Transformacija bakterij Campylobacter jejuni z genom za luciferazo in priprava sevov mutant z izbitimi geni vključenimi v tvorbo biofilma

Čukajne, Tjaša (author), Klančnik, Anja (mentor), Berlec, Aleš (comentor)

Patogene bakterije vrste Campylobacter jejuni so že vrsto let glavni povzročitelj bakterijskega gastroenteritisa pri ljudeh po vsem svetu. Velik problem predstavljajo biofilmi, ki jih tvorijo na površinah v živilsko predelovalni industriji, saj jim omogočajo preživetje kljub neugodnim razmeram v okolju. Posledično so biofilmi glavno tveganje za širjenje bakterij C. jejuni v prehranjevalni verigi. Študije biofilmov so ključnega pomena za razumevanje odpornosti bakterij in razvoj novih strategij za obvladovanje bakterijskih okužb. Pomemben je razvoj novih, hitrih, specifičnih in ponovljivih metod za odkrivanje in kvantifikacijo bakterij v biofilmih. To je še posebnega pomena za bakterije C. jejuni, saj lahko neugodne razmere v okolju preživijo v živi, vendar ne kultivabilni obliki. V magistrskem delu smo oblikovali plazmid pMW10_nLuc s konstitutivnim izražanjem luciferaze NanoLuc. Plazmid smo uspešno transformirali v bakterijo C. jejuni in vpeljali novo specifično in ponovljivo metodo kvantifikacije bakterij z merjenjem bioluminiscence. Vpeljano metodo smo uporabili za kvantifikacijo planktonskih celic in določanje adhezivnosti ter filmotovornosti bakterij tako na polistirenski površini, kot tudi na površini prevlečeni z mucinom. Vpeljano metodo smo primerjali z metodo kvantifikacije kultivabilnih celic preko štetja kolonijskih enot, metodo kvantifikacije živih celic z resazurinom in barvanjem biomase biofilma s kristal vijoličnim. Specifičnost vpeljane metode smo dokazali v mešani kulturi s sevom Salmonella enterica, uporabnost pa v razmerah in vitro v modelu govejega fecesa in v piščančjem soku kot modelu živila. Razvito metodo kvantifikacije planktonskih celic in določanja adhezivnosti ter filmotvornosti smo uporabili tudi po tretiranju celic z antibiotikom ciprofloksacinom in tremi naravnimi protiadhezivnimi snovmi (epigalokatehin galat, karvakrol, timol). Vpeljana metoda predstavlja pomemben korak pri hitrejši in bolj celoviti kvantifikaciji adheriranih celic C. jejuni na površini in v biofilmu. Poleg tega smo uspešno pripravili seva mutant z izbitima genoma Cj0794 in Cj0736, ki sta domnevno pomembna pri tvorbi biofilma bakterij C. jejuni.

• Language: Slovenian
• Keywords: Campylobacter jejuni, luciferaza NanoLuc, bioluminiscenca, kvantifikacija bakterij, adhezija, biofilm, izbitje genov
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Year: 2023
• PID: 20.500.12556/RUL-151711
• COBISS.SI-ID: 168914179
• Publication date in RUL: 18.10.2023

Secondary language

• Language: English
• Title: Transformation of Campylobacter jejuni bacteria with the luciferase gene and preparation of mutant strains with knocked-out genes involved in biofilm formation.

Abstract

Campylobacter jejuni, a leading cause of bacterial gastroenteritis worldwide, presents a significant challenge due to its ability to form tenacious biofilms in the food processing industry. These biofilms enable its survival in adverse environments, posing a grave threat to food safety and public health. Understanding biofilms is essential for tackling bacterial resistance and the development of effective infection control strategies. In master's degree thesis, we addressed the pressing need for novel, rapid, specific, and reproducible methods to detect and quantify bacteria within biofilms which is particularly crucial for C. jejuni, which can survive harsh conditions in a viable but non-culturable state. We designed the pMW10_nLuc plasmid with a constitutive NanoLuc luciferase system. The plasmid was successfully transformed into C. jejuni, introducing a precise and consistent method for quantifying bacteria through bioluminescence. Our innovative approach allowed us to quantify planktonic cells and assess bacterial adhesion and biofilm formation on polystyrene and mucin-coated surfaces. We compared our method to conventional quantification techniques, including colony counting, resazurin-based live cell quantification, and crystal violet staining of biofilm biomass. To establish specificity, we validated our method in mixed cultures with Salmonella enterica strain. Its applicability was demonstrated in vitro, using bovine feces, and with chicken juice as a food model. Furthermore, we employed our method to quantify planktonic cells and evaluate adhesion/biofilm formation after treating cells with the antibiotic ciprofloxacin and three natural anti-adhesive substances (epigallocatechin gallate, carvacrol, thymol). In summary, our introduced method represents a significant advancement in the rapid and comprehensive quantification of adhered C. jejuni cells within biofilms. Additionally, we successfully generated mutant strains featuring Cj0794 and Cj0736 gene knockouts, shedding light on their roles in C. jejuni biofilm formation.

• Keywords: Campylobacter jejuni, NanoLuc luciferase, bioluminescence, bacteria quantification, adhesion, biofilm, gene knock-out