Disinfection, sterilization and preservation. Denyer S. A and Gorman S. P American Journal of Infectious Diseases. Finch R. Antibiotic and chemotherapy, 8 th edition. Churchill Livingstone, London and Edinburg. Russell A. D and Chopra I Understanding antibacterial action and resistance. Save my name, email, and website in this browser for the next time I comment. Spread the love. Figure 1: Broth dilution method for determining the MIC of antibiotic. Leave a Reply Cancel reply Your email address will not be published.
Up to 7 compounds and one quality control QC antibiotic are serially diluted from column 1 to column 11 of a well microplate to form a concentration gradient. Column 12 serves as a positive growth control.
Click on the image below to watch a video of our Biology scientists explaining the basics of the MIC assay! All Rights Reserved. Minimum Inhibitory Concentration Assay. Principles The Minimum Inhibitory Concentration Assay is a technique used to determine the lowest concentration of a particular antibiotic needed to kill bacteria. Note: MIC values will differ between bacteria. However, currently, the gradient test is not allowed for fosfomycin MIC determinations by American regulators [ 4 ] and EUCAST does not recommend it as a reference method either [ 3 ], although some national recommendations like, e.
The gradient method requires a 0. Reading the MIC value using the E-test gradient strip is more complicated than performing the test itself. It may depend on the tested bacterial strain, the antibiotic in particular whether it is bacteriostatic or bactericidal , the resistance mechanism, the presence of a heterogeneously resistant population and even on the way in which the E-test gradient strip test is performed.
The above-mentioned fosfomycin can be an example here, for which the growth of individual colonies in the growth inhibition zone should not be taken into account in determining MIC value [ 32 ]. The determined MIC value must be compared with MIC clinical breakpoints to assess whether the strain is susceptible or resistant to the antibiotic.
Evaluation of antibiotic resistance based on the MIC value does not mean the identification of the resistance mechanism. However, due to epidemiological reasons, qualifying such a strain to resistant category according to the MIC value may be a trigger to undertake further research on the detection of resistance mechanism. The determination of clinical breakpoints BP requires the cooperation of specialists in various fields: microbiologists, pharmacologists, infectious diseases physicians, but also experts in data processing and statistical analysis [ 33 , 34 ].
BP determination requires taking into account antibiotic doses including the maximum doses for which these values will be established, clinical indications for which they will be applied and reference to the specific micro-organisms.
ECOFF distinguishes between bacterial strains without any phenotypically established acquired antibiotic resistance mechanism and those displaying such mechanisms [ 35 , 36 , 37 ]. The presence of resistance mechanisms to various antibiotics is also subject to phenotypic and genotypic evaluation. Literature reports also need to be analyzed and clinical breakpoints for MIC need to be finally set but in such a manner so that they do not separate MIC values for wild type strains [ 34 ].
MIC clinical breakpoints in turn are used to establish the clinical breakpoints for the disc-diffusion method correlating with the BP for MIC. The clinical MIC breakpoints for susceptible strains are usually not equal to the epidemiological cut-off values for susceptible strains. The former is most often higher and includes not only wild-type strains but also those with low levels of resistance, which, however, does not affect clinical efficacy [ 1 ].
In addition, the established values are not constant, so they are updated periodically, as microbes and dosing rules change. Therefore, when comparing the incidence of susceptible and resistant strains across the world, it is necessary to take into account the impact of recommendations on cumulative data. Like the bacterial growth inhibition zone in the qualitative method, the MIC value serves as the basis for assessing the category of susceptibility or resistance of the pathogen to a given antibiotic.
According EUCAST [ 39 ] recommendations, two susceptibility categories and one resistance category have been introduced since Susceptible S , standard dosing regimen: there is a high likelihood of therapeutic success using a standard dosing regimen of the agent. Susceptible I , increased exposure: there is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection.
Resistant: there is a high likelihood of therapeutic failure even when there is increased exposure. The major shift in the clinical interpretation of results, concerns those bacterial strains classified by the end of as intermediate susceptible to antibiotics I.
Such strains were previously included in the epidemiological reports as resistant. Both, an antibiotic with the new susceptibility category —I and one with the -S category, contributes to the same degree of clinical efficacy. However, EUCAST has prepared a table with the dosing rules taking into account standard dosing for category S and high doses for the new category I doses were updated in ,version This facilitates the decision-making with regard to dosing or with regard of the method of drug administration.
