Staphylococcus aureus is a major causative agent of infections associated with hospital environments, where antibiotic-resistant strains have emerged as a significant threat. Phage therapy could offer a safe and effective alternative to antibiotics. Phage preparations should comply with quality and safety requirements; therefore, it is important to develop efficient production control technologies. This study was conducted to develop and evaluate a rapid and reliable method for identifying staphylococcal bacteriophages, based on detecting their specific proteins using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling that is among the suggested methods for meeting the regulations of pharmaceutical authorities. Five different phage purification techniques were tested in combination with two MALDI-TOF MS matrices. Phages, either purified by CsCl density gradient centrifugation or as resuspended phage pellets, yielded mass spectra with the highest information value if ferulic acid was used as the MALDI matrix. Phage tail and capsid proteins yielded the strongest signals whereas the culture conditions had no effect on mass spectral quality. Thirty-seven phages from Myoviridae, Siphoviridae or Podoviridae families were analysed, including 23 siphophages belonging to the International Typing Set for human strains of S. aureus, as well as phages in preparations produced by Microgen, Bohemia Pharmaceuticals and MB Pharma. The data obtained demonstrate that MALDI-TOF MS can be used to effectively distinguish between Staphylococcus-specific bacteriophages.

Central European Institute of Technology Masaryk University Kamenice 5 62500 Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Kotlářská 2 61137 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

Zobrazit více v PubMed

Melo L.D.R., Oliveira H., Santos S.B., Sillankorva S., Azeredo J. Bioprospecting. Volume 16. Springer; Cham, Switzerland: 2017. Phages against infectious diseases; pp. 269–294. Topics in Biodiversity and Conservation.

Sybesma P., Pirnay J.-P. Expert round table on acceptance and re-implementation of bacteriophage therapy Silk route to the acceptance and re-implementation of bacteriophage therapy. Biotechnol. J. 2016;11:595–600. doi: 10.1002/biot.201600023. PubMed DOI

Bárdy P., Pantůček R., Benešík M., Doškař J. Genetically modified bacteriophages in applied microbiology. J. Appl. Microbiol. 2016;121:618–633. doi: 10.1111/jam.13207. PubMed DOI

Ackermann H.-W. Phage classification and characterization. Methods Mol. Biol. 2009;501:127–140. doi: 10.1007/978-1-60327-164-6_13. PubMed DOI

Del Rio B., Binetti A.G., Martín M.C., Fernández M., Magadán A.H., Alvarez M.A. Multiplex PCR for the detection and identification of dairy bacteriophages in milk. Food Microbiol. 2007;24:75–81. doi: 10.1016/j.fm.2006.03.001. PubMed DOI

Kahánková J., Pantůček R., Goerke C., Růžičková V., Holochová P., Doškař J. Multilocus PCR typing strategy for differentiation of Staphylococcus aureus siphoviruses reflecting their modular genome structure. Environ. Microbiol. 2010;12:2527–2538. doi: 10.1111/j.1462-2920.2010.02226.x. PubMed DOI

Brüssow H., Hendrix R.W. Phage genomics: Small is beautiful. Cell. 2002;108:13–16. doi: 10.1016/S0092-8674(01)00637-7. PubMed DOI

Deghorain M., Van Melderen L. The staphylococci phages family: An overview. Viruses. 2012;4:3316–3335. doi: 10.3390/v4123316. PubMed DOI PMC

Howard-Varona C., Hargreaves K.R., Abedon S.T., Sullivan M.B. Lysogeny in nature: Mechanisms, impact and ecology of temperate phages. ISME J. 2017;11:1511–1520. doi: 10.1038/ismej.2017.16. PubMed DOI PMC

Zeman M., Mašlaňová I., Indráková A., Šiborová M., Mikulášek K., Bendíčková K., Plevka P., Vrbovská V., Zdráhal Z., Doškař J., et al. Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction and package mecA gene. Sci. Rep. 2017;7:46319. doi: 10.1038/srep46319. PubMed DOI PMC

