{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T02:33:23Z","timestamp":1771036403021,"version":"3.50.1"},"reference-count":62,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2018,3,8]],"date-time":"2018-03-08T00:00:00Z","timestamp":1520467200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>The occurrence of infections by pathogenic bacteria is one of the main sources of financial loss for the aquaculture industry. This problem often cannot be solved with antibiotic treatment or vaccination. Phage therapy seems to be an alternative environmentally-friendly strategy to control infections. Recognizing the cellular modifications that bacteriophage therapy may cause to the host is essential in order to confirm microbial inactivation, while understanding the mechanisms that drive the development of phage-resistant strains. The aim of this work was to detect cellular modifications that occur after phage AS-A treatment in A. salmonicida, an important fish pathogen. Phage-resistant and susceptible cells were subjected to five successive streak-plating steps and analysed with infrared spectroscopy, a fast and powerful tool for cell study. The spectral differences of both populations were investigated and compared with a phage sensitivity profile, obtained through the spot test and efficiency of plating. Changes in protein associated peaks were found, and these results were corroborated by 1-D electrophoresis of intracellular proteins analysis and by phage sensitivity profiles. Phage AS-A treatment before the first streaking-plate step clearly affected the intracellular proteins expression levels of phage-resistant clones, altering the expression of distinct proteins during the subsequent five successive streak-plating steps, making these clones recover and be phenotypically more similar to the sensitive cells.<\/jats:p>","DOI":"10.3390\/antibiotics7010021","type":"journal-article","created":{"date-parts":[[2018,3,8]],"date-time":"2018-03-08T12:07:33Z","timestamp":1520510853000},"page":"21","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Protein Expression Modifications in Phage-Resistant Mutants of Aeromonas salmonicida after AS-A Phage Treatment"],"prefix":"10.3390","volume":"7","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0352-2616","authenticated-orcid":false,"given":"Catarina","family":"Moreirinha","sequence":"first","affiliation":[{"name":"Departament of Biology &amp; CESAM, Campus Universit\u00e1rio de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal"}]},{"given":"N\u00e1dia","family":"Os\u00f3rio","sequence":"additional","affiliation":[{"name":"Escola Superior de Tecnologia da Sa\u00fade, Rua 5 de Outubro, SM Bispo. Instituto Polit\u00e9cnico de Coimbra, Apartado 7006, 3046-854 Coimbra, Portugal"}]},{"given":"Carla","family":"Pereira","sequence":"additional","affiliation":[{"name":"Departament of Biology &amp; CESAM, Campus Universit\u00e1rio de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal"}]},{"given":"Sara","family":"Sim\u00f5es","sequence":"additional","affiliation":[{"name":"Escola Superior de Tecnologia da Sa\u00fade, Rua 5 de Outubro, SM Bispo. Instituto Polit\u00e9cnico de Coimbra, Apartado 7006, 3046-854 Coimbra, Portugal"}]},{"given":"Ivonne","family":"Delgadillo","sequence":"additional","affiliation":[{"name":"Departament of Chemistry, QOPNA, University of Aveiro, Campus Universit\u00e1rio de Santiago, 3810-193 Aveiro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8422-8664","authenticated-orcid":false,"given":"Adelaide","family":"Almeida","sequence":"additional","affiliation":[{"name":"Departament of Biology &amp; CESAM, Campus Universit\u00e1rio de Santiago, Universidade de Aveiro, 3810-193 Aveiro, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2018,3,8]]},"reference":[{"key":"ref_1","unstructured":"FAO (2014). The State of World Fisheries and Aquaculture, FAO."},{"key":"ref_2","unstructured":"Iwama, G.K., Pickering, A.D., Sumpter, J.P., and Schreck, C.B. (2011). Fish Stress and Health in Aquaculture, Cambridge University Press."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5336","DOI":"10.1128\/AEM.69.9.5336-5342.2003","article-title":"Similarity of Tetracycline Resistance Genes Isolated from Fish Farm Bacteria to Those from Clinical Isolates","volume":"69","author":"Furushita","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_4","unstructured":"World Health Organization (WHO) (2002). Use of Antimicrobials Outside Medicine and Resultant Antimicrobial Resistance in Humans, WHO."