KR20220066727A - Method for evaluating antibiotic susceptibility of microorganisms by image analysis - Google Patents

Abstract

The present application relates to a method for evaluating antibiotic susceptibility through image analysis. According to the method for evaluating antibiotic susceptibility according to one aspect, antibiotic susceptibility in a sample can be evaluated within several hours by quantifying and analyzing the level of test microorganisms, specifically, the amount of the test microorganisms in a sample through a probe-based analysis method.

Description

{Method for evaluating antibiotic susceptibility of microorganisms by image analysis}

It relates to a method for evaluating antibiotic susceptibility through image analysis.

The conventional antibiotic susceptibility evaluation method includes the steps of isolating a pure colony of the test microorganism, culturing the isolate on a solid medium or in a liquid phase, and finally analysis of the biochemical and / or phenotypic characteristics of the test microorganism By performing the antibiotic sensitivity was evaluated. The traditional drug susceptibility test method as described above is characterized by performing sensitivity evaluation of the culture in which the isolate is cultured by using a liquid medium dilution or agar diffusion analysis after the separation step of microorganisms.

Specifically, broth dilution involves inoculating a pure isolate of a test microorganism into a growth medium containing a series of specified concentrations of a specific antibiotic for which a minimum inhibitory concentration (MIC), or MIC-like measure, is determined. The inoculated medium is incubated for 18-24 hours and visible growth is observed as measured by turbidity, pellet size, and/or release of chromogenic or fluorescent moieties. In addition, the agar diffusion assay is to place an antibiotic-containing disc or an antibiotic gradient strip on the surface of an agar medium inoculated with a pure isolate of the test microorganism. The plate is incubated for 18 to 24 hours, during which time the antibiotic material diffuses from the disc or strip, observing that the effective concentration of the antibiotic changes as a function of radius from the disc or strip.

Current FDA-approved antibiotic susceptibility testing methods require inoculation of approximately 10 ⁵ CFU/mL microorganisms. Because clinical samples typically contain much less than 10 ⁵ CFU/mL, it is difficult to apply FDA-approved tests directly to clinical samples. Typically, clinical samples should be inoculated into culture medium and allowed to grow until the number of microorganisms reaches about 10 ⁸ CFU/mL. Therefore, the traditional microbial antibiotic susceptibility evaluation method as described above requires 48 to 72 hours to obtain results. However, during the above period, microorganisms suspected of not being sensitive to antibiotics spread to the patient and the environment, and endanger the health of the patient.

According to a report analyzed in 2014, there is an analysis that in 2050, there will be more deaths from antibiotic-resistant bacteria than deaths from cancer. There is also a prediction that the specific number of deaths will reach 10 million per year. As the risk of antibiotic resistance increases and the development of antibiotics to prepare for it becomes important, the importance of developing a method for evaluating antibiotic susceptibility is also increasing.

The traditional antibiotic sensitivity evaluation method has a problem in that it takes a long time to obtain the results, since it is necessary to perform the steps of isolating microorganisms and culturing the isolates for evaluation. Therefore, shortening the time required to identify infectious microorganisms and select effective antibiotic therapy could significantly reduce microbial morbidity and mortality, prevent communicable diseases, and reduce the cost of treating patients with aggressive microbial infections. have.

Under this technical background, research on a technology that can evaluate antibiotic susceptibility in a short time is being actively conducted (Korea Patent No. 10-1774995), but it is still incomplete.

One aspect is to provide a method for evaluating the antibiotic sensitivity in a sample within a few hours through probe-based image analysis, without processes such as identification of a test microorganism and growing the identified microorganism.

One aspect includes calculating an amount of a test microorganism in a first microbial community and a second microbial community; And as a method of evaluating the antibiotic sensitivity of the test microorganism in the sample, comprising the step of comparing the calculated amount of the test microorganism,

The first microbial community is obtained by culturing a community containing the test microorganism in a medium to which an antibiotic is added,

The second microbial community provides a method for evaluating antibiotic sensitivity, which is obtained by culturing a community containing the test microorganism in a medium not containing antibiotics.

