Determining the Minimum Inhibitory Concentration of Bacteriophages: Potential Advantages


The minimum inhibitory concentration (MIC) is the concentration at which an antibacterial agent experiences the complete inhibition of organism growth. Bacteriophages represent a rich and unique resource of anti-infectives to counter the growing world-wide problem of antibiotic resistance. In this study, we compared the host range of lytic bacteriophages and temperate phagesbelonging to various genera, namely Staphylococcus, E. coli and Salmonella, with a range of clinical isolates using two methods: the classical agar overlay method and a newly developed MIC method. MIC was only observed with isolates that were susceptible to phage infection, which correlated with the agar overlay assay, whereas no MIC was detected with isolates that were resistant to phage infection. The simple MIC method was useful in determining phage adsorption and host range, and detecting possible prophage contamination in phage preparations. Interestingly, this method was also applicable to strain differentiation through phage susceptibility testing using a 96-well, high throughput format that proved to be easy, cost-effective, fast and reliable.

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A. Vipra, S. Desai, R. Junjappa, P. Roy, N. Poonacha, P. Ravinder, B. Sriram and S. Padmanabhan, "Determining the Minimum Inhibitory Concentration of Bacteriophages: Potential Advantages," Advances in Microbiology, Vol. 3 No. 2, 2013, pp. 181-190. doi: 10.4236/aim.2013.32028.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] D. H. Duckworth, “Who Discovered Bacteriophage?” Bacteriology Reviews, Vol. 40, No. 4, 1976, pp. 793-802.
[2] A. Sulakvelidze and P. Barrow, “Phage Therapy in Animals and Agri Business,” In: E. Kutter and A. Sulakvelidze, Eds., Bacteriophages: Biology and applications, CRC Press, Boca Raton, 2005, pp. 335-380.
[3] A. Sulakvelidze and E. Kutter, “Bacteriophage Therapy in Humans,” In: E. Kutter and A. Sulakvelidze, Eds., Bacteriophages: Biology and application, CRC Press, Boca Raton, 2005, pp. 381-436.
[4] V. D. Paul, S. Sudarson, R. S. Saravanan, S. Hariharan, K. Nanjundappa, S. Padmanabhan, B. Sriram and J. Ramachandran, “Lysis Deficient Phages for Controlling Bacterial Infection,” BMC Microbiology, Vol. 11, 2011, p.195. doi:10.1186/1471-2180-11-195
[5] H.-W. Ackermann and D. Prangishvili, “Prokaryote Viruses Studied by Electron Microscopy,” Archives of Virology, Vol. 157, No. 10, 2012, pp. 1-78. doi:10.1007/s00705-012-1383-y
[6] L. Prescott, “Microbiology,” Wm. C. Brown Publishers, Dubuque, 1993.
[7] J. C. Adams, J. A. Gazaway Jr., M. D. Brailsford, P. A. Hartman and N. L. Jacobson, “Isolation of Bacteriophages from the Bovine Rumen,” Experientia, Vol. 22, No. 11, 1966, pp. 717-718. doi:10.1007/BF01901335
[8] M. J. B. Paynter, D. L. Ewert and W. Chalupa, “Some Morphological Types of Bacteriophages in Bovine Rumen Contents,” Applied Microbiology, Vol. 18, No. 5, 1969, pp. 942-943.
[9] A. E. Ritchie, I. M. Robinson and M. J. Allison, “Rumen Bacteriophage: Survey of Morphological Types,” In: P. Favard, Ed., Microscopie electronique, Societe Francaise de Microscopie Electronique, Vol. 3, Societe Francaise de Microscopie Electronique, Paris, 1970, pp. 333-334.
[10] A. V. Klieve and T. Bauchop, “Morphological Diversity of Ruminal Bacteriophages from Sheep and Cattle,” Applied and Environmental Microbiology, Vol. 54, No. 6, 1988, pp. 1637-1641.
