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The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested <jats:italic>in vitro<\/jats:italic> and <jats:italic>in vivo<\/jats:italic> against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against <jats:italic>Salmonella enterica<\/jats:italic> subsp. <jats:italic>enterica<\/jats:italic> serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 \u03bcg\u202fml<jats:sup>\u22121<\/jats:sup> against <jats:italic>Pseudomonas aeruginosa<\/jats:italic> ATCC 27853, 16.25 \u03bcg\u202fml<jats:sup>\u22121<\/jats:sup> against <jats:italic>S<\/jats:italic>. <jats:italic>enterica<\/jats:italic> Typhimurium ATCC 13311, and 32.50 \u03bcg\u202fml<jats:sup>\u22121<\/jats:sup> against <jats:italic>Klebsiella pneumoniae<\/jats:italic> ATCC BAA-2146 and <jats:italic>Enterobacter cloacae<\/jats:italic> P2224. LysKpV475 showed bactericidal activity only for <jats:italic>P. aeruginosa<\/jats:italic> ATCC 27853 (32.50 \u03bcg\u202fml<jats:sup>\u22121<\/jats:sup>) and <jats:italic>P. aeruginosa<\/jats:italic> P2307 (65.00 \u03bcg\u202fml<jats:sup>\u22121<\/jats:sup>) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against <jats:italic>K. pneumoniae<\/jats:italic> ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive\/combined effect] and <jats:italic>S<\/jats:italic>. <jats:italic>enterica<\/jats:italic> Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against <jats:italic>S<\/jats:italic>. <jats:italic>enterica<\/jats:italic> Typhimurium was also observed when the lyophilized LysKpV475 at \u2154 MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant S<jats:italic>almonella<\/jats:italic> reduction of 2 logs after 6\u2009h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.<\/jats:p>","DOI":"10.1099\/mic.0.001462","type":"journal-article","created":{"date-parts":[[2024,5,13]],"date-time":"2024-05-13T15:17:07Z","timestamp":1715613427000},"source":"Crossref","is-referenced-by-count":12,"title":["Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5"],"prefix":"10.1099","volume":"170","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6605-3402","authenticated-orcid":false,"given":"Marco","family":"Gontijo","sequence":"first","affiliation":[{"name":"Departamento de Gen\u00e9tica, Evolu\u00e7\u00e3o, Microbiologia e Imunologia, Instituto de Biologia, 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