Xia G., Wolz C. Phages of Staphylococcus aureus and their impact on host evolution. Infect. Genet. Evol. 2014;21:593–601. doi: 10.1016/j.meegid.2013.04.022. PubMed DOI

Goerke C., Pantůček R., Holtfreter S., Schulte B., Zink M., Grumann D., Bröker B.M., Doškař J., Wolz C. Diversity of prophages in dominant Staphylococcus aureus clonal lineages. J. Bacteriol. 2009;191:3462–3468. doi: 10.1128/JB.01804-08. PubMed DOI PMC

Brüssow H., Canchaya C., Hardt W.-D. Phages and the evolution of bacterial pathogens: From genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 2004;68:560–602. doi: 10.1128/MMBR.68.3.560-602.2004. PubMed DOI PMC

Haaber J., Leisner J.J., Cohn M.T., Catalan-Moreno A., Nielsen J.B., Westh H., Penadés J.R., Ingmer H. Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells. Nat. Commun. 2016;7:13333. doi: 10.1038/ncomms13333. PubMed DOI PMC

Mašlaňová I., Doškař J., Varga M., Kuntová L., Mužík J., Malúšková D., Růžičková V., Pantůček R. Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies. Environ. Microbiol. Rep. 2013;5:66–73. doi: 10.1111/j.1758-2229.2012.00378.x. PubMed DOI

Mašlaňová I., Stříbná S., Doškař J., Pantůček R. Efficient plasmid transduction to Staphylococcus aureus strains insensitive to the lytic action of transducing phage. FEMS Microbiol. Lett. 2016;363:fnw211. doi: 10.1093/femsle/fnw211. PubMed DOI

Adams M.J., Lefkowitz E.J., King A.M.Q., Harrach B., Harrison R.L., Knowles N.J., Kropinski A.M., Krupovic M., Kuhn J.H., Mushegian A.R., et al. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2016) Arch. Virol. 2016;161:2921–2949. doi: 10.1007/s00705-016-2977-6. PubMed DOI PMC

Doškař J., Pallová P., Pantůček R., Rosypal S., Růžičková V., Pantůčková P., Kailerová J., Klepárník K., Malá Z., Boček P. Genomic relatedness of phages of the International Typing Set and detection of serogroup A, B and F prophages in lysogenic strains. Can. J. Microbiol. 2000;46:1066–1076. doi: 10.1139/cjm-46-11-1066. PubMed DOI

Cui Z., Guo X., Dong K., Zhang Y., Li Q., Zhu Y., Zeng L., Tang R., Li L. Safety assessment of Staphylococcus phages of the family Myoviridae based on complete genome sequences. Sci. Rep. 2017;7:41259. doi: 10.1038/srep41259. PubMed DOI PMC

Bizzini A., Greub G. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification. Clin. Microbiol. Infect. 2010;16:1614–1619. doi: 10.1111/j.1469-0691.2010.03311.x. PubMed DOI

Fenselau C., Demirev P.A. Characterization of intact microorganisms by MALDI mass spectrometry. Mass Spectrom. Rev. 2001;20:157–171. doi: 10.1002/mas.10004. PubMed DOI

Thomas J.J., Falk B., Fenselau C., Jackman J., Ezzell J. Viral characterization by direct analysis of capsid proteins. Anal. Chem. 1998;70:3863–3867. doi: 10.1021/ac9802372. PubMed DOI

Madonna A.J., Van Cuyk S., Voorhees K.J. Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2003;17:257–263. doi: 10.1002/rcm.900. PubMed DOI

Rees J.C., Voorhees K.J. Simultaneous detection of two bacterial pathogens using bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2005;19:2757–2761. doi: 10.1002/rcm.2107. PubMed DOI

McAlpin C.R., Cox C.R., Matyi S.A., Voorhees K.J. Enhanced matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of bacteriophage major capsid proteins with β-mercaptoethanol pretreatment. Rapid Commun. Mass Spectrom. 2010;24:11–14. doi: 10.1002/rcm.4349. PubMed DOI

