Minimum overwhelming concentration. Determination of the minimum overwhelming concentration on a dense medium
Methods for determining sensitivity to antimicrobial means. Minimum inhibiting concentration (MIC). Method of serial dilutions in liquid media.
The criteria of activity of this or that drug are minimum inhibiting concentration (Mick) - the smallest concentration of the drug, which is inhibiting the growth of test culture and minimum bactericidal concentration (MBK) - the smallest concentration of the drug, which causes a bactericidal effect.
Method of serial dilutions in liquid media
Method of serial dilutions in liquid media Allows you to install minimal inhibiting concentration (Mick) I. minimal bactericidal concentric (MBK) The drug for the dedicated pathogen. Studies can be performed in different volumes of the nutrient medium (1-10 ml). Use liquid nutrient media corresponding to the food needs of the causative agent. In test tubes (usually eight) prepare a series of double dilutions of the drug on the nutrient medium. The concentration is reduced by 128 to 0.06 μg / ml, respectively (base concentration may vary depending on the activity of the drug). The final volume of the medium in each tube is 1 ml. Control serves a test tube containing a pure nutrient medium. In each test tube, 0.05 ml of a physiological solution containing 106 / ml microbial cells are brought. The tubes are incubated for 10-18 hours at 37 ° C (or before the appearance of bacterial growth in the control tube). Upon expiration of the specified period, the results are taken into account by changing the optical density of the medium visually or nephowerometric. You can also use a modified method using a medium supplemented with glucose and indicator. The growth of microorganisms is accompanied by a change in the pH of the medium and, accordingly, the color of the indicator.
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Name of substances |
The level of threshold |
|---|---|
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Amphetamine Group |
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Amphetamine |
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Methamphetamine |
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MetylandioxyMofthemine (MDA) |
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Other substances amphetamine group |
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Group opiate |
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Morphine |
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Codeine |
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6-monoacetylmorphin |
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Group of benzodiazepines |
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Ocasazepam |
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Diazepam |
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Nordiazepam |
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Midazolam |
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Penazepam |
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Other substances of the group of benzodiazepine |
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Group of barbiturates |
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Barbamil |
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Sodium etame |
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Chemicals of other groups |
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11-Nor-Δ 9 -thetrahydrokannabolic acid (main metabolite δ 9-tetra-hydrocannabinol) |
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Cocaine and its metabolites |
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Methadon and its metabolites |
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Propoxyphen and its metabolites |
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Bucrenorphine and its metabolites |
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d-LIZERGID (LSD, LSD-25) |
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Fentanyl and its metabolites |
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Metaqual |
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Fencyclidin |
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Table 2 Annexes to the procedure for conducting medical examination on the state of intoxication (alcoholic, narcotic or other toxic), approved by the order of the Ministry of Health Russian Federation (Project), "Levels of threshold content values narcotic drugs, psychotropic substances, other chemical substances and their metabolites defined by the methods of confirming analysis. "
Note: the level of threshold - This is the minimum concentration of the substance (its metabolite) in the biological object, determined by the methods of preliminary or confirming analysis, when the result of the study is considered positive.
In fact, a similar table called " Threshold levels for confirming analysis methods in urine study"is provided in the Central Chemical-toxicological Laboratory at the Department of Analytical and Forensic Medical Toxicology of State educational institution Higher Professional Education First Moscow State Medical University named after I. M. Sechenov (Tshtl GOU VPO First MGMU. I. M. Sechenov) Ministry of Health of the Russian Federation dated August 30, 2011 No. 179-25 / 12I, where, however, Other things are indicated by the concentrations of phenobarbital (1 "000 ng / ml), other substances from the barbiturate group (100 ng / ml) and the seabe (100 ng / ml). According to this TCCHL GOU VPO, the first MGMU them. I. M. Sechenov, in accordance with paragraph 2 of the Order of the Ministry of Health and Social Development of the Russian Federation of January 27, 2006 No. 40 "On the organization of conducting chemical-toxicological studies at analytical diagnosis presence in the human body of alcohol, narcotic drugs, psychotropic and other toxic substances"Developed and approved the requirements for conducting chemical-toxicological studies in the analytical diagnosis of the presence of narcotic drugs, psychotropic and other toxic substances in the human body.
