CN112176024B

CN112176024B - Method for detecting bacterial inhibition of antibiotics by single cell counting

Info

Publication number: CN112176024B
Application number: CN202010793224.8A
Authority: CN
Inventors: 崔璟; 唐明忠; 张会翠
Original assignee: Shenzhen Aier Biotechnology Co ltd
Current assignee: Beijing Xingyuanhui Technology Co.,Ltd.; Tang Mingzhong
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2023-07-14
Anticipated expiration: 2040-08-07
Also published as: CN112176024A

Abstract

The invention provides a method for detecting bacterial inhibition by antibiotics through single cell counting, which comprises the following steps: adding antibiotics with preset concentration into bacteria to be detected, setting the bacteria to be detected as a bacterial antibiotic mixture, and setting the bacteria to be detected without adding the antibiotics as positive control; obtaining a current number of said bacteria of said bacterial antibiotic mixture and a current number of said bacteria of said positive control when a first predetermined time period is reached between the moment of addition of said antibiotic; determining a minimum inhibitory concentration of the antibiotic against the bacteria, and determining a degree of inhibition of the antibiotic against the bacteria. The method solves the technical problem of accurately determining the relation between the single cell count value and the drug sensitivity experimental result on the basis of realizing drug sensitivity by single cell count, and has the advantages of reliable result, strong clinical practicality, low cost and easy automation.

Description

Method for detecting bacterial inhibition of antibiotics by single cell counting

Technical Field

The invention relates to the field of biological medicine, in particular to a method for detecting bacterial inhibition by antibiotics through single cell counting.

Background

Bacterial drug resistance is more and more serious and rapidly spread widely in the world, governments of various countries attach great importance to the public, china is also out of the table for a large number of management regulations, reasonable use of antibiotics is the most core work for coping with bacterial drug resistance, and rapid antibiotic sensitivity tests are of great importance.

In view of the development of mass spectrometry technology and nucleic acid technology, bacterial identification has been substantially achieved (as the result of the day, completed within 1-2 hours), and thus the development of new rapid antibiotic susceptibility tests is more urgent and practical.

The switching of empirical broad-spectrum antibiotic treatment to targeted treatment as soon as possible is the basis for antibiotic management, but the current drug sensitivity test reporting time restricts clinical practice, and the traditional manual method reporting time is too long, so that the full-automatic drug sensitivity test method is mostly adopted clinically at present, wherein the VITEK system of the company Mei Liai in France and the Phoeni x system of the company BD in America are the fastest detection systems, the reliability and accuracy of the two systems have been proven, but the average time Phoeni x is 12.1 hours, and the Vit ek2 is 9.8 hours. This reporting time, taking into account the daily workflow and the work and rest time, is actually the only time for the doctor to choose the medication for the next day.

At present, in order to shorten the report time of drug sensitive tests, a great deal of research is carried out at home and abroad, and various methods have been developed, such as mass spectrometry, flow cytometry, vibrating cantilever microbial cell weighing, isothermal trace heat generation, magnetic bead rotation, droplet detection, real-time PCR, microarray, conductivity, surface plasmon resonance, RNA sequencing, phage, real-time microscopy and microscopic acoustic wave. However, these techniques are only research stages, perform only small sample analyses, and all require skilled personnel to operate, are expensive to implement, are non-traditional specialized equipment, and are complex to operate. Unstable performance, high cost, inconvenient use and poor practical prospect.

Therefore, it is important to provide an economical and rapid drug sensitivity test scheme.

Disclosure of Invention

The invention provides a method for detecting bacterial inhibition by antibiotics through single cell counting, which at least solves the technical problem that the method for detecting bacterial inhibition by antibiotics in the prior art has long result time.

The invention provides a method for detecting bacterial inhibition by antibiotics through single cell counting, which comprises the following steps:

adding antibiotics with preset concentration into bacteria to be detected, setting the bacteria to be detected as a bacterial antibiotic mixture, and setting the bacteria to be detected without adding the antibiotics as positive control;

obtaining a current number of said bacteria of said bacterial antibiotic mixture and a current number of said bacteria of said positive control when a first predetermined time period is reached between the moment of addition of said antibiotic;

determining a minimum inhibitory concentration of said antibiotic against said bacteria based on said current number of said bacteria of said bacterial antibiotic mixture and said current number of said bacteria of said positive control, and determining a degree of inhibition of said antibiotic against said bacteria.

