KR101210590B1 - Monitoring of susceptibility of biofilms for a sample and apparatus therefor - Google Patents

Abstract

The present invention relates to a method for measuring sample sensitivity of a biofilm which can quickly and easily and reproducibly measure the sample sensitivity to bacteria in a biofilm state, and more specifically, (A) forming a biofilm of bacteria in a microchannel; (B) culturing the biofilm by injecting a sample such that the sample concentration is gradient to the cross-sectional side in the microchannel in which the biofilm is formed; And (C) detecting whether the cultured biofilm is inhibited from forming.
The present invention also relates to an apparatus capable of measuring the sample sensitivity of a biofilm by the above method.

Description

Monitoring of susceptibility of biofilms for a sample and apparatus therefor}

The present invention is a sample for the bacteria in the biofilm state The present invention relates to a method for measuring sample sensitivity of a biofilm, which can measure sensitivity quickly, easily and reproducibly, and to an apparatus capable of measuring sample sensitivity of a biofilm by the above method.

Most bacteria attach to a variety of surfaces, including human tissues, plastics, metals, and the surface of medical implant materials to form bacterial aggregates called biofilms. Biofilms are a common form of the natural environment and are responsible for chronic bacterial infections in humans.

Bacteria in biofilms are highly resistant to antibiotics and the external environment because they are surrounded by an irregular and complex network structure called extracellualr polymeric substances (EPS) consisting of proteins, nucleic acids, lipids, and exopolysaccharides. . EPS also provides mechanical stability, high adhesion and viscoelasticity to biofilms. On the other hand, bacteria that are not surrounded by biofilms, such as floating bacteria, are more likely to be affected by antibiotics or the external environment.

Pseudomonas aeruginosa is a causative agent of hospital infection and is widely used as a model for biofilm research. In particular, one of the major causes of death in patients with cystic fibrosis is infection of the lungs by Pseudomonas aeruginosa, which is due to the formation of Pseudomonas aeruginosa biofilm. Most cystic fibrosis patient is to receive suffering from chronic bacterial respiratory tract infections, to prevent the characteristic of the biofilm with the multidrug resistance to these infections is very difficult. Drug resistance is due to the inability of antibiotics to penetrate through biofilm matrices, the slow growth of biofilm cells, and the expression of multidrug efflux pumps to release antibiotics in vitro. Bacterial biofilm formation triggers the expression of virulence genes and blocks the host's defense mechanism by blocking the phagocytic pathway of immune cells. Due to the protective properties of these biofilms, the sensitivity of bacteria to antibiotics in actual dynamic micro-formatting environments such as blood vessels, respiratory organs, and infected tissues of patients differs from that of suspended cells. One study found that biofilms had more than 100-fold resistance to antibiotics when compared to suspended bacteria.

Several methods have been proposed to measure the antibiotic sensitivity of bacteria in biofilms. For example, Calgary Biofilm Device (CBD) published in Korean Patent Application Publication No. 10-2008-81891 is an efficient method for screening antibiotic sensitivity of biofilms and measuring minimal biofilm eradication concentration (MBEC). However, this method has to rely on the size of the bacterial inoculation, which greatly affects the outcome of antibiotic sensitivity (J. Bacteriol., 2001, 183, 6746). In addition, in the above experiments, the biofilm must be recovered from the equipment in order to count the viable cells of the biofilm, and bacteria must be separated and counted from the recovered biofilm. Bacteria isolated from biofilms have a problem that it is difficult to accurately measure the antibiotic sensitivity of biofilms because they lose the physiological state specific to biofilms.

Separately, a method for detecting the formation and development of a biofilm by detecting the detected radiation using an electromagnetic radiation beam as image data or by placing an electric field, magnetic field or electromagnetic field particles in the medium and measuring the viscosity of the microbial culture medium through the development It became. This method can be measured only in a medium of homogeneous viscosity, but since the bacterial culture medium is usually milky, cloudy and opaque, it is difficult to test the formation of the biofilm in the culture medium. Therefore, there is a need for the development of a method capable of reproducibly measuring the sensitivity of the biofilm to the sample while saving time, cost and effort.

Republic of Korea Patent Publication No. 10-2008-81891

 J. Bacteriol., 2001, 183, 6746.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a method capable of measuring the sensitivity of a biofilm sample to a more accurate and reproducible measurement in a simple process.

