AiM  Vol.3 No.1 , March 2013
Therapeutic Potential of Staphylococcal Bacteriophages for Nasal Decolonization of Staphylococcus aureus in Mice
ABSTRACT

Bacteriophages represent a rich and unique resource of anti-infectives to counter the global problem of antibiotic resis- tance. In this work, we assessed the bactericidal activity of two virulent staphylococcal phages, K and 44AHJD, against S. aureus isolates of clinical significance, and tested their efficacy in vivo. The phage cocktail lysed >85% of the clinical isolates tested. Both the phages were purified by ion-exchange column chromatography following propagation in bioreactors. The purity profiles of the ion-exchange purified phages were compared with those of phages purified using cesium chloride density gradient ultracentrifugation, and infectiousness of the purified phages was confirmed by plaque forming assay. The in vivo efficacy of a phage cocktail was evaluated in an experimental murine nasal colonization model, which showed that the phage cocktail was efficacious. To our knowledge, this is the first report of phage use in an in vivo model of nasal carriage.


Cite this paper
M. H. Narasimhaiah, J. Y. Asrani, S. M. Palaniswamy, J. Bhat, S. E. George, R. Srinivasan, A. Vipra, S. N. Desai, R. Patil Junjappa, P. Roy, B. Sriram and S. Padmanabhan, "Therapeutic Potential of Staphylococcal Bacteriophages for Nasal Decolonization of Staphylococcus aureus in Mice," Advances in Microbiology, Vol. 3 No. 1, 2013, pp. 52-60. doi: 10.4236/aim.2013.31008.
References
[1]   T. L. Bannerman and S. J. Peacock, “Staphylococcus, Micrococcus, and Other Catalase-Positive Cocci,” In: P. R. Baron, E. J. Jorgensen, J. H. Landry, M. L. Pfaller and M. A. Murray, Eds., Manual of Clinical Microbiology, ASM Press, Washington DC, 2007, pp. 384-404.

[2]   G. Lina, Y. Piémont, F. Godail-Gamot, M. Bes, M. O. Peter, V. Gauduchon, F. Vandenesch and J. Etienne, “Involvement of Panton-Valentine Leukocidin Producing Staphylococcus aureus in Primary Skin Infections and Pneumonia,” Clinical Infectious Diseases, Vol. 29, No. 5, 1999, pp. 1128-1132. doi:10.1086/313461

[3]   A. L. Casey, P. A. Lambert and T. S. J. Elliott, “Staphylococci,” Journal of Antimicrobial Agents, Vol. 29, No. 3, 2007, pp. S23-S32. doi:10.1016/S0924-8579(07)72175-1

[4]   P. A. C. Maple, J. M. T. Hamilton-Miller and W. Brumfitt, “World-Wide Antibiotic Resistance in Methicillin-Resistant Staphylococcus aureus,” The Lancet, Vol. 333, No. 8637, 1989, pp. 537-540. doi:10.1016/S0140-6736(89)90076-7

[5]   H. L. Evans and R. G. Saywer, “Cycling Chemotherapy: A Promising Approach to Reducing the Morbidity and Mortality of Nosocomial Infections,” Drugs Today, Vol. 39, No. 9, 2003, pp. 733-738. doi:10.1358/dot.2003.39.9.799480

[6]   J. C. Gould, J. H. Smith and H. Moncur, “Mupirocin in General Practice: A Placebo Controlled Trial. In: D. S. Wilkinson and J. D. Price, Eds., International Congress and Symposium Series, Mupirocin—A Novel Topical Antibiotic, Royal Society of Medicine, London, 1984, pp. 85-93.

[7]   T. Coates, R. Bax and A. Coates, “Nasal Decolonization of Staphylococcus aureus with Mupirocin: Strengths, Weaknesses and Future Prospects,” Journal of Antimicrobial Chemotherapy, Vol. 64, No. 1, 2009, pp. 9-15.