Optionally, the current number of the bacteria of the positive control is multiplied by any one of coefficients 0.2 to 0.6 as a reference point, and the first of the antibiotic concentrations corresponding to the reference point is a Minimum Inhibitory Concentration (MIC) of the bacteria at the antibiotic concentration.

Alternatively, the above coefficient is 0.4.

Alternatively, the calculation is performed according to the following inflection point judgment method:

r 1-r0=k0, r 2-r1=k1, and when the value of k1-k0 is greater than a predetermined threshold value, the antibiotic concentration is a Minimum Inhibitory Concentration (MIC) of the bacterium at the antibiotic concentration, wherein r1 is a count value of a medium concentration gradient, r0 is a count value of a low concentration gradient, and r2 is a count value of a high concentration gradient.

Optionally, the threshold is determined by an inhibitory effect of the antibiotic against the bacteria.

Alternatively, when the antibiotic is tetracycline and the bacteria is Pseudomonas, the threshold is less than a predetermined threshold for a majority of the bacteria other than Pseudomonas.

Optionally, the method uses a multipoint prediction method of Minimum Inhibitory Concentration (MIC) of the bacteria at the antibiotic concentration, wherein the multipoint prediction method is a positive control method of setting not less than two gradients of the antibiotic concentration, comparing a calculated value of the measured inhibitory concentration of the antibiotic with a percentage of the positive control, and regarding the inhibitory concentration of the antibiotic satisfying the percentage of the positive control as a corresponding gradient of the percentage of the positive control.

Alternatively, when the bacterium is a coccus, the Minimum Inhibitory Concentration (MIC) of the coccus is increased by 1 to 2 gradients on the basis of the inflection point determining method calculation as the Minimum Inhibitory Concentration (MIC) of the coccus.

Alternatively, a current number of the bacteria of the bacterial antibiotic mixture and a current number of the bacteria of the positive control are obtained using a resistance count method.

Alternatively, the current number of said bacteria of said bacterial antibiotic mixture and the current number of said bacteria of said positive control are obtained using a flow-type bacterial counting method or a microscopic bacterial counting method or a counter assay or an electronic counter counting method or a cell weight measurement method.

In order to solve the problem of quick drug sensitivity, the observation that the antibiotics sensitivity can be observed by waiting 18 to 24 hours for the population to pass through is changed into the quick drug sensitivity realization by a single cell counting mode, because the bacterial propagation speed is about 20 minutes at a proper culture temperature.

The essence of the drug sensitivity test is to observe the influence of antibiotics on the growth, metabolism and reproduction of bacteria, and according to the conditions of the influence of drugs on the growth, metabolism and reproduction of bacteria (namely the inhibition conditions of bacteria) observed by in vitro tests, the effectiveness of future drug administration is deduced by combining the conditions of clinic and pharmacokinetics, the traditional method monitors the killing effect of antibiotics on bacteria through the change of the bacterial number in a liquid or solid culture medium, the bacterial population is observed, if each bacterial individual can be accurately monitored, but the change trend of the sum of the bacterial population is not detected, the influence of the drugs on the bacteria can be detected early and quickly, so that the drug sensitivity test has great breakthrough in time, but the technical scheme of the invention creatively invents a method for rapidly detecting the bacterial drug sensitivity by using the bacterial counting from another angle because the prior art lacks accurate, practical and automatable individual bacterial microscopic detection technology.

