Spatio-Temporal Evolution of Sporulation in Bacillus thuringiensis Biofilm

Abstract

Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls – the ring – is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed.

Metal-Adapted Bacteria Isolated From Wastewaters Produce Biofilms by Expressing Proteinaceous Curli Fimbriae and Cellulose Nanofibers

“…Importantly, the ability of these bacteria to produce the biofilms in the glass test tubes with SOBG broth was not examined yet. Definitely, certain strains of E. coli ( Weiss-Muszkat et al, 2010 ; Hung et al, 2013 ), P. fluorescens ( Spiers et al, 2003 ; Koza et al, 2009 ), K. pneumoniae ( Wang et al, 2016 ), A. lwoffii ( Martí et al, 2011 ), and B. thuringiensis ( El-Khoury et al, 2016 ) from other than wastewater origins have been reported to form the AL biofilms in defined laboratory systems. In this study, E. asburiae ENSD102 (from dyeing wastewater), uncultured Vitreoscilla sp.…”

Mutanofactin promotes bacterial adhesion and biofilm formation of cariogenicStreptococcus mutans

“…5f). In addition, 5 induced macroscopically visible aggregation and classical biofilm ring formation of Δmuf mutant 49 . Furthermore, the addition of 5 to the wild-type or the Δmuf mutants of Smu56 and Smu57 significantly decreased the polar electron-donor (γ -) parameter of bacterial surface ( Fig.…”

Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54

“…Additionally, in certain conditions this may also result in higher total numbers of spores eventually formed in the biofilms compared to the surrounding planktonic phase, as observed in Chapter 2. The onset of sporulation in the ring of an air-liquid interface biofilm of B. thuringiensis was also shown to precede the sporulation in the pellicle type biofilm in the same setting, presumably linked to the earlier nutrient limitation experienced in the ring compared to the pellicle (El-Khoury et al 2016). …”

“…2. Recently, El-Khoury et al (El-Khoury et al 2016) analysed sporulation efficacy in B. thuringiensis air liquid interface biofilms and presented a model describing spore formation in the biofilm, the impact of mechanical washing and cleaning causing only partial removal of biofilm spores, and the role of residual spores in starting a new biofilm after germination and outgrowth. Since the environment is known to affect the onset and efficiency of sporulation (Driks 2002, Piggot and Hilbert 2004, Abbas et al 2014, the conditions that biofilm cells encounter in the production environment, such as fluctuations in nutrient availability or air exposure, may affect the amount of spores formed, which in turn will affect the risk related to those biofilms.…”

“…For example, within one hour an 8 log CFU/coupon biofilm of B. cereus (formed in BHI) released 5 log CFU/ml cells into the standing phosphate buffer surrounding it (not shown). The number of spores in the biofilm (El-Khoury et al 2016) and their heat resistance is influenced by the phase of the biofilm development and environmental parameters encountered such as dry or wet conditions (summarised in Fig. 2).…”