[8]   S. Fujimura and A. Watanabe, “Survey of High- and Low-Level Mupirocin-Resistant Strains of Methicillin-Resistant Staphylococcus aureus in 15 Japanese Hospitals,” Chemotherapy, Vol. 49, No. 1-2, 2003, pp. 36-38. doi:10.1159/000069780

[9]   L. Djouhri-Bouktab, K. Alhanout, V. Andrieu, D. Raoult, J. M. Rolain and J. M. Brunel, “Squalamine Ointment for Staphylococcus aureus Skin Decolonization in a Mouse Model,” Journal of Antimicrobial Chemotherapy, Vol. 66, No. 6, 2011, pp. 1306-1310.

[10]   T. Iwase, Y. Uehara, H. Shinji, A. Tajima, H. Seo, K. Takada, T. Agata and Y. Mizunoe, “Staphylococcus epidermidis Esp inhibits Staphylococcus aureus Biofilm Formation and Nasal Colonization,” Nature, Vol. 465, No. 7296, 2010, pp. 346-349. http://dx.doi.org/10.1038/nature09074PMid:20485435

[11]   B. Park, T. Iwase and G. Y. Liu, “Intranasal Application of S. epidermidis Prevents Colonization by Methicillin-Resistant Staphylococcus aureus in Mice,” PLoS ONE, Vol. 6, No. 10, 2011, p. e25880. doi:10.1371/journal.pone.0025880

[12]   T. K. Lu and J. J. Collins, “Dispersing Biofilms with Engineered Enzymatic Bacteriophage,” Proceedings of National Academy of Sciences USA, Vol. 104, No. 27, 2007, pp. 11197-11202. doi:10.1073/pnas.0704624104

[13]   S. Chibani-Chennou, A. Bruttin, M.-L. Dillmann and H. Brussow, “Phage-Host Interaction: An Ecological Perspective,” Journal of Bacteriology, Vol. 186, No. 12, 2004, pp. 3677-3686. doi:10.1128/JB.186.12.3677-3686.2004

[14]   S. M. Shasha, N. Sharon and M. Inbar, “Bacteriophages as Antibacterial Agents,” Harefuah, Vol. 143, No. 2, 2004, pp. 121-125.

[15]   B. Weber-Dabrowska, M. Mulczyk and A. Górski, “Bacteriophage Therapy of Bacterial Infections: An Update of Our Institute’s Experience,” Archives of Immunological Therapies and Experiments (Warsz), Vol. 48, No. 6, 2000, pp. 547-551.

[16]   D. Kelly, O. McAuliffe, R. P. Ross, J. O’Mahony and A. Coffey, “Development of a Broad-Host-Range Phage Cocktail for Biocontrol,” Bioengineered Bugs, Vol. 4, No. 2, 2011, pp. 31-37. doi:10.4161/bbug.2.1.13657

[17]   A. J. Synnott, Y. Kuang, M. Kurimoto, K. Yamamichi, H. Iwano and Y. Tanji, “Isolation from Sewage Influent and Characterization of Novel Staphylococcus aureus Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities,” Applied and Environmental Microbiology, Vol. 75, No. 13, 2009, pp. 4483-4490. doi:10.1128/AEM.02641-08

[18]   K. Sau, S. K. Gupta, S. Sau and T. C. Ghosh, “Synonymous Codon Usage Bias in 16 Staphylococcus aureus Phages: Implication in Phage Therapy,” Virus Research, Vol. 113, No. 2, 2005, pp. 123-131. doi:10.1016/j.virusres.2005.05.001

[19]   H. Hoshiba, J. Uchiyama, S.-I. Kato, T. Ujihara, A. Muraoka, M. Daibata, H. Wakiguchi and S. Matsuzaki, “Isolation and Characterization of a Novel Staphylococcus aureus Bacteriophage, ?MR25, and Its Therapeutic Potential,” Archives of Virology, Vol. 155, No. 4, 2010, pp. 545-552. doi:10.1007/s00705-010-0623-2