[11] C. A. Suttle, “The Significance of Viruses to Mortality in Aquatic Microbial Communities,” Microbial Ecology, Vol. 28, No. 2, 1994, pp. 237-243. doi:10.1007/BF00166813
[12] K. Holmfeldt, M. Middelboe, O. Nybroe and L. Riemann, “Large Variabilities in Host Strain Susceptibility and Phage Host Range Govern Interactions between Lytic Marine Phages and Their Flavobacterium Hosts,” Applied and Environmental Microbiology, Vol. 73, No. 21, 2007, pp. 6730-6739. doi:10.1128/AEM.01399-07
[13] P. Hyman and S. T. Abedon, “Bacteriophage Host Range and Bacterial Resistance,” Advances in Applied Microbiology, Vol. 70, 2010, pp. 217-248. doi:10.1016/S0065-2164(10)70007-1
[14] G. P. Nirmal Kumar, S. Sudarson, V. D. Paul, S. Nandini, S. R. Sanjeev, S. Hariharan, S. Bharathi and S. Padmanabhan, “Use of Prophage Free Host for Achieving Homogenous Population of Bacteriophages: New findings,” Virus Research, Vol. 169, No. 1, 2012, pp. 182-187. doi:10.1016/j.virusres.2012.07.026
[15] M. R. J. Clokie and A. M. Kropinski, “Bacteriophages: Methods and Protocols,” 1st Edition, Humana Press, New York, 2008.
[16] D. Refardt, “Within-Host Competition Determines Reproductive Success of Temperate Bacteriophages,” The ISME Journal, Vol. 5, No. 9, 2011, pp. 1451-1460. doi:10.1038/ismej.2011.30
[17] M. M. Ijzerman and C. Hagedorn, “Improved Method for Coliphage Detection Based on β-Galactosidase Induction,” Journal of Virological Methods, Vol. 40, No. 1, 1992, pp. 1-36. doi:10.1016/0166-0934(92)90004-W
[18] M. D. Sobsey, “Quality of Currently Available Methodology for Monitoring Viruses in the Environment,” Environment International, Vol. 7, No. 1, 1982, pp. 39-51. doi:10.1016/0160-4120(82)90149-0
[19] S. J. Labrie, J. E. Samson and S. Moineau, “Bacteriophage Resistance Mechanisms,” Nature Reviews in Microbiology, Vol. 8, No. 5, 2010, pp. 317-327. doi:10.1038/nrmicro2315
[20] Z. Csiszovszki, Z. Buzás, S. Semsey, T. Ponyi, P. P. Papp and L. Orosz, “Imm X Immunity Region of Rhizobium Phage. 16-3: Two Overlapping Cistrons of Repressor Function,” Journal of Bacteriology, Vol. 185, No. 15, 2003, pp. 4382-4392. doi:10.1128/JB.185.15.4382-4392.2003
[21] F. N. Enikeeva, K. V. Severinov and M. S. Gelfand, Restriction-modification systems and bacteriophage invasion: Who wins?” Journal of Theoretical Biology, Vol. 266, No. 4, 2010, pp. 550-559. doi:10.1016/j.jtbi.2010.07.006
[22] S. O’Flaherty, R. P. Ross, W. Meaney, G. F. Fitzgerald, M. F. Elbreki and A. Coffey, “Potential of the Polyvalent Anti-Staphylococcus Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals,” Applied and Environmental Microbiology, Vol. 71, No. 4, 2005, pp. 1836-1842. doi:10.1128/AEM.71.4.1836-1842.2005
[23] D. Vybiral, M. Takac, M. Loessner, A. Witte, U. von Ahsen and U. Blasi, “Complete Nucleotide Sequence and Molecular Characterization of Two Lytic Staphylococcus Aureus Phages: 44AHJD and P68,” FEMS Microbiology Letters, Vol. 219, No. 2, 2003, pp. 275-283. doi:10.1016/S0378-1097(03) 00028-4
[24] C. B. Appaiah, S. Sudarson, B. R. Anupama, S. Bharathi and S. Padmanabhan, “Staphylococcus Bacteriophage Tails with Bactericidal Properties: New Findings,” Biotechnology and Applied Biochemistry, Vol. 59, No. 6, 2012, pp. 495-502. doi:10.1002/bab.1052
[25] G. Mosig, and F. Eiserling, “T4 and Related Phages: Structure and Development,” In: R. Calender, Ed., The Bacteriophages, 2nd Edition, Oxford University Press, New York, 2006, pp. 1-131.
[26] B. Anderson, M. H. Rashid, C. Carter, G. Pasternack, C. Rajanna, T. Revazishvili, T. Dean, A. Senecal and A. Sulakvelidze, “Enumeration of Bacteriophage Particles: Comparative Analysis of the Traditional Plaque Assay and Real-Time QPCR- and Nanosight-Based Assays,” Bacteriophage, Vol. 1, No. 2, 2011, pp. 86-93. doi:10.4161/bact.1.2.15456
[27] D. A. Scudiero, R. H. Shoemaker, K. D. Paull, A. Monks, S. Tierney, T. H. Nofziger, M. J. Currens, D. Seniff and M. R. Boyd, “Evaluation of a Soluble Tetrazolium/Formazan Assay for Cell Growth and Drug Sensitivity in Culture Using Human and Other Tumor Cell Lines,” Cancer Research, Vol. 48, No. 17, 1988, pp. 4827-4833.
[28] N. J. Marshall, C. J. Goodwin and S. J. Holt, “A Critical Assessment of the Use of Microculture Tetrazolium Assays to Measure Cell Growth and Function,” Growth Regulation, Vol. 5, No. 2, 1995, pp. 69-84.
[29] M. G. Stevens and S. C. Olsen, “Comparative Analysis of Using MTT and XTT in Colorimetric Assays for Quantitating Bovine Neutrophil Bactericidal Activity,” Journal of Immunological Methods, Vol. 157, No. 1-2, 1993, pp. 225-231. doi:10.1016/0022-1759(93)90091-K
[30] A. Sulakvelidze, Z. Alavidze and G. J. Morris Jr., “Bacteriophage Therapy,” Antimicrobial Agents and Chemotherapy, Vol. 45, No. 3, 2001, pp. 649-659. doi:10.1128/AAC.45.3.649-659.2001
[31] V. A. Fischetti, “Bacteriophage Endolysins: A Novel Anti-Infective to Control Gram-Positive Pathogens,” International Journal of Medical Microbiology, Vol. 300, No. 6, 2010, pp. 357-362. doi:10.1016/j.ijmm.2010.04.002
[32] V. D. Paul, S. S. Rajagopalan, S. Sundarrajan, S. E. George, J. Y. Asrani, R. Pillai, R. Chikkamadaiah, M. Durgaiah, B. Sriram and S. Padmanabhan, “A Novel Bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and Its Development into a Potent, Antitaphylococcal Protein,” BMC Microbiology, Vol. 11, 2011, p. 226. doi:10.1186/1471-2180-11-226
[33] S. E. George, R. Chikkamadaiah, M. Durgaiah, A. A. Joshi, U. P. Thankappan, S. N. adhusudhana and B. Sriram, “Biochemical Characterization and Evaluation of Cytotoxicity of Anti Staphylococcal Chimeric Protein P128,” BMC Research Notes, Vol. 5, 2012, p. 280. doi:10.1186/1756-0500-5-280
[34] A. A. Vipra, S. N. Desai, P. Roy, R. Patil, J. M. Raj, N. Narasimhaswamy, V. D. Paul, R. Chikkamadaiah and B. Sriram, “Anti-Staphylococcal Activity of Bacteriophage Derived Chimeric Protein P128,” BMC Microbiology, Vol. 12, 2012, p. 41.doi:10.1186/1471-2180-12-41
[35] L. Gravitz, “Turning a New Phage,” Nature Medicine, Vol. 18, 2012, pp. 1318-1320. doi:10.1038/nm0912-1318
[36] F. Schumacher-Perdreau, G. Pulverer and K. H. Schleifer, “The Phage Adsorption Test: A Simple Method for Differentiation between Staphylococci and Micrococci,” Journal of Infectious Diseases, Vol. 138, No. 3, 1978, pp. 392-395. doi:10.1093/infdis/138.3.392
[37] K. Watanabe and S. Takesue, “Use of L-Rhamnose to Study Irreversible Adsorption of Bacteriophage PL-1 to a Strain of Lactobacillus casei,” Journal of General Virology, Vol. 28, No. 1, 1975, pp. 29-35. doi:10.1099/0022-1317-28-1-29
[38] I. Rabbani, U. Radoja, S. Ray, A. Selim and C. Weston, “The Dynamics of Bacteriophage T4 Binding to Escherichia coli,” Journal of Experimental Microbiology and Immunology, Vol. 5, 2004, pp. 61-64.
