Bifidobacterial biofilm formation is a multifactorial adaptive phenomenon in response to bile exposure

Abstract

In the current study, we show that biofilm formation by various strains and species belonging to Bifidobacterium, a genus that includes gut commensals with reported health-promoting activities, is induced by high concentrations of bile (0.5% (w/v) or higher) and individual bile salts (20 mM or higher), rather than by acid or osmotic stress. The transcriptomic response of a bifidobacterial prototype Bifidobacterium breve UCC2003 to such high bile concentrations was investigated and a random transposon bank of B. breve UCC2003 was screened for mutants that affect biofilm formation in order to identify genes involved in this adaptive process. Eleven mutants affected in their ability to form a biofilm were identified, while biofilm formation capacity of an insertional mutation in luxS and an exopolysaccharide (EPS) negative B. breve UCC2003 was also studied. Reduced capacity to form biofilm also caused reduced viability when exposed to porcine bile. We propose that bifidobacterial biofilm formation is an adaptive response to high concentrations of bile in order to avoid bactericidal effects of high bile concentrations in the gastrointestinal environment. Biofilm formation appears to be a multi-factorial process involving EPS production, proteins and extracellular DNA release, representing a crucial strategy in response to bile stress in order to enhance fitness in the gut environment. Biofilms are microbial communities that are attached to a surface and are enclosed/structured by an extracellular matrix 1. Biofilms may also form when free floating cells clump together or adhere to particulate matter, this being referred to as microcolony formation 2. Biofilm formation is a complex process involving multiple steps, including initial attachment, accumulation, maturation and dispersal 3. Initial attachment is reversible and can be driven by electrostatic interactions; attachment to a surface can also be mediated by cell wall-associated proteins that bind to a substrate-covered surface or extracellular DNA (eDNA) release, where DNA released by cell lysis coats the surface and changes surface properties to allow attachment 4,5. The accumulation phase of a biofilm can be mediated by cell wall associated protein interactions or extracellular matrix (EM) secretion 5,6. The EM of biofilms may be composed of extracellular polysaccharide (EPS), DNA and/or proteins 7-9. Finally, following maturation, individual cells may disperse from the biofilm to resume planktonic growth 10. Biofilm formation is often triggered in response to environmental stresses, such as nutrient starvation, antibiotics, pH and bile, or induced through quorum sensing systems, such as the Agr or autoinducer-2 (AI-2) systems 11-14. Bifidobacteria are non-motile gut commensals, some of which are purported to exert health-promoting or probiotic properties (see review 15). Particular bifidobacterial strains are for this reason included in certain 'functional foods' so as to bestow these benefits to the host that ingests them 16. However, whether bifidobacteria ...

The genus Bifidobacterium: from genomics to functionality of an important component of the mammalian gut microbiota

“…Although in silico investigations aimed at predicting the presence of the lux S gene in bifidobacteria has been limited to only a small number of species, its conservation in all analysed genomes suggests the possible presence of such a gene in all bifidobacterial species [8] , [142] , [143] . Despite the as yet incomplete characterization of this cell density-dependent regulatory mechanism within the genus Bifidobacterium , bifidobacterial AI-2 production seems to be involved in biofilm formation, suggesting that AI-2 plays a crucial role in influencing the colonization of these symbiotic microorganisms in the gut ecosystem [96] , [142] . This hypothesis was further corroborated by the observation that a B. breve UCC2003 lux S insertion mutant strain is much more sensitive to multiple iron chelators, is not able to colonize the murine GIT and was shown to confer less protection against a pathogen infection in Caenorhabditis elegans [143] .…”

“…Specifically, bile salt hydrolases confer protection by catalysing the de-conjugation of glycine and taurine from bile salts generating unconjugated acids that can be metabolized by other intestinal bacteria [94] , [95] . In addition, biofilm formation has been proposed as a crucial adaptive strategy in response to bile stress relying on a multi-factorial process involving exopolysaccharide (EPS) production as well as protein and extracellular DNA release, as observed in the bifidobacterial prototype B. breve UCC2003 [96] .…”

Transcriptome Analysis Reveals the Genes Involved in Bifidobacterium Longum FGSZY16M3 Biofilm Formation

“…All Bifidobacterium strains sequenced harbor luxS genes, which are involved in the production of the classic QS signaling molecule autoinducer-2 (AI-2) [ 22 , 23 , 24 ], yet no AI-2 receptor has been reported in this genus [ 25 ]. Despite a vast amount of research on biofilm formation [ 26 ], its mechanism has not yet been fully elucidated in many bacterial species, and it is especially true for Bifidobacterium species [ 27 ].…”

Bacteroides thetaiotaomicron uses a widespread extracellular DNase to promote bile-dependent biofilm formation

“…Some studies tested purified bile acids, rather than bile extract, and showed that all bacteria did not induce biofilm in response to the same bile acid. For instance, C. difficile reacted to deoxycholate (Dubois et al, 2019), B. breve to conjugated bile acids (taurocholate, glycocholate, taurodeoxycholate, glycodeoxycholate) (Kelly et al, 2020), and Lactobacillus to taurocholate (Ambalam et al, 2012). Conversely, taurine-conjugated bile acids disperse biofilms of V. cholerae or P. aeruginosa (Hay and Zhu, 2015; Sanchez et al, 2016).…”