It is another object of the present invention to provide a method for measuring the sensitivity of a sample of a biofilm in various micro-formatting environments to provide quantitative physiological activity data in conditions similar to the actual site of infection of the patient.

Still another object of the present invention is to provide an apparatus capable of measuring the sample sensitivity of a biofilm by the above method.

The present invention for achieving the above object comprises the steps of (A) forming a biofilm of bacteria in the microchannel; (B) culturing the biofilm by injecting a sample such that the sample concentration is gradient to the cross-sectional side in the microchannel in which the biofilm is formed; And (C) detecting whether the cultured biofilm is inhibited from forming.

According to the measuring method of the present invention, it is possible to simply and qualitatively measure the sample sensitivity of the biofilm simply by comparing the inhibition of the biofilm formation according to the concentration. Furthermore, the sample sensitivity of the biofilm can be quantitatively determined by calculating the sample concentration at the point where the biofilm is removed in step (C) from the calibration curve for the concentration gradient of the sample in the microchannel. It is possible to measure. According to the present invention, the biofilm formation and the sensitivity of the biofilm according to the sample concentration can be measured in a simple and reproducible manner with only one experiment without performing each experiment for various concentrations in one sample.

Step (A) comprises the steps of (a) attaching bacteria to the microchannel; (b) washing the bacteria not attached to the microchannel; And (c) culturing the attached bacteria to form a biofilm. First, when the bacterial suspension is flowed into the microchannel at a slow flow rate, most of the bacteria introduced into the microchannel are attached. However, some bacteria remain suspended or weakly attached without being attached to microfluidics. If the bacteria remain in a suspended state, errors may occur in the detection process, so it is desirable to remove them before biofilm formation. After removing the suspended bacteria, the culture solution is flowed to form a biofilm.

The flow rates of the bacterial suspension, wash solution and culture medium for attachment, washing and culture of bacteria may vary depending on the width of the microchannel, the material, and the type of the fluid, but it is natural that the flow rate is faster in the order of suspension <culture solution <washing solution. Do. The flow rate in the following examples was determined by preliminary experiments, and similar conditions can be used to optimize the numerical value with reference to this. If the flow rate of the bacterial suspension is too fast at the time of attachment of the bacteria, the bacteria are not effectively attached. If the flow rate is too slow, the floating bacteria or weakly coupled bacteria cannot be effectively removed, and if the flow rate is too fast, the washing liquid may exfoliate the bacteria attached to the microchannel. The flow rate of the culture fluid affects the formation of the biofilm. If the culture fluid is introduced at too high a rate, the biofilm formed by shear stress acting on the biofilm may peel off.

The sensitivity of the biofilm to the sample can be measured by injecting a sample into the microchannel in which the biofilm is formed so that the concentration of the sample is gradient to the cross-sectional side, and culturing the biofilm, and detecting whether the biofilm is inhibited from forming. If the injection rate of the sample is too slow, the concentration gradient due to diffusion in the microchannel may not be linear, so it should be appropriately controlled. In the following examples, the sample and the medium were injected at a rate of 0.1 μl / ml, respectively, but are also influenced by the width of the microchannel, the material, the type of fluid, and the like.

Whether to inhibit the formation of the biofilm can be easily detected through an optical microscope, but if the bacteria are labeled, it is possible to more easily measure the degree of formation of the biofilm. In the following example, the inhibition of the formation of the biofilm was detected by measuring fluorescence, for example, bacteria recombined with a fluorescence gene, but in addition to the conventional biotechnology, such as fluorescent dyes, magnetic particles, radioisotopes or quantum dots. Labeling is possible by being used as a labeling substance. At this time, the detection method may be appropriately selected according to the labeling method. In addition, although the following examples are directed to Pseudomonas aeruginosa, the method of the present invention is not limited to Pseudomonas aeruginosa, and it is natural that any bacterium forming a biofilm can be used.

The sample may be an antibiotic in a narrow sense. That is, by measuring the sample sensitivity of the biofilm using an antibiotic as a sample, it is possible to develop an effective antibiotic and to calculate an effective dose for treating a disease caused by bacterial infection.

However, the sample is not limited to antibiotics and may be targeted to acids, bases, oxidants or reducing agents. By measuring the sensitivity of the biofilm to the sample, it is possible to effectively evaluate the environmental factors affecting the formation of the biofilm, it can be used to suppress the formation of the biofilm or to create an environment that can keep the biofilm stable.

