Investigation of bacterial social interactions in two species biofilms composed of Salmonella enterica serovar Typhimurium and Bacillus subtilis

Podnar, Eli (author), Mandić Mulec, Ines (mentor), Danevčič, Tjaša (comentor)

Salmonella enterica serovar Typhimurium is one of the most common foodborne pathogens affecting humans and animals. Its pervasive nature allows it to thrive in environments beyond its host. The emergence of antimicrobial resistance within Salmonella strains is a major concern, prompting the exploration of alternative strategies to curtail infections. Probiotic bacteria, such as Bacillus subtilis, are a promising new strategy to combat foodborne pathogens. However, existing literature on B. subtilis-Salmonella interactions mostly focuses on probiotic effects in live animals leaving a gap in understanding the mechanisms of their interplay. In this thesis, we tested the competition between B. subtilis PS-216 and S. Typhimurium SL1344 under different environmental conditions, focusing on nutrient availability. The results show that under nutrient-rich conditions, B. subtilis PS-216 inhibits the growth of S. Typhimurium SL1344 and reduces its adhesion and biofilm thickness. The B. subtilis antagonistic potential is lost in the Δpks mutant, in which the synthesis of the polyketide antibiotic bacillaene was impaired. S. Typhimurium increased the PpksC activity, which controls bacillaene production. This suggests that B. subtilis senses and responds to a Gram-negative competitor. However, nutrient limitation reduced B. subtilis antagonism against S. Typhimurium and inhibited the efficacy of B. subtilis sporulation in coculture. The results also showed that sporulation inhibition by the pathogen is dependent on iron limitation and sigma B (SigB)-controlled general stress response in B. subtilis. Furthermore, interactions under nutrient limitation were dependent on cell-cell contact and the type VI secretion system (T6SS) of S. Typhimurium. The thesis reveals novel molecular determinants of competition between the two bacteria but also underscores the pivotal role played by environmental conditions and cell-cell contact. The thesis also highlights the need to assess probiotic strains in conditions resembling their intended use, which is crucial for enhancing strategies to control pathogens.

• Language: English
• Keywords: B. subtilis, S. Typhimurium, biofilm, competition, secondary metabolites, polysaccharides, sporulation, probiotics, pathogens
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: BF - Biotechnical Faculty
• Publisher: [E. Podnar]
• Year: 2023
• PID: 20.500.12556/RUL-152875
• UDC: 579.22/.26:579.842.1/.2:579.852.1
• COBISS.SI-ID: 176990979
• Publication date in RUL: 10.12.2023

Secondary language

• Language: Slovenian
• Title: Raziskava socialnih interakcij v vrstno mešanih biofilmih bakterij Salmonella enterica serovar Typhimurium in Bacillus subtilis

Abstract

Bakterija Salmonella enterica serovar Typhimurium je eden najpogostejših patogenov pri ljudeh in živalih, ki se prenaša s hrano. Bakterije iz rodu Salmonella so vseprisotne in lahko prebivajo tudi v okoljih zunaj gostitelja, ki so revna s hranili. Razvoj odpornosti proti antibiotikom je pogost med sevi bakterije Salmonella in potrebni so alternativni pristopi za omejevanje okužb. Probiotične bakterije, kot je bakterija Bacillus subtilis, so obetavna nova strategija za boj proti patogenom, ki se prenašajo s hrano. V literaturi so interakcije med bakterijama B. subtilis-Salmonella preučevali večinoma in vivo na živalih in niso obravnavali mehanizmov, ki usmerjajo kompeticijo med bakterijama. V doktorski disertaciji smo raziskali interakcije med bakterijama B. subtilis PS-216 in S. Typhimurium SL1344, v pogojih z različno vsebnostjo hranil. Rezultati kažejo, da v pogojih obilja bakterija B. subtilis PS-216 zavira rast bakterije S. Typhimurium SL1344 in zmanjša njeno adhezijo ter debelino biofilma. Mutanta B. subtilis Δpks, ki ne sintetizira poliketidnega antibiotika bacilena ne zavira rast patogena. V kokulturi je bakterija S. Typhimurium sprožila aktivacijo PpksC bakterije B. subtilis, ki uravnava izražanje bacilenskega operona, kar kaže, da bakterija B. subtilis zaznava in se odziva na prisotnost patogena. Ugotovili smo tudi, da ob pomanjkanju hranil bakterija B. subtilis izgubi antagonistični učinek proti bakteriji S. Typhimurium in tvori manj spor v kokulturi kot v monokulturi. Inhibicija sporulacije je povezana s pomanjkanjem železa, s stresnim odzivom bakterije B. subtilis, ki ga uravnava sigma B (SigB). Inhibicija sporulacije je tudi pogojena z direktnim celičnim stikom obeh vrst in sistema za izločanje tipa VI (T6SS) bakterije S. Typhimurium. Doktorsko delo razkriva nove molekularne determinante kompeticije med probiotikom in patogenom ter izpostavi vpliva okoljskih pogojev in tesnih medceličnih interakcij na izid kompeticije. Delo razkriva pomen vrednotenja probiotičnih sevov proti patogenom pod pogoji, ki ustrezajo predvideni uporabi in prinaša uvide, pomembne za izboljšanje kontrole patogenov s probiotiki.

• Keywords: B. subtilis, S. typhimurium, biofilm, kompeticija, sekundarni metaboliti, polisaharidi, sporulacija, probiotiki, patogeni mikroorganizmi