Bourdin G., Schmitt B., Guy L.M., Germond J.-E., Zuber S., Michot L., Reuteler G., Brüssow H. Amplification and purification of T4-like Escherichia coli phages for phage therapy: From laboratory to pilot scale. Appl. Environ. Microbiol. 2014;80:1469–1476. doi: 10.1128/AEM.03357-13. PubMed DOI PMC

Borecká P., Rosypal S., Pantůček R., Doškař J. Localization of prophages of serological group B and F on restriction fragments defined in the restriction map of Staphylococcus aureus NCTC 8325. FEMS Microbiol. Lett. 1996;143:203–210. doi: 10.1111/j.1574-6968.1996.tb08481.x. PubMed DOI

Li X., Gerlach D., Du X., Larsen J., Stegger M., Kühner P., Peschel A., Xia G., Winstel V. An accessory wall teichoic acid glycosyltransferase protects Staphylococcus aureus from the lytic activity of Podoviridae. Sci. Rep. 2015;5:17219. doi: 10.1038/srep17219. PubMed DOI PMC

Botka T., Růžičková V., Konečná H., Pantůček R., Rychlík I., Zdráhal Z., Petráš P., Doškař J. Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus. Virus Genes. 2015;51:122–131. doi: 10.1007/s11262-015-1223-8. PubMed DOI

Pantůček R., Rosypalová A., Doškař J., Kailerová J., Růžičková V., Borecká P., Snopková Š., Horváth R., Götz F., Rosypal S. The polyvalent staphylococcal phage φ812: Its host-range mutants and related phages. Virology. 1998;246:241–252. doi: 10.1006/viro.1998.9203. PubMed DOI

Nováček J., Šiborová M., Benešík M., Pantůček R., Doškař J., Plevka P. Structure and genome release of Twort-like Myoviridae phage with a double-layered baseplate. Proc. Natl. Acad. Sci. USA. 2016;113:9351–9356. doi: 10.1073/pnas.1605883113. PubMed DOI PMC

Yoshizawa Y. Isolation and characterization of restriction negative mutants of Staphylococcus aureus. Jikeikai Med. J. 1985;32:415–421.

Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967;33:155–166. doi: 10.1016/0042-6822(67)90105-5. PubMed DOI

Moša M., Boštík J., Pantůček R., Doškař J. Medicament in the Form of Anti-Staphylococcus Phage Lysate, Process of Its Preparation and Use. CZ201200668-A3. Patent Application. 2012 Sep 27;

Kramberger P., Honour R.C., Herman R.E., Smrekar F., Peterka M. Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths. J. Virol. Methods. 2010;166:60–64. doi: 10.1016/j.jviromet.2010.02.020. PubMed DOI

Sambrook J., Maniatis T., Fritsch E.F. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY, USA: 1987.

Aziz R.K., Bartels D., Best A.A., DeJongh M., Disz T., Edwards R.A., Formsma K., Gerdes S., Glass E.M., Kubal M., et al. The RAST server: Rapid annotations using subsystems technology. BMC Genom. 2008;9:75. doi: 10.1186/1471-2164-9-75. PubMed DOI PMC

Frottin F., Martinez A., Peynot P., Mitra S., Holz R.C., Giglione C., Meinnel T. The proteomics of N-terminal methionine cleavage. Mol. Cell. Proteom. 2006;5:2336–2349. doi: 10.1074/mcp.M600225-MCP200. PubMed DOI

Martinez A., Traverso J.A., Valot B., Ferro M., Espagne C., Ephritikhine G., Zivy M., Giglione C., Meinnel T. Extent of N-terminal modifications in cytosolic proteins from eukaryotes. Proteomics. 2008;8:2809–2831. doi: 10.1002/pmic.200701191. PubMed DOI

Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. The Proteomics Protocols Handbook. Humana Press; New York, NY, USA: 2005. Protein identification and analysis tools on the ExPASy server; pp. 571–607.