In particular, according to paragraph 12 of the information letter in the medical examination of drivers of vehicles, testing students, conducting chemical-toxicological studies when dealing with citizens and other cases established by the legislation, the study of fluid oral cavity (saliva) is unacceptable, since it does not allow reliably to establish the fact of the presence in the human body of narcotic drugs, psychotropic and other toxic substances. Controlled substances can be determined in the fluid of the oral cavity (saliva) in the time interval not exceeding several hours from the moment of use.
Requirements for technical means used to detect narcotic drugs, psychotropic and other toxic substances (their metabolites) in urine samples, during preliminary chemical-toxicological studies (Appendix No. 1 to Methodical recommendations: Rules for conducting chemical-toxicological studies for the existence of students in general educational organizations and professional educational organizations, as well as educational organizations higher education In order to early detecting illegal consumption of narcotic drugs and psychotropic substances of narcotic drugs, psychotropic and other toxic substances (their metabolites) / Developer: Association of specialists and organizations of the Laboratory Medicine Federation of Laboratory Medicine, edited by the Main Freelance Specialist in the Analytical and Forensic Medical Toxicology Ministry of Health Russia, D.Kh.n., Professor B.N. Isota and the main freelance specialist in clinical laboratory diagnostics Ministry of Health of Russia, D.M., Professor A. G. Kochetova // Moscow, 2015)
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Name of groups of substances |
Concentration (NG / ml) |
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Opiates (6 monoacetylmorphine, morphine, codeine, dehomorphine, etc.) |
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Cannabinoids |
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Phenylalkylamin (amphetamine, methamphetamine, mefedron, etc.) |
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Methadone |
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Benzodiazepines |
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MDMA |
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Cocaine |
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Barbiturates |
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Sotinine |
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Synthetic cannabinoids |
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Katinonon |
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Ethylglucuronid |
Requirements for technical means used to detect narcotic drugs, psychotropic and other toxic substances (their metabolites) in urine samples, during confirmation of chemical-toxicological studies (Appendix No. 2 to the above source)
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Name of groups of substances |
Concentration (NG / ml) |
|---|---|
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Amphetamine Group |
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Amphetamine |
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Methamphetamine |
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MetylandioxyMofthemine (MDA) |
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Metiladioxymethamfetamine (MDMA) |
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Other substances amphetamine group |
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Group opiate |
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Morphine |
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Codeine |
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6-monoacetylmorphin |
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Other substances of opium group |
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Group of benzodiazepine |
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Ocasazepam |
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Diazepam |
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Nordiazepam |
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Midazolam |
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Penazepam |
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Other substances of the group of benzodiazepine |
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Group of barbiturates |
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Phenobarbital |
1000 |
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Barbamil |
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Sodium etame |
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Other substances of the Barbiturate Group |
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Substances of other groups |
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11-Nor-Δ9-tetrahydrokannabolic acid (main metabolite Δ9-tetra-hydrocannabinol) |
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BenzoylEkgonine (cocaine metabolite) |
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Methadone |
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Propoxyphen |
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Buprenorphin |
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LSD |
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Fentanyl |
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Metaqual |
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Fencyclidin |
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Sotinine |
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Synthetic cannabinoids |
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Katinonon |
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Ethylglucuronid |
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At the same time, when implementing protection for administrative offenses under articles 12.8 and 12.27, part 3 of the Code of Code of the Russian Federation, as well as on cases involving criminal liability in managing the vehicle in a state of intoxication (Articles 264 and 264.1 of the Criminal Code of the Russian Federation) should not be forgotten That administrative responsibility occurs in the case of the presence of narcotic drugs or psychotropic substances in the human body, regardless of their concentration in the human body, in the blood and in the urine.