As used herein, the term "test microorganism" is a microorganism for which the presence or absence of sensitivity to antibiotics is to be confirmed, and may be bacteria, viruses, fungi, fungi, or a combination thereof. The test microorganism may be one comprising Gram-negative or Gram-positive bacteria, for example, Enterococcus faecium , Staphylococcus aureus , Klebsiella species , Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Enterobacter species It may include, but is not limited to microorganisms that can acquire antibiotic resistance.

As used herein, the term “microbial community” refers to a microbial community in which one or more microorganisms are mixed. The microbial community may be obtained from a sample containing a test microorganism, and optionally, the microbial community may refer to a microbial community including the test microorganism and an antibiotic-resistant microorganism.

As used herein, the term "sample" refers to a sample containing at least one microorganism, and may be a biological sample, an environmental sample, or a food sample. The biological sample refers to a liquid sample obtained from a living organism, and may be blood, plasma, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, urine, sputum, or a combination thereof. The environmental sample may include a water quality sample or a soil sample.

As used herein, the term "antibiotic sensitivity" refers to the degree to which a target microorganism is killed or inhibited by antibiotics, and microorganisms with low or no antibiotic sensitivity, that is, microorganisms with antibiotic resistance, are public health or clinically relevant. The importance is very high. As the antibiotic, for example, amikacin, amoxicillin, ampicillin, aztreonam, benzylpenicillin, calburanic acid (Clavulanic Acid), cefazolin ), Cefepime, Cefotaxime, Cefotetan, Cefoxitin, Cefpodoxime, Ceftazidime, Ceftriaxone, Cefuroxime (Cefuroxime), Ciprofloxacin, Dalfopristin, Doripenem, Daptomycin, Ertapenem, Arithromycin, Gentamicin Imipenem), Levofloxacin, Linezolid, Meropenem, Minocycline, Moxifloxacin, Nitrofurantoin, Norfloxacin, Piperacillin (Piperacillin), Quinupristin, Rifampicin, Streptomycin, Sulbactam, Sulfamethoxazole, Telithromycin, Tetracycline, Tetracycline It may be one or more selected from the group consisting of Ticarcillin, Tigecycline, Tobramycin, Trimethoprim, and Vancomycin, but is not limited thereto.

As used herein, the term “evaluation” may mean judging the level of a certain phenomenon. The evaluation in the present invention may be to determine whether and/or the degree of antibiotic sensitivity of the test microorganism, for example, whether the antibiotic-resistant microorganism.

In one embodiment, the step of comparing the calculated amount of the test microorganism may mean quantitatively comparing the calculated test microorganism, and when two or more types of microorganisms exist, comparing the ratio of each microorganism it could be

In one embodiment, in the step of comparing the calculated amount of the test microorganism, when the ratio of the test microorganism in the second microbial community is lower than that of the first microbial community, the test microorganism is evaluated as having antibiotic sensitivity, or the second When the ratio of the test microorganisms in the second microbial community is equal to or higher than that of the first microbial community, the method may further include evaluating the test microorganism as being resistant to the antibiotic.

The first microbial community is obtained by culturing a community containing the test microorganism in a medium to which an antibiotic is added, and the second microbial community is obtained by culturing a community containing the test microorganism in a medium not containing an antibiotic. can Here, the second microbial community is cultured in a buffer-added medium by replacing the antibiotic added to the first microbial community, for example, cultured in a medium containing 1X PBS, physiological saline, or 1X TBS. can

In one embodiment, the first microbial community may be obtained by culturing each community including the test microorganism in a medium in which the concentration of the added antibiotic is changed. The concentration of the antibiotic may, for example, be adjusted to be sequentially increased or decreased, and quantitative antibiotic susceptibility determination may be performed through this concentration change.

