibiotic screening and diagnostic methods using the above test structure. Background Art

[0003] Tuberculosis (TB) is an infectious disease that threatens the health of one-third of the world's population. Despite the existence of anti-TB drugs, about 1% of the world's population is infected with TB and 1.3 million people die from TB each year. Due to poor management of TB patients or suspected TB populations, multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are gradually increasing. In recent years, the first case of completely drug-resistant tuberculosis (TDR-TB) has been reported in India. In order to reduce the spread of tuberculosis and improve the efficacy of tuberculosis patients, rapid and accurate drug treatment is needed after drug sensitivity testing (DST). Diagnosis of TB resistance to certain drugs in standard first-line anti-TB agents is very important in determining MDR-TB, XDR-TB and XDR-TB and avoiding ineffective drug treatment.

[0004] Commonly used DST methods are based on culturing cells on solid and liquid culture media. According to the solid culture media culture method, mycobacterium tuberculosis (MTB) is inoculated in a solid culture media comprising antibiotics, and is observed with naked eye test whether bacterium colonies are formed. Due to the slow growth of MTB (cell differentiation for about 24 hours), conventional DST methods take at least 4-6 weeks. For liquid culture media, MTB growth is accelerated, and the time required for DST can be reduced. The commercial DST platform of the liquid culture media referred to as the MGIT960 system can be utilized. The MGIT system tests the fluorescent signal generated as MTB grows and consumes oxygen. The DST time of the MGIT system is as short as about 1 week, but this method causes an increase in error rate, because the growth of MTB cells is not directly observed, but indirectly determined.

[0005] In order to shorten the DST time, a direct observation microscope observation drug sensitivity (MODS) method was developed. According to the method, sputum samples are inoculated in 24-well plates together with drugs, and colony formation is monitored every day, which plays an important role in determining whether MTB grows. The method can produce DST results within a week. However, since the single cell of MTB cannot be fixed in a liquid culture medium, it is impossible to track the single cell. The method can produce DST results within a week. However, since the single cell of MTB cannot be fixed in a liquid culture medium, it is impossible to track the single cell. In order to obtain DST results, microscopic observation in the entire well area is essential for testing colony formation, and long-term observation is inevitable.

[0006] Many lab-on-a-chip technologies have been developed for single-cell tracking: arrays using microscale wells, passive microfluidic capture, active valve microfluidic systems, and droplet microfluidics. In array approaches using microscale wells and passive microfluidic capture, cells settle by gravity and are captured in a weir plate by fluid flow. However, the difficulties associated with controlling the fluid environment hinder multiplexed assays for drug testing. To better control cell and drug loading, active valve microfluidic approaches use computer-controlled pneumatically actuated capture methods to accurately control small amounts of liquid through multiple control elements. However, this approach relies on complex controls, which come with high costs and excessive operational efforts. Therefore, this approach is not suitable for routine clinical drug testing. Droplet microfluidics uses a small number of chambers for multiplexed drug testing. However, these approaches cannot guarantee the immobilization of bacterial cells within the droplets, which is necessary for single-cell tracking.

[0007] Inoculum effect (IE) is an undesirable phenomenon in the laboratory and may lead to an overestimation of resistance in vitro, resulting in an increase in the minimum inhibitory concentration (MIC). Therefore, inoculum effect worsens experimental reproducibility. Moreover, since MIC is the parameter that determines the microbial efficacy of antibiotics in pharmacodynamics, different MIC values ​​across cell density can limit the clinical evaluation of antibiotics. Summary of the invention

[0008] Problems to be solved by the present invention

[0009] The inventors have made careful and intensive research to develop effective and rapid diagnostic methods for bacteria, particularly Mycobacterium tuberculosis, DST and screening, resulting in the invention of a new chamber (referred to as the "DAC system") to make a thin layer of agarose mixture with an unobstructed surface above the thin layer for supplying liquid culture medium and drugs. In addition, the inventors have found that the DAC system is effective for rapid and simple drug DST, drug screening and tuberculosis diagnosis. Based on these findings, the present invention was achieved.

[0010] Therefore, an object of the present invention is to provide a novel bioactivity test structure and a bioactivity test system comprising the same.

[0011] In addition, another object of the present invention is to provide a method for bioactivity testing, antibiotic sensitivity testing of bacteria, antibiotic screening, and bacterial diagnosis using a bioactivity test structure.

[0012] Means of solving the problem

[0013] One aspect of the present invention provides a biological activity test structure, which includes a plate body, one or more culture units 100 arranged on the plate body, and a bottom plate 110, under which a solid film is formed on the bottom surface of each culture unit.

[0014] Biological activity tests may be drug sensitivity, drug screening, or bacterial culture diagnostic tests.

[0015] Each culture unit may further have a receiving recess 120 formed on the bottom plate.

[0016] Each culture unit may have an inlet 111 formed in the bottom plate, and a mixed solution of a liquid culture medium containing a gelling agent and a biological agent is introduced through the inlet 111 .

[0017] Each culture unit 100 may have one or more connection members 113 through which the base plate 110 is connected to the blocking portion (partition wall) of the plate body and a cutting groove 114 formed between the connection members.

[0018] Each culture unit may have one or more through holes 112 formed in the substrate 110 .

[0019] Preferably, each culture unit 100 has a size of 1-50 mm×1-50 mm and a height of 1-50 mm.

[0020] Preferably, the receiving recess 120 has a width of 1-5 mm and a height of 1-3 mm.

[0021] The mixed solution is solidified under the base plate to form a solid film in which the biological agent is fixed.

[0022] The film may have a thickness of 1 μm to 10 mm, and the density of the bioagent in the mixed solution may be 10 1 Up to 10 80 cfu / ml.

[0023] The gelling agent is not limited to a specific kind, and it may be agar, agarose, gelatin, alginate, collagen or fibrin.

[0024] The bioactive agent may be further introduced into the culture unit, and more preferably, introduced into the containing recess in a freeze-dried form.

[0025] The plate body, the culture unit and the bottom plate may be made of transparent or translucent materials.

[0026] Another aspect of the present invention provides a bioactivity test method, which includes: providing a mixed solution of a liquid culture medium containing a gelling agent and a biological agent to a bottom surface of a bioactivity test structure; solidifying the mixed solution to form a solid film, wherein the biological agent is fixed in the solid film; supplying the biological active agent to the solid film and allowing the biological active agent to diffuse into the solid film; and observing the individual response of a single cell of the biological agent to the biological active agent or observing whether the biological agent forms colonies or colony forming units (CFU).

[0027] The method may further include imaging the individual response of a single cell to the biological agent, a colony or a colony forming unit (CFU) of the biological agent, and analyzing the image. More preferably, the method further includes determining the minimum inhibitory concentration (MIC) of the biological agent based on the analysis of the image.

[0028] The present invention also provides an antibiotic sensitivity test method for bacteria, which comprises: providing a mixed solution containing a gelling agent and a liquid culture medium of a bacterial strain to the bottom surface of a biological activity test structure; solidifying the mixed solution to form a solid film, wherein the bacterial strain is fixed in the solid film; providing an antibiotic to the solid film and allowing the antibiotic to diffuse into the solid film; and observing the individual response of a single cell of the bacterial strain to the antibiotic or observing whether the bacterial strain forms a colony or a colony forming unit (CFU).

[0029] Preferably, the method further comprises imaging the individual responses of single cells, colonies or colony forming units (CFU), analyzing the images and determining the minimum inhibitory concentration (MIC) of the antibiotic based on the image analysis.

[0030] The bacterial strain can be Mycobacterium tuberculosis or nontuberculous mycobacteria (NTM). Mycobacterium tuberculosis can be multidrug-resistant (MDR) tuberculosis, extensively drug-resistant (XDR) tuberculosis, or totally drug-resistant (TDR) tuberculosis.