By virtue of the identification of drug-toxicological studies, judicial research of drugs within the framework of narcological examinations, chemical-toxicological research, funds, psychotropic and toxic substances, and intoxication substances, even at the level, so to speak, the "method of detection of the method used" is the basis for attracting the driver vehicle to administrative or criminal liability for the relevant articles of the Codex of the Russian Federation on administrative offenses and / or criminal code of the Russian Federation.
For information - " "
7. The essence of the concepts is the minimum inhibitory (overwhelming) concentration and minimum bactericidal concentration.
Minimum inhibiting concentration (MIC)- minimum concentration of chemotherapeutic or antiseptic substance causing Full suppressionnoticeable with naked eye Heostthis microorganism on media under standard conditions of experience.
Measured in μg / ml or in units. actions. It is established by sowing a tested culture on dense or liquid media containing various concentrations of the drug.
Minimum bactericidal concentration (MBC)- the minimum concentration of chemotherapeutic or antiseptic agent causing Full deathbacteria in standard experience conditions.
Measured in μg / ml or units. actions. It is established by sowing a tested culture on dense or liquid nutrient media containing different concentrations of the drug. To distinguish between MIC from sterile zones or transparent tubes, it is made on the medium without a drug (the appearance of growth indicates a static action, the absence of it is on a zeal).
MBK and MICs are used in chemotherapy and antiseptics to select efficient drugs and doses for this patient.
8. The concept of concepts sensitivity and resistance of the pathogen, an additional libeotic effect.
Sensitivity of the pathogen- the absence of the mechanisms of resistance to xs; In this case, the reproduction of the pathogen is suppressed by the average therapeutic doseexceeding the minimum inhibitory concentration of 2-4 times.
Resistant pathogen- the presence of mechanisms of resistance to xc; The growth of the pathogen is not suppressed by the concentration of the drug that has the toxic effect in vivo.
Old-proteic effect- persistent inhibition of the vital activity of bacteria after their short-term contact with the antibacterial preparation.
9. Determinants of selective toxicity of chemotherapeutic agents.
1) HS accumulates in microbial cells in concentrations many times large than in mammalian cells
2) Xs act on the structures that are available only in the microbial cell (cell wall, DNA girase II type) and are absent in the mammalian cell
3) Xc act on biochemical processes flowing exclusively in microbial cells and missing mammalian cells.
10. The essence of the difference in pharmacodynamic and chemotherapeutic properties.
1. Pharmacodynamic therapy operates at the level of architecture of functional systems, its effects are usually reversible. For chemotherapy, agents are most valuable with the most irreversible action.
2. Pharmacodynamic agents cause a gradual response of the body system, for chemotherapeutic means the most desirable effects "all or nothing".
3. Chemotherapy has a etiotropic strategy aimed at the destruction of the pathogen or on transformed organism cells, and pharmacodynamic therapy can be both etiotropic and pathogenetic.
11. Basic principles of rational chemotherapy.
1. The pathogen must be sensitive to ab
The "best sentence" rule is reference tables, taking into account regional population features of antibacterial sensitivity.
2. AB must compare therapeutic concentration in the focus.
3. Preferably adequate dosing mode, depending on:
ü pathogen
ü Dynamics of the clinical course of infection
ü Localization of infection
ü The duration and nature of the flow of infection (acute, chronic or bacterianesis)
4. Optimal duration of antimicrobial chemotherapy (example: Streptococcal pharyngitis healing for 10 days, acute uncompressed gonoxococcal urethritis for 1-3 days, sharp low cystitis in 3 days).
To prevent adverse reactions, the development of superinfection or resistance, the duration of treatment must correspond to the trace agent eradication period.