[20]   S. O’Flaherty, A. Coffey, R. Edwards, W. Meaney, G. F. Fitzgerald and R. P. Ross, “Genome of Staphylococcal Phage K: A New Lineage of Myoviridae Infecting Gram-Positive Bacteria with a Low G + C Content,” Journal of Bacteriology, Vol. 186, No. 9, 2004, pp. 2862-2871. doi:10.1128/JB.186.9.2862-2871.2004

[21]   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

[22]   A. Bruttin and B. Brussow, “Human Volunteers Receiving Escherichia coli Phage T4 Orally: A Safety Test of Phage Therapy,” Antimicrobial Agents and Chemotherapy, Vol. 49, No. 7, 2005, pp. 2874-2878. doi:10.1128/AAC.49.7.2874-2878.2005

[23]   M. Merabishvili, J. P. Pirnay, G. Verbeken, N. Chanishvili, M.Tediashvili, N. Lashkhi, T. Glonti, V. Krylov, J. Mast, L. V. Parys, R. Lavigne, G. Volckaert, W. Mattheus, G. Verveen, P. De Corte, T. Rose, S. Jennes, M. Zizi, D. De Vos and M. Vaneechoutte, “Quality-Controlled Small-Scale Production of a Well-Defined Bacteriophage Cocktail for Use in Human Clinical Trials,” PLos ONE, Vol. 4, No. 3, 2009, p. e4944. doi:10.1371/journal.pone.0004944

[24]   A. Wright, C. H. Hawkins, E. E. Angg?rd and D. R. Harper, “A Controlled Clinical Trial of a Therapeutic Bacteriophage Preparation in Chronic Otitis Due to Antibiotic-Resistant Pseudomonas aeruginosa—A Preliminary Report of Efficacy,” Clinical Otolaryngology, Vol. 34, No. 4, 2009, pp. 349-357. doi:10.1111/j.1749-4486.2009.01973.x

[25]   D. D. Rhoads, R. D. Wolcott, M. A. Kuskowski, B. M. Wolcott, L. S. Ward and A. Sulakvelidze, “Bacteriophage Therapy of Venous Leg Ulcers in Humans: Results of a Phase I Safety Trial,” Journal of Wound Care, Vol. 18, No. 6, 2009, pp. 237-243.

[26]   S. Guenther, D. Huwyler, S. Richard and M. J. Loessner, “Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-to-Eat Foods,” Applied and Environmental Microbiology, Vol. 75, No. 1, 2009, pp. 93-100. doi:10.1128/AEM.01711-08

[27]   K. G. P. Nirmal, S. Sudarson, V. D. Paul, S. Nandini, S. R. Sanjeev, S. Hariharan, B. Sriram 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

[28]   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

[29]   J. Sambrook, A. Fritsch and T. Maniatis, “Bacteriophage Lambda and Its Vectors,” In: N. Ford and C. Nolan, Eds., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, pp. 2.1-2.117.

[30]   O. H. Lowry, N. J. Rosenbrough, A. L. Farr and R. J. Randall, “Protein Measurement with Folin Phenol Reagent,” Journal of Biological Chemistry, Vol. 193, No. 1, 1951, pp. 265-275.

[31]   M. S. Rouse, M. Rotger, K. E. Piper, J. M. Steckelberg, M. Scholz, J. Andrews and R. Patel, “In Vitro and in Vivo Evaluations of the Activities of Lauric Acid Monoester Formulations against Staphylococcus aureus,” Antimicrobial Agents Chemotherapy, Vol. 49, No. 8, 2005, pp. 3187-3191. doi:10.1128/AAC.49.8.3187-3191.2005

[32]   K. B. Kiser, J. M. Cantey-Kiser and J. C. Lee, “Development and Characterization of a Staphylococcus aureus Nasal Colonization Model in Mice,” Infection and Immunity, Vol. 67, No. 10, 1999, pp. 5001-5006.

[33]   H. M. R. T. Parracho, B. H. Burrowes, M. C. Enright, M. L. McConville and D. R. Harper, “The Role of Regulated Clinical Trials in the Development of Bacteriophage Therapeutics,” Journal of Molecular and Genetic Medicine, Vol. 6, 2012, pp. 279-286.