Specifically, the method adopts the technical scheme that antibiotics with different concentrations are added when bacteria grow, the antibiotics with a concentration above a certain concentration can be found to inhibit the growth and reproduction of the bacteria, so that the minimum inhibitory concentration is measured, the minimum inhibitory concentration is measured by the change of turbidity of broth after the bacteria grow, which takes a long time, usually 18 hours, is optimized by commercial companies in recent years, the method also takes 10 hours to report when the bacteria grow or inhibit condition is found early by using a more sensitive turbidimeter or adding a redox indicator, and because the antibiotics can influence the bacteria in a short time, the method or the scheme for finding the fastest definite effect of the medicine on the bacteria has very practical significance, the sensitivity of the bacteria on the medicine can be measured in a short time. The realization of drug sensitivity through single cell counting is the basis of the solution of the invention, on the basis of the realization of the drug sensitivity, how to accurately determine the relation between the single cell counting value and the drug sensitivity experimental result is the key link of realizing the rapid drug sensitivity through the single cell counting.

Drawings

The above, as well as additional purposes, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description when read in conjunction with the accompanying drawings. Several embodiments of the present invention are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which:

FIG. 1 is a schematic diagram showing the variation of bacterial amounts of an alternative Escherichia coli broth cultured for different times according to an embodiment of the present invention;

FIG. 2 is a schematic diagram showing the turbidity change of an alternative E.coli broth culture for different times according to an embodiment of the present invention;

FIG. 3 is a schematic diagram showing the results of observation of the change in turbidity and the number of bacteria cultured in an alternative broth according to the embodiment of the present invention;

FIG. 4 is a schematic diagram showing the results of the growth experiment in example 3 of the present invention;

FIG. 5 is a graph showing the results of comparison of 2h and 24h in example 3 of the present invention;

FIG. 6 is a graph showing the sensitivity compliance rate in example 3 of the present invention;

FIG. 7 is a graph showing the results of Table 12 in example 5 of the present invention;

FIG. 8 is a graph showing the results of Table 13 in example 5 of the present invention;

FIG. 9 is a graph showing the results of Table 14 in example 5 of the present invention;

FIG. 10 is a graph showing the results of Table 15 in example 5 of the present invention;

FIG. 11 is a schematic diagram showing the results of Table 17 in example 5 of the present invention.

The following detailed description is provided to further illustrate, but not limit, the invention, and the following examples are merely one preferred embodiment of the invention.

Detailed Description

The principles and spirit of the present invention will be described below with reference to several exemplary embodiments. It should be understood that these embodiments are presented merely to enable those skilled in the art to better understand and practice the invention and are not intended to limit the scope of the invention in any way. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1

The purpose is as follows: when searching broth bacteria culture, determining a theoretical basis for a rapid drug sensitivity test by using a bacterial change sensitivity index, and establishing a bacterial drug sensitivity detection method.

Materials and methods:

1. preparing strains:

three standard strains (ATOC 25922 E.coli, ATOC 25923 Staphylococcus aureus, ATOC27853 Pseudomonas aeruginosa) were transferred for use and incubated at 37℃for 18 hours.

2. Preparation of broth:

the strain was prepared and ground on the wall of an AST broth (antibiotic susceptibility test) bottle, mixed well, and the bottle cap was capped and turbidity was measured with a turbidimeter (BD company Phoeni xSpec Nephel omter) at 0.5 mahalanobis unit for use. Bacteria to be tested were inoculated at the inoculation concentration of CLSI (clinical laboratory standard tissue) broth dilution drug sensitivity test, and incubated in an incubator at 37 ℃ (degrees celsius).

3. Bacterial count

Incubation in incubator at 37℃was performed at 0min, 10 min, 30 min, 60min, 90 min, 120 min by resistance counting (RC-3000 resistance method (Coulter) particle counter Zhuhai Euramerican). The average was taken 2 times and the data was recorded.

4. Turbidity measurement

Incubates at 37℃for 2 turbidity measurements with a turbidimeter (BD company Phoeni xSpec Nephel omter) at 0min, 10 min, 30 min, 60min, 90 min, 120 min, and the data were recorded.

Results

Table 1 observation of culture turbidity and change in bacterial count of broth:

TABLE 1

The amount of bacteria in the Escherichia coli broth cultured for various times is shown in FIG. 1;

the turbidity changes of the E.coli broth culture for different times are shown in FIG. 2.