5. Accounting patient factors:
ü Allergianamnez, immunocompetence
ü liver and kidney function
ü portability oral reception; compliance
ü heaving condition
ü Age, gender, pregnancy or child feeding, oral contraceptives
ü Side Effects
6. Combined antibiotic therapy.
In principle, this procedure is similar to the definition of the IPC in the liquid "medium. Several Petri dishes are prepared with stepped antibiotic dilutions dissolved in the dinous medium. Add molten agar, the cups are cooled so that the culture of one or more bacteria is cooked on the surface of the medium and after Incubations in the thermostat for the required time determine the minimum concentration of the antibiotic, at which the growth of bacteria is suppressed. One of the advantages of this method before the titration method in the liquid medium is that different sections of the same cup can be sized. different species or bacteria strains. As a result, the MPK values \u200b\u200bfor several bacteria can be defined in one experiment (Fig. 2.2).
- Determining the activity of antibiotics
This method is used to determine the concentration of the antibiotic in the solution. It consists next. The filter paper discs moistened with the test antibiotic solution are placed on the surface of the agar medium containing the diluted suspension of bacteria. After the corresponding incubation, the surface of the agar, which was initially translucent, purre due to the scattering of the light by the grew by bacteria. Translucent zones remain only around the filter paper disks, since the antibiotic diffuses in agar and suppresses the growth of bacteria. If the diameter of these zones is to determine under strictly standard conditions, it will be a function of a logarithm of the antibiotic concentration. Using known antibiotic concentrations, build a standard curve at which an antibiotic concentration can be determined in unknown solutions (Fig. 2.3). Sometimes instead of discs from filter paper use small hollow cylinders, put on the surface of the agar medium and the antibiotic solution containing a solution.
- Factors affecting the definition
The activity of antibiotics in vitro depends on the definition conditions (Table 2.1). The definition of activity is influenced by such factors as the composition of the medium, the density of inoculum, the number of bacterial cells in Inoku Luma (the value of inoculum).
/
Table 2.1. Factors affecting the definition of antibiotic activity
Test organism
Composition of the medium (pH, ions, serum, antagonists)
The magnitude and density of inoculum (the heterogeneity of the population, the mechanisms of inactivation, the ingress of antagonist substances)
Incubation conditions (time, temperature, aeration)
A. The composition of the environment
Consider as an example an antibiotic that overwhelming the biosynthesis of one of the amino acids. If the antibiotic is experiencing in a medium without this amino acid, it seems that it has a very high activity, in other words, has a low MPK. When testing in a "medium containing amino acid, which he suppresses the synthesis and which bacterium can receive from the medium, the antibiotic will seem inactive.
In addition to these specific effects, there is often a less specific effect of the medium that is not directly related to the mechanism of action or with the chemical structure of the antibiotic. One particularly interesting case is the presence of serum in the medium. It is introduced on Wednesday when determining the IPC to create physiological conditions resembling conditions in the blood. Many antibiotics are binding to serum proteins (especially with albumin), as a result, the number of free antibiotic molecules that can penetrate bacterial cells decreases. The binding of an antibiotic with serum proteins usually correlates with the lipophobor of some substituents in its molecule.
Obviously, the conditions for determining the IPC in a liquid medium differ from the conditions in a dense medium, if only because agar is present in a dense medium. Agar, containing S03-RPYN, can adsorb the antibiotic, changing its diffusing ability, or adsorb dissolved oxygen and some components of the nutrient medium. It is not surprising, therefore, that the magnitude of the antibiotic IPC in relation to this bacteria depends on whether it is determined in a liquid or in a dense medium, even if these two media, with the exception of the presence or absence of agar, are identical in composition. It should also be remembered that cell physiology may vary depending on whether they are growing in a liquid or in the form of colonies on the surface of the dense medium.
A very strong effect on the activity of antibiotics is the pH of the medium. In addition to the secondary effects, as the effect of pH on the growth rate of microorganisms and, following
teshzyno, indirect effect on their susceptibility, the pH value has a very strong and direct effect on the ability of the drug to penetrate into the bacterial cell. For example, substances in a non-ionized form are better diffundated through a cellular steak and plasma membrane than substances in ionized form. Thus, the pH of the medium, determining the degree of ionization of the main or. Clear antibiotic, can directly affect the rate of its penetration into the bacteria and, therefore, effectiveness.
B. Density and magnitude of bacterial inoculum
Inoculum density is the number of sowing bacteria, referred to the volume in which they grow. It is usually expressed as the number of cells in 1 ml of culture. The value of inoculum is the total number of snowy bacteria. At the IPC, many antibiotics do not affect the variations in the density of the commonly used inoculum (103-106 bacterium / ml). Indeed, even with very low concentrations An antibiotic, for example 0.01 μg / ml, the ratio of the number of antibiotic molecules to the number of bacterial cells remains very large (at the concentration of an antibiotic with mol. Weight 1000, equal to 0.01 μg / ml, in 1 ml of solution contains ~ 1012 molecules). However, there are some exceptions. For example, often a lot of antibiotic molecules is adsorbed on the outer surface. bacterial cell. If the density of bacteria is high, the number of free antibiotic molecules that can penetrate into the cell is very much reduced. In addition, a large number of antibiotic molecules is often needed to suppress the growth of one cell. As the bacteria grows, the enzymes can be synthesized and isolated on Wednesday, which can destroy the antibiotic (for example, p-lacta-maza acting on p-lactam antibiotics; see ch. 4). The amount of destroyed antibiotic is a function of the concentration of the enzyme in the nutrient medium and, therefore, depends on the value of inoculum.
At first glance, it seems that if the density of culture of bacteria is the same, it does not matter whether the activity of the antibiotic in small volumes is investigated, for example 0.25 ml and less in miniaturized systems, or in a volume of 10 ml in conventional laboratory test tubes. If all bacteria in the population are identical, there will be no difference in the results.
When the total number of bacteria in Inoculum is very large, the likelihood increases cells less susceptible to the antibiotic in the medium. The growth of all susceptible cells will be depressed, but less susceptible (in principle, it may even be a single cell) will multiply, and after incubation, it is formed for 18 hours
Table 2.2. Factors affecting the activity of some antibiotics
pouution of bacteria with high density. Significant variability of the magnitude of the IPC with a change in the number of inoculated cells typically indicates a high frequency of mutants resistant to an antibiotic (ch. 4). The frequency of the appearance of stable mutants is non-etinakov for different antibiotics. In the case of antibiotics used in the clinic, it varies from 10 ~ 7 to 10-10.
This section describes not all factors that may affect the activity of the antibiotic with respect to a certain bacterium (Table 2.2). Therefore, in order to use the suppression of the growth of bacteria to quantify the activity of antibiotics and obtain data that can be reproduced in different laboratories, it is necessary that all the conditions be accurately defined and as standardized.
Analysis of the ability of bacteria to multiply and grow on media containing a decreasing concentration medicinal substanceIt allows you to determine the minimum inhibitory concentration of the antibiotic (MIC), the oppressive role of in vitro bacteria (Table 3 (Vet7)). This dose determines the choice of a drug that can achieve similar concentration values \u200b\u200bin vivo, and is the basis for comparing the relative sensitivity of the body with respect to other drugs. It is believed that to ensure the effect of exposure, the concentration of the medicinal substance in the focus of the infection should be at least equal to the value of the minimum inhibitory concentration of the antibiotic. On the other hand, the concentration of the drug in plasma should usually be higher to ensure adequate concentration in tissues. However, an unjustified increase in doses of antimicrobial drugs in order to achieve a minimum dose of an antibiotic, oppressing the growth of bacteria of a certain type in vitro, can lead to accumulating drug in the recipient body in toxic doses.
"Critical Mick" for a particular drug substance is the greatest safe concentration of the drug, which can be achieved using a clinically acceptable dose and the method of use of the drug (Table 3 (Vet7)). Mick depends on the specific type of bacterial culture and a specific type of medicinal substance. At the same time, the critical micro is specific to a specific recipient and a particular drug substance. Thus, the critical mica will be the same for any organism (Table 3 (Vet7)). The critical value of the concentration for a particular organism may differ depending on the type of animal (due to differences in sensitivity or in the nature of the distribution drug) And a specific laboratory. It is necessary to contact the laboratory providing data on the methods of cultivation and sensitivity to antibiotics, in order to obtain critical values \u200b\u200bused in the course of research conducted there.