Based on new rules of MIC values interpretation, the use of, e. Wantia et al. Proper clinical interpretation will result in inclusion of antibiotics with category I to therapeutic possibilities, therefore, there is an increased need of healthcare professionals training on newly introduced rules of MIC values clinical interpretation.
For some antibiotics and bacteria, the determination of MIC is the only reliable phenotypic method for assessing drug sensitivity because qualitative methods have provided false results Table 3 [ 3 , 4 ].
Reason might be poor penetration of an antibiotic into agar, like it happens in case of polymyxin or daptomycin; alternatively, due to impossible distinctions between wild type isolates and those with non-vanA-mediated glycopeptide resistance in Staphylococcus spp. For anaerobic bacteria false results may occur due to nondefined breakpoints [ 40 , 42 , 43 ].
With regard to daptomycin, it is also difficult to obtain appropriate concentration of calcium ions in agar, which are necessary for the evaluation of bacterial susceptibility. All of the facts mentioned above make the disc-diffusion method not recommended [ 44 ].
Antibiotics and pathogens for which susceptibility can only be derived from quantitative phenotype methods. In addition, contrary to qualitative methods, the MIC value allows to assess the degree of susceptibility or resistance to the antibiotic. Information on the degree of susceptibility carries great epidemiological and clinical value, but it must be properly interpreted.
Such an interpretation leads to the erroneous belief that the strain is most sensitive to the antibiotic for which the MIC is the lowest. EUCAST has greatly facilitated the assessment of susceptibility degree of micro-organisms to antibiotics by introducing new criteria for result interpretation. These criteria distinguish between two levels of susceptibility of strains.
The first level concerns standard dosing whereas the second level is referred to higher MIC values and requires increased exposure to the antibiotic.
It must not be forgotten that in some cases higher levels of antibiotic MIC close to breakpoints may indicate therapy failure, although the strain might be considered as sensitive at standard doses. This may be the first signal of resistance to the medicine.
Such a phenomenon was found, for example, in Salmonella enterica serovar Typhi S. The relationship between the MIC value of susceptible strain and the effectiveness of the medicine is also described when vancomycin is used in S. Why does the degree of susceptibility matter? The more susceptible the strain to the antibiotic, the greater the likelihood that its MIC is below the ECOFF and therefore the strain does not develop any drug-resistant subpopulation, so there is no increased risk of bacterial survival during treatment.
In addition, high susceptibility of the strain increases the chance for reaching the therapeutic concentration of the antibiotic and for effective eradication of the pathogen using the standard dosage even in patients with significant changes in pharmacokinetic parameters. The knowledge of the degree of susceptibility of strains to various antibiotics used in the hospital may also be a tool used in antibiotic stewardship [ 47 , 48 , 49 , 50 ].
Thus, antibiotics whose MIC value for most strains MIC 90 is close to the breakpoint could be transferred to a group of medications with a limited access to empirical therapy, unless of course there are other factors in favor of using such an antibiotic. Doing so could contribute to reducing the selection of antibiotic-resistant strains, although this would need to be confirmed in the results of studies which are difficult to perform. It is virtually impossible to obtain data about the distribution of MIC values of antibiotics in most hospitals, because the actual values of MIC and not the approximate MIC values offered by automated systems such as VITEK or Phoenix are not routinely determined and if they are, they are performed for selected antibiotics only.
Thus, there is usually no cumulative data on the distribution of MIC values for dominant pathogens in specific infections. The obtained data could be used as a basis for assessing whether the list of antibiotics dedicated for empirical therapy and the dosage regimens adopted are appropriate for the degree of susceptibility of the strains. Based on such cumulative data, Kuti et al.
In addition, by establishing MIC values in the range 1. After 12 months, this procedure contributed to reducing mortality from The introduction of such solutions simply by transferring them from other hospitals without any reference to the local epidemiological situation is unjustified and will not produce the expected results.
The use of low MIC values antibiotics during infections treatment may improve therapy efficacy. However, it should be taken into consideration that in some cases, even when correct dosing is applied, pathogen eradication will not be achieved. There may be various reasons of such a condition including heterogeneous antibiotic resistance heteroresistance , tolerance or persistence of micro-organisms in the environment of certain antibiotics.