Conclusion:

1. when bacteria are cultured in a broth, the bacteria are observed to be greatly different from each other by different methods, the sensitivity of the currently commonly used bacterial colony growth turbidimetry is poor, and the difference can not be detected within 120 minutes.

2. The bacterial count method can determine significant differences within 30 minutes of broth culture, demonstrating the feasibility of the bacterial drug susceptibility assay.

Example 2

Method for detecting the inhibition of ATOC25922 Escherichia coli by ampicillin (by resistance count method):

materials and methods:

1. preparing strains:

the standard strain ATOC25922 E.coli was transferred for use and incubated at 37℃for 18 hours.

2. Preparation of broth:

10 of the drug sensitive test tubes (or cups) contained the required antibiotic at a double dilution concentration (different drug concentrations refer to the CLSI standard in the united states); the eleventh tube contained no antibiotic as a Positive Control (PC); the twelfth tube without bacterial suspension served as a Negative Control (NC).

The prepared strain was ground on the wall of MH (antibiotic susceptibility) broth, mixed well, and the bottle cap was capped and turbidity was measured with a turbidimeter (BD company Phoeni xSpec Nephel omter) at 0.5 McO for use.

Bacteria to be tested were inoculated at the inoculation concentration of the CLSI (clinical laboratory standard tissue) broth dilution drug sensitivity test and incubated in an incubator at 37 ℃.

Preparing bacterial liquid: the alternate colonies were picked up to make a bacterial suspension at a concentration of 0.5 McO. The colony suspension is added into broth with different concentrations (MH) of various antibiotics, and after each tube is inoculated, the bacterial content is 1X 10-4 cfu/ml to 5X 10-7 cfu/ml, and the bacterial content is most suitable for 5X 10-6 cfu/ml (colony forming units/ml), namely the bacterial content is most suitable for 5X 10-6 colony forming units/ml.

3. Resistance counting method (Coulter principle)

Incubator incubation at 37℃was performed at 0min, 10 min, 30 min, 60min, 90 min, 120 min, and cell counts were performed by resistance counting (RC-3000 resistance (Coulter) particle counter, european and American Pearl sea). The average was taken 2 times and the data was recorded.

Table 2 table oc25922 the bacterial count changes of escherichia coli on the ampicillin rapid drug sensitive assay are shown in the following table:

TABLE 2

The results of observation of the change in turbidity of the culture of the broth bacteria and the change in the number of bacteria are shown in FIG. 3, wherein the abscissa represents the concentration value of the antibiotic drug, and the unit of the first horizontal line in Table 2 is μg/ml (microgram per milliliter), and the ordinate represents the number of bacteria in units of mu.l (each microliter).

A total of 12 tubes were tested, the eleventh tube as Positive Control (PC), the twelfth tube as Negative Control (NC), and 10 additional tubes as test tubes, 12 tubes were assayed at each test time point.

The results of the resistance drug sensitivity test are shown in FIG. 3, namely, the inhibition of ampicillin on the bacterium ATOC25922 E.coli in this example was detected, and the detection result was a minimum inhibitory concentration of 4. Mu.g/ml.

In addition, the bacteria in this example were also subjected to a drug susceptibility test in which the inhibition of ampicillin against the bacteria ATOC25922 E.coli in this example was measured according to the operating manual of the drug susceptibility system for VI TEK microorganism identification by French Mei Liai, with a minimum inhibitory concentration of 4 μg/ml (microgram per milliliter); according to the Et est method, the method is described in an operation manual of Et est kit of the company Sieimer, and the inhibition of ampicillin on the bacterium ATOC25922 Escherichia coli in this example is detected, and the detection result is a minimum inhibitory concentration of 2 mug/ml; the inhibition of ampicillin by the bacterium ATOC25922 E.coli in this example was tested according to the broth dilution method drug sensitivity test, which was described in detail in the American clinical laboratory standards institute broth dilution method drug sensitivity test, and the minimum inhibitory concentration of 4. Mu.g/ml was found to be consistent and sensitive.