[34]   J. J. Gill, J. C. Pacan, M. E. Carson, K. E. Leslie, M. W. Griffiths and P. M. Sabour, “Efficacy and Pharmacokinetics of Bacteriophage Therapy in Treatment of Subclinical Staphylococcus aureus Mastitis in Lactating Dairy Cattle,” Antimicrobial Agents and Chemotherapy, Vol. 50, No. 9, 2006, pp. 2912-2918. doi:10.1128/AAC.01630-05

[35]   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

[36]   J. Gu, X. Liu, Y. Li, W. Han, L. Lei, Y. Yang, H. Zhao, Y. Gao, J. Song, R. Lu, C. Sun and X. Feng, “A Method for Generation of Phage Cocktail with Great Therapeutic Potential,” PLoS ONE, Vol. 7, No. 3, 2012, pp. 1-8. doi:10.1371/journal.pone.0031698

[37]   B. Biswas, S. Adhya, P. Washart, B. Paul, A. N. Trostel, B. Powell, R. Carlton and C. R. Merril, “Bacteriophage Therapy Rescues Mice Bacteremia from a Clinical Isolate of Vancomycin-Resistant Enterococcus faecium,” Infection and Immunity, Vol. 70, No. 1, 2002, pp. 204-210. doi:10.1128/IAI.70.1.204-210.2002

[38]   P. Kramberger, R. C. Honour, R. E. Herman F. Smrekar and M. Peterka, “Purification of the Staphylococcus aureus Bacteriophages VDX-10 on Methacrylate Monoliths,” Journal of Virological Methods, Vol. 166, No. 1-2, 2010, pp. 60-64. doi:10.1016/j.jviromet.2010.02.020

[39]   M. Y. Zakharova, A. V. Kozyr, A. N. Ignatova, I. A. Vinnikov, I. G. Shemyakin and A. V. Kolesnikov, “Purification of Filamentous Bacteriophage for Phage Display Using Size-Exclusion Chromatography,” BioTechniques, Vol. 38, No. 2, 2005, pp. 194-198. doi:10.2144/05382BM04

[40]   J. W. Uhr, M. S. Finkelstein and J. B. Baumann, “Antibody Formation: III. The Primary and Secondary Antibody Response to Bacteriophage ?X 174 in Guinea Pigs,” Journal of Experimental Medicine, Vol. 115, No. 3, 1962, pp. 655-670. doi:10.1084/jem.115.3.655

[41]   H. D. Ochs, S. D. Davis and R. J. Wedgwood, “Immunologic Responses to Bacteriophage ?X 174 in Immunodeficiency Diseases,” Journal of Clinical Investigation, Vol. 50, No. 12, 1971, pp. 2559-2568. doi:10.1172/JCI106756

[42]   R. Monjezi, B. T. Tey, C. C. Sieo and W. S. Tan, “Purification of Bacteriophage M13 by Anion Exchange Chromatography,” Journal of Chromatography B. Analytical Technologies in the Biomedical and Life Sciences, Vol. 878, No. 21, 2010, pp. 1855-1859. doi:10.1016/j.jchromb.2010.05.028

[43]   A. A. Elshayeb, S. O. Yagoub, A. S. Yousif, E. A. Abedalkareem, S. M. E. Hag and A. A. Elagib, “Identification of Protein Profiles of Escherichia coli, Staphylococcus aureus and Their Corresponding Phages,” American Journal of Biotechnology and Molecular Sciences, Vol. 1, No. 2, 2011, pp. 39-44. doi:10.5251/ajbms.2011.1.2.39.44

[44]   P. F. Roslansky, M. E. Dawson and T. J. Novitsky, “Plastics, Endotoxins, and the Limulus Amebocyte Lysate Test,” Journal of Parenteral Science and Technology, Vol. 45, No. 2, 1991, pp. 83-87.

