Interactions between Campylobacter jejuni and Bacillus subtilis

Erega, Andi (author), Smole Možina, Sonja (mentor), Mandić Mulec, Ines (comentor)

The gram-negative, foodborne pathogen Campylobacter jejuni is typically found in animals, such as birds, as an intestinal commensal, and also in food products, especially fresh poultry meat. Probiotics are one of the strategies that can be easily applied in animal feeding to attenuate Campylobacter problem in the food production and human nutrition. Here, we present a natural strain Bacillus subtilis PS-216, gram-positive, beneficial, spore forming potentially probiotic strain and reference strain C. jejuni NCTC 11168 in in vitro co-culture experimental model system. We hypothesize that B. subtilis secretes metabolites that reduce C. jejuni survival in a co-culture and that this interaction changes the growth dynamics and spatial distribution of both species in a multi-species biofilm during static, microaerobic conditions at 42 㓋C. We confirm this hypothesis by using microscale (confocal laser scanning microscopy), C. jejuni and B. subtilis mutants and colony counts of fluorescently labelled strains. We confirm that B. subtilis decreases the fitness of C. jejuni, reduces its biofilm prominence, and decreases the adhesion of C. jejuni to abiotic surfaces. Moreover, we show that the addition of B. subtilis cells to a pre-established C. jejuni biofilm induces its dispersal, that the antagonistic effect of B. subtilis is contact independent and involves non-ribosomal peptides bacillaen and bacylisin as a fully functional two-component system response regulator (ComA) for full antagonistic strength of B. subtilis PS-216 against C. jejuni during co-culture assay. Furthermore, B. subtilis two-time diluted CFS affects the faster formation of biofilm in response to B. subtilis extracellular components. Also, we showed importance of efflux apparatus system, quorum sensing and stress response of C. jejuni during interaction with B. subtilis PS-216 in co-culture and adhesion to abiotic surfaces. Altogether results indicate that mechanism of competitive exclusion is driven by interference competition between two competing strains which is in line with all three hypotheses. In conclusion, we show a strong potential of B. subtilis PS-216 to limit C. jejuni biofilm growth and adhesion to abiotic surfaces, which makes this strain interesting for application in pathogen biofilm control and as a potentially probiotic strain to be applied in animal feeding.

• Language: English
• Keywords: Campylobacter jejuni, Bacillus subtilis, biofilm formation, confocal microscopy, food safety, secondary metabolites
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [A. Erega]
• Year: 2021
• PID: 20.500.12556/RUL-131715
• UDC: 579.22/.26:579.6
• COBISS.SI-ID: 81643523
• Publication date in RUL: 01.10.2021

Secondary language

• Language: Slovenian
• Title: Interakcije bakterij Campylobacter jejuni in Bacillus subtilis

Abstract

Po Gramu negativne patogene bakterije Campylobacter jejuni, ki se prenašajo s hrano, običajno najdemo pri živalih, kot so ptice, kot črevesni komenzal, pa tudi v prehrambenih izdelkih, zlasti svežem perutninskem mesu. Probiotiki so ena izmed strategij, ki jih je mogoče zlahka uporabiti pri krmljenju živali, da bi rešili problem kontaminacije pri pridelavi hrane in prehrani ljudi. V tej študiji smo raziskali Bacillus subtilis PS-216, po Gramu pozitiven, sporotvoren in potencialno probiotičen sev in referenčni sev C. jejuni NTCT 11168 v in vitro eksperimentalnem modelnem sistemu s kokulturo. Predvidevamo, da B. subtilis izloča presnovke, ki zmanjšujejo preživetje C. jejuni v kokulturi in da ta interakcija spreminja dinamiko rasti in prostorsko porazdelitev obeh vrst v večvrstnem biofilmu med statičnimi mikroaerobnimi pogoji pri temperaturi 42 㓋C. To hipotezo potrjujemo z uporabo konfokalne laserske mikroskopije, mutantov C. jejuni in B. subtilis ter števila kolonij fluorescentno označenih sevov. Potrjujemo, da B. subtilis zmanjšuje sposobnost rasti C. jejuni, zmanjšuje vzdrževanje biofilma in zmanjšuje oprijem C. jejuni na abiotske površine. Poleg tega dokazujemo, da dodajanje celic B. subtilis predhodno vzpostavljenemu biofilmu C. jejuni povzroči njegovo razpršitev, da je antagonistični učinek B. subtilis neodvisen od stikov in vključuje ne-ribosomske peptide bacilen in bacilizin. Za popolno antagonistično učinkovitost B. subtilis PS-216 proti C. jejuni v ko-kulturi smo dokazali, da B. subtilis uporablja funkcionalni dvokomponentni sistemski regulator odziva (ComA). Poleg tega zunajcelične komponente dvakrat razredčenega izrabljenega gojišča B. subtilis pospešijo filmotvornost C. jejuni. Pokazali smo tudi vpletenost sistema efluksnih črpalk, zaznavanje kvoruma in stresni odziv C. jejuni med interakcijo z B. subtilis PS-216 pri ko-kulturi in adheziji na abiotske površine. Rezultati skupaj kažejo, da mehanizem konkurenčne izključenosti vodi interferenčna kompeticija med dvema konkurenčnima vrstama, kar je v skladu z vsemi tremi hipotezami. Dokazali smo velik potencial naravnega seva B. subtilis PS-216 za omejevanje rasti in oprijema C. jejuni na abiotskih površinah, zaradi česar je sev PS-216 zanimiv za uporabo pri nadzoru biofilma te patogene bakterije.

• Keywords: Campylobacter jejuni, Bacillus subtilis, biofilm, konfokalna mikroskopija, varnost hrane, sekundarni metaboliti