Study of Bacillus thuringiensis behaviour in food environment by genome – wide transcriptome analysis

Bacillus thuringiensis is a spore forming bacterium that belongs to the Bacillus cereus family. It was first characterized for its ability to produce a parasporal crystal active against several insect species, especially Lepidoptera, Diptera, and Coleoptera. Due to its insect activities it is worldwide used in forestry and agriculture to control pests. Recent studies showed that most of the genetic determinants for B. cereus virulence, such as haemolysin BL (HBL), non haemolytic enterotoxin (NHE), cytotoxin K, and bc-D-ENT enterotoxin, are harboured by B. thuringiensis strains. Since B. thuringiensis can contaminate food, being residual in spore form after treatment in the fields, it is ever more urgent to deepen investigate the potential risks arising from the presence of B. thuringiensis in food industry. Phylogenetic studies based on the analysis of chromosomal genes bring controversial results, and it is unclear whether B. cereus and B. thuringiensis are varieties of the same species or different species (Ivanova et al. 2003). Hence, what may seem to be a minor problem of taxonomy may therefore have serious implications for virulence and pathogenicity. This work of thesis was aimed to achieve a deeper scientific information on the food-associated Bacilli, taking the advantage of new genome based molecular approaches, focusing the attention on B. thuringiensis strains used as commercial biopesticides. The in vitro pathogenic profile of ten commercial B. thuringiensis strains, was characterised by the high distribution of the nhe, hbl, bceT and cytK genes, coding for respectively four B. cereus associated virulence factors. Enterotoxin genes were detected by PCR in all the strains analyzed. RT-PCR analysis confirmed the enterotoxin genes expression. Toxin productions was detected by RPLA test in the strains belonging to the widely used subsp. kurstaki. These features and the difficult discrimination between B. thuringiensis and B. cereus, suggested that the role of B. thuringiensis in outbreaks of foodborne disease may have been underestimated. The development of a vegetable based food model, that would allow to asses the behaviour of B. thuringiensis spores, after the simulation of an industrial processing treatment, was an important point in this study. The analysis of Bacillus spore envelope, and its ability to interact with food environment, have been performed using SEM and SEM X-ray microanalysis applied to the food model proposed. In more detail, particular attention was devoted to morphological and chemical changes of B. thuringiensis spores during germination process in food. We observed a rapid evolution of the B. thuringiensis biological cycle compared to that of other spore forming bacteria like Clostridium spp. (Bassi et al. 2008, personal communication). Interesting was that only two hours after spore activation, cell outgrowth was completed and cell division was at the maximum level. RT-qPCR analysis were performed to quantify the expression, in food, of the major virulence genes involved in B. cereus-associated food borne disease. Toxin mRNAs were detected, in variable amounts, at all investigated growth stages of B. thuringiensis, with a strong increase during the log phase of microorganism growth. Although no information on the B. cereus toxin expression in food are available, previous in vitro studies on B. cereus enterotoxins production, reported that the highest toxin level is achieved during the late log/early stationary phase. The production of the L2 component of HBL enterotoxin, involved in the diarrhoeal syndrome was detected in food model, even in low amount, during the early log phase. We concluded that B. thuringiensis can complete an entire life cycle in food systems after an industrial processing simulation, producing enterotoxins as observed in broth cultures. Given this finding, the need to identify systems for manage the risks associated with B. thuringiensis in industrial fields has became clear. An experimental approach was described in this work of thesis. Identification and inactivation of general systems for regulating virulence, through null mutants construction, were considered to evaluate changes in growth performance, cellular metabolism and toxins expression, in the studied microorganism. Besides homologous recombination, the mobility mechanism of group II introns were assessed to generate highly specific chromosomal gene disruption in B. thuringiensis. A novel approach and several experiments were performed to achieve the desired chromosomal inactivation, however no attempts gave the expected results. In order to manage risks associated with B. thuringiensis outgrowth in foodstuffs, and to gain more information on its life cycle, a microarray transcriptome analysis of B. thuringiensis in four different stages of the biological cycle, was performed from dormant spore to vegetative/sporulating cells. We could emphasized that mRNA is a component of bacterial spores. We discovered that spores are equipped with a large amount of transcripts probably useful to front the next steps of outgrowth. Dormant spores contained populations of ribosomes; during the first 40 minutes after spore activation, rate of both rRNA and ribosomal proteins synthesis strongly increased. A basic and strong activation of polyfunctional genes seemed to begin in germinant spores: most of the genes involved in the metabolic activity (house-keeping genes, translation initiation factor, ribosomal proteins, and elongation factors) were overrepresented at this time in microarray analysis. A large number of transcripts for protein involved in the regulation of different biological process, including resistance to different antimicrobial compounds and oxidative stress agents, were found to be present in B. thuringiensis vegetative cells. We hypothesized that B. thuringiensis cells may activate these systems in response to external stimuli for cell defence and adaptation to changing environmental conditions in food model. The transcripts for germination proteins (ger type) found in spore, are an index of the expression of this genes in previous sporulation stage and suggested the importance during dormancy, to monitor the environment for proper outgrowth conditions. This finding could explain the ability of B. cereus-like microrganism to occupy and complete a full life cycle within several different environmental niches. According to literature data, all the associated virulence genes, represented in microarray analysis, were up-regulated especially during the late stage of cell growth. Transcriptomic has been demonstrated to be not only a powerful tool to study the germination and outgrowth of B. thuringiensis spores, but also a suitable method to assess the environmental response to bacterial pathogens in food. Data obtained, provide new basic knowledge on Bacillus cereus group. These data extends our knowledge on the metabolic versatility of B. thuringiensis and also added to our view of virulence traits of this potential food-pathogen. Since B. thuringiensis is widely used and popular in biological farming, a careful monitoring of the strains used should be justified. Literature reports widespread the risks associated with the food-pathogen B. cereus, but those related to B. thuringiensis are often underestimated. From data obtained in this study we could assume that B. thuringiensis could actually be responsible for many of the food borne outbreaks previously attributed to B. cereus; taking this enterotoxigenic potential into account, as well as the fact that B. thuringiensis cannot be separated from B. cereus at the chromosomal level, food producers and food authorities, responsible for food safety, should consider the risk of B. thuringiensis insecticide residue in the food chain.