In microbiology, the minimum inhibitory concentration (MIC) is the lowest concentration of a chemical, usually a drug, which prevents visible growth of bacterium. MIC depends on the microorganism, the culture environment and the antimicrobial.[1]
The MIC is determined by preparing solutions of the chemical in vitro at increasing concentrations, incubating the solutions with the separate batches of cultured bacteria, and measuring the results using agar dilution or broth microdilution. Results have been graded into susceptible (often called sensitive), intermediate, or resistant to a particular antimicrobial by using a breakpoint. Breakpoints are agreed upon values, published in guidelines of a reference body, such as the U.S. Clinical and Laboratory Standards Institute (CLSI), the British Society for Antimicrobial Chemotherapy (BSAC) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST).[2] Though, there have been major discrepancies between the breakpoints from various European countries over the years, and between those from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the US Clinical and Laboratory Standards Institute (CLSI). [3]
This is different than the minimum bactericidal concentration (MBC), which is the concentration resulting in microbial death as defined by the inability to re-culture bacteria. The closer the MIC is to the MBC, the more bactericidal the compound.[4]
The first step in drug discovery is often the screening of a library drug candidate for MICs against bacteria of interest.[5] As such, MICs are usually the starting point for larger pre-clinical evaluations of novel antimicrobial agents.[6]
Background edit
History edit
After the discovery and commercialization of antibiotics, Alexander Fleming developed the broth dilution technique using the turbidity of the broth for assessment.[7] This is commonly believed to be the conception point of minimum inhibitory concentrations.[8] Later in the 1980s, Clinical and Laboratory Standards Institute has consolidated the methods and standards for MIC determination and clinical usage. Following the discovery of new antibacterials, pathogens and their evolution, the protocols by CLSI are also continually updated to reflect that change.[9] The protocols and parameters set by CLSI are considered to be the "gold standard" in the United States, and are used by regulatory authorities including FDA on evaluations[10]
Clinical usage edit
Nowadays, the MIC is used in antimicrobial susceptibility testing. In clinics, more often than not, exact pathogens cannot be easily determined by symptoms of the patient. Even if the pathogen is determined, different serovars of pathogens, such as Staphylococcus aureus, have differing resistances to antimicrobials, so it is hard to prescribe correct antimicrobials.[11] The MIC is determined in such cases by growing the pathogen isolate from the patient on plate or broth, which is later used in the assay. [12] So, by knowing the MIC of the pathogen, the physician will have a better idea on which antimicrobial(s) in doses to prescribe. Accurate and precise usage of antimicrobials is also important in the context of multi-drug resistant bacteria. Microbes, such as bacteria, have been gaining resistance to antimicrobials they were previously susceptible to.[13] This evolution of resistance in bacterial pathogens are hastened due to selective pressure from usage of incompatible or sub-MIC levels of antimicrobials.[14] As such, determining the MIC and using the best choice antimicrobials has been gaining importance.