[0031] This method showed no inoculation effect.

[0032] Another aspect of the present invention provides an antibiotic screening method, which includes: providing a mixed solution containing a gelling agent and a liquid culture medium of a bacterial strain to the bottom surface of a biological activity test structure; solidifying the mixed solution to form a solid film, wherein the bacterial strain is fixed in the solid film; providing an antibiotic to the solid film and allowing the antibiotic to diffuse into the solid film; and observing the individual response of a single cell of the bacterial strain to the antibiotic or observing whether the bacterial strain forms a colony or a colony forming unit (CFU).

[0033] The method may also include imaging the individual responses of single cells, colonies, or colony forming units (CFU), analyzing the images, and determining the minimum inhibitory concentration (MIC) of the antibiotic based on the image analysis.

[0034] Another aspect of the present invention provides a method for bacterial diagnosis, which includes: providing a mixed solution containing a gelling agent and a liquid culture medium of a bacterial strain to the bottom surface of a biological activity test structure; solidifying the mixed solution to form a solid film, wherein the bacterial strain is fixed in the solid film; providing a culture medium of the bacterial strain to the solid film and allowing the culture medium to diffuse into the solid film; and observing the individual response of a single cell of the bacterial strain or observing whether the bacterial strain forms colonies or colony forming units (CFU).

[0035] Culture media of bacterial strains can be further supplemented with antibiotics, and diagnostic methods can be performed simultaneously with drug sensitivity testing.

[0036] Another aspect of the present invention provides a bioactivity test system, which includes: a bioactivity test structure, the bioactivity test structure includes a culture unit and a bottom plate, the culture unit is provided with a bioactive agent, and a mixed solution of a liquid culture medium containing a gelling agent and a bioactive agent is solidified to form a thin film on the bottom surface of each culture unit; a table for supporting and observing the test structure; and a measurement system, which is used to image the individual response of a single cell of the bioactive agent or the formation of a colony of the bioactive agent, or to image the colony forming unit (CFU) when the bioactive agent is delivered by diffusion, analyze the image and determine the minimum inhibitory concentration (MIC) of the bioactive agent based on the image analysis.

[0037] The measurement system may include an imaging system and an image processing system.

[0038] Effects of the Invention

[0039] The bioactive test structure of the present invention realizes very fast and simple bacteria, particularly drug sensitivity test, drug screening and bacterial diagnosis of Mycobacterium tuberculosis. In particular, using the test structure, DST and bacterial diagnosis can be performed only by pretreatment, without the need to concentrate human sputum samples, without considering the inoculation effect, thereby ensuring fast, accurate and simple testing compared with conventional tuberculosis diagnosis or DST systems. In addition, the test structure of the present invention can simultaneously perform tuberculosis diagnosis and drug sensitivity test. Therefore, the present invention provides an effective alternative to the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 A schematic diagram of a DAC chip according to the present invention is shown.

[0041] Figure 2a and 2b 2 are a perspective view and a plan view, respectively, of a first embodiment of a test structure according to the invention.

[0042] Figure 3a and 3b are a plan view and a perspective view, respectively, of a second embodiment of a test structure according to the invention.

[0043] Figure 4 A detailed diagram showing a DAC chip and a DST method using the DAC chip is shown.

[0044] Figure 5 The diffusion of Rhodamine B as an antibiotic from agarose is shown.

[0045] Figure 6 Shown are time-lapse and processed images of Mycobacterium tuberculosis after drug susceptibility testing.

[0046] Figure 7 shows that by using continuous digital data processing Figure 6 Growth curves of resistant and sensitive samples were plotted based on the images.

[0047] Figure 8 The time required to detect the growth of M. tuberculosis in 49 H37Rv sputum samples by the N-acetyl-L-cysteine-NaOH (NALC-NaOH) method performed on the DAC system and the MGIT 960 system was compared.

[0048] Fig. 9 The results of the NALC-NaOH method or the direct inoculation method (without concentration) performed on a DAC system and an MGIT 960 system were compared.

[0049] Fig.10 The number of colonies counted in 7H11 medium used to calculate the inoculum concentration is shown.

[0050] Fig.11 Images showing the growth of the first clinical strain isolated from TDR 102 patient at different concentrations of drugs to calculate the MIC of the drugs.

[0051] Fig.12 It is through quantification Fig.11 The image obtained is shown in Figure .

[0052] Fig.13 Images showing the growth of a second clinical strain isolated from TDR 103 patient at different concentrations of drugs to calculate the MIC of the drugs.

[0053] Fig.14 It is through quantification Fig.13 The image obtained is shown in Figure . DETAILED DESCRIPTION

[0054] The present invention will be described in more detail with reference to the following examples. However, these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0055] Example 1: Development of DAC system and verification of DST

[0056] 1.1. Sample

[0057] Agarose was used as the MTB fixation material. The agarose solution was mixed with the TB stock solution, and there was a risk of heat shock if a high temperature agarose solution was used. To reduce this risk, a 0.5% concentration of agarose was used, as this concentration was low enough to prevent solidification at 37°C. The lower concentration of agarose enhanced the diffusion of the culture medium and drugs. The drugs used for DST were purchased from Sigma-Aldrich. The liquid culture medium was Middlebrook 7H9 broth supplemented with 10% OADC.

[0058] 1.2. Strains

[0059] The standard strain, Mycobacterium tuberculosis H37Rv, and the clinical strains, MDR and XDR-TB, were obtained from the Korean Tuberculosis Research Institute. The tuberculosis strains were pre-cultured on LJ medium until several colonies were present for the DST process.

[0060] 1.3. Chip design and fabrication

[0061] For microscopic time-lapse imaging of TB, a test chip was needed to immobilize TB in agarose to ensure adequate drug delivery and optical transparency. To meet these requirements, a new chip, the disc agarose channel (DAC) chip, was designed and fabricated.

[0062] The schematic diagram of the DAC chip is as follows Figure 1 shown.

[0063] Figure 2a and 2b as well as Figure 3a and 3b Detailed perspective and plan views of the DAC chip are shown.

[0064] The DAC chip consists of a disk-shaped channel (bottom plate) filled with a mixture of agarose and TB. The chip also has structures that enhance the diffusion of culture medium and drugs, such as wells containing culture medium, drugs, open spaces and holes.

[0065] Specifically, the DAC chip is basically composed of one or more culture units and a bottom plate arranged on a plate body, and a solid film will be formed on the bottom surface of each culture unit below the bottom plate. Each culture unit can also have a accommodating recess formed on a disc-shaped channel (bottom plate). Each culture unit can have an inlet formed in the bottom plate, through which a mixture of agarose and TB is introduced. Each culture unit has one or more connecting members, through which the bottom plate is connected to the blocking portion of the plate body and the cutting groove formed between the connecting members. Each culture unit can have one or more through holes formed in the bottom plate. The depth of the disc-shaped channel (e.g., 300 μm) is determined by the height of the spacer. The DST of TB requires a long incubation time and a sufficient amount of culture medium should be provided to TB. However, it is inconvenient to use a syringe pump to provide culture medium and limits the processing capacity of the system. Therefore, each hole is designed to have the same size as a single hole in a 24-well plate. Specifically, the diameter of the hole is 11 mm and the height of the hole is 10 mm. These holes contain 1 ml of culture medium, which is suitable for incubation for more than 1 month.

[0066] Fabricating the chip using PDMS limits the size of the chip and has low manufacturing yields. Therefore, injection molding is used for DAC chip fabrication. After designing the chip using a 3D design tool (SolidWorks), the aluminum mold is processed. Poly(methyl methacrylate) (PMMA) is used to produce the chip. After the chip is manufactured, a polycarbonate membrane is bonded to the bottom of the chip using a solvent bonding method. The chamber is manufactured by a 3D design tool and an injection molding process. In addition, the lower membrane, polycarbonate, is bonded under pressure (80% by weight ethanol and 20% by weight 1,2-dichloroethane, Sigma-Aldrich). The chip is then treated with O2 plasma to ensure hydrophilicity. The chip is then treated with gamma rays for 5 hours for sterilization.