According to one embodiment, from a sample containing a test microorganism ( E. coli K-12 or ER7 ) and an antibiotic-resistant microorganism, E. faecalis , the first and second microbial communities according to an aspect are obtained in the sample By measuring the ratio of the test microorganisms, variables such as the growth rate of each microorganism could be excluded, and as a result, it was possible to easily determine whether or not antibiotic susceptibility was achieved with only a culture process of about 1 hour.

Therefore, the step of comparing the ratio of the test microorganisms of the first microbial community and the second microbial community is compared to the conventional antibiotic sensitivity evaluation method, which essentially includes the process of identifying the microorganisms in the sample and growing the identified microorganisms. , by combining it with the calculation of the proportion of microorganisms through image-based analysis, it is possible to determine whether or not antibiotic sensitivity is present in a short time. Specifically, the first microbial community and the second microbial community may be obtained by culturing for 0.2 hours to 24 hours, and the incubation time is, for example, 0.2 hours to 20 hours, 0.2 hours to 15 hours, 0.2 hours to 10 hours, 0.2 hours to 6 hours, 0.5 hours to 4 hours, 0.2 hours to 3 hours, 0.5 hours to 24 hours, 0.5 hours to 20 hours, 0.5 hours to 15 hours, 0.5 hours to 10 hours, 0.5 hours to 8 hours hours, 0.5 hours to 6 hours, 0.5 hours to 4 hours, 0.5 hours to 3 hours, 1 hour to 20 hours, 1 hour to 15 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 5 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 20 hours, 2 hours to 15 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours It may be from 6 hours to 6 hours, from 2 hours to 5 hours, from 2 hours to 4 hours, or from 2 hours to 3 hours, but this can be changed according to the need.

In one embodiment, calculating the ratio of the test microorganism may include quantifying the signal emitted from the probe hybridized with the microorganism through a probe-based assay.

As used herein, the term "probe" refers to a part of a microorganism, for example, a polysaccharide expressed on the surface of the microorganism, genetic material in the microorganism (DNA, RNA, etc.), an antibody that binds to the protein of the microorganism, aptamer, DNA , RNA, Peptide Nucleic Acid (PNA), oligosaccharide, peptide or protein.

The probe may be labeled with a fluorescent material, a coloring material, or a chemiluminescent material. In addition, the fluorescent material is from the group consisting of FAM, VIC, HEX, Cy5, Cy3, SYBR Green, DAPI, TAMRA, FITC, RITC, rhodamine, fluoredite, Fluoredite, FluorePrime, and ROX. It may be at least one selected type.

The probe-based assay may include hybridizing a genetic material of a microorganism with a probe, and detecting a signal emitted from the hybridized probe. Specifically, by comparing the signal of the portion where the signal emitted from the probe hybridized with the microorganism exists with the signal of the other portion, the presence or absence of the microorganism or the type of the microorganism may be determined. In addition, it may be to obtain the concentration of the microorganisms contained in the sample by comparing using a standard sample containing the microorganisms of known concentrations in advance.

The probe-based assay may be performed by histochemistry, immunohistochemistry, immunofluorescence, chromogenic in situ hybridization, fluorescence in situ hybridization (FISH), etc., for example, Single-molecule RNA FISH, Fiber FISH, Q-FISH, Flow -FISH, MA-FISH, or Hybrid Fusion-FISH.

In one embodiment, the method may further include concentrating the sample containing the test microorganism.

As used herein, the term “concentration” may refer to any activity that increases the concentration of microorganisms in a sample. The concentration, which enables the detection of microorganisms by separating and concentrating bacteria from the sample, may be performed before obtaining a first microbial community, a second microbial community, and the like.

The concentrating may be performed by, but not limited to, known techniques known in the art, for example, by contacting the sample with the magnetic particles and combining the microorganisms included in the sample with the magnetic particles. (Korean Patent Registration 10-2020-0084688).