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"57","DOI":"10.2165\/00063030-200216010-00006","article-title":"Bacteriophages","volume":"16","author":"Duckworth","year":"2002","journal-title":"BioDrugs"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1016\/S0044-8486(97)00114-2","article-title":"The use of immunostimulation in marine larviculture: Possibilities and challenges","volume":"155","author":"Vadstein","year":"1997","journal-title":"Aquaculture"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"49","DOI":"10.3354\/dao032049","article-title":"Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: A review","volume":"32","author":"Wiklund","year":"1998","journal-title":"Dis. Aquat. Organ."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Uhland, F.C., Martineau, D., Mikaelian, I., and Canada, S.-L.V. (2000). Maladies des Poissons D\u2019eau Douce du Qu\u00e9bec: Guide de Diagnostic, University Press of Montreal.","DOI":"10.2307\/j.ctv69t4zz"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/S0147-619X(03)00058-1","article-title":"Three small, cryptic plasmids from Aeromonas salmonicida subsp","volume":"50","author":"Boyd","year":"2003","journal-title":"salmonicida A449. Plasmid"},{"key":"ref_10","first-page":"2111","article-title":"Attenuated virulence of an Aeromonas salmonicida subsp","volume":"151","author":"Burr","year":"2005","journal-title":"salmonicida type III secretion mutant in a rainbow trout model. Microbiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"268","DOI":"10.3390\/md7030268","article-title":"Phage therapy and photodynamic therapy: Low environmental impact approaches to inactivate microorganisms in fish farming plants","volume":"7","author":"Almeida","year":"2009","journal-title":"Mar. Drugs"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3241","DOI":"10.1007\/s10096-012-1691-x","article-title":"Phage therapy to control multidrug-resistant Pseudomonas aeruginosa skin infections: In vitro and ex vivo experiments","volume":"31","author":"Vieira","year":"2012","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2","DOI":"10.2174\/138920110790725311","article-title":"Phage Choice, Isolation, and Preparation for Phage Therapy","volume":"11","author":"Gill","year":"2010","journal-title":"Curr. Pharm. Biotechnol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Silva, Y.J., Costa, L., Pereira, C., Mateus, C., Cunha, A., Calado, R., Gomes, N.C.M., Pardo, M.A., Hernandez, I., and Almeida, A. (2014). Phage Therapy as an Approach to Prevent Vibrio anguillarum Infections in Fish Larvae Production. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0114197"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.virusres.2016.04.020","article-title":"Bacteriophages with potential to inactivate Salmonella Typhimurium: Use of single phage suspensions and phage cocktails","volume":"220","author":"Pereira","year":"2016","journal-title":"Virus Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1016\/j.virusres.2016.09.019","article-title":"Characterization and in vitro evaluation of new bacteriophages for the biocontrol of Escherichia coli","volume":"227","author":"Pereira","year":"2017","journal-title":"Virus Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1038\/nrmicro2315","article-title":"Bacteriophage resistance mechanisms","volume":"8","author":"Labrie","year":"2010","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1007\/BF00245239","article-title":"Molecular interaction between bacteriophage and the gram-negative cell envelope","volume":"158","author":"Heller","year":"1992","journal-title":"Arch. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Bull, J.J., Vegge, C.S., Schmerer, M., Chaudhry, W.N., and Levin, B.R. (2014). Phenotypic Resistance and the Dynamics of Bacterial Escape from Phage Control. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0094690"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Laanto, E., Bamford, J.J.K.H., Laakso, J., and Sundberg, L.L.-R. (2012). Phage-driven loss of virulence in a fish pathogenic bacterium. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0053157"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6306","DOI":"10.1021\/es400660g","article-title":"Effects of UV Radiation on the Lipids and Proteins of Bacteria Studied by Mid-Infrared Spectroscopy","volume":"47","author":"Santos","year":"2013","journal-title":"Environ. Sci. Technol."},{"key":"ref_22","unstructured":"Taillandier, E., and Liquier, J. (1992). DNA Structures Part A: Synthesis and Physical Analysis of DNA, Elsevier. Methods in Enzymology."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1111\/j.1574-6968.1995.tb07393.x","article-title":"Identification of some bacterial cell components by FT-IR spectroscopy","volume":"126","author":"Helm","year":"1995","journal-title":"FEMS Microbiol. Lett."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1111\/jfs.12176","article-title":"Evaluation of the potential of Mid-infrared spectroscopy to assess the microbiological quality of ham","volume":"35","author":"Moreirinha","year":"2015","journal-title":"J. Food Saf."