[0067] 1.4.DST Process

[0068] For DST in DAC chip, the chip was treated with O2 plasma to ensure hydrophilicity and then sterilized with gamma irradiation. TB stock solution was prepared by collecting colonies from LJ medium using a sterilized loop. Aggregated colonies were dispersed in a tube with a U-shaped bottom by vortexing with 2mm diameter glass beads. For safety reasons, the vortexed tube was left to stand for 15 minutes. After stabilization, 7H9 medium containing 10% OADC was added to prepare a stock solution containing McFarland 1.5-3.0. The stock solution was mixed with 0.5% agarose at 37°C by vortexing. Subsequently, 40μl of 0.375% agarose mixture containing TB stock solution was loaded into the inlet of the DAC chip. The agarose was then allowed to solidify at room temperature for 1 minute. A 0.5ml aliquot of 7H9 containing 10% OADC and TB drugs was then added to the DAC chip. The drugs in the culture medium were then diffused into the agarose. After this process, the DAC chip was sealed with a gas-permeable membrane to prevent evaporation of the culture medium and incubated in a temperature-controlled culture chamber at 37°C. Using the time-lapse method, an area at the edge of the agarose was imaged every day on an inverted microscope with a ×40 lens. These procedures were all performed on a clean bench.

[0069] 1.5. Using 3D Culture DAC Chips for DST of MTB

[0070] In the present invention, agarose is used as a 3D culture matrix for DST of MTB. In order to culture MTB for a long time, a stable 3D culture matrix is ​​required.

[0071] Figure 4 Detailed diagrams of the DAC chip and the DST method using the DAC chip are shown. To verify the stability of the agarose matrix, 0.5% agarose was mixed with microbeads in a volume ratio of 3:1 in phosphate buffered saline. The mixture was added to the inlet of the DAC chip, and 7H9 culture medium was added to the well. For the DST conditions of MTB, the chip was cultured in the chamber at 37°C. In the same area, the beads in the agarose matrix were well observed for 3 weeks, indicating that the 3D culture matrix is ​​sufficiently stable for the DST of MTB. When H37Rv was cultured in the agarose matrix, the structure was also maintained well during the 3-week single cell tracking. In order to ensure a uniform supply of liquid culture medium and drugs, the agarose matrix was suitably formed in a mold. In order to visualize the formation of the agarose matrix, a 0.5% agarose mixture containing a 3 (food dye): 1 (agarose) volume ratio of food dye was loaded into the chip and imaged. The agarose matrix is ​​well formed on the DAC chip.

[0072] 1.6. Diffusion characteristics of DAC chips

[0073] Agarose is used to fix TB. TB drugs are dissolved in liquid culture medium, 7H9 broth, to be delivered to agarose matrix. In the present invention, the drug concentration (imaging area) at the corner of the DAC chip needs to be uniform to ensure the accuracy of the DST results. The time required for small molecules (molecular weight 200) to diffuse into the imaging area is calculated. The concentration of molecules in the imaging area is the same. Rhodamine B (molecular weight 479.02g / mol) is used to visualize the diffusion properties of the agarose matrix in the DAC chip because its molecular weight is similar to that of TB drugs. The chip is treated with these molecules at various concentrations and imaged at 0, 10, 30 and 60 minutes to evaluate the uniformity of diffusion. The fluorescent signal becomes uniform within 30 minutes of loading the fluorescent dye, which means that the system is suitable for DST (considering the cell division of MTB, about 20-24 hours). The growth rate of MTB is the same throughout the entire area of ​​the chip, which shows that the liquid culture medium and the drug diffuse at the same level in the chip.

[0074] Figure 5 The diffusion of the antibiotic Rhodamine B from agarose is shown. Specifically, Figure 5 In the figure, A shows an image taken immediately after loading with rhodamine B, and B, C, and D show images taken sequentially at 10-minute intervals, respectively. The diffusion time was 0.1 seconds.

[0075] 1.7. Tracking single cells in DAC chips

[0076] Conventional DST methods rely on measuring MTB growth by visual observation of colony formation on solid culture media or on fluorescence-based indirect detection methods that measure oxygen consumption in liquid culture media.

[0077] The single cell growth tracking method of the present invention is used to measure the growth of MTB under various drug conditions. The MTB cells on the DAC chip are tracked every day and for several days. One imaging area (200 μm×300 μm) contains many TBs of different sizes because MTB cells are prepared from colonies (large cluster forms) on solid culture media.

[0078] 1.8. Image Processing for Automatic Sensitivity Determination

[0079] The conventional DST method on solid medium (LJ medium) relies on the visual assessment of MTB growth by experienced experimenters. Therefore, this method is subject to human error.

[0080] In the present invention, automatic image processing is used to determine the drug sensitivity of MTB and avoid human error. Microscopic time-lapse images with a CCD camera are processed to digitize the growth of MTB under TB drug conditions. The raw images of the CCD camera are processed into binary form. Although not all white areas represent growing MTB cells because the boundaries of the MTB colonies are unclear and the imaging area contains some debris, most of the white areas are due to the growth of MTB in the agarose matrix and are therefore proportional to this parameter.

[0081] Figure 6 Time-lapse images and processed images are shown.

[0082] A) and A') are examples of drug resistance, and B) and B') are examples of sensitivity. In the drug resistance example, the formation of many colonies in the imaged area was found. After image processing, the white area represents the MTB colonies growing in the image. In the sensitivity example, there is no difference in the growth of MTB in the image.

[0083] Calculate the white area in the image by counting the number of pixels. Using continuous digital data, Figure 7 The growth curves over time were plotted in Figure 2. The drug sensitivity of TB strains at different concentrations of TB drugs was determined by setting thresholds.

[0084] Figure 7 Quantified growth curves for the resistant and sensitive examples are shown. The growth curves were constructed by measuring the area occupied by the growth region of MTB in the image. The area occupied by the bacterial region in the resistant example increased continuously, while there was no obvious change in the sensitive example.

[0085] 1.9. DST for MTB using DAC system

[0086] The MTB DST is based on the estimation or measurement of the growth or non-growth of an MTB strain in the presence of a single “critical concentration” of a drug. If growth is observed, the critical concentration of the anti-TB drug represents clinically relevant resistance, and sensitive MTB strains are inhibited at that critical concentration. In the DAC system, the critical concentration of each anti-TB drug is determined by the critical concentration of the MGIT because the DAC system is more similar to a liquid culture system than a solid culture system. After the critical concentration of each anti-TB drug is determined, the DST is performed using the DAC system at serial two-fold concentrations close to the critical concentration to obtain the minimum inhibitory concentration (MIC) and susceptibility of MTB.

[0087] The MIC value of an anti-tuberculosis drug can be determined by adequate testing at each drug concentration. Thereafter, when the MIC value is equal to or lower than the critical concentration, the strain is considered sensitive, while a resistant strain is defined by a MIC value exceeding the critical concentration. Specifically, the critical concentration of rifampicin is 1.0 μg / ml in the DAC system. DST using the DAC system was performed at rifampicin concentrations of 0.25 μg / ml, 0.5 μg / ml, 1.0 μg / ml, and 2.0 μg / ml. A growth curve of the concentration of each anti-tuberculosis drug as a function of incubation time was plotted. Sensitivity was determined by measuring the MIC value based on the critical concentration.