Another aspect of the invention relates to an apparatus for measuring sample sensitivity of a biofilm, comprising two inlets; A gradient forming region for separating each fluid injected into the injection hole into a plurality of microchannels having different concentration gradients; A detection channel through which a fluid separated into a plurality of microchannels passes through a gradient forming region and moves; And a discharge port through which the fluid passing through the detection channel is discharged. The present invention relates to a microfluidic device for measuring sample sensitivity of a biofilm. The gradient forming region is a region in which the concentration gradient generated by mixing the sample and the mobile phase injected in each of the two inlets can be linearly formed in the detection channel. More specifically, the sample injected from each inlet and the mobile phase are mixed and divided into a multi-flow stream in which the concentration gradient of the sample is formed. do. The composition of the gradient forming region can be variously configured with reference to the prior art, and thus, detailed descriptions thereof are omitted in the present invention (NL Jeon, H. Baskaran, SKW Dertinger, GM Whitesides, LVD Water and M. Toner, Nat. Biotechnol., 2002, 20, 826 .; P. Hung, P. Lee and LP Lee, Biotechnol. Bioeng., 2005, 89, 1 .; J. Diao, L. Young, S. Kim, EA Fogarty, SM Heilman , P. Zhou, ML Shuler, M. Wu and MP DeLisa, Lab Chip, 2006, 6, 381.)

The width of the micro channel may be designed in an appropriate range depending on the purpose of the experiment, but is preferably 100 to 500 microns, more preferably 200 to 300 microns. If the width of the microchannel is too narrow, it is difficult to inject bacteria or samples, and if the width of the microchannel is too wide, it is difficult to form a concentration gradient due to stable laminar flow. In addition, there is a disadvantage that the observation becomes cumbersome when the width of the microchannel is too wide and out of the microscope detection window.

As described above, according to the present invention, the method for measuring antibiotic sensitivity of microfluidic equipment and biofilms can be efficiently used for setting the dosage of antibiotics because it is possible to measure biofilm formation and antibiotic sensitivity in a simple manner by in-situ. have.

In addition, effective dosages can be easily measured at the development stage of new drugs as well as existing antibiotics, providing more accurate information on the rational design of antibiotics.

In addition, the present invention can be easily carried out to evaluate the environmental factors such as pH, oxidative stress affecting the formation of biofilms as well as antibiotics to suppress the formation of biofilms to build a more hygienic environment or For example, wastewater treatment that uses biofilms industrially can make it easier to create an environment in which biofilms can be formed efficiently.

1 is a photograph and a schematic view showing a microfluidic device for measuring the antibiotic sensitivity of the biofilm according to an embodiment of the present invention.
Figure 2 is a schematic view showing the specifications of the microfluidic device according to an embodiment of the present invention.
Figure 3 is a graph showing that the concentration gradient of the drug is linearly generated in the detection channel of the microfluidic device of FIG.
Figure 4 is a schematic diagram showing a process of forming a biofilm in a microfluidic device according to an embodiment of the present invention.
Figure 5 is a fluorescence micrograph showing the formation of the biofilm in the microfluidic device according to an embodiment of the present invention.
Figure 6 is a schematic diagram showing the principle of measuring the MBEC value in the microfluidic device according to an embodiment of the present invention.
Figure 7 is a graph of the sensitivity measurement of Pseudomonas aeruginosa biofilm for kanamycin according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and examples. However, the drawings and the embodiments are only illustrative of the contents and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

Example 1 Preparation of Microfluidic Equipment

The microfluidic device was fabricated to have a microchannel with two inlets, a gradient generator, a detection channel and one outlet as shown in FIG. 1 by soft lithography. .

More specifically, the negative photoresist (SU-8, Microchem Co., USA) was evenly applied on the silicon wafer, and then spin-coated at 1,000 rpm to raise the photoresist having a height of 40 μm. The photosensitive agent was irradiated with UV through a mask having a micro flow path shape as shown in FIG. 1 to prepare a master mold having a shape opposite to that of the channel. In the microchannel of FIG. 1, the width of the detection channel is 300 μm, and the area of the other microchannels is 50 μm, and the size of the entire microchannel is shown in FIG. 2.

Then, PDMS (Sylgard 184; Dow Corning, Midland, MI) was poured into the prepared master mold and then 4 hours at 65 ° C. Curing to produce a PDMS mold having a microchannel of the desired shape. The glass substrate was attached to the PDMS mold thus prepared through an oxygen plasma treatment to produce a microfluidic device.

