Abstract
The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick’s laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.
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References
Wheat P F. History and development of antimicrobial susceptibility testing methodology. J Antimicrob Chemother, 2001, 48: 1–4
Bauer A W, Kirby W M, Sherris J C, et al. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol, 1966, 45: 493–496
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. Twentieth. Informational Supplement. CLSI Document M100—S20. Wayne: Clinical and Laboratory Standard Institute, 2010
World Health Organization. Standardization of Methods for Conducting Microbiology Sensitivity Tests. Second Report of the Expert Committee on Antibiotics. WHO Technical Report Series, No. 210. WHO, Geneva, 1961
Gould I M. Towards a common susceptibility testing method? J Antimicrob Chemother, 2000, 45: 757–762
Yang Q W, Wang H, Xu Y C, et al. Introduction current situation and research of antimicrobial resistance surveillance in teaching hospitals of China. Chin J Clin Pharm, 2008, 24: 570–573
Xiao Y H. Introduction of Mohnarin (Ministry of Health National Antimicrobial Resistance Investigation Net. Chin J Antibiotics, 2008, 33: 577–588
Taubes G. The Bacteria fight back. Science, 2008, 18: 356–361
Richard G W, Lewis K, Salyers A A, et al. Bacterial Resistance to Antimicrobials. 2nd ed. Boca Raton: CRC Press, 2007
Zhu X L, Qi J, Bai H, et al. Multi-drug resistance and hereditability of Escherichia coli isolated from animals in Shandong. Chin J Health Labor Tech, 2009, 19: 1473–1476
Bryson V, Szybalski W. Microbial selection. Science, 1952, 116: 45–51
Shimkets L J, Dworkin M, Keller K H. A method for establishing stable concentration gradients in agar suitable for studying chemotaxis on a solid surface. Can J Microbiol, 1979, 25: 1460–1467
Zhang J E, Zhu J P, Liu Y X, et al. Screening of high gibberellin-producing strain from chloramphenicol resistant mutants. Chin J Antibiotics, 2005, 30: 301–303
Chen S Y, Deng Z X. Development of transformation system for Strepomyces hygroscopicus var Jingganshan JG5008. Chin J Appl Environ Biol, 2000, 6: 267–270
Nelson P (author), Li M, Dai L R (translation). Biological Physics: Energy, Information and Life. Shanghai: Shanghai Science and Technology Press, 2006. 116-118
Liu Y Q, Zhang Y Z, Gao P J. Novel concentration—killing curve method for estimation of bactericidal potency of antibiotics in an in vitro dynamic model. Antimicrob Agents Chemother, 2004, 48: 3884–3891
Wang S J, Shen X Z, Zong J. Spectrofluormetric determination of norfloxacin. Chin Medi Pharm, 1993, 24: 411–413
Bagel S, Hullen V, Wiedemann B, et al. Impact of gyrA and parC mutations on quinolone resistance, doubling time, and supercoiling degree of Escherichia coli. Antimicrob Agents Chemother, 1999, 43: 868–875
Liu Y Q, Zhang H Q, Hu M, et al. The methodological limitation of antibiotic susceptibility test and its improvement. J Shandong University (Health Science), 2011, 49: 124–132
Brown D F. Determination of MICs by the E test. J Antimicrob Chemother, 1992, 29: 455
Hill G B, Schalkowsky S. Development and evaluation of the spiral gradient endpoint method for susceptibility testing of anaerobic gram-negative bacilli. Rev Infect Dis, 1990, 12: S200–S209
Cooper K E. Theory of antibiotic inhibition zones in agar media. Nature, 1995, 175: 510–511
Finn R K. Theory of agar diffusion methods for bioassay. Anal Chem, 1959, 31: 975–977
Awerbuch T E, Lustman L. A mathematical model for determining minimum inhibitory concentration (MIC) via diffusion assay. J Theory Biol, 1987, 129: 219–230
Ringertz S, Kronvall G. On the theory of the disk diffusion test. APMIS, 1988, 96: 484–490
Drugeon H B, Juvin M E, Caillon J, et al. Assessment of formulas for calculating critical concentration by the agar diffusion method. Antimicrob Agents Chemother, 1987, 31: 870–875
Anderson B. Improved susceptibility disk assay method employing an agar overlay technique. Antimicrob Agents Chemother, 1978, 14: 761–764
Bonev B, Hooper J, Parisot J. Principles of assessing bacterial susceptibility to antibiotics using the agar diffusion method. J Antimicrob Chemother, 2008, 61: 1295–1301
Andrews J M. The development of the BASA standardized method of disk diffusion testing. Antimicrob Agent Chemoth, 2001, 48: 29–42
Turnidge J, Paterson D L. Setting and revising antibacterial susceptibility breakpoints. Clin Microbiol Rev, 2007, 20: 391–408
Chen X S. Disk susceptibility test—the limitation and some additional method. Chin J Clin Lab Sci, 1994, 12: 2–5
Jorgensen J H, Crawford S A, Fulcher L C, et al. Multilaboratory evaluation of disk diffusion antimicrobial susceptibility testing of Neisseria meningitidis isolates. J Clin Microb, 2006, 44: 1744–1754
Zhao X, Drlica K. Restricting the selection of antibiotic—resistant mutants: measurement and potential uses of the mutant selection window. J Infect Dis, 2002, 185: 561–565
Zhang H Q, Liu Y Q, Liu B, et al. A novel approach for estimating growth phase and parameters of bacterial population in batch culture. Sci China Ser C-Life Sci, 2006, 49: 130–140
Liu Y Q, Zhang H Q, Shen J Z, et al. Effect of physiological heterogeneity of E. coli population on antibiotic susceptibility test. Sci China Ser C-Life Sci, 2007, 50: 808–813
Zhang H Q, Zhao Y, He X L, et al. A novel approach for assessing the susceptibility of Escherichia coli to antibiotics. Sci China Life Sci, 2010, 53: 1346–1355
Cooper K E, Linton A H, Sehgal S N. The effect of inoculum size on inhibition zones in agar media using staphylococci and streptomycin. J Gen Microbiol, 1958, 18: 670–687
Firsov A A, Vostrov S N, Kononenko O V, et al. Prediction of the effects of inoculum size on the antimicrobial action of trovafloxacin and ciprofloxacin against Staphylococcus aureus and Escherichia coli in an in vitro dynamic model. Antimicrob Agents Chemoth, 1999, 43: 498–502
Yang J K, Qi X L, Chen L. Biomathematics Overview. Beijing: Science Press, 1982. 640-722
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Liu, Y., Li, J., Du, J. et al. Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate. Sci. China Life Sci. 54, 953–960 (2011). https://doi.org/10.1007/s11427-011-4230-6
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DOI: https://doi.org/10.1007/s11427-011-4230-6