[0088] With the increasing number of patients infected with MDR, XDR and TDR worldwide, rapid DST is urgently needed for public health and diagnosis. The DAC system is based on the concept of microfluidic channels to achieve rapid DST. To validate the DAC system for rapid SDT, a virulent standard strain of MTB, H37Rv, and clinical strains of MDR and XDR strains were tested to determine the susceptibility to isoniazid, rifampicin, streptomycin, and ethambutol.

[0089] The commonly used LJ medium-based DST was used for quality control and as a comparison target because it is considered the “gold standard” for DST. The MIC values ​​of isoniazid, rifampicin, streptomycin, and ethambutol against H37Rv, MDR, and XDR were determined by using the DAC system.

[0090] From these MIC values, the susceptibility of H37Rv, MDR, and XDR to isoniazid, rifampicin, streptomycin, and ethambutol was determined to be below the critical concentration or by measuring the MIC values ​​at concentrations other than the critical concentration.

[0091] Images of all tested strains were collected at the same location every other day for 5 days using the time-lapse method. Images were processed using daily time-lapse data for 5 days and curves were drawn to determine the MIC values ​​of the four anti-TB drugs.

[0092] The results are shown in Table 1.

[0093] [Table 1] DST results measured using the DAC system

[0094]

[0095] The susceptibility of H37Rv, MDR and XDR TB strains to four primary TB drugs, isoniazid (INH), rifampicin (RFP), streptomycin (SM) and ethambutol (EMB), was tested. The test concentrations were determined by the critical concentrations of the corresponding drugs.

[0096] After the MIC is determined in the time-lapse images, the values ​​are tested by restraint points and the drug susceptibility is determined.

[0097] In Table 1 , A shows the MIC values ​​of DST obtained from the DAC system, and B shows the sensitivity determined from the MIC data and the drug cutoff concentration.

[0098] As can be seen from Table 1A, the MIC values ​​of isoniazid, rifampicin, streptomycin and ethambutol are 0.05μg / ml, 0.25μg / ml, 2.0μg / ml and 5.0μg / ml, respectively. From the MIC data, it was found that strain H37Rv was sensitive to isoniazid, rifampicin, streptomycin and ethambutol. The same results were obtained in the LJ culture test.

[0099] Two additional test strains from MDR and XDR patients were found to be resistant to all drugs in the DAC system. The sensitivity results were confirmed using the LJ culture method, which is considered the gold standard. All sensitivity results in the DAC system were consistent with the results of the gold standard method. From these validated results, it was demonstrated that the DAC system can shorten the DST time and be used to accurately diagnose the sensitivity and resistance of anti-TB drugs.

[0100] Example 2: Comparison of DAC system with conventional culture diagnostic products

[0101] By comparing the DAC system of the present invention with conventional TM culture diagnosis methods, the usefulness of the DAC system can be confirmed.

[0102] 2.1. Comparison of recovery rates

[0103] Three divided portions of the same sputum sample were tested in the three systems and the results were compared.

[0104] Specifically, 6 / 25 smear-positive pulmonary tuberculosis patients were tested. Among 25 clinical sputum samples, 3 positive samples were found (positive rate was 12.0%). Among the three positive samples, one was positive in Ogawa culture medium, Middlebrook 7H11 culture medium and DAC system, another was positive in 7H11 culture medium and DAC system, and another was positive in DAC system. The number of negative samples in Ogawa culture medium, Middlebrook 7H11 culture medium and DAC system were 23 (negative rate 92.0%), 22 (negative rate 88.0%) and 21 (negative rate 84.0%) respectively. In all methods, one sample was contaminated (4.0%).

[0105] The recovery rates of Mycobacterium tuberculosis in the three culture methods were compared. As a result, the recovery rate obtained in the DAC system (12.0%) was higher than that of Ogawa medium (4.0%) and Middlebrook 7H11 medium (8.0%). The contamination rates of the three methods were the same.

[0106] The detailed comparison results are shown in Table 2.

[0107] [Table 2]

[0108]

[0109] 2.2. Comparison of colony forming units (CFU) with existing technologies

[0110] After pretreatment of 49 H37Rv sputum samples, the samples were inoculated in DAC system and Middlebrook 7H11 medium and their colony forming units (CFU) were counted. The initial CFU was 1×10 4 / ml. CFU values ​​in sputum samples were counted by culture method. It was found that CFU values ​​in DAC system were similar to those obtained by conventional method in Middlebrook 7H11 medium. However, CFU results in DAC system were obtained in 9 days, while CFU results in Middlebrook 7H11 medium were obtained in 21 days. The median CFU in DAC system was 4.27log 10 CFU / ml (SD±0.75), the median CFU in Middlebrook7H11 medium is 4.32log 10 CFU / ml (SD±0.64). Fig.15 )

[0111] 2.3. Comparison of culture detection time with existing technologies

[0112] The tuberculosis culture diagnosis or LJ culture method used in the DST system takes 4-6 weeks. TM ) is currently the fastest product in the culture diagnosis of Mycobacterium tuberculosis. The culture diagnosis time of the DAC system according to the present invention is compared with that of the prior art.

[0113] The results are as follows Figure 8 shown.

[0114] Figure 8 The time required to detect the growth of Mycobacterium tuberculosis in 49 H37Rv sputum samples by the N-acetyl-L-cysteine-NaOH (NALC-NaOH) method performed on the DAC system and the MGIT 960 system was compared.

[0115] like Figure 8 As shown, TTD was 5-13 days in the MGIT 960 system and 5-9 days in the DAC system. The highest detection level of M. tuberculosis in both the MGIT 960 system and the DAC system was 7 days.

[0116] The TTD values ​​of Mycobacterium tuberculosis at different inoculum concentrations were compared and the results are shown in Table 3.

[0117] [Table 3]

[0118]

[0119] Specifically, a total of 49 H37Rv sputum samples were used to detect M. tuberculosis in order to determine TTD values ​​based on CFU values ​​in Middlebrook 7H11 medium.

[0120] In the DAC system, the time required for detection of Mycobacterium tuberculosis was 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, and 42 days, respectively.

[0121] The median TTD value based on the CFU values ​​in Middlebrook 7H11 medium is 7 days (5-6 log 10 CFU / ml) to 9 days (2-3log 10 CFU / ml).

[0122] The TTD value based on the CFU value in Middlebrook 7H11 medium is 6 days (5-6 log 10 CFU / ml) to 12 days (2-3log 10 CFU / ml).

[0123] When the count of Mycobacterium tuberculosis is 2-3log 10 CFU / ml, the median TTD values ​​of M. tuberculosis in the MGIT 960 system and the DAC system were 12 days and 9 days, respectively.

[0124] When the count of Mycobacterium tuberculosis is 3-4log 10 At 144 CFU / ml, the median TTD values ​​of M. tuberculosis in the MGIT 960 system and DAC system were 8 days and 7 days, respectively.

[0125] When the count of Mycobacterium tuberculosis is 4-5log 10 At 40 CFU / ml, the median TTD values ​​of M. tuberculosis in the MGIT 960 system and DAC system were 7 days and 6 days, respectively.

[0126] When the count of Mycobacterium tuberculosis is 5-6log 10 CFU / ml, the median TTD values ​​of M. tuberculosis in the MGIT 960 system and the DAC system were 6 and 7 days, respectively.

[0127] When the count of Mycobacterium tuberculosis is 2-5log 10 CFU / ml, the detection of M. tuberculosis growth was faster in the DAC system (range, 6 to 9 days) compared with the MGIT 960 system (range, 7 to 12 days). 10 CFU / ml, the detection of M. tuberculosis growth was faster in the MGIT 960 system (6 days) compared to the DAC system (7 days).

[0128] Four of the 49 samples (8.2%) were contaminated in the MGIT 960 system. M. tuberculosis in liquid culture showed a higher false positive rate than in the DAC system.