Example 2 Antibiotic Sensitivity Measurement of Biofilm

1) Preparation of concentration gradient calibration curve

Visualizing and confirming that the gradient of the chemical occurs in the detection channel of the microfluidic device prepared in Example 1, a calibration curve for the concentration gradient was obtained.

Connect a Tygon tube to each inlet of the microfluidic device manufactured in Example 1, connect a micro syringe (Norm-Ject, Germany) to each tube and use a syringe pump (Harvard Apparatus, USA) To allow the solution in the microsyringe to be pumped into the microchannel. By the above method, one injection hole was simultaneously injected with 0.1 mM solution of Rhodamine B, a fluorescent dye, and the other injection hole was injected with PBS (phosphage buffer saline) at a rate of 0.1 μl / min ⁻¹ .

Fluorescence of rhodamine B at various locations in the detection channel was measured using an inverted fluorescence microscope equipped with a CCD camera (Coolsnap cf, Photometrics, USA), TE2000, Nikon, Japan) and the results are shown in FIG. 3. Figure 3 (a) shows the length of the detection channel and the fluorescence intensity in the cross section of the channel in a three-dimensional graph, Figure 3 (b) shows the fluorescence intensity of the cross section at a specific length of the specific detection channel graphically will be.

Injecting rhodamine B and PBS through the microfluidic device of Example 1, since the concentration of rhodamine B splits into five different flows through the gradient generating region and continuously flows into the detection channel, the cross section of the detection channel There is a concentration gradient of rhodamine B. As shown in (a) and (b) of FIG. 3, at the inflow point (0 mm) of the detection channel where the flows of the five branches passing through the gradient generation region are combined, the difference between the concentrations introduced in the five branches flows into a curve. Although apparent, the concentration gradient changes linearly as it passes through the detection channel. This concentration gradient is maintained to the end of the detection channel, i.e. 8 mm position. 2 (a) and 2 (b), it can be seen that the concentration gradient of the chemical is linearly detected in the detection channel in the microfluidic device of Example 1, and the graph of the chemical introduced into the calibration curve. The concentration at a specific position of the detection channel can be calculated from the concentration.

2) formation of biofilm

Biofilm was formed using P. aeruginosa PAO1 wild type strain (ATCC number: 47085D-5) in the microchannel of the microfluidic device manufactured in Example 1.

Science Vol. A plasmid pMRP9-1 expressing green fluorescent protein (GFP) was prepared according to the method described in 280 10 April 1998, and the plasmid was introduced according to a conventional method. P. aeruginosa PAO1 was transformed. Transformed P. aeruginosa PAO1 was selected in LB (Luria-Bertani) medium containing 150 μg / ml carbenicillin, inoculated in 37 ° C. LB medium and incubated overnight. The cultured bacteria were harvested by centrifugation at 5000 ㅧ g, washed twice with 50 mM potassium phosphate buffer (pH 7.5), and then resuspended in 1 ml LB medium so that the concentration of bacteria was 1 × 10 ⁹ cells / ml. The process of forming the biofilm in the microfluidic device using the suspension is shown in FIG. 4.

More specifically, a Tygon tube was connected to the outlet of Example 1, and a micro syringe (Norm-Ject, Germany) containing the bacterial suspension was connected to the tube. A syringe pump (Harvard Apparatus, USA) was used to inject the bacterial suspension in the micro syringe at a rate of 0.05 μl / min for 1 minute, then the syringe pump was turned off and allowed to stand for 10 minutes (FIG. 3 (a)). The bacteria injected in this process are attached to the surface of the microchannel of the detection channel.

Thereafter, the sterilized PBS buffer was flowed through one of the injection strains at a rate of 5 μl / min for 30 minutes to remove bacteria not attached or weakly attached to the microchannel (FIG. 3 (b)). In order to form a biofilm from the attached bacteria, LB medium was incubated for 24 hours while flowing at one of the inlets at a rate of 0.1 μl / min (FIG. 4C).

The formation of the biofilm was observed over time using an inverted fluorescence microscope (TE2000, Nikon, Japan) equipped with a CCD camera (Coolsnap cf, Photometrics, USA) and the results are shown in FIG. Referring to Figure 5, at the start of the culture (0 h) attached bacteria grow over time (4 h), after 12 h to form a bacterial cluster and after 24 h the entire substrate of the microfluidic channel It was confirmed that a biofilm was formed on the substratum and covered.