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1016\/S0165-9936(02)00404-1","article-title":"NIR spectroscopy: A rapid-response analytical tool","volume":"21","author":"Blanco","year":"2002","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Brereton, R. (2003). Chemometrics: Data Analysis for the Laboratoty and Chemical Plant, Wiley.","DOI":"10.1002\/0470863242"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1016\/j.bbabio.2007.06.004","article-title":"Infrared spectroscopy of proteins","volume":"1767","author":"Barth","year":"2007","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"613","DOI":"10.4155\/fmc-2015-0008","article-title":"Overall biochemical changes in bacteria photosensitized with cationic porphyrins monitored by infrared spectroscopy","volume":"8","author":"Alves","year":"2016","journal-title":"Future Med. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1046","DOI":"10.1039\/c1pp05028a","article-title":"Alterations of Escherichia coli envelope as a consequence of photosensitization with tetrakis(N-ethylpyridinium-4-yl)porphyrin tetratosylate","volume":"10","author":"Pudziuvyte","year":"2011","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Naumann, D. (1998). Infrared and NIR Raman Spectroscopy in Medical Microbiology. Proc. SPIE, 3257.","DOI":"10.1117\/12.306089"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1016\/S0039-9140(00)00462-8","article-title":"FTIR spectroscopy studies of nucleic acid damage","volume":"Volume 53","author":"Dovbeshko","year":"2000","journal-title":"Talanta"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1263","DOI":"10.1016\/j.bbamem.2009.02.016","article-title":"IR spectroscopy as a new tool for evidencing antitumor drug signatures","volume":"1788","author":"Gasper","year":"2009","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_33","unstructured":"Smith, B.C. (1999). Infrared Spectral Interpretation: A Systematic Approach, CRC Press."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4606","DOI":"10.1021\/ac034571w","article-title":"Analysis of Microbial Components Using LC-IR","volume":"75","author":"Huffman","year":"2003","journal-title":"Anal. Chem."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Salzer, R., and Siesler, H.W. (2014). Infrared and Raman Spectroscopic Imaging, Wiley-VCH.","DOI":"10.1002\/9783527678136"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Stuart, B. (2004). Infrared Spectroscopy: Fundamentals and Applications, John Wiley & Sons, Ltd.. [2nd ed.].","DOI":"10.1002\/0470011149"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3767","DOI":"10.1128\/AEM.65.9.3767-3773.1999","article-title":"Biocontrol of Escherichia coli O157 with Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages","volume":"65","author":"Kudva","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1111\/lam.12074","article-title":"Phage biocontrol of enteropathogenic and shiga toxin-producing escherichia coli during milk fermentation","volume":"57","author":"Tomat","year":"2013","journal-title":"Lett. Appl. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"33","DOI":"10.3354\/dao053033","article-title":"Bacteriophage control of Pseudomonas plecoglossicida infection in ayu","volume":"53","author":"Park","year":"2003","journal-title":"Dis. Aquat. Organ."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1111\/j.1365-2672.2005.02792.x","article-title":"The newly isolated lytic bacteriophages st104a and st104b are highly virulent against Salmonella enterica","volume":"101","author":"Coffey","year":"2006","journal-title":"J. Appl. Microbiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.aquaculture.2015.07.025","article-title":"Biological control of Aeromonas salmonicida infection in juvenile Senegalese sole (Solea senegalensis) with Phage AS-A","volume":"450","author":"Silva","year":"2016","journal-title":"Aquaculture"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Filippov, A.A., Sergueev, K.V., He, Y., Huang, X.-Z., Gnade, B.T., Mueller, A.J., Fernandez-Prada, C.M., and Nikolich, M.P. (2011). Bacteriophage-resistant mutants in Yersinia pestis: Identification of phage receptors and attenuation for mice. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0025486"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1038\/nrmicro822","article-title":"Population and evolutionary dynamics of phage therapy","volume":"2","author":"Levin","year":"2004","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1111\/1574-6976.12072","article-title":"Bacteria-phage coevolution as a driver of ecological and evolutionary processes in microbial communities","volume":"38","author":"Koskella","year":"2014","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e1050153","DOI":"10.1080\/21597081.2015.1050153","article-title":"Experimental evolution and bacterial resistance: (Co)evolutionary costs and trade-offs as opportunities in phage therapy research","volume":"5","author":"Scanlan","year":"2015","journal-title":"Bacteriophage"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"34997","DOI":"10.1074\/jbc.M606628200","article-title":"Heterogeneous Nucleotide Occupancy Stimulates Functionality of Phage Shock Protein F, an AAA+ Transcriptional Activator","volume":"281","author":"Joly","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"55707","DOI":"10.1074\/jbc.M408994200","article-title":"Identification of a New Member of the Phage Shock Protein Response in Escherichia coli, the Phage Shock Protein G (PspG)","volume":"279","author":"Lloyd","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1111\/j.1365-2958.2005.04694.x","article-title":"The phage-shock-protein response","volume":"57","author":"Darwin","year":"2005","journal-title":"Mol. Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1073\/pnas.87.3.862","article-title":"Phage shock protein, a stress protein of Escherichia coli","volume":"87","author":"Brissette","year":"1990","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"43679","DOI":"10.1074\/jbc.M111.292565","article-title":"Early Contacts between Substrate Proteins and TatA Translocase Component in Twin-arginine Translocation","volume":"286","author":"Rose","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Bageshwar, U.K., VerPlank, L., Baker, D., Dong, W., Hamsanathan, S., Whitaker, N., Sacchettini, J.C., and Musser, S.M. (2016). High Throughput Screen for Escherichia coli Twin Arginine Translocation (Tat) Inhibitors. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0149659"},{"key":"ref_52","doi-asserted-by":"crossref","unstructured":"Friman, V.-P., Hiltunen, T., Jalasvuori, M., Lindstedt, C., Laanto, E., \u00d6rm\u00e4l\u00e4, A.-M., Laakso, J., Mappes, J., and Bamford, J.K.H. (2011). High Temperature and Bacteriophages Can Indirectly Select for Bacterial Pathogenicity in Environmental Reservoirs. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0017651"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Domingues, S., Harms, K., Fricke, W.F., Johnsen, P.J., da Silva, G.J., and Nielsen, K.M. (2012). Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002837"},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Schuster, C.F., Mechler, L., Nolle, N., Krismer, B., Zelder, M.-E., G\u00f6tz, F., and Bertram, R. (2015). The MazEF Toxin-Antitoxin System Alters the \u03b2-Lactam Susceptibility of Staphylococcus aureus. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0126118"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"6089","DOI":"10.1128\/JB.00958-07","article-title":"Hypothetical functions of toxin-antitoxin systems","volume":"189","author":"Magnuson","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"4590","DOI":"10.1093\/nar\/gkt1419","article-title":"A widespread bacteriophage abortive infection system functions through a Type IV toxin-antitoxin mechanism","volume":"42","author":"Dy","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_57","doi-asserted-by":"crossref","unstructured":"Mirzaei, K.M., and Nilsson, A.S. (2015). Isolation of Phages for Phage Therapy: A Comparison of Spot Tests and Efficiency of Plating Analyses for Determination of Host Range and Efficacy. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0127606"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"46","DOI":"10.4161\/bact.1.1.13980","article-title":"Lysis from without","volume":"1","author":"Abedon","year":"2011","journal-title":"Bacteriophage"},{"key":"ref_59","unstructured":"Adams, M.H. (1959). Bacteriophages, John Wiley and Sons Inc."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1016\/S0042-6822(03)00012-6","article-title":"Identification of the host determinant of two prolate-headed phages infecting lactococcus lactis","volume":"309","author":"Janzen","year":"2003","journal-title":"Virology"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4","volume":"227","author":"Laemli","year":"1970","journal-title":"Nature"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1002\/elps.1150090603","article-title":"Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250","volume":"9","author":"Neuhoff","year":"1988","journal-title":"Electrophoresis"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/7\/1\/21\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T14:58:00Z","timestamp":1760194680000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/7\/1\/21"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,3,8]]},"references-count":62,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2018,3]]}},"alternative-id":["antibiotics7010021"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics7010021","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,3,8]]}}}