For example, the "magnetic particle" may refer to a particle exhibiting paramagnetic properties. Paramagnetic refers to the property of being weakly magnetized in the direction of a magnetic field when a magnetic field is present. The magnetic particles may be manufactured by a known method or may be commercially available. The magnetic particles may be coated with a microorganism adhesion factor on the surface. In the step of contacting the sample with the magnetic particles and the microorganisms contained in the sample combine with the magnetic particles, a magnetic field may be applied to the sample including the microorganisms bound to the magnetic particles to immobilize and concentrate the microorganisms. Here, the microorganism adhesion factor is mannose binding lectin (MBL), C-reactive protein (CRP), opsonin, CD14, Toll like receptor 4 (Toll like receptor 4: TLR4), NET ( Neutrophil extracellular trap), and may be at least one selected from the group consisting of cytokine receptors.

In one embodiment, the method comprises calculating a proportion of the test microorganisms in the first microbial community and the third microbial community; and comparing the calculated ratio of the test microorganisms, wherein the third microbial community may be a non-cultured, non-cultured community including the test microorganism.

Comparing the ratio of the test microorganisms in the first microbial community and the third microbial community may serve to evaluate errors that may occur in the culturing process of the microbial community. For example, if there is little or no deviation in the proportion of the test microorganisms in the first and third microbial communities, the antibiotic susceptibility-related results based on the proportions of the test microorganisms in the first and third microbial communities are not reliable. On the other hand, if the deviation of the ratio of the test microorganisms in the first microbial community and the third microbial community is large, it is a decrease in the survival of the test microorganisms according to the culture conditions, and the first and third microbial communities Results related to antibiotic susceptibility based on the proportion of test microorganisms in the microbial community may be interpreted as unreliable.

Therefore, the method for evaluating the antibiotic sensitivity of a test microorganism in a sample according to an aspect can not only evaluate the antibiotic sensitivity in a short time without the steps of identifying the microorganism and growing the identified microorganism, but also a self-verification process Through this, the accuracy of antibiotic susceptibility results can be further improved.

According to a method for evaluating antibiotic sensitivity according to an aspect, the level of a test microorganism in a sample, specifically, by quantifying and analyzing the ratio of the test microorganism through a probe-based analysis method, it is possible to evaluate the antibiotic sensitivity in a sample within a few hours.

1 is a diagram schematically illustrating a process for obtaining a first microbial community and a second microbial community including a test microorganism in the antibiotic sensitivity evaluation method according to an embodiment.
2 is a result of evaluating a sample containing a test microorganism having no antibiotic sensitivity through the antibiotic susceptibility evaluation method according to an embodiment.
3 is a result of evaluating a sample containing a test microorganism with antibiotic sensitivity through the antibiotic sensitivity evaluation method according to an embodiment.
4 is a diagram illustrating a method for evaluating antibiotic sensitivity using first, second, and third microbial communities according to an embodiment.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Classification and Preparation of Test Microorganisms

In this example, as a test microorganism with antibiotic sensitivity, E. coli K-12, as a test microorganism with antibiotic resistance, a total of two experiments were performed on E. coli ER7 bacteria, and each group was It was set to include resistant E. faecalis bacteria.

1.1. Classification of microbial communities

In this Example, in order to evaluate the antibiotic sensitivity of the test microorganism, the microbial community including the test microorganism was classified into three.

Specifically, as shown in Figure 1, the first microbial community is a group cultured for about 1 hour by adding the antibiotic Cefotaxime to the medium containing the test microorganism; The second microbial community is a group cultured for about 1 hour by adding saline to a medium containing the test microorganism; And the third microbial community was obtained by classifying the medium containing the test microorganism into a group not cultured, respectively. The number or ratio of the initial microorganisms of the first microbial community, the second microbial community, and the third microbial community was set to be the same.

1.2 Preparation of Samples Containing Test Microorganisms

In this example, a sample containing the test microorganism was prepared so that the antibiotic sensitivity of the test microorganism could be evaluated.

