Comparative Genomic and Phenotypic Analyses of Bacillus cereus sensu stricto biovar Thuringiensis and Non-Thuringiensis
Restricted (Penn State Only)
- Author:
- Chung, Taejung
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 06, 2023
- Committee Members:
- Jasna Kovac, Chair & Dissertation Advisor
Istvan Albert, Outside Unit & Field Member
Edward Dudley, Major Field Member
Josephine Wee, Major Field Member
Robert Roberts, Program Head/Chair - Keywords:
- Food Microbiology
Food Safety
Bacillus cereus
Bacillus thuringiensis
Food borne pathogen - Abstract:
- The Bacillus cereus group, consisting of eight genomospecies based on genomic similarity calculated by average nucleotide identity value of < 92.5% with diverse genomic and phenotypic characteristics, presents a complex challenge in the field of food safety and public health due to the foodborne pathogenic potential of some strains. The ability to form spores is a critical survival feature of Bacillus cereus group strains, enabling them to survive harsh environmental conditions, such as food processing environments or the human gastrointestinal (GI) tract. Bacillus cereus sensu stricto (s.s.) is one of the eight genomospecies within the Bacillus cereus group, which contains strains capable of causing foodborne illness, as well as strains capable of producing insecticidal crystal proteins. The latter are classified into biovar Thuringiensis due to their entomopathogenic characteristics. Traditionally, Bacillus cereus s.s. biovar Thuringiensis (Bt) has been considered a non-toxic biocontrol agent and has been utilized as a bioinsecticide in food crop production for over a century. However, concerns about its food safety have recently emerged. They have been prompted by the detection of human enterotoxin encoding genes in strains of biovar Thuringiensis or the ability to produce human enterotoxin associated with foodborne illness. Nevertheless, it remains unclear whether strains of biovar Thuringiensis that carry enterotoxin genes can survive GI-tract associated stressors and subsequently germinate and produce enterotoxins, which is required to cause toxicoinfection in humans. Throughout this dissertation, I present three studies investigating genomic and phenotypic differences between B. cereus s.s. biovar Thuringiensis and non-Thuringiensis strains. Specifically, I focus on assessing the methods for genome-based identification of biovar Thuringiensis, identifying genetic markers predictive of biovar Thuringiensis, and phenotypic assessment of differences in spore germination in response to human GI tract related stresses and potential germinants present in the human GI tract. In Chapter 2, I compared the performance of multiple whole-genome sequencing-based bioinformatics programs for identification of biovar Thuringiensis. Currently, two methods are used to distinguish biovar Thuringiensis from non-Thuringiensis strains: standard microbiological technique based on microscopy to screen for crystal protein production and genomic identification based on the detection of crystal protein-encoding genes. Due to the significant variability in crystal protein-encoding gene sequences, the accuracy of genomic identification of biovar Thuringiensis was unknown and required phenotypic validation. We found that among the four tested bioinformatics tools, IDOPS performed best, with sensitivity and specificity exceeding 90%. To understand genomic differences between B. cereus s.s. biovar Thuringiensis and non-Thuringiensis strains, I applied a pangenome-wide association study (pan-GWAS) to identify genes significantly associated with biovar Thuringiensis among B. cereus s.s. strains (Chapter 3). Additionally, I identified germination-related genes associated with biovar Thuringiensis to gain a better understanding of genomic differences in the germination of biovar Thuringiensis. I identified two genes significantly associated with biovar Thuringiensis, comR (a copper-inducible transcriptional repressor) and ydhP (inner membrane transport protein), with relatively high sensitivity (> 85%) and specificity (> 70%). Of these, the comR gene may play a role in regulating crystal protein production by controlling intracellular copper concentration. However, no germination-related genes were significantly associated with biovar Thuringiensis. This does not exclude the possibility of genes being differentially expressed between biovar Thuringiensis and non-Thuringiensis and necessitates further phenotypic germination analyses. Lastly, I assessed the effects of human gastrointestinal (GI) tract related stresses and nutrients on germination of strains of B. cereus biovar Thuringiensis and non-Thuringiensis (Chapter 4). I found that exposure of spores to human GI tract-related stresses resulted in insignificant germination. Conversely, specific nutrients, including inosine, and L-alanine in conjunction with L-phenylalanine, induced germination of B cereus s.s. Importantly, there was no evidence to suggest differences in spore germination between B. cereus s.s. biovar Thuringiensis and non-Thuringiensis strains. In conclusion, the findings from this dissertation indicate minimal genomic differences between B. cereus biovar Thuringiensis and non-Thuringiensis and no phenotypic differences in germination between biovar Thuringiensis and non-Thuringiensis strains in tested conditions. These results highlight potential food safety risk associated with the human exposure to B. cereus s.s. biovar Thuringiensis, which are similar to those of other pathogenic B. cereus s.s. strains.