Based on data on dilution in the tube tube, bacteria refer to sensitive (S) to a specific drug substance, if MIC is significantly lower than the critical value of this indicator. The growth of pathogenic microorganisms with an average (MS) or intermediate (IS) sensitivity is oppressed at the concentration of the drug approaching the critical value of MIC. Such bacteria can cause negative patient's body reactions or not affect it. MIC for resistant (R) bacteria exceeds the critical value of the minimum dose. The effective importance of the concentration in the patient's body of such a drug, affecting a certain microorganism, is unlikely to be achieved. In such cases, the danger of the cumulation of the drug in toxic doses may also translate the potential benefit from the use of therapy. The critical value of the minimum dose of new generation antibiotics, oppressing the growth of bacteria, in some cases it is more difficult to determine in connection with the transition to a professional flexible labeling of dosage ranges.
Drugs need to be chosen in such a way that when applied in accordance with the scheme that prevents the accumulation of a substance in toxic doses, it was possible to achieve the limiting concentration of the drug in plasma significantly exceeding MIC. Many bacteria will be sensitive to the effects of a certain medicinal substance at concentrations much lower than the critical value of the minimum dose. The difference between the critical value and own meaning Mick can be used to compare the relative efficiency of various antimicrobial drugs. For example, for amikacin, the critical value is 32 μg / ml, so E. coli with a value of Mick 2 μg / ml has a relatively greater sensitivity to amikacin than E. coli with a Mick value of 16 μg / ml. Both types should be considered sensitive (although the second form can be considered as having an average sensitivity), but the growth of the bacteria of the first type, apparently, is depressing to a greater extent. If the same type of E. coli with the value of MIC 2 μg / ml relative to amoxiciline is MIC 16 μg / ml (with a critical value of 32 μg / ml), then, apparently, the growth of this microorganism will be easier to prevent the use by applying Amicacin, and not amoxicillin, since the value of Mick Amikacin will continue from its critical value of Mick than the value of Mick Amoxicillin.
Despite the fact that the differences between the MIC values \u200b\u200bfor a particular type of bacteria and a particular drug substance (16 or 32) may seem quite large (especially in the context of the limiting concentration of the drug in plasma), such a difference corresponds to only one solution in the test tube. This is an example of the risk of revaluation of sensitivity data. If the value of Mick a specific organism is sufficiently close to the critical value, then due to possible discrepancies in the interpretation of this microorganism, the degree of sensitivity "S" or "MS" can be assigned in one laboratory, and in the other - "R". Such possible discrepancies in the assessment are one of the reasons why the use of medicinal substances should be avoided in respect of which a certain body has a sensitivity "MS" (or if Mick is close to critical), except in cases where the concentration of the drug in the field of infection can It is much higher than the value of Mick defined in vitro analysis. Visual example It may be the use of drugs derived by the kidneys for the treatment of infection urinary tract Or the use of medicinal substances with bile, for the treatment of infection bile paths. The accumulation of certain medicinal substances with leukocytes (fluoroquinolones, macrolides) can also lead to a concentration of the drug in tissues, a significant exceeding Mick (or the critical value of Mick), despite the lower concentration in the plasma.
Mick bacteria may vary in subsequent infections caused by the bacterium of the same type, and also can change in the process of infectious disease. An increase in the MIC value may simply reflect a different approach to an assessment of the analysis results (especially if the differences are detected only during dilution in the tube), however, it may also be considered a consequence of the production of resistance relative to a certain drug substance. In such cases, the course of antimicrobial therapy can be changed due to the use of an additional drug or transition to a new, more effective drug. In polyimicrobial infections, the Mick value of a particular drug substance is likely to be different for each infecting bacterium. It is believed to be easier to prevent the growth of bacteria with a low value of MIC relative to a certain drug substance than the growth of the microorganism with more high meaning MIC relative to the same medicinal substance.