Specifically, E. coli K-12 bacteria, antibiotic-resistant E. coli ER7 bacteria, and E. faecalis resistant to cefotaxime were used, and each strain was cultured up to OD ₆₀₀ = 0.5, and samples containing the test microorganism were prepared. prepared. On the other hand, after the sample was treated with antibiotics, the amount of the test microorganism was quantified by OD ₆₀₀ to imaging. In this case, in the imaging process, the sample was concentrated 10 times through a centrifuge at 3,000 g for 3 minutes and Vectabond An experiment was performed by fixing it to a glass substrate coated with .

Example 2. Antibiotic susceptibility evaluation through FISH assay

In this example, in the microbial community obtained and classified, the ratio of the test microorganism was quantified, and based on this, the antibiotic sensitivity of the test microorganism was evaluated.

For bacterial sample fixation, a 50:1 mixed solution of acetone and Vectabond was dropped into the sample dish. Next, the sample dish was incubated at room temperature for 5 minutes, and then washed 2-3 times with tertiary water to completely remove moisture. Thereafter, the test microorganism sample of Example 1 was dropped on the coated sample dish, and then incubated at room temperature for 30 minutes, the remaining sample was washed 3 times. Next, a 3.7% formaldehyde solution was dropped on the sample dish on which the sample was fixed, fixed at room temperature for 30 minutes, and washed 2-3 times with tertiary water without nuclease. Next, in order to increase the bacterial permeability of the PNA probe, it was treated with 5 mg/ml of lysozyme prepared in tertiary water without nuclease and incubated at 37 °C for 30 minutes. Next, 10% (wt/vol) Dextran sulfate, 10 mM NaCl, 30% (vol/vol) formamide, 0.1% (vol/vol) Triton X-100, 50 mM Tris-HCl (pH 7.5), 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) Ficoll, 0.1% (wt/vol) sodium pyrophosphate, 5 mM disodium EDTA, 100 nM E. coli PNA probe and 100 nM E. faecalis PNA probe 100 µL of the solution was treated at 55 °C for 30 min to the bacterial sample fixed on the bottom of the sample dish. Thereafter, 200 µL of washing solution containing 15% (vol/vol) formamide, 5 mM Tris base, 15 mM NaCl and 0.1% Hoechst 33342 was treated twice at 55 °C for 15 min to wash off the residual PNA probe, It was washed with 1X PBS solution diluted with tertiary water without nuclease. Finally, after immersing the sample in a 2xSSC solution diluted with tertiary water without nuclease, a fluorescence image of the sample was acquired with a confocal fluorescence microscope.

2 is a result of evaluating a sample containing a test microorganism without antibiotic sensitivity through the antibiotic sensitivity evaluation method according to an embodiment; 3 is a result of evaluating a sample containing a test microorganism with antibiotic sensitivity.

As shown in FIG. 2 , in the case of the test microorganism without antibiotic sensitivity, the ratio of the test microorganism in the first microbial community was maintained, while as shown in FIG. 3 , in the case of the test microorganism with antibiotic sensitivity, the first microorganism It was confirmed that the ratio of the test microorganisms in the community decreased. These results indicate that the sensitivity of the test microorganisms can be confirmed within a short time by measuring the ratio of the test microorganisms to the total bacteria.

In addition, FIG. 4 is a result showing a method for evaluating antibiotic sensitivity using the first, second and third microbial communities, as in Example 1 above.