Conclusion:

MIC (minimum inhibitory concentration) was determined within 1.60 minutes.

2. The MIC (minimum inhibitory concentration) and VITEK (Mei Liai public drug sensitivity test) methods are measured within 60 minutes, and the Eest method and the broth dilution method have consistent drug sensitivity test results, namely the feasibility of the bacterial drug sensitivity detection method is demonstrated again, and the beneficial effects of remarkably and rapidly obtaining the bacterial drug sensitivity result are achieved.

From examples 1 and 2, it is understood that the inhibition results of the inhibition detection method of bacteria by the antibiotic of the present invention are compared with those of the conventional method: the results were compared to VI TEK (Mei Liai public drug sensitive assay), et est and BMD (broth dilution method) results, and the comparison standard was determined according to FDA (american food and drug administration) regulations.

The observation of 20%, 40%, 60% or 80% decrease in cell count compared to the positive control was used as a decision point for drug sensitivity and compared to the consistency of the traditional method, which concluded that the correct rate reached 100% when cell count was decreased by 40% to 60% or more, where the optimal time for different bacteria to reach 40% to 60% decrease in cell count was different, and that clinically common bacteria could all be completed within 90 to 120 minutes, and most clinically common bacteria could all be completed within 90 minutes.

Example 3

1. Growth experiment

1.1 purpose of experiment

Six common strains (ATOC 29212, ATOC29213, ATOC27853, ATOC25922, klebsiella pneumoniae ATOC700603 and Acinetobacter baumannii) commonly used in clinic are inoculated according to the CLSI standard requirement, and the growth trend is recorded to determine whether the possibility of growth is determined for 2 hours.

1.2 Experimental methods

1.2.1 negative control: detecting the culture medium without bacteria, detecting by a resistance bacteria counter, and recording the particle number;

1.2.2 Strain preparation: according to the CLSI standard requirement, picking up a fresh strain within 0.5 McAb 24h, and adding 100ul of the fresh strain into 10ml of culture medium;

1.2.3 recording results at 0h/0.5h/1h/1.5h respectively, removing negative background at the same time, and analyzing the results.

2.3 experiment results are shown in fig. 4, and the growth experiment results show that the bacteria have changed obviously in 2h, and the change can be captured clearly by a resistance counting method, so that the feasibility of resistance counting is shown in the view of observation and detection.

2.2h and 24h drug susceptibility comparison results (the feasibility of performing bacterial drug susceptibility by the resistance method was determined from the results of drug susceptibility for both time periods).

2.1 purpose of experiment

And (3) respectively selecting a group of A drugs for comparison of results aiming at clinical common standard strains, and examining consistency results of 2h and 24 h. Strains and corresponding antibiotics are shown in Table 3.

TABLE 3 Table 3

Strain name	Antibiotics
		Pseudomonas aeruginosa (ATOC 27853)	Ceftazidime
Staphylococcus aureus (ATOC 29213)	Erythromycin
		Enterococcus faecalis (ATOC 29212)	Penicillin
Klebsiella pneumoniae	Gentamicin
		Acinetobacter baumannii	Meropenem

2.2 Experimental methods

2.2.1 Strain preparation: respectively picking fresh strains within 0.5 McAb 24h according to the CLSI standard requirement and the strain name list 3;

2.2.2 inoculation: respectively taking 100ul of 0.5 M.sp suspension, adding the suspension into a gradient antibiotic 48-well plate prepared in advance, and adding 500ul of the suspension into each well;

2.2.3 incubation and detection: incubating at 37deg.C for 2 hr, detecting with resistance bacteria counter, adjusting the instrument to optimal sensitivity state, counting bacteria, recording 24 hr conventional drug sensitivity result, and performing comparative analysis.

2.3 analysis of experimental results

TABLE 4 Table 4

As can be seen from the results of fig. 5 and table 4, the first-choice drug on CLSI requirements was selected for five strains common in clinic for antibiotic susceptibility testing, the MIC obtained by the resistance counting method (determination of MIC according to 60% of 2h positive count value as critical point) was compared with the conventional susceptibility testing method by visual inspection of 24h susceptibility results, the difference was in an acceptable range, thereby proving the feasibility of the resistance counting method on bacterial susceptibility testing.