[0129] 2.4. Comparison of the MGIT960 system and the DAC system using the direct inoculation method

[0130] After sputum decontamination, the efficiency of the MGIT960 system and the DAC system were compared using a direct inoculation method (without concentration).

[0131] The results are as follows Fig. 9 shown.

[0132] Fig. 9 The results of the NALC-NaOH method (with concentration) or the direct inoculation method (without concentration) performed on the MGIT 960 system and the DAC system were compared.

[0133] It takes 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42 days to test the growth of M. tuberculosis using the DAC system.

[0134] Before inoculation, 18 H37Rv sputum samples were cleaned up with 2% NALC-NaOH and divided into 2 samples. One sample was centrifuged at 3,000g for 15 minutes according to the standard NALC-NaOH method, and the other sample was used in the direct method (without concentration). The samples pre-treated with the NALC-NaOH method (with concentration) and the direct method (without concentration) were cultured in the MGIT 960 system and the DAC system, respectively.

[0135] It was found that the above two sputum pretreatment methods, such as the DAC system culture method using the direct pretreatment method (without concentration) and the MGIT 960 culture method using the NALC-NaOH method (with concentration), had similar culture morphologies.

[0136] The median TTD was 7 days in the DAC1 (5-7 days), DAC2 (5-7 days), and MGIT1 systems (6-7 days).

[0137] In contrast, in the MGIT2 system, the median TTD required to detect M. tuberculosis in samples was 14 days (7-24 days).

[0138] No contamination was observed in the DAC system, whereas 5.6% and 27.8% were contaminated in the MGIT1 and MGIT2 systems, respectively.

[0139] That is, positive samples were found between 5 and 7 days in the DAC culture method, regardless of sputum pretreatment. In contrast, when sputum samples were pretreated according to the NALC-NaOH method recommended by the supplier, positive samples were found between 6 and 7 days, and a low false positive rate was obtained in the MGIT 960 culture method.

[0140] 2.5. Comparison of time required to obtain true negative results

[0141] For the liquid culture-based MGIT 960 system, 6 weeks are required to detect mycobacteria.

[0142] The time required to obtain a true negative result using the DAC system was measured. The results are shown in Table 4.

[0143] [Table 4] Test time required for the growth of Mycobacterium tuberculosis

[0144]

[0145] The time required to detect the growth of Mycobacterium tuberculosis in three clinical sputum samples (3 positive samples out of 25 clinical sputum samples) and 49 H37Rv sputum samples depended on the recovery rate of mycobacteria in the DAC system, as shown in Table 4 above. The time required to test Mycobacterium tuberculosis in the DAC system was 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35 and 42 days, respectively.

[0146] Of the total 74 samples, 52 samples were positive, 22 samples were negative, and no sample was contaminated. When measured on the 5th day, 6 (11.5%) of the 52 positive samples changed from negative to positive, when measured on the 9th day, 50 (96.2%) of the 52 positive samples changed from negative to positive, and when measured on the 14th day, all samples changed from negative to positive. 22 negative samples did not change for up to 42 days. From these results, it was determined that in the DAC system, significant culture results (p<0.05) were obtained on the 9th day, and all accurate culture results were obtained on the 14th day. It seems that the DAC system is about 28 days faster than the MGIT 960 system to determine true negative results.

[0147] Example 3: Identifying the Presence of Inoculation Effect (IE) in DAC Systems

[0148] The present inventors evaluated the antibacterial activity of rifampicin, isoniazid, ethambutol and streptomycin as primary drugs against tuberculosis using the DAC system and whether the DAC system is affected by changes in cell density. The system can draw reproducible and reliable MIC values ​​of drugs regardless of the inoculum size.

[0149] 3.1. Samples and growth conditions

[0150] Mycobacterium tuberculosis was isolated from pulmonary tuberculosis patients at the Korea Institute of Tuberculosis (KIT). A total of 150 clinical strains, including 134 broadly sensitive strains and 59 drug-resistant strains, were identified using the LJ method before use. All isolates were freshly pre-cultured on LJ medium before use.

[0151] Sputum samples were digested and cleaned by the N-acetyl-L-cysteine-NaOH method described by Kent and Kubica. The processed samples were cultured in MGIT vials for 42 days. On the day of testing, all positive tubes were checked for the presence or absence of AFB by ZN staining, and parallel tubes were used for drug sensitivity testing.

[0152] Three clinical ESBL E. coli strains and one non-ESBL E. coli ATCC strain were used for lactamase substrate analysis. The E. coli strains were grown, subcultured, and analyzed on Mueller-Hinton agar (Difco) in Mueller-Hinton broth (Difco Laboratories, Detroit, MI).

[0153] 3.2. Antibiotics

[0154] All drugs were purchased from Sigma-Aldrich (St. Louis, MO). Powders were stored in a desiccator before use according to the supplier's recommendations. Isoniazid (INH), ethambutol (EMB), streptomycin (SM), and cefepime were dissolved in deionized water (DW), and rifampicin (RIF) was dissolved in DMSO. All stock solutions were sterilized by membrane filtration through a 0.22 μm pore Millex-GS filter unit (Millipore, Bedford, MA). Small aliquots of each stock antibiotic solution were stored at -80°C. Freeze-dried drug solutions were used immediately after thawing, and the remaining material was discarded without storage in a freezer. Working solutions were freshly prepared from stock solutions and serially diluted to achieve the desired concentrations.

[0155] 3.3. DST using DAC system

[0156] M. tuberculosis was identified by ZN staining and grown from LJ slopes or MGIT tubes and subjected to DST. To prepare inoculum from LJ cultures, colonies up to 14 days old were extracted from growing cultures. Colonies were vibrated with glass beads to break up large clumps. To obtain high density (~5 × 10 9 CFU / ml), the suspension was centrifuged at 3000 g for 10 min, and the pellet was resuspended in sterile PBS. Subsequently, the suspension was added to liquid agarose and inoculated in the bacteria-agarose mixture in the range of 10 1 Up to 5×10 8 CFU / ml and loaded into the main channel. After solidification, 7H9 broth containing each antibiotic was applied to the chamber. Cells of all bacteria were imaged using an inverted optical microscope (IS71, Olympus).

[0157] 3.4. LJ ratio method and DST using MGIT 960 system

[0158] The LJ ratio method was used as a reference. All strains were tested at concentrations of 40 μg / ml (RIF), 0.1 μg / ml (INH), 2.0 μg / ml (EMB) and 1.0 μg / ml (SM) according to the standard procedure performed on LJ medium, except that they were tested at 10 1 Up to 5×10 8 A range of CFU / ml was inoculated and all strains were tested according to the supplier's recommendations except for the inoculum size in the DST using the MGIT 960 system.

[0159] To prepare the inoculum for the day of testing, all positive tubes were checked for the presence or absence of AFB by ZN staining. Drug susceptibility testing was performed in parallel in the MGIT 960 and DAC systems at 0, 3, 10 and 15 days after the positive reaction.

[0160] 3.5. DST by microdilution

[0161] In addition to bacterial concentration, the MIC of cefepime against E. coli was determined according to standard CLSI guidelines. Broth microdilution wells were examined after 16 hours of incubation at 37°C.

[0162] 3.6. β-lactamase activity analysis

[0163] The culture of E. coli was grown to the initial logarithmic phase (OD 600 = 0.4). Thereafter, the bacteria were separated from the supernatant by centrifugation at 3,000 g for 10 minutes. The supernatant was filtered through a 0.45 mm filter and the culture filtrate was stored. The pellet was washed twice with sterile PBS and sonicated on ice at 60% power and 30 second pulses for more than 6 minutes and lysed with ice. The crude lysate was centrifuged at 4°C and 20,000 g for 30 minutes and the pellet was resuspended in the same volume of PBS. The corresponding volume of 10 7 Each fraction of CFU / ml bacteria was inoculated in a 96-well plate for analysis. 4.5 μM Fluorocillin Green (Invitrogen) was added to each well, and the plate was gently shaken for 5 minutes at room temperature and cultured at 37°C for 0.5 to 20 hours. Fluorescence changes were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

[0164] 3.7. Analysis of the effective residual concentration of antibiotics in the culture medium

[0165] Two bacterial cultures at high or low density were treated with each antibiotic in liquid culture.