Each of these phenomena is different from commonly described antibiotic resistance. Typical bacterial resistance is displayed in many mechanisms originated from stable genetic mutation or as a result of expression of acquired, by entire cell population, resistance genes. Heteroresistance means the resistance of a very small subpopulation of cells, which begins to grow rapidly in the presence of an antibiotic while the vulnerable population is killed.
Antibiotic discontinuation reduces replication of a resistant subpopulation [ 51 ]. The detection of resistant cells in the tests is usually not possible due to the low frequency of their occurrence in the population, which may lead to wrong conclusions about antibiotic susceptibility. Sometimes, such a heterogeneous subpopulation may be observed in gradient tests as a presence of bacterial colonies in a growth-inhibition zone.
The detection of such strains usually requires an analysis of the population profile—which is not performed routinely. Heteroresistance was described for bacteria, such as S.
Difficult detection of heteroresistance to colistin, one of the last active antibiotic against resistant Gram-negative bacteria, has led to recommendations update. It is advised to administer properly It is advised to administer properly adjusted first loading dose of colistin and frequently use it in combination with other antibiotics [ 52 ]. Moreover, Fernandez et. Another phenomenon is tolerance, which means the survival of entire population of a strain in a presence of high antibiotic concentration despite the absence of cells resistant to antibiotic.
Bacteria with such a tolerance do not grow or divide but stay viable. The tolerance may arise from genotype and be associated with mutation that enables bacteria to avoid bactericidal activity of antibiotic.
It may also be of phenotypic character when growth inhibition or retardation is a result of poor nutritional conditions. The tolerance applies only to bactericidal antibiotics. Its bactericidal activity decreases or disappears while bacteriostatic properties are maintained. Bacteria tolerating and these nontolerating antibiotics can have the same MIC.
The strain is considered to display tolerance when an antibiotic at a concentration 32 times higher than the MIC does not contribute to Persistence is yet another bacterial mechanism that can contribute to ineffective therapy despite the apparent strain sensitivity.
Those three phenomena discussed above are very difficult to detect and may be the explanation of treatment inefficacy. Subsequently, survival of the bacterial population despite antibiotic presence can promote the spread of resistance mechanisms. The MIC value is regarded to have the greatest importance in the optimization of targeted antibiotic therapy.
For this purpose however, it must be analyzed together with pharmacokinetic PK parameters that describe the fate of the drug in the host organism. In addition, PK parameters change with time.
In the case of antibiotic-resistant strains, identification of the mechanism of bacterial resistance, may have an additional impact on the significance of the MIC value. This topic is particularly discussed in case of Gram-negative bacteria producing carbapenemases.
It is widely known that carbapenemases may possess a different spectrum and strength of hydrolytic activity against particular beta-lactams, including carbapenems. According to both EUCAST and CLSI, the detection of the resistance mechanism does not eliminate the possibility of carbapenems use in the treatment of infections caused by carbapenemase-producing bacteria.
Regardless of the type of carbapenemase or extended spectrum beta-lactamases ESBL produced, clinical interpretation and S, I or R classification of the strain should be based on the obtained MIC value.
Currently, meropenem is considered the most important carbapenem, active against Gram-negative Enterobacterales. Usually, elicitation of full susceptibility indicates effective therapy with the use of standard doses of a drug, but in this case, due to detection of carbapenemase production, meropenem should be used in high doses, i.
The most difficult selection of antibiotics is when pan drug resistance is identified. In that case, the only solution is a combination therapy with minimum of three drugs. Does the MIC of the antibiotics matter in such a case? The authors of the International Consensus Guidelines for the Optimal Use of the Polymyxins from [ 52 ] suggest to choose drugs with the lowest degree of resistance, where MIC value is closest to the breakpoints. Therefore, even in extreme situations, the MIC value may be important, although there is no evidence of the effectiveness of such a procedure.
The achievement of this value is especially important in immunosuppressed patients and in the case of Gram-negative infections [ 63 , 65 , 66 ]. It is easy to deduce that the lower the MIC value of an antibiotic, the easier it is to achieve the required parameter with a standard dosage.
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