Author:

Abstract:

Clostridium botulinum is a strictly anaerobic spore-forming bacterium, and produces the most deadly toxin known, the botulinum neurotoxin (BoNT). Therefore, food contaminated with (spores of) this pathogen constitutes an important threat to humans and animals. Bacterial spores are highly resistant dormant structures that are resilient against many hostile conditions like heat, dryness, lack of nutrients, UV, chemicals and oxygen. When conditions become favorable, they can grow out and resume vegetative growth, which is accompanied with toxin production. Since spores first have to complete a complex cascade of events designated as germination, before they can grow out, outgrowth and toxin production can in principle be prevented by inhibition of germination. However, the germination mechanism of C. botulinum spores has only been unraveled in very little detail, mainly due to biosafety and bioterrorism restrictions and because a strict anaerobic environment is required to grow the pathogen. In this PhD thesis, we focused on psychrotrophic group II C. botulinum (gIICb), the major pathogen of concern in minimally processed chilled ready-to-eat foods with extended shelf life. Due to biosafety reasons, nontoxigenic mutants were first constructed to facilitate the research on the germination mechanism. To this end, a novel deletion strategy was developed and used to create a ∆bontE mutant in C. botulinum NCTC 11219. This was the first report of a deletion mutant in gIICb, a species group that is known to be less genetically tractable than other clostridia, and for which the genetic toolbox is still very limited. In addition, a bontE insertional knockout mutant was created by the ClosTron system. While most characteristics of the mutants showed to be identical to the parental strain, also some differences were noted. Presumably, these changes are due to some spontaneous mutations that were identified by whole genome sequence analysis. It could be excluded that the changed properties are associated with BoNT inactivation, since the same atoxigenic mutants were constructed (by insertion and deletion) in a different parental strain (NCTC 8266) and these did not show any altered properties, except for a growth defect at lowered temperatures for the ClosTron mutant. The deletion mutants of these two strains offer safe surrogate organisms for gIICb in challenge testing of foods. In a second part of this PhD research, the spore germination process was investigated. First, the ability to induce germination of the nutrient mixture L-alanine/L-lactate/NaHCO3, high pressure treatment, calcium dipicolinic acid (Ca2+-DPA) and dodecylamine was assessed. Based on the loss of heat resistance and the release of DPA, it was concluded that only the nutrient mixture and the surfactant dodecylamine could induce germination. However, dodecylamine germinated spores did not loose their refractility in phase-contrast microscopy, indicating that they had not rehydrated. In addition, these spores only became heat sensitive when dodecylamine was present, whereas they remained resistant when dodecylamine was washed away before the heat treatment. This suggests that dodecylamine does not induce genuine germination. Furthermore, four natural antimicrobial compounds (carvacrol, cinnamaldehyde, carrot seed essential oil and hop β-acids) were tested for their inhibitory action on germination. Although these compounds have mainly been studied for their growth inhibiting action, the inhibition of spore germination could also contribute to the control of spore-formers in foods. The results demonstrated that the minimal concentration of carvacrol, cinnamaldehyde and carrot seed essential oil that could significantly reduce nutrient germination, was many times lower than the minimal inhibitory concentration (MIC) on vegetative growth. This could be of interest because the low sensory- and odor thresholds often interfere with the application of these compounds in foods. In a more mechanistically oriented part, specific genes that are predicted to encode proteins involved in germination of gIICb, were investigated via deletion analysis. The presence of only one Ger-type germinant receptor, GerX3b, in these strains is unusual because other spore-formers generally contain more receptors. Moreover it is intriguing, because it is unclear how a single receptor can respond to the many different nutrients that can induce germination in gIICb. The gerBAC gene cluster, encoding the three subunits of the GerX3b receptor, was successfully deleted. Germination experiments with this mutant showed that the GerX3b receptor is not involved in germination induced by different nutrient mixtures. Furthermore, two predicted cortex hydrolases, SleB and SleC, were investigated via the same knockout strategy, but a deletion mutant could only be successfully created for SleB. Because spores lacking SleB could still complete cortex hydrolysis, it was concluded that SleB is not an essential cortex hydrolase in gIICb, similar to what has been reported for SleB of C. difficile. Nevertheless, an auxiliary role in other germination events can not be excluded since the germination assays showed a large variability.