[39] I. Katsura, “Tail Assembly and Injection,” In: R. W. Hendrix, J. W. Roberts, F. W. Stahl and R. A. Weisberg, Eds., Lambda II, Cold Spring Harbor Laboratory Press, New York, 1983, pp. 331-346.
[40] E. Goldberg, L. Grinius and L. Letellier, “Recognition, Attachment, and Injection,” In: J. W. Drake, K. N. Kreuzer, G. Mosig, D. H. Hall and F. A. Eiserling, Eds., Molecular Biology of Bacteriophage T4, American Society for Microbiology, Washington DC, 1994, pp. 347-356.
[41] S. T. Abedon, “Lysis from Without,” Bacteriophage, Vol. 1, No. 1, 2011, pp. 46-49. doi;10.4161/bact.1.1.13980
[42] L. M. Kasman, A. Kasman, C. Westwater, J. Dolan, M. G. Schmidt and J. S. Norris, “Overcoming the Phage Replication Threshold: A Mathematical Model with Implications for Phage Therapy,” Journal of Virology, Vol. 76, No. 1, 2002, pp. 5557-5564.
[43] L. Rodriguez-Rubio, B. Martinez, D. M. Donovan, A. Rodriguez and P. Garcia, “Bacteriophage Virion-Associated Peptidoglycan Hydrolases: Potential New Enzybiotics,” Critical Reviews in Microbiology, 2012, pp. 1-8. doi;10.3109/1040841X.2012.723675
[44] M. L. Tortorello, K. M. Trotter, S. M. Angelos, R. A. Ledford and G. M. Dunny, “Microtiter Plate Assays for the Measurement of Phage Adsorption and Infection in Lactococcus and Enterococcus,” Analytical Biochemistry, Vol. 192, No. 2, 1991, pp. 362-366. doi;10.1016/0003-2697(91)90549-9
[45] P. Roslev and G. M. King, “Application of a Tetrazolium Salt with a Water-Soluble Formazan as an Indicator of Viability in Respiring Bacteria,” Applied and Environmental Microbiology, Vol. 59, No. 9, 1993, pp. 2891-2896.
[46] R. Zimmermann, R. Iturriaga and J. Becker-Birck, “Simultaneous Determination of the Total Number of Aquatic Bacteria and the Number Thereof Involved in Respiration,” Applied and Environmental Microbiology, Vol. 36, No. 6, 1978, pp. 926-935.
[47] T. T. Packard, “Measurement of Electron Transport Activity of Microplankton,” In: H. Jannasch and P. J. Leb Williams, Eds., Advances in Aquatic Microbiology, Vol. 3, Academic Press, London, 1985, pp. 207-261.
[48] H. G. Von Bielig, G. A. Kausche and H. Haardick, “Uber den NachweiB von Reduktion in Bakterian,” Zeitschrift für Naturforschung, Vol. 46, 1949, pp. 80-91.
[49] P. Dufour and M. Colon, “The Tetrazolium Reduction Method for Assessing the Viability of Individual Bacterial Cells in Aquatic Environments: Improvements, Performance and Applications,” Hydrobiologia, Vol. 232, No. 3, 1992, pp. 211-218. doi;10.1007/BF00013706

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