[0166] At each time point, the culture medium was filtered to remove cells and then added to different DST groups to measure the residual efficacy of antibiotics. Broth microassay and DAC protocols were performed in the same manner as described above.

[0167] 3.8. Non-generational inoculation effect on the standard strain H37Rv in the DAC system and comparison between the LJ system and the MGIT system

[0168] To confirm the absence of IE in the DAC system, DST was performed on the standard strain H37Rv at various inoculation concentrations on the DAC system. At the same time, DST was performed on the LJ and MGIT systems at the same concentrations as in the DAC system. 3 Up to 107 ) were used. The inoculum size indicates the concentration of TB in agarose for the DAC system, the concentration of TB stock solution for the LJ system, and the final concentration of TB in the culture bottle for the MGIT system. Four primary drugs, rifampicin, isoniazid, ethambutol, and streptomycin, were used for DST.

[0169] In the DAC system, the MIC values ​​were the same or differed by about 2-fold at various inoculum concentrations (see Table 1).

[0170] In the MGIT system, all strains were confirmed to be ≤10 5 CFU / ml, but due to the high initial inoculum concentration, DST was at 10 6 CFU / ml and 10 7 CFU / ml determination was not possible. Therefore, this was considered an error in the MGIT system. In the LJ system (MKIT), all strains were found to be ≤10 6 CFU / ml is sensitive, but at 10 7 CFU / ml becomes resistant.

[0171] In short, only the DAC system in 10 3 Up to 10 7 The same DST results were shown at inoculum sizes of CFU / ml.

[0172] [Table 5] Comparison of inoculation effect test results on standard strain H37Rv

[0173]

[0174] 3.9. Identification of the absence of inoculum effects on clinical strains in the DAC system

[0175] The standard strain H37Rv showed sensitivity to four anti-tuberculosis drugs. Clinical strains expressed various phenotypes, especially in drug sensitivity. To confirm that the DAC system did not show an inoculation effect on various drug-sensitive strains, five clinical strains obtained from KIT were inoculated with 10 3 Up to 10 7 Different inoculum concentrations of CFU / ml were tested, and this concentration range was proven to be effective in testing standard strains. Rifampicin, isoniazid, streptomycin, and ethambutol were used as test drugs. The actual CFU values ​​of clinical strains are shown in Table 6. For 131 strains, the highest concentration was 1.3×10 9 .

[0176] From 10 3 -10 7The DST results obtained for the five clinical strains with CFU / ml are shown in Table 6. Three strains, KHS, CMS and CUD, showed susceptibility to the three primary drugs. 3 -10 7 The MIC values ​​of the strains were the same or differed only by about two-fold under the inoculum concentration. Therefore, the sensitivity determination was not affected by the change in inoculum concentration, and all strains were found to be sensitive. The 1088 and 131 strains were resistant to all four primary drugs. All MIC values ​​were the same regardless of the inoculum concentration. Overall, no inoculum effect on drug sensitivity was found in the DAC system.

[0177] [Table 6] Results of the inoculation effect on clinical strains on the DAC system

[0178]

[0179]

[0180] 3.10. Accurate DST without adjusting the inoculum using the DAC system

[0181] Accurate DST in the LJ (Mkit) system and MGIT (an automated DST based on liquid culture medium) requires accurate inoculum volume. Therefore, a cumbersome procedure should be performed to adjust the inoculum concentration.

[0182] From previous results, it has been demonstrated that no adjustment of the inoculum concentration is required in the DAC system. In this example, the adjustment of the inoculum concentration was omitted in this test, and the DST was performed directly on the prepared stock solution in the DAC system. Therefore, the inoculum concentration was used for various ranges. Fig.10 As shown in Figure 1, the actual inoculum concentration was obtained by the number of colonies in 7H11 medium. 5 -10 8 CFU / ml. The DST results obtained in the DAC system were compared with those in the LJ (Mkit) system, and their error rates were calculated. A total of 117 strains were tested, with a total of 516 cases.

[0183] No differences were observed for PanS strains that were sensitive to all drugs. In the case of MDR, 8 cases were confirmed to be sensitive (major errors (ME)) and 4 cases were confirmed to be resistant (very major errors (VME)) in the LJ system. A total of 516 tests, the overall error rate was only 6.2%.

[0184] [Table 7] Comparison of DST results and error rates using LJ (Mkit) and DAC systems

[0185] PanS MDR XDR total Total samples 26 50 41 117 Total Test 108 216 192 516 ME 0 8 15 23 VME 0 4 2 6 NG 4 16 28 48 Error rate 0.0％ 6.0％ 10.4％ 6.2％

[0186] 3.11. No inoculum effect based on the use of agarose-encapsulated bacteria

[0187] The two main reasons for the inoculum effect are as follows.

[0188] The first reason is that a large inoculum of antibiotic-degrading proteins derived from bacteria may destroy the antibiotic and lead to an increase in the MIC value. Another reason is that a large inoculum of antibiotic-degrading proteins derived from bacteria may reduce the number of antibiotic molecules relative to a single cell of bacteria and lead to an increase in the MIC. To verify the reason for the absence of inoculum effect in the DAC system, ESBL-negative E. coli (ATCC 35218), ESBL-positive E. coli (ATCC 25922), and clinically obtained E. coli strains (which are well-known bacterial strains that secrete antibiotic-degrading proteins) were incubated at 5×10 7 , 5×10 6 and 5×10 5 The tests were performed at inoculum concentrations of CFU / ml. These concentrations represent the concentration of bacteria in agarose for the DAC system and in liquid for the MDT system.

[0189] Cefepime is a fourth generation cephalosporin antibiotic that was tested to demonstrate the presence of an inoculum effect. The MICs for ESBL-negative and ESBL-positive E. coli ranged from 0.015 μg / ml to 0.12 μg / ml.

[0190] [Table 8] Comparison of AST results from DAC system and MDT system at various inoculation concentrations

[0191]

[0192] In the DAC system, no inoculum effect was observed in various inoculum sizes. In contrast, in the MDT system, ESBL+ and #5 strains showed gradually increasing MIC values ​​with increasing inoculum sizes. These results indicate that, unlike the MDT system, there is no inoculum effect in the DAC system.

[0193] The present inventors hypothesized that the absence of an inoculum effect in the DAC system could be due to the fact that proteins released from the bacteria were captured by the agarose and, unlike in broth culture conditions, were unable to degrade the antibiotics. To demonstrate the efficacy of this hypothesis, additional tests were performed using an ESBL-positive E. coli strain at an inoculum concentration of 5 × 10 7 , 5×10 6 and 5×10 5CFU / ml. These concentrations represent the bacterial concentration in all wells of the DAC system and in the liquid of the MDT system. Therefore, the final concentration of bacteria in the DAC system and the MAC system is the same. Cefepime was used for these tests. After overnight incubation, the antibiotic solutions in the wells of the DAC and MDT systems were extracted and filtered to obtain pure antibiotics. MDT using ESBL-positive E. coli strains was performed using antibiotics. The results are shown in Table 9.

[0194] [Table 9] Comparison of AST results using the DAC system and the MDT system after overnight culture in antibiotic solution

[0195]

[0196] In the DAC system, no differences in MIC values ​​were produced in various inoculum sizes, indicating that the degradation rate was similar to the inoculum size. In contrast, in the MDT system, MIC values ​​increased with increasing inoculum size, indicating enhanced degradation of the antibiotic. These results suggest that the reduced degradation of the antibiotic in the DAC system is attributed to the capture of bacteria in agarose.