Minimum bactericidal concentration (MBC), which is the concentration resulting in microbial death, as defined by the inability to re-culture bacteria, as compared to minimum inhibitory concentration, which is when there is no visible growth of the bacteria. However, MIC is used clinically over MBC because MIC is more standard to determine.[9] Also, drug effectiveness is generally similar when taken at both MIC and MBC concentrations. Because, the host immune system, typically a human, can expel the pathogen when bacterial proliferation is at a standstill.[15] When the MBC is much higher than the MIC, prescribing the drug at the MBC is detrimental to patient due to drug toxicity. Antimicrobial toxicity can come in many forms, such as immune hypersensitivity and off-target toxicity.[16]
Methods edit
Broth dilution assay edit
There are three main reagents necessary to run this assay which are the media, antimicrobial and the microbe being tested. Most commonly used media is Cation adjusted-Mueller Hinton Broth. This is due to its ability to support growth of most pathogens and the lack of inhibitors towards common antibiotics.[17] Depending on the pathogen and antibiotics being tested, the media can be changed and/or adjusted. For example, some pathogens such as Streptococci are considered fastidious and won't grow on CA-MHB, so the media is supplemented with lyzed blood in correlation to pathogen.[18]The antimicrobial concentration is adjusted into the correct concentration by mixing stock antimicrobial with media. The adjusted antimicrobial is serially diluted into multiple tubes (or wells) to get a gradient. The dilution rate can be adjusted depending on the breakpoint and the practitioner's needs. The microbe, or the inoculating agent, needs to come from the same colony forming unit per sample, and needs to be at the correct concentration. This can be adjusted by incubation time and dilution. For verification, the positive control is plated in a hundred fold dilution to count colony forming units. The microbes inoculate the tubes (or plate) and are incubated for 16-20 hours. The MIC is determined by turbidity.[17][19][18]
Depending on the pathogen and antimicrobial, the practitioner can also use an agar dilution assay, where the media is supplanted with agar.[17][18]
Kirby–Bauer test edit
For more information please find the Wikipedia page at this link: Kirby–Bauer test
Caveats edit
There are drawbacks to the determination of MICs in this manner. Due to the main media, CA-MHB, not mimicking the host body well enough, the MIC of the microbial pathogen to the antibiotics can vary between in clinical situations and in vitro assays.[20] This is partly due to the pathogen making phenotypical adjustments once detecting host environment, leading to gain or loss of antimicrobial resistance.[14][20]
References edit
- ^ McKinnon, PS and Davis, SL. Pharmokinetic and pharmacodynamic issues in the treatment of bacterial infectious diseases. in: VL Yu, G Edwards, PS McKinnon, C Peloquin, G Morse (Eds.) Antimicrobial therapy and vaccines, volume II: antimicrobial agents. ESun Technologies, Pittsburgh, PA; 2005: 5–19
- ^ Andrews, J. M. (1 July 2001). "Determination of minimum inhibitory concentrations". Journal of Antimicrobial Chemotherapy. 48 (suppl 1): 5–16. doi:10.1093/jac/48.suppl_1.5. PMID 11420333.
- ^ Seydina M Diene, Cédric Abat, Jean-Marc Rolain, Didier Raoult. How artificial is the antibiotic resistance definition? Lancet Infectious Diseases, Volume 17, No. 7, p690, July 2017. DOI: https://dx.doi.org/10.1016/S1473-3099(17)30338-9
- ^ Tripathi, K.D. (2013). Essentials of Medical Pharmacology (7th ed.). New Delhi, India: Jaypee Brothers Medical Publishers. pp. 696, 697.
- ^ Turnidge JD, Ferraro MJ, Jorgensen JH (2003) Susceptibility Test Methods: General Considerations. In PR Murray, EJ Baron, JH Jorgensen, MA Pfaller, RH Yolken. Manual of Clinical Microbiology. 8th Ed. Washington. American Society of Clinical Microbiology. p 1103 ISBN 1-55581-255-4
- ^ O'Neill, AJ; Chopra, I (August 2004). "Preclinical evaluation of novel antibacterial agents by microbiological and molecular techniques". Expert Opinion on Investigational Drugs. 13 (8): 1045–63. doi:10.1517/13543784.13.8.1045. PMID 15268641.
- ^ 1881-1955., Fleming, Alexander, Sir, ([1944]). On the antibacterial action of cultures of a penicillium, with special reference to their use in the isolation of B. influenzae. H.K. Lewis. OCLC 25424051.
{{cite book}}:|last=has numeric name (help); Check date values in:|date=(help)CS1 maint: extra punctuation (link) CS1 maint: multiple names: authors list (link) - ^ Wheat, Philip F. (2001-07). "History and development of antimicrobial susceptibility testing methodology". Journal of Antimicrobial Chemotherapy. 48 (suppl_1): 1–4. doi:10.1093/jac/48.suppl_1.1. ISSN 1460-2091.
{{cite journal}}: Check date values in:|date=(help) - ^ a b Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard. Cockerill, Franklin R., Clinical and Laboratory Standards Institute. (9th ed ed.). Wayne, Pa: Clinical and Laboratory Standards Institute. 2012. ISBN 1562387847. OCLC 1016466655.
{{cite book}}:|edition=has extra text (help)CS1 maint: others (link) - ^ Performance standards for antimicrobial susceptibility testing : twenty-second informational supplement. Cockerill, F. (Franklin), Clinical and Laboratory Standards Institute. Wayne, PA: Clinical and Laboratory Standards Institute. 2012. ISBN 1562387855. OCLC 795927370.
{{cite book}}: CS1 maint: others (link) - ^ Cameron, D. R.; Howden, B. P.; Peleg, A. Y. (2011-08-24). "The Interface Between Antibiotic Resistance and Virulence in Staphylococcus aureus and Its Impact Upon Clinical Outcomes". Clinical Infectious Diseases. 53 (6): 576–582. doi:10.1093/cid/cir473. ISSN 1058-4838.
- ^ Antimicrobial susceptibility testing protocols. Schwalbe, Richard., Steele-Moore, Lynn., Goodwin, Avery C. Boca Raton: CRC Press. 2007. ISBN 9781420014495. OCLC 666899344.
{{cite book}}: CS1 maint: others (link) - ^ Global antimicrobial resistance surveillance system : manual for early implementation. World Health Organization,. Geneva, Switzerland. ISBN 9241549408. OCLC 950637154.
{{cite book}}: CS1 maint: extra punctuation (link) CS1 maint: others (link) - ^ a b Geisinger, Edward; Isberg, Ralph R. (2017-02-15). "Interplay Between Antibiotic Resistance and Virulence During Disease Promoted by Multidrug-Resistant Bacteria". The Journal of Infectious Diseases. 215 (suppl_1): S9–S17. doi:10.1093/infdis/jiw402. ISSN 0022-1899. PMC 5853982. PMID 28375515.
{{cite journal}}: CS1 maint: PMC format (link) - ^ author., Gallagher, Jason C.,. Antibiotics simplified. ISBN 9781284111293. OCLC 952657550.
{{cite book}}:|last=has generic name (help)CS1 maint: extra punctuation (link) CS1 maint: multiple names: authors list (link) - ^ Guengerich, F. Peter (2011). "Mechanisms of Drug Toxicity and Relevance to Pharmaceutical Development". Drug metabolism and pharmacokinetics. 26 (1): 3–14. ISSN 1347-4367. PMC 4707670. PMID 20978361.
{{cite journal}}: CS1 maint: PMC format (link) - ^ a b c Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard. Cockerill, F. (Franklin), Clinical and Laboratory Standards Institute. (Tenth edition ed.). Wayne, Pa.: Clinical and Laboratory Standards Institute. 2015. ISBN 1562387839. OCLC 932608948.
{{cite book}}:|edition=has extra text (help)CS1 maint: others (link) - ^ a b c "Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by broth dilution". Clinical Microbiology and Infection. 9 (8): ix–xv. 2003-08. doi:10.1046/j.1469-0691.2003.00790.x. ISSN 1198-743X.
{{cite journal}}: Check date values in:|date=(help) - ^ Andrews, Jennifer M. (2001-07). "Determination of minimum inhibitory concentrations". Journal of Antimicrobial Chemotherapy. 48 (suppl_1): 5–16. doi:10.1093/jac/48.suppl_1.5. ISSN 1460-2091.
{{cite journal}}: Check date values in:|date=(help) - ^ a b Ersoy, Selvi C.; Heithoff, Douglas M.; Barnes, Lucien; Tripp, Geneva K.; House, John K.; Marth, Jamey D.; Smith, Jeffrey W.; Mahan, Michael J. (2017-06). "Correcting a Fundamental Flaw in the Paradigm for Antimicrobial Susceptibility Testing". EBioMedicine. 20: 173–181. doi:10.1016/j.ebiom.2017.05.026. ISSN 2352-3964.
{{cite journal}}: Check date values in:|date=(help)