Isolamento e caracterização de óleos essenciais e bacteriófagos como tratamentos de controle biológico para Salmonella enterica

Abstract

Background: Foodborne diseases are a global public health issue with 1 in 10 people falling ill after eating contaminated food every year. In Brazil, Salmonella enterica serovar Enteritidis is a significant health threat. Bacteriophages (phages; bacteria viruses), have potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. Objetives: The thesis has five chapters, the aims of every chapter were: 1) Develop a narrative review about the applications of phage therapy for various infectious diseases, phage pharmacology, immunological responses to phages, legal concerns, and the potential benefits and disadvantages of this novel treatment. 2) Develop a systematic review and meta-analysis that evaluated the efficiency of phages patented as a biological control for foodborne pathogens and determined the physical-chemical characteristics of the antimicrobial effect. 3) Determine the antimicrobial activity of seventeen plant extracts, commercial essential oils, carvacrol and thymol against Salmonella ATCC 14028. 4) Describe the fenotipic and genomic characterization of Salmonella SE3 strain isolate from Subaé River, Santo Amaro, BA-Brazil. 5) Describe the fenotipic and genomic characterization of SF1 phage, isolate from Subaé River, Santo Amaro, BA-Brazil. Determine the host range of SF1 phage against Salmonella strains and other species of bacteria. Results: The systematic review was developed using scientific article and patent databases with inclusion and exclusion criteria applied by automatic and manual processes. A random-effects meta-analysis was carried out and revealed: (i) significant antimicrobial effect of Listeria phages in apple, apple juice, pear, and pear juice (ii) significant antimicrobial effect of Salmonella phages in eggs, apple and ready to cook chicken, (iii) no heterogeneity was identified in either meta-analysis, (iv) publication bias was detected in Listeria phages but not in Salmonella phages. (v) ListShield and Felix01 phages showed the best result for Listeria and Salmonella biological control, respectively, (vi) concentration of phage and bacteria, time and food had significant effect in the biological control of Listeria, (vii) temperature and time had a significant effect on the antimicrobial activity of Salmonella phages. Moreover, the antibacterial activity of Seventeen plants extracts from the semi-arid region of the northeast Brazil were macerated using hexane, ethyl acetate and ethanol to produce 51 extracts. Six commercially produced essential oils and the essential oils of Croton heliotropiifolius (obtained by hydrodistillation), thymol and carvacrol against Salmonella using in vitro approaches were evaluated. Clove bud essential oil and thymol showed activity against Salmonella at a concentration of 1mg/ml. Salmonella SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. De novo hybrid sequencing assembly of Salmonella SE3 from Illumina HiSeq and ONT MinION whole genome sequencing yielded 10 contigs and showed 99.98% of identity with Salmonella enterica subsp. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven defense systems, seven prophages and a heavy metal resistance gene (arsC) were identified. Pangenome analysis of the S. enterica clade, including SE3, revealed an open pangenome, with a core genome of 2,137 genes. The accessory genome comprised 3,390 shell genes and 69,352 cloud genes. Furthermore, SF1 phage was isolated and characterized, Furthermore, phage genome were sequenced by ONT MiION and Illumina Hiseq sequencing, three genomes assemblies, no Hybrid (MinION and Hiseq) and hybrid (MinION + Hiseq), was tested, the genomes were annotated and analyzed, and their genomes were compared with the reference Salmonella phage. MiION assembly showed the best results. Besides, no lysogenic cycle, antimicrobial resistance and virulence genes were identified in our work. SF1 phage showed activity against twenty seven strains: Salmonella var. Enteritidis, Salmonella var. Typhimurium, Salmonella var. Minnesota, Shigella flexneri, Escherichia coli, Escherichia cloacae, Escherichia fergusonii, Citrobacter europeus, Citrobacter freundii, Corynebacterium pseudotuberculosis, Corynebacterium striatum, Glutamicibacter creatinolíticus, Klebsiella oxytoca, Listeria monocytogenes and Rodococos iaqui. Conclusions: In summary, we evaluated the efficiency of phages previously patented as a biological control for fruits and vegetables, and meat. Most of the natural products extracts tested in this study did not show significant antimicrobial activity against Salmonella enterica subsp. Typhimurium ATCC 14028. However, Clove bud essential oil and thymol showed activity against Salmonella at a concentration of 1mg/ml. On the other hand, we showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. The hybrid genome assembly enabled identification of virulence and resistance genes, mobile genetic elements and pangenome analysis. No obstant, MinION platform showed the best assembly for SF1 phage. The host range of SF1 phage showed activity against twenty seven strains. SF1 phage showed to be a polyvalent phage. Two receptors were identified: receptor b and tail tube protein.