Combined effect of SAR- endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5

abstract

Endolysins are bacteriophage (or phage)- encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. 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 in vitro and in vivo 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 Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 pg ml -1 against Pseudomonas aeruginosa ATCC 27853, 16.25 pg ml -1 against S . enterica Typhimurium ATCC 13311, and 32.50 pg ml -1 against Klebsiella pneumoniae ATCC BAA- 2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 pg ml -1 ) and P. aeruginosa P2307 (65.00 pg ml -1 ) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA- 2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S . enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S . enterica Typhimurium was also observed when the lyophilized LysKpV475 at 2 / 3 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 almonella reduction of 2 logs after 6 h 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.

keywords

MEMBRANE; LIPOPOLYSACCHARIDE; FILMS

subject category

Microbiology

authors

Gontijo, M; Teles, MP; Correia, HM; Jorge, GP; Goes, ICRS; Flores, AJF; Braz, M; Ceseti, LD; Ramos, PZ; Silva, IRE; Vidigal, PMP; Kobarg, J; Couñago, RM; Alvarez-Martinez, CE; Pereira, C; Freire, CSR; Almeida, A; Brocchi, M

Groups

G4 - Renewable Materials and Circular Economy

Projects

Collaboratory for Emerging Technologies, CoLab (EMERGING TECHNOLOGIES)

CICECO - Aveiro Institute of Materials (UIDB/50011/2020)

CICECO - Aveiro Institute of Materials (UIDP/50011/2020)

Associated Laboratory CICECO-Aveiro Institute of Materials (LA/P/0006/2020)

acknowledgements

M.G. was supported by the Brazilian funding agency Fundacao de Amparo a Pesquisa do Estado de Saeo Paulo (FAPESP) through an M.Sc. scholarship (grant 2020/01535-9) . M.G. was supported by a grant (88881.625374/2021-01) from the Fulbright Association and the Coordenacaeo de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) . M.P.T. was also supported by FAPESP through an undergraduate research assistant scholarship (2020/09815-0) and a research internship abroad (FAPESP - BEPE) scholarship (2022/03682-4) . M. Braz was supported by the Portuguese Foundation for Science and Technology with a Fundacaeo para a Ciencia e a Tecnologia/Ministerio da Ciencia, Tecnologia e Ensino Superior (FCT/MCTES) PhD scholarship (2020.06571.BD) . H.M.C. and I.C.R.S.G. were supported by a master's degree CAPES fellowship (88887.666189/2022-00 and 88887.800759/2023-00, respectively) . Research at CQMED was supported by FAPESP (2013/50724-5 and 2014/50897-0) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) (465651/2014-3) . C.P. acknowledges the FCT for a Junior Research contract (CEEC Individual/03974/2017) . FCT is also acknowledged for a research contract under Scientific Employment Stimulus to C.S.R.F. (CEECIND/00464/2017) . Research in the Department of Biology, University of Aveiro, was supported by FCT/MCTES, which finances the Centre for Environmental and Marine Studies (CESAM) (UIDP/50017/2020 + UIDB/50017/2020 + LA/P/0094/2020) . Part of this work was developed within the scope of the project CICECO-Aveiro Instituteof Materials (UIDB/50011/2020, UIDP/50011/2020 and LA/P/0006/2020) , financed by national funds through the FCT/MECTES (PIDDAC) . Research in the Brocchi laboratory was supported by FAPESP (2021/00465-0 and 2021/10577-0) . M. Brocchi is a research fellow of CNPq (309380/2019-7 and 309678/2022-6) .r of Materials (UIDB/50011/2020, UIDP/50011/2020 and LA/P/0006/2020) , financed by national funds through the FCT/MECTES (PIDDAC) . Research in the Brocchi laboratory was supported by FAPESP (2021/00465-0 and 2021/10577-0) . M. Brocchi is a research fellow of CNPq (309380/2019-7 and 309678/2022-6) .