Bonilla N., Rojas M.I., Netto Flores Cruz G., Hung S.-H., Rohwer F., Barr J.J. Phage on tap—A quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ. 2016;4:e2261. doi: 10.7717/peerj.2261. PubMed DOI PMC

Adriaenssens E.M., Lehman S.M., Vandersteegen K., Vandenheuvel D., Philippe D.L., Cornelissen A., Clokie M.R.J., Garría A.J., De Proft M., Maes M., et al. CIM® monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles. Virology. 2012;434:265–270. doi: 10.1016/j.virol.2012.09.018. PubMed DOI PMC

Valentine N., Wunschel S., Wunschel D., Petersen C., Wahl K. Effect of culture conditions on microorganism identification by matrix-assisted laser desorption ionization mass spectrometry. Appl. Environ. Microbiol. 2005;71:58–64. doi: 10.1128/AEM.71.1.58-64.2005. PubMed DOI PMC

Kwan T., Liu J., DuBow M., Gros P., Pelletier J. The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA. 2005;102:5174–5179. doi: 10.1073/pnas.0501140102. PubMed DOI PMC

Salmond G.P.C., Fineran P.C. A century of the phage: Past, present and future. Nat. Rev. Microbiol. 2015;13:777–786. doi: 10.1038/nrmicro3564. PubMed DOI

Pirnay J.-P., Merabishvili M., Raemdonck H.V., Vos D.D., Verbeken G. Bacteriophage production in compliance with regulatory requirements. Methods Mol. Biol. 2018;1693:233–252. doi: 10.1007/978-1-4939-7395-8_18. PubMed DOI

Cox C.R., Rees J.C., Voorhees K.J. Modeling bacteriophage amplification as a predictive tool for optimized MALDI-TOF MS-based bacterial detection. J. Mass Spectrom. 2012;47:1435–1441. doi: 10.1002/jms.3087. PubMed DOI

Pierce C.L., Rees J.C., Fernández F.M., Barr J.R. Viable Staphylococcus aureus quantitation using 15N metabolically labeled bacteriophage amplification coupled with a multiple reaction monitoring proteomic workflow. Mol. Cell. Proteom. 2012;11:M111.012849. doi: 10.1074/mcp.M111.012849. PubMed DOI PMC

Swatkoski S., Russell S., Edwards N., Fenselau C. Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry. Anal. Chem. 2007;79:654–658. doi: 10.1021/ac061493e. PubMed DOI

Rees J.C., Barr J.R. Detection of methicillin-resistant Staphylococcus aureus using phage amplification combined with matrix-assisted laser desorption/ionization mass spectrometry. Anal. Bioanal. Chem. 2017;409:1379–1386. doi: 10.1007/s00216-016-0070-3. PubMed DOI PMC

Cargile B.J., McLuckey S.A., Stephenson J.L. Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions. Anal. Chem. 2001;73:1277–1285. doi: 10.1021/ac000725l. PubMed DOI

Wick C.H., Elashvili I., Stanford M.F., McCubbin P.E., Deshpande S.V., Kuzmanovic D., Jabbour R.E. Mass spectrometry and integrated virus detection system characterization of MS2 bacteriophage. Toxicol. Mech. Methods. 2007;17:241–254. doi: 10.1080/15376510601123195. PubMed DOI

Serafim V., Pantoja L., Ring C.J., Shah H., Shah A.J. Rapid identification of E. coli bacteriophages using mass spectrometry. J. Proteom. Enzymol. 2017;6:1000130. doi: 10.4172/2470-1289.1000130. DOI

Goerke C., Köller J., Wolz C. Ciprofloxacin and trimethoprim cause phage induction and virulence modulation in Staphylococcus aureus. Antimicrob. Agents Chemother. 2006;50:171–177. doi: 10.1128/AAC.50.1.171-177.2006. PubMed DOI PMC