Specifically, as shown in FIG. 4, a sample in which two microbial ( E. coli and E. faecalis ) communities are mixed is classified into first, second and third microbial communities, and antibiotics ( cefotaxime) was treated and cultured for 1 hour, showing the results of changing the ratio of E. coli and E. faecalis . In general, there may be differences in the growth rate depending on the growth rate and physiological state of each microorganism, but 1 hour of incubation is insufficient time to confirm individual differences in the population, as a result, the first microorganism of an aspect of the antibiotic sensitivity evaluation method The step of comparing the proportion of the test microorganisms in the community and the third microbial community may serve to evaluate errors that may occur in the culturing process of the microbial community. Therefore, in the case of the third microbial community, which is the initial microbial community, and the second microbial community cultured without antibiotics, there was no significant difference in the amount or ratio of microorganisms, so it was determined that the culture was carried out without errors and antibiotic sensitivity evaluation was performed. As a result, as shown in Figure 4, in the case of the first microbial community treated with antibiotics and cultured, the number of E. coli K-12 showing antibiotic sensitivity is rapidly reduced, whereas E. faecalis showing resistance continues It was confirmed that the ratio changed significantly as it grew.

These results indicate that when the ratio of test microorganisms in the first microbial community is lower than that of the second microbial community, the test microorganism is sensitive to antibiotics, and the ratio of test microorganisms in the second microbial community is equal to the first microbial community or high, it means that the test microorganism is resistant to antibiotics. In addition, when the second microbial community and the third microbial community are compared, if there is a large difference in the microbial ratio, it means that it can be determined that there is a problem in culture.

Therefore, according to the evaluation method of the present invention, there is no problem of reducing the accuracy of the antibiotic susceptibility test due to the problem of culture, and even in a sample in which one or more types of microorganisms are present, the antibiotic sensitivity of the test microorganism is quickly and accurately determined only by culturing for about 1 hour. can judge

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (12)

calculating an amount of a test microorganism in the first microbial community and the second microbial community; And as a method of evaluating the antibiotic sensitivity of the test microorganism in the sample, comprising the step of comparing the calculated amount of the test microorganism,
The first microbial community is obtained by culturing a community containing the test microorganism in a medium to which an antibiotic is added,
The second microbial community is a method for evaluating antibiotic sensitivity, which is obtained by culturing a community containing the test microorganism in a medium that does not contain antibiotics. The method of claim 1 , further comprising: calculating an amount of a test microorganism in the first microbial community and the third microbial community; and comparing the calculated amount of the test microorganism,
The third microbial community is not cultured, a method for evaluating antibiotic susceptibility to a community containing the test microorganism. The method according to claim 1, wherein the calculating of the amount of the test microorganism comprises quantifying the signal emitted from the probe hybridized with the microorganism through a probe-based assay, the method for evaluating antibiotic sensitivity. The method according to claim 3, wherein the probe-based assay is a Fluorescence in situ Hybridization (FISH) assay method, the method for evaluating antibiotic sensitivity. The method according to claim 1, Comparing the calculated amount of the test microorganism, when the ratio of the test microorganism in the second microbial community is lower than that of the first microbial community, the test microorganism is further evaluated as having antibiotic sensitivity A method for assessing antibiotic susceptibility, comprising: The method according to claim 1, wherein the comparing of the calculated amount of the test microorganism is when the ratio of the test microorganism in the second microbial community is equal to or higher than that of the first microbial community, evaluating the test microorganism as being resistant to antibiotics Further comprising, a method for evaluating antibiotic sensitivity. The method according to claim 1, wherein the first microbial community and the second microbial community include microorganisms that are not susceptible to antibiotics. The method according to claim 1, wherein the first microbial community and the second microbial community is obtained by culturing for 0.2 hours to 24 hours, the method for evaluating antibiotic sensitivity. The method according to claim 1, wherein the second microbial community is obtained by culturing in a buffer-added medium to replace the antibiotic, the method for evaluating antibiotic sensitivity. The method for evaluating antibiotic sensitivity according to claim 1, wherein the first microbial community is obtained by culturing a community containing the test microorganism in a medium in which the concentration of the added antibiotic is changed, respectively. The method of claim 1, wherein the sample is a biological sample, an environmental sample, or a food sample. The method according to claim 1, further comprising the step of concentrating the test microorganism prior to obtaining the first microbial community and the second microbial community.

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