[0197] That is, the DAC system of the present invention has the ability to control the molecular interactions and dynamics of bacterial populations, which are caused by the excessive binding of drugs to bacteria and the resulting products when the cell density increases. The ability of the DAC system is because the bacterial population is fixed within the matrix and the culture medium containing each drug is supplied to the chamber in a gradient mode.

[0198] This system facilitates DST without requiring strict limits on the inoculum on mycobacteria, since the inoculum is greater than 10 7 No IE was observed when the inoculum size was 1.5 mycobacterium. Therefore, the DAC system can accurately predict the microbial efficacy of anti-tuberculosis drugs and is expected to effectively replace the commonly used methods.

[0199] Example 4: Application of DAC system in antibiotic screening

[0200] The DAC system of the present invention is applied to antibiotic screening.

[0201] Specifically, the MIC of the anti-tuberculosis drug Q203 developed by the applicant's subcontractor was measured to confirm the sensitivity of the TDR clinical strains.

[0202] The MIC of the drug against the first clinical strain from TDR 102 patient was determined by treating it with 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500 and 1000 ng / ml of Q203 on the DAC system. Fig.11 The image is quantified and plotted. The result is shown in Fig.12 shown.

[0203] The MIC of the drug against the second clinical strain from the TDR 103 patient was determined by treating it with Q 2O 3 in the same manner as above. Fig.13 The image is quantified and plotted. The result is shown in Fig.14 shown.

[0204] Mode for Carrying Out the Invention

[0205] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are provided so that the disclosure will be thorough and complete, and the scope of the present invention will be fully conveyed to those skilled in the art. Therefore, the present invention can be implemented in many different forms, and should not be construed as being limited to the exemplary embodiments set forth herein. In the accompanying drawings, for the sake of clarity, the size of the element, such as width, length and thickness, may be exaggerated. The accompanying drawings are explained from the viewpoint of the observer. It should be understood that when an element is referred to as being "on" another element, it may be directly on another element, or one or more intermediate elements may also be present. It will be understood by those skilled in the art that many modifications and variations may be made without departing from the spirit of the present invention. In all drawings, the same reference numerals are used to represent substantially the same elements.

[0206] One aspect of the present invention provides a biological activity test structure, which includes a plate body, one or more culture units 100 arranged on the plate body, and a bottom plate 110, under which a solid film will be formed on the bottom surface of each culture unit. Figure 2a and 2b 2 are a perspective view and a plan view, respectively, of a first embodiment of a test structure according to the invention.

[0207] refer to Figure 2a and 2b , each culture unit may have an inlet 111 formed in the bottom plate, and a mixed solution of a liquid culture medium containing a gelling agent and a biological agent is introduced through the inlet 111. Each culture unit 100 may have one or more connecting members 113, through which the bottom plate 110 is connected to a blocking portion of the plate body and a cutting groove 114 formed between the connecting members. Each culture unit may have one or more through holes 112 formed in the bottom plate 110. The bioactive agent may be uniformly diffused into the solid film through the cutting groove 114 and the through hole 112.

[0208] The thickness and width of the solid film depend on the depth and width of the accommodating recess 120. The term "film" used herein refers to a thin layer having a thickness sufficient to fix the biological agent and observe a single cell. For example, the thickness of the film may be in the range of 1 μm to 5 mm, 1 μm to 3 mm, 1 μm to 2 mm, 1 μm to 1.5 mm, 1 μm to 1 mm, 1 μm to 800 μm, 1 μm to 500 μm, 1 μm to 100 μm, 10 μm to 3 mm, 10 μm to 1 mm, 100 μm to 1 mm, 200 μm to 1 mm, or 500 μm to 1 mm, but is not particularly limited to this range. The thickness of the solid film may correspond to the dimension in the vertical direction of the surface of the solid film to be observed. Within the above-mentioned range of the thickness of the solid film, a single cell of the biological agent fixed in the solid film may be observed, the formation of colonies may be effectively observed, or the colony forming units may be counted.

[0209] The width (size) of each culture unit is not limited and can vary depending on the type and characteristics of the biological agent. Preferably, the size of each culture unit is 1-50mm×1-50mm, 1-30mm×1-30mm, 5-50mm×5-50mm, 5-30mm×5-30mm, 10-50mm×10-50mm, 10-30mm×10-30mm or 1-20mm×1-20mm. Most preferably, the size of the culture unit is 12mm×12mm.

[0210] The height of each culture unit is also not limited and can vary according to the type and properties of the biological agent and bioactive agent. For example, each culture unit can have a height of 1 to 50 mm, 1 to 30 mm, 5 to 30 mm, or 5 to 15 mm.

[0211] There is no limitation on the size of the through hole 112. The diameter of the through hole 112 is usually 1 mm or less, for example, 0.99 mm or less, 0.90 mm or less, 0.85 mm or less, 0.80 mm or less, 0.75 mm or less, 0.70 mm or less, 0.65 mm or less, 0.60 mm or less, 0.55 mm or less, 0.50 mm or less, 0.45 mm or less, 0.40 mm or less, 0.35 mm or less, 0.30 mm or less, or 0.25 mm or less.

[0212] In one embodiment of the present invention, a mixed solution of a liquid culture medium containing a gelling agent and a biological agent may be solidified under a substrate to form a solid film in which the biological agent is immobilized.

[0213] The liquid culture medium may contain about 80% or more of water or a buffer solution. The liquid culture medium may be solidified due to the presence of a gelling agent. As a gelling agent, agar, agarose, gelatin, alginate, collagen or fibrin may be cited. Agar or agarose is preferably used. For example, agar may be used in an amount of 0.5% to 5% by weight in the liquid culture medium. The liquid culture medium generally does not require nutrients. However, in some embodiments, the liquid culture medium may include nutrients.

[0214] Examples of suitable biological agents include viruses, bacteria, fungi, algae, protozoa, parasitic pathogens, human and mammalian cells, and biofilms. Biological agents can be grown in liquid or solid culture media, and their growth may be affected by the type and concentration of external physiologically active agents.

[0215] The density of the biological agent in the mixed solution is not limited. In particular, the present invention is characterized in that the density of the biological agent is independent of the DST or the diagnostic result. That is, the DAC system of the present invention shows no seeding effect. The density of the biological agent can be 10 1 Up to 10 80 cfu / ml, for example 10 70 cfu / ml or lower, 10 60 cfu / ml or lower, 10 50 cfu / ml or lower, 10 40 cfu / ml or lower, 10 30 cfu / ml or less 10 20 cfu / ml or lower, 10 10 cfu / ml or lower, 10 9 cfu / ml or lower, 10 8 cfu / ml or lower, 10 7 cfu / ml or lower, 10 6 cfu / ml or lower, 10 5 cfu / ml or less or 10 4 cfu / ml or lower.

[0216] The mixed solution can be solidified under the base plate to form a solid film in which the biological agent is fixed. As the temperature of the liquid culture medium decreases, the culture medium is solidified, resulting in reduced movement of the biological agent. Therefore, immobilization helps to continuously observe the moving biological agent.

[0217] Figure 3a and 3b are a plan view and a perspective view, respectively, of a second embodiment of a test structure according to the invention.

[0218] refer to Figure 3a and 3bEach culture unit may further include a receiving recess 120 formed on the bottom plate.

[0219] The width of the accommodating recess 120 is not limited, and is preferably 1-5 mm, such as 1-4.5 mm, 1-4.0 mm or 1-3.5 mm. The height of the accommodating recess is preferably 1-3 mm, such as 1-2.5 mm.

[0220] Bioactive agents may further be introduced into the culture unit. Bioactive agents may physiologically affect biological agents, examples of which include: drugs such as antibiotics, anticancer agents and immunosuppressants, nutrients, cell secretions, signal transducers, viruses, cells, microRNAs, proteins, antigens, antibodies and DNA.