3. Sensitivity compliance rate

A96-well enterobacter drug-sensitive reagent plate produced by 11 clinically common enterobacteria (escherichia coli 2, morganella, shigella, enterobacter aerogenes, strabismus, klebsiella pneumoniae 2, proteus mirabilis, enterobacter cloacae and salmonella) is selected, the reagent plate is inoculated according to CLSI requirement, the reagent plate is incubated for 2 hours at 37 ℃, two reagent plates are inoculated, one reagent plate is subjected to bacterial counting result interpretation for 2 hours, and is compared with a positive control, and a positive result value with a result value not less than 60% is initially selected as a positive value, so that the result is recorded. One reagent plate is used for recording the experimental result of 24 hours and judging the sensitivity coincidence rate of 2 hours and 24 hours, and the result is shown in figure 6, which shows that the clinical common antibiotics have higher coincidence rate compared with the traditional CLSI result for carrying out the drug sensitivity test by the resistance counting method for 11 common enterobacteria.

Example 4

Results of drug sensitivity test of living bacteria and consistency with traditional method

1.1 purpose of experiment

For the clinical common standard strain, a group of A drugs are respectively selected for result comparison, the consistency results of 2h and 24h are inspected, the possibility of determining the growth of the strain in 2h is recorded, and the strain and the corresponding antibiotics are shown in Table 5.

TABLE 5

1.2 Experimental methods

1.2.1 Strain preparation: fresh strains within 24h of 0.5 McRapid were picked according to the CLSI standard requirements and according to the list of strain names in Table 6.

1.2.2 inoculation: 100ul of 0.5 M.sp suspension was added to a 48-well plate of gradient antibiotic prepared in advance, with 500ul of each well.

1.2.3 incubation and detection: live bacteria were counted by incubation at 37℃for 2h and 24h of conventional drug sensitivity was recorded for comparative analysis.

1.3 analysis of the results of the experiments,

1.3.1 experimental results

Drug sensitivity results of pseudomonas aeruginosa to ceftazidime:

quick drug sensitivity results of living cells: mic=2; traditional drug sensitivity results: mic=2.

Specific data on the rapid drug sensitivity of living cells are shown in Table 6.

TABLE 6 concentration of antibiotic drug in μg/ml for the first lateral and bacteria in μg/μl for the second lateral

Drug sensitivity results of staphylococcus aureus to erythromycin:

quick drug sensitivity results of living cells: MIC = 0.25; traditional drug sensitivity results: mic=0.25.

Specific data on the rapid drug sensitivity of living cells are shown in Table 7.

TABLE 7 concentration of antibiotic drug in μg/ml for the first lateral and bacteria in μg/μl for the second lateral

Drug sensitivity results of enterococcus faecalis to penicillin:

quick drug sensitivity results of living cells: mic=1; traditional drug sensitivity results: mic=2.

Specific data on the rapid drug sensitivity of living cells are shown in Table 8.

TABLE 8 concentration of antibiotic drug in μg/ml for the first lateral and bacteria in μg/μl for the second lateral

Drug sensitivity results of klebsiella pneumoniae to gentamicin:

quick drug sensitivity results of living cells: mic=8; traditional drug sensitivity results: mic=8.

Specific data on the rapid drug sensitivity of living cells are shown in Table 9.

TABLE 9 concentration of antibiotic drug in μg/ml for the first lateral and bacteria in μg/μl for the second lateral

Drug sensitivity results of acinetobacter baumannii on meropenem:

quick drug sensitivity results of living cells: MIC = 0.12; traditional drug sensitivity results: mic=0.12.

Table 10 of the live cell rapid drug sensitivity specific data.

Table 10 (wherein the first traverse number is the concentration of antibiotic drug in μg/ml and the second traverse number is the bacterial number in μg/ml)

From the results in tables 6 to 10, it can be seen that the MIC obtained by the living cell count method (determination of MIC according to 60% of the 2h positive count value as the critical point) was compared with the conventional drug sensitivity test method by visually observing the drug sensitivity results for 24h, and the difference was in an acceptable range, so as to prove the feasibility of the living cell count method in the bacterial drug sensitivity test, and the consistency with the conventional method.