[45]   J. Kluytmans, A. VanBelkum and H. Verbrugh, “Nasal Carriage of Staphylococcus aureus: Epidemiology, Underlying Mechanisms, and Associated Risks,” Clinical Microbiology Reviews, Vol. 10, No. 3, 1997, pp. 505-520.

[46]   M. Fenton, P. G. Casey, C. Hill, C. G. M. Gahan, R. P. Ross, O. McAuliffe, J. O’Mahony, F. Maher and A. Coffey, “The Truncated Phage Lysin CHAPk Eliminates Staphylococcus aureus in the Nares of Mice,” Bioenginneered Bugs, Vol. 1, No. 6, 2010, pp. 404-407. doi:10.4161/bbug.1.6.13422

[47]   S. S. Chatterjee, P. Ray, A. Agarwal, A. Das and M. Sharma, “A Community-Based Study on Nasal Carriage of Staphylococcus aureus,” Indian Journal of Medical Research, Vol. 130, No. 6, 2009, pp. 742-748.

[48]   R. P. Lamers, J. W. Stinnett, G. Muthukrishnan, C. L. Parkinson and A. M. Cole, “Evolutionary Analyses of Staphylococcus aureus Identify Genetic Relationships Between Nasal Carriage and Clinical Isolates,” PLoS ONE, Vol. 6, No. 1, 2011, p. e16426. doi:10.1371/journal.pone.0016426

[49]   M. Alfadhela, U. Puapermpoonsiria, S. J. Fordb, F. J. McInnesa and C. F. van der Walle, “Lyophilized Inserts for Nasal Administration Harboring Bacteriophage Selective for Staphylococcus aureus: In Vitro Evaluation,” International Journal of Pharmaceutics, Vol. 416, No. 1, 2011, pp. 280-287. doi:10.1016/j.ijpharm.2011.07.006

[50]   L. Golshahi, K. D. Seed, J. J. Dennis and W. H. Finlay, “Towards Modern Inhalational Bacteriophage Therapy: Nebulization of Bacteriophages of Burkholderia cepacia Complex,” Journal of Aerosol Medicine and Pulmonary Drug Delivery, Vol. 21, No. 4, 2008, pp. 351-360. doi:10.1089/jamp.2008.0701

[51]   S. Deresinski, “Bacteriophage Therapy: Exploiting Smaller Fleas,” Clinical Infectious Diseases, Vol. 48, No. 8, 2009, pp. 1096-1101. doi:10.1086/597405

[52]   P. A. Barrow and J. S. Soothill, “Bacteriophage Therapy and Prophylaxis: Rediscovery and Renewed Assessment of Potential,” Trends in Microbiology, Vol. 5, No. 7, 1997, pp. 268-271. doi:10.1016/S0966-842X(97)01054-8

[53]   V. D. Paul, S. Sundarrajan, S. S. Rajagopalan, S. Hariharan, N. Kempashanaiah, S. Padmanabhan, B. Sriram and J. Ramachandran, “Lysis-Deficient Phages as Novel Therapeutic Agents for Controlling Bacterial Infection,” BMC Microbiology, Vol. 11, 2011, p. 195. doi:10.1186/1471-2180-11-195

[54]   C. Goerke, R. Pantucek, S. Holtfreter, B. Schulte, M. Zink, D. Grumann, B. M. Br?ker, J. Doskar and C. Wolz, “Diversity of Prophages in Dominant Staphylococcus aureus Clonal Lineages,” Journal of Bacteriology, Vol. 191, No. 11, 2009, pp. 3462-3468. doi:10.1128/JB.01804-08

[55]   D. C. Coleman, D. J. Sullivan, R. J. Russell, J. P. Arbuthnott, B. F. Carey and H. M. Pomeroy, “Staphylococcus aureus Bacteriophages Mediating the Simultaneous Lysogenic Conversion of Beta-Lysin, Staphylokinase and Enterotoxin A: Molecular Mechanism of Triple Conversion,” Journal of General Microbiology, Vol. 135, No. 6, 1989, pp. 1679-1697.

 
 
Top