Selva L., Viana D., Regev-Yochay G., Trzcinski K., Corpa J.M., Lasa Í., Novick R.P., Penadés J.R. Killing niche competitors by remote-control bacteriophage induction. Proc. Natl. Acad. Sci. USA. 2009;106:1234–1238. doi: 10.1073/pnas.0809600106. PubMed DOI PMC

Tang Y., Nielsen L.N., Hvitved A., Haaber J.K., Wirtz C., Andersen P.S., Larsen J., Wolz C., Ingmer H. Commercial biocides induce transfer of prophage Φ13 from human strains of Staphylococcus aureus to livestock CC398. Front. Microbiol. 2017;8:2418. doi: 10.3389/fmicb.2017.02418. PubMed DOI PMC

Kahánková J., Španová A., Pantůček R., Horák D., Doškař J., Rittich B. Extraction of PCR-ready DNA from Staphylococcus aureus bacteriophages using carboxyl functionalized magnetic nonporous microspheres. J. Chromatogr. B. 2009;877:599–602. doi: 10.1016/j.jchromb.2009.01.006. PubMed DOI

Czyz A., Los M., Wrobel B., Wegrzyn G. Inhibition of spontaneous induction of lambdoid prophages in Escherichia coli cultures: Simple procedures with possible biotechnological applications. BMC Biotechnol. 2001;1:1. doi: 10.1186/1472-6750-1-1. PubMed DOI PMC

O’Flaherty S., Ross R.P., Meaney W., Fitzgerald G.F., Elbreki M.F., Coffey A. Potential of the polyvalent anti-Staphylococcus bacteriophage K for control of antibiotic-resistant staphylococci from hospitals. Appl. Environ. Microbiol. 2005;71:1836–1842. doi: 10.1128/AEM.71.4.1836-1842.2005. PubMed DOI PMC

Kvachadze L., Balarjishvili N., Meskhi T., Tevdoradze E., Skhirtladze N., Pataridze T., Adamia R., Topuria T., Kutter E., Rohde C., et al. Evaluation of lytic activity of staphylococcal bacteriophage Sb-1 against freshly isolated clinical pathogens. Microb. Biotechnol. 2011;4:643–650. doi: 10.1111/j.1751-7915.2011.00259.x. PubMed DOI PMC

Vandersteegen K., Mattheus W., Ceyssens P.-J., Bilocq F., De Vos D., Pirnay J.-P., Noben J.-P., Merabishvili M., Lipinska U., Hermans K., et al. Microbiological and molecular assessment of bacteriophage ISP for the control of Staphylococcus aureus. PLoS ONE. 2011;6:e24418. doi: 10.1371/journal.pone.0024418. PubMed DOI PMC

Łobocka M., Hejnowicz M.S., Dąbrowski K., Gozdek A., Kosakowski J., Witkowska M., Ulatowska M.I., Weber-Dąbrowska B., Kwiatek M., Parasion S., et al. Genomics of staphylococcal Twort-like phages—Potential therapeutics of the post-antibiotic era. Adv. Virus Res. 2012;83:143–216. doi: 10.1016/B978-0-12-394438-2.00005-0. PubMed DOI

Międzybrodzki R., Kłak M., Jończyk-Matysiak E., Bubak B., Wójcik A., Kaszowska M., Weber-Dąbrowska B., Łobocka M., Górski A. Means to facilitate the overcoming of gastric juice barrier by a therapeutic staphylococcal bacteriophage A5/80. Front. Microbiol. 2017;8 doi: 10.3389/fmicb.2017.00467. PubMed DOI PMC

Staphylococcus epidermidis Phages Transduce Antimicrobial Resistance Plasmids and Mobilize Chromosomal Islands

Structure and mechanism of DNA delivery of a gene transfer agent

Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification

Lytic and genomic properties of spontaneous host-range Kayvirus mutants prove their suitability for upgrading phage therapeutics against staphylococci