Example 5

5.1 determination of the onset of sensitization determination:

negative control: particle count of bacteria inoculated in nutrient-free environment;

positive control: bacterial or fungal inoculation in the absence of antibiotic medium in natural growth of particle number;

starting point judgment: the number of positive control cells for bacterial growth which satisfies 2-8 times of the negative control is used as an initial determination point of the drug sensitivity result.

Negative control: particle number variation of bacterial inoculation in nutrient-free environment

Table 11 (Klebsiella pneumoniae)

Cultivation time (min)	Negative control cell count	Positive control cell number
			0	1636	1711
6	1733	1952
			12	1814	2164
18	1756	2437
			24	1737	2580
30	1825	2982
			36	1970	3590
42	1868	4186
			48	1733	4207
54	1665	4993
			60	1871	5074
AVERAGE	1782.545455
			SD	97.99935065
CV	5.5

As is clear from Table 11, the bacteria were not grown in the bacterial growth count of Klebsiella pneumoniae without nutrients for 0 to 60min, and thus served as a reference point for negative control.

Positive control: from the positive control, the count value of the klebsiella pneumoniae naturally growing in 36min shows that the bacteria are doubled in 36min, which lays a foundation for realizing the drug sensitivity of cell count.

5.2 correlation between single cell count and drug sensitivity

5.2.1 fixed threshold method

Taking the positive control multiplied by a coefficient (0.2-0.6) as a reference point, taking the first antibiotic concentration corresponding to the count value exceeded as the MIC of the bacteria at the antibiotic concentration, and then determining the sensitivity of the bacteria to the antibiotic by referring to the CLSI standard.

5.2.2 inflection point determination method

According to a certain algorithm:

r 1-r0=k0, r 2-r1=k1, and when the value of k1-k0 is larger than a predetermined threshold value, a positive point is indicated, that is, the antibiotic concentration is the Minimum Inhibitory Concentration (MIC) of the bacteria at the antibiotic concentration, where r1 is the count value of the medium concentration gradient, r0 is the count value of the low concentration gradient, and r2 is the count value of the high concentration gradient.

Table 12 (Enterobacter cloacae-levofloxacin)

Drug name	Gradient of	Count value
			Levofloxacin	0.008	69404
Levofloxacin	0.015	69064
			Levofloxacin	0.03	59692
Levofloxacin	0.06	9529
			Levofloxacin	0.12	6680
Levofloxacin	0.25	8352
			Levofloxacin	0.5	8023
Levofloxacin	1	8443
			Levofloxacin	2	6983
Levofloxacin	4	4796
			Levofloxacin	8	3988
Positive control		70413

The results of Table 12 are shown in FIG. 7.

TABLE 13 Enterobacter cloacae-ciprofloxacin

Drug name	Gradient of	Count value
			Ciprofloxacin	0.004	70499
Ciprofloxacin	0.008	69458
			Ciprofloxacin	0.015	65047
Ciprofloxacin	0.03	31476
			Ciprofloxacin	0.06	6528
Ciprofloxacin	0.12	7461
			Ciprofloxacin	0.25	8997
Ciprofloxacin	0.5	9502
			Ciprofloxacin	1	8540
Ciprofloxacin	2	6398
			Ciprofloxacin	4	6008
Positive control	-1	70413

The results of Table 13 are shown in FIG. 8.

TABLE 14 Enterobacter cloacae-gentamicin

Drug name	Gradient of	Count value
			Gentamicin	0.03	67172
Gentamicin	0.06	67875
			Gentamicin	0.12	60382
Gentamicin	0.25	5901
			Gentamicin	0.5	2358
Gentamicin	1	1201
			Gentamicin	2	1010
Gentamicin	4	911
			Gentamicin	8	907
Gentamicin	16	893
			Positive control	70413

The results of Table 14 are shown in FIG. 9.