[0221] The bioactive agent mixed with the liquid culture medium may be introduced into each culture unit. Alternatively, the bioactive agent may be introduced into the receiving recess 120. In this case, the bioactive agent may be in a freeze-dried form that is easy to transport and store.

[0222] The plate body, the culture unit and the bottom plate may be partially or entirely made of transparent or translucent materials. Preferably, the test structure is a light-transmitting substrate, which is desirable in terms of optical imaging. The test structure is not particularly limited as long as it has surface properties suitable for forming a thin film by applying a liquid culture medium.

[0223] Another aspect of the present invention provides a bioactivity test method, comprising: providing a mixed solution of a liquid culture medium containing a gelling agent and a biological agent to a bottom surface of a bioactivity test structure; solidifying the mixed solution to form a solid film, in which the biological agent is fixed; providing the biologically active agent to the solid film and allowing the biologically active agent to diffuse into the solid film; and observing the individual response of a single cell of the biological agent to the biologically active agent or observing whether the biological agent forms colonies or colony forming units (CFU).

[0224] The method may further include imaging the individual response of a single cell to the biological agent, a colony or a colony forming unit (CFU) of the biological agent, and analyzing the image. More preferably, the method further includes determining the minimum inhibitory concentration (MIC) of the biological agent based on the analysis of the image.

[0225] The observation may be made using an optical measurement system. The optical measurement system may include an imaging system such as a CCD or CMOS camera. The optical measurement system may include optical units or devices required for focusing and light imaging, such as lenses, illuminators, and light guides. The optical measurement system may include an image processing system for processing and analyzing image data observed by the imaging system. The optical measurement system quickly records and analyzes changes in the growth of the biological agent observed during the test to obtain test results.

[0226] Bioactivity test methods can be used in a variety of applications including antibiotic susceptibility testing of bacteria, antibiotic screening, and bacterial diagnostics.

[0227] Another aspect of the present invention provides an antibiotic screening method, comprising: providing a mixed solution containing a gelling agent and a liquid culture medium of a bacterial strain to the bottom surface of a biological activity test structure; solidifying the mixed solution to form a solid film, in which the bacterial strain is fixed; providing an antibiotic to the solid film and allowing the antibiotic to diffuse into the solid film; and observing the individual response of a single cell of the bacterial strain to the antibiotic or observing whether the bacterial strain forms a colony or a colony forming unit (CFU).

[0228] The method may also include imaging the individual responses of single cells, colonies, or colony forming units (CFU), analyzing the images, and determining the minimum inhibitory concentration (MIC) of the antibiotic based on the image analysis.

[0229] Another aspect of the present invention provides a bacterial diagnosis method, comprising: providing a mixed solution containing a gelling agent and a liquid culture medium of a bacterial strain to the bottom surface of a biological activity test structure; solidifying the mixed solution to form a solid film, in which the bacterial strain is fixed; providing a culture medium of the bacterial strain to the solid film, allowing the culture medium to diffuse into the solid film; and observing the individual response of a single cell of the bacterial strain or observing whether the bacterial strain forms colonies or colony forming units (CFU).

[0230] The culture medium of the bacterial strain can be further supplemented with antibiotics, and the diagnostic method can be performed simultaneously with drug sensitivity testing.

[0231] Biological activity tests may be drug sensitivity, drug screening, or bacterial culture diagnostic tests.

[0232] There is no limitation on the type of bacterial strain. Most preferably, the bacterial strain is Mycobacterium tuberculosis or non-tuberculous mycobacteria (NTM). Mycobacterium tuberculosis may be multi-drug resistant (MDR) tuberculosis, extensively drug resistant (XDR) tuberculosis or completely drug resistant (TDR) tuberculosis.

[0233] Another aspect of the present invention provides a bioactivity test system, comprising: a bioactivity test structure, the bioactivity test structure including culture units and a bottom plate, the culture units being provided with a bioactive agent, below the bottom plate, a mixed solution of a liquid culture medium containing a gelling agent and a bioactive agent being solidified to form a thin film on the bottom surface of each culture unit; a stage for supporting and observing the test structure; and a measurement system for imaging the individual response of a single cell of the bioactive agent or the colony formation of the bioactive agent when the bioactive agent is delivered by diffusion, or imaging colony forming units (CFU), analyzing the image and determining the minimum inhibitory concentration (MIC) of the bioactive agent based on the image analysis.

[0234] The measurement system may include an imaging system and an image processing system. The imaging system may be a CCD or CMOS camera. The image processing system is suitable for processing and analyzing image data observed by the imaging system.

[0235] Description of Reference Numerals

[0236] 100: Training unit

[0237] 120: Accommodating recess

[0238] 110: Base plate

[0239] 111: Entrance

[0240] 112: Through hole

[0241] 113: Connecting components

[0242] 114: Cutting grooves

Claims

1. A biological activity test structure, comprising a plate body, one or more culture units arranged on the plate body, and a bottom plate, wherein a solid film is formed on the bottom surface of each culture unit under the bottom plate, wherein each of the culture units has an inlet formed in the bottom plate, and a mixed solution of a liquid culture medium containing a gelling agent and a biological agent is introduced through the inlet; wherein the mixed solution of the liquid culture medium containing the gelling agent and the biological agent is solidified under the bottom plate to form a solid film in which the biological agent is fixed; Each of the culture units has one or more connecting components, and the bottom plate is connected to the blocking portion of the plate body and the cutting grooves formed between the connecting components through the connecting components.

2. The biological activity test structure according to claim 1, characterized in that: The bioactivity test is a drug sensitivity, drug screening or bacterial culture diagnostic test.

3. The biological activity test structure according to claim 1, characterized in that: Each of the culture units also has a receiving recess formed on the bottom plate.

4. The biological activity test structure according to claim 1, characterized in that: Each of the culture units has one or more through holes formed in the bottom plate.

5. The biological activity test structure according to claim 1, characterized in that: Each of the culture units has a size of 1-50 mm×1-50 mm and a height of 1-50 mm.

6. The biological activity test structure according to claim 3, characterized in that: The accommodating recess has a width of 1-5 mm and a height of 1-3 mm.

7. The biological activity test structure according to claim 1, characterized in that: The thickness of the film is 1 μm to 10 mm.

8. The biological activity test structure according to claim 1, characterized in that: The density of the biological agent in the mixed solution is 101 cfu / ml to 1080 cfu / ml.

9. The biological activity test structure according to claim 1, characterized in that: The gelling agent is selected from the group consisting of agar, agarose, gelatin, alginate, collagen, fibrin and mixtures thereof.

10. The biological activity test structure according to claim 3, characterized in that: A bioactive agent is further introduced into the culture unit.

11. The biological activity test structure according to claim 10, characterized in that: The bioactive agent is introduced into the receiving recess in freeze-dried form.

12. The biological activity test structure according to claim 1, characterized in that: The plate body, the culture unit and the bottom plate are made of transparent or translucent materials.

13. A biological activity testing system, comprising: The bioactive test structure according to any one of claims 1 to 12, comprising culture units and a bottom plate, wherein the culture units are provided with bioactive agents, and below the bottom plate, a mixed solution of a liquid culture medium containing a gelling agent and a biological agent is solidified to form a thin film on the bottom surface of each culture unit; a table for supporting and observing the test structure; and a measurement system for imaging the individual response of a single cell of the biological agent or the colony formation of the biological agent when the biological agent is delivered by diffusion, or imaging colony forming units (CFU), analyzing the image and determining the minimum inhibitory concentration (MIC) of the biological agent based on the analysis of the image.

14. The biological activity testing system according to claim 13, characterized in that: The measuring system includes an imaging system and an image processing system.

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