Vpliv hranil na interakcije med bakterijama Bacillus subtilis in Salmonella enterica serovar Typhimurium

Požar, Maruša (author), Mandić Mulec, Ines (mentor), Podnar, Eli (comentor)

V magistrskem delu smo preučevali interakcije med bakterijama Bacillus subtilis in Salmonella enterica serovar Typhimurium, da bi ugotovili, kako na njun odnos vplivajo hranila ter različni sevi B. subtilis. Bakteriji smo sočasno gojili v bogatem gojišču TSB, bogatem sporulacijskem gojišču, ki spodbuja sporulacijo bakterije B. subtilis, ter revnem gojišču 1/20 TSB. Po kokultivaciji smo z metodo določanja kolonijskih enot okarakterizirali njune interakcije: ugotavljali smo, kako močno bakterija B. subtilis inhibira rast bakterije S. Typhimurium ter kako močno bakterija S. Typhimurium inhibira sporulacijo bakterije B. subtilis. Ugotovili smo, da vsi testirani sevi B. subtilis inhibirajo rast bakterije S. Typhimurium SL1344 v bogatem gojišču TSB (NCIB 3610, TA7-1, TA19-1) ter sporulacijskem gojišču (NCIB 3610, PS-216, PS-218, PS-196), medtem ko v revnem gojišču (1/20 TSB) inhibicije rasti bakterije S. Typhimurium SL1344 ni izkazal noben sev B. subtilis. Pokazali smo, da bakterija S. Typhimurium SL1344 inhibira sporulacijo v revnem gojišču 1/20 TSB, ne pa v sporulacijskem gojišču pri vseh testiranih sevih B. subtilis (NCIB 3610, PS-216, PS-218, PS-196). Z merjenjem izražanja fluorescenčnih poročevalskih fuzij dveh sporulacijskih genov Pspo0A-yfp ter PspoIIQ-yfp bakterije B. subtilis PS-218 smo merili dinamiko sporulacije v monokulturi in kokulturi z bakterijo S. Typhimurium SL1344. Ugotovili smo nižjo aktivnost izražanja genov spo0A in spoIIQ v kokulturi kot monokulturi, kar je skladno z meritvami frekvence sporulacije po 24 urah. Določali smo tudi prostorsko razporejanje celic v vzorcu in debelino biofilma bakterije S. Typhimurium z metodo konfokalne laserske mikroskopije. Pokazali smo, da debelino biofilma bakterije S. Typhimurium SL1344 zmanjša le sev B. subtilis NCIB 3610, medtem ko se prostorsko razporejanje celic v kokulturi ne spremeni pri nobenem sevu B. subtilis (NCIB 3610, TA7-1, TA19-1).

• Language: Slovenian
• Keywords: Bacillus subtilis, Salmonella enterica, hranila, mikrobne interakcije, sporulacija, probiotiki.
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Požar]
• Year: 2024
• PID: 20.500.12556/RUL-163848
• UDC: 579.22/.26
• COBISS.SI-ID: 211357955
• Publication date in RUL: 13.10.2024

Secondary language

• Language: English
• Title: The influence of nutrients on interactions between Bacillus subtilis and Salmonella enterica serovarTyphimurium

Abstract

In this Master's thesis, we investigated the interactions between the potential probiotic, Bacillus subtilis, and the pathogen Salmonella enterica serovar Typhimurium to determine how nutrients and different strains of B. subtilis affect their relationship. The bacteria were cocultured in nutrient-rich TSB medium, where sporulation is inhibited, nutrient-rich sporulation medium that promotes the sporulation of B. subtilis, and nutrient-poor 1/20 TSB medium, which also promotes sporulation. After incubation, pahogen-probiotic interactions were characterized using colony-forming units (CFU/mL) and B. subtilis sporulation frequency. We also determined the spatial distribution of cells in static medium and the biofilm thickness of S. Typhimurium by confocal laser scanning microscopy. We demonstrated that only B. subtilis NCIB 3610 reduces the biofilm thickness of S. Typhimurium SL1344 and that the spatial distribution of cells in the coculture is not altered by any of the other B. subtilis strains tested (NCIB 3610, TA7-1, TA19-1). We found that all tested strains of B. subtilis inhibited the growth of S. Typhimurium SL1344 in nutrient-rich TSB medium (NCIB 3610, TA7-1, TA19-1) and sporulation medium (NCIB 3610, PS-216, PS-218, PS-196), but no B. subtilis strain inhibited the growth of S. Typhimurium SL1344 in nutrient-poor 1/20 TSB. We have shown that S. Typhimurium SL1344 inhibits sporulation in nutrient-poor 1/20 TSB medium in all B. subtilis strains (NCIB 3610, PS-216, PS-218, PS-196) but not in sporulation medium. We monitored the dynamics of sporulation of B. subtilis PS-218 over time in coculture with S. Typhimurium SL1344 compared to monoculture by measuring the expression of fluorescent reporter fusions for the Pspo0A and PspoIIQ promoters. The results showed that the expression of both genes was lower in coculture, consistent with sporulation inhibition. As the spo0A expression curve increased slightly towards the end of the experiment, we cannot exclude that sporulation is merely delayed.

• Keywords: Bacillus subtilis, Salmonella enterica, nutrients, microbial interactions, sporulation, probiotics.