3) MBEC measurement

MBEC values for various antibiotics were measured to determine the antibiotic sensitivity of Bacteria in biofilms. The principle of MBEC measurement in the microfluidic device in which the biofilm is formed is shown in FIG. 6.

Using Kanamycin (Km) as an example, the MBEC measurement method is described. In 2), the kanamycin solution was injected into one inlet of the microfluidic device in which the biofilm was formed in the detection channel, and the LB medium was injected into the other inlet at a rate of 0.1 μl / min. To increase the precision of the experiment, kanamycin was separately tested using 100, 200, or 300 µg / ml, respectively.

After 30 seconds of antibiotic flow, the intensity of fluorescence was measured using an inverted fluorescence microscope (TE2000, Nikon, Japan) equipped with a CCD camera (Coolsnap cf, Photometrics, USA) and the results are shown in FIG. . As shown in (A) of FIG. 7, when kanamycin is injected into the detection channel in which the biofilm is formed through the gradient generating region, the biofilm is maintained in the region where the concentration of kanamycin is low, but in the region where the concentration of the antibiotic is high, the biofilm bacteria are killed. Therefore, the fluorescence is weak. The position indicated by the arrow in (A) indicates the position where the fluorescence intensity becomes the same as the fluorescence intensity of the background without the biofilm. From the concentration of kanamycin at the arrow position, it was confirmed that the MBEC was 57 ± 4.7 μl / ml, which was in good agreement with all values measured for various concentrations of kanamycin. 7 (B) is a graph showing the fluorescence intensity and the concentration of kanamycin according to the position of the channel cross section, and it can be seen that the fluorescence intensity decreases as the concentration of kanamycin increases.

For antibiotics other than kanamycin, MBEC was measured by the same method as that described above, and the results are shown in Table 1. In addition, MIC values were measured by standard doubling dilution according to the "Clinical and Laboratory Standards Institute guidelines" for comparison with MIC values for suspended bacteria (Can. J. Vet. Res., 2002, 66, 86). Are listed together in Table 1. For all antibiotics measured in Table 1, the MBEC value was high MIC value for suspended bacteria, it was confirmed that the antibiotic sensitivity of the bacteria in the actual biofilm can be easily measured by only one experiment.

Claims (10)

(A) forming a biofilm of bacteria in the microchannel;
(B) culturing the biofilm by injecting a sample such that the sample concentration is gradient to the cross-sectional side in the microchannel in which the biofilm is formed; And
(C) detecting whether the cultured biofilm is inhibited from forming;
Sample sensitivity measurement method of a biofilm comprising a.
The method of claim 1,
After step (C), from the calibration curve (D), the sample concentration of the biofilm is calculated by calculating the sample concentration at the point of destruction of the biofilm detected in step (C) to obtain the minimum biofilm extinction concentration (MBEC). Way.
3. The method according to claim 1 or 2,
The step (A)
(a) attaching bacteria to the microchannel;
(b) washing the bacteria not attached to the microchannel; And
(c) culturing the attached bacteria to form a biofilm;
Sample sensitivity measurement method of a biofilm comprising a.
The method of claim 3, wherein
The sample sensitivity measurement method of the biofilm, characterized in that the sample is an antibiotic.
The method of claim 3, wherein
A method of measuring sample sensitivity of a biofilm, characterized in that the effect of environmental factors on the biofilm is evaluated using an acid, an alkali, an oxidizing agent or a reducing agent as the sample.
3. The method according to claim 1 or 2,
In the step (C), the detection of the biofilm formation is inhibited by the optical microscope.
3. The method according to claim 1 or 2,
The bacterium is a sample sensitivity measurement method of a biofilm, characterized in that the labeled bacteria.
The method of claim 7, wherein
The labeling method for measuring the sample sensitivity of the biofilm, characterized in that using a fluorescent material, magnetic particles, radioisotopes or quantum dots.
Two inlets;
A gradient forming region for separating each fluid injected into the injection hole into a plurality of microchannels having different concentration gradients;
A detection channel through which a fluid separated into a plurality of microchannels passes through a gradient forming region and moves; And
A discharge port through which the fluid passing through the detection channel is discharged;
Microfluidic equipment for measuring the sample sensitivity of the biofilm comprising a.
The method of claim 9,
Microfluidic equipment, characterized in that the width of the detection channel is 100 ~ 500 microns.

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