TABLE 15 Enterobacter cloacae-tobramycin

The results of Table 15 are shown in FIG. 10.

5.2.3 orthogonal threshold and inflection point determination method of bacteria and antibiotics;

because different antibiotics have different action mechanisms or inhibition effects corresponding to different bacteria, different thresholds are selected for different bacteria, and the thresholds selected by tetracycline for pseudomonas aeruginosa are much smaller than those of normal bacteria.

5.2.4 empirical correction

In the single cell counting process, part of bacteria react slowly with antibiotics in a short time, the inflection point of the coccus is more gentle relative to the bacillus, and the inflection point of the coccus needs to be adjusted on the basis of the original algorithm until the curve is smoothed and then is increased by 1-2 gradients, so that the inflection point is used as a calculation point of MIC.

Table 16

Enterococcus faecalis	2hMIC	24hMIC
			Tetracycline
	2	8

TABLE 17 enterococcus faecalis-tetracycline

Antibiotics	Gradient of	Count value
			Tetracycline	0.06	13659
Tetracycline	0.12	13802
			Tetracycline	0.25	11742
Tetracycline	0.5	7912
			Tetracycline	1	3637
Tetracycline	2	2402
			Tetracycline	4	1838
Tetracycline	8	1768
			Tetracycline	16	1747
Tetracycline	32	2196

The results of Table 17 are shown in FIG. 11, and it is clear from Table 16 that the MIC was 2 as determined by the algorithm of inflection points, but the conventional drug susceptibility was 8, and the experiment was concluded through experiments of 16 enterococcus faecalis strains against tetracycline, so that the correction of the drug concentration was performed to correct the MIC2 to 8, which is a systematic deviation. 5.2.5MIC multipoint prediction method

The method has the advantage that the MIC of the drug sensitivity can be estimated by calculating more drug gradient values through the drug concentration of fewer points. And estimating the concentration gradient which is not on the board card according to the concentration gradient which is related on the board card. For example: taking the example of colibacillus aiming at ampicillin, the count value of a 2h positive growth hole is 30000, and only drug gradients of 0.5, 2 and 8 are arranged on a board, if the count value of a 0.5 drug gradient hole is less than or equal to 3000, the MIC is predicted to be less than 0.25; if the positive control for a 0.5 drug gradient well is equal to or greater than 30000 a.0.6=18000, then its drug concentration is determined to be 0.5. The count value is determined to be equal to 0.25 between 10% and 60%, and so on.

TABLE 18

6. Drug sensitivity results

And determining the sensitivity of antibiotics corresponding to bacteria according to the MIC corresponding to the CLSI standard determined by the algorithm.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method of detecting bacterial inhibition by an antibiotic using single cell counting, said method for non-disease diagnosis comprising:

obtaining a current number of the bacteria of the bacterial antibiotic mixture and a current number of the bacteria of the positive control when a first predetermined time period is reached from a time of addition of the antibiotic;

determining a minimum inhibitory concentration of the antibiotic on the bacteria based on the current number of the bacteria of the bacterial antibiotic mixture and the current number of the bacteria of the positive control, determining a degree of inhibition of the antibiotic on the bacteria;

obtaining a current number of the bacteria of the bacterial antibiotic mixture and a current number of the bacteria of the positive control using a resistance count method;

the calculation is performed according to the following inflection point judgment method:

r 1-r0=k0, r 2-r1=k1, and when the value of k1-k0 is greater than a predetermined threshold, the antibiotic concentration is the minimum inhibitory concentration of the bacteria at the antibiotic concentration, wherein r1 is the count value of the medium concentration gradient, r0 is the count value of the low concentration gradient, and r2 is the count value of the high concentration gradient.

2. The method of claim 1, wherein the threshold is determined by an inhibitory effect of the antibiotic against the bacteria.

3. The method according to claim 1, wherein when the bacterium is a coccus, the minimum inhibitory concentration of the coccus is increased by 1 to 2 gradients on the basis of the inflection point judgment method calculation as the minimum inhibitory concentration of the coccus.