Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
(N/A)
Non Technical Summary
This project will investigate Bacillus cereus, a foodborne pathogen commonly found in the natural environment and food systems. Bacillus cereus group bacteria are particularly problematic because they can form spores that resist heat and sanitizers, which helps them persist in food processing environments. These bacteria have been of growing concern in baby formula and plant-based food alternatives, highlighting the need for research that can inform risk-based food safety decision-making. Strains of B. cereus group vary in their ability to produce diarrheal enterotoxins, such as Hemolysin BL, in human gut. However, the capacity of a strain to express enterotoxins remains challenging to predict. To improve our ability to identify high-risk strains, this research focuses on identifying and validating genetic variants in enterotoxin genes that provide B. cereus with an increased ability to produce enterotoxins and cause toxicity towards human gut cells. Identified variants will be validated and integrated into an exposure assessment model that will also provide qualitative information about food safety risk posed by individual strains of B. cereus. We will further investigate the role of enterotoxin Hemolysin BL in surface colonization and biofilm formation to assess whether Hemolysin BL helps B. cereus survive and outcompete other bacteria in food processing environments. By understanding how Hemolysin BL contributes to B. cereus survival and competition, we can develop strategies to mitigate the persistence of virulent strains in food systems.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
The goal of this project is two-fold: (i) to evaluate the effect of polymorphisms in Bacillus cereus gene encoding for an enterotoxin hemolysin BL on transcription and cytotoxicity, and (ii) to assess how the expression of Hemolysin BL affects isolates' ability to colonize surfaces and biofilms. These goals will be completed using the following two objectives:Evaluate the effects of single nucleotide polymorphisms in the hbl genes and corresponding regulatory sequence PlcR-box on the transcription of Hemolysin BL and cytotoxicity towards human colon cells CaCo-2.Examine the effect of Hemolysin BL on surface colonization and biofilm formation.
Project Methods
We identified nonsynonymous SNPs in hbl genes that were significantly associated with cytotoxicity towards CaCo-2 cells. We will additionally identify SNPs in the PlcR box that are associated with cytotoxicity. Mutants in identified SNPs in the hbl genes and PlcR box will be created for use in Obj. 1 and a mutant with the entire hbl operon deletion will be created for use in Obj. 2. The B. cereus type strain ATCC 14579 will be used to create mutants using CRISPR-Cas9 system. The effects of mutations on transcription and expression of a hbl genes will be quantified using SYBR green RT-qPCR and CaCo-2 cytotoxicity assay. Data will be normalized by subtracting the average absorbance of the tested mutant by the ΔhblABCD control and dividing it by the difference between the B. cereus wild type and ΔhblABCD control. Differences in cytotoxicity between mutants and the wild type will be evaluated using a one-way ANOVA followed by Tukey's HSD. SNPs with confirmed effects on cytotoxicity will be incorporated as a variable into an existing exposure assessment model. Users of the exposure assessment model can provide genomic data and concentration of B. cereus group strain in HTST milk to obtain data on the percentage of HTST milk units in which B. cereus exceeds 105 CFU/ml, as well as the qualitative assessment of food safety risk associated with a strain.In Obj. 2, the B. cereus ATCC 14579 ΔhblABCD and wild type strain will be evaluated for their surface colonization abilities in competition with each other and with other motile spore-forming Bacilli commonly found in dairy processing facilities. Skim milk agar (0.3%) will be used to simulate nutrients in a dairy food processing environment and promote surface expansion. Isolates will be grown in skim milk broth and then inoculated onto skim milk agar at set distances from each other and incubated at 25°C. The area of growth will be imaged using a Bio-RAD gel documentation station and quantified using ImageJ software. The significance of differences in colonization areas between the wild type vs. the mutant strains, the wild type vs. Bacilli, and the mutant strain vs. Bacilli will be assessed using ANOVA followed by Tukey's HSD. This will allow me to determine whether the expression of Hbl provides B. cereus with a competitive advantage in surface colonization. The B. cereus ΔhblABCD mutant and wild type strain will be grown in both single-species biofilms (wild type, mutant, and wild type vs. mutant) and in multi-species biofilms with other Bacilli species (wild type vs. other Bacilli, mutant vs. other Bacilli). Biofilm formation will be measured in vitro using two complementary methods: (1) crystal violet staining to quantify total biofilm biomass and (2) confocal laser scanning microscope (CLSM) to quantify the structure and relative abundance of B. cereus in a formed biofilm. A one-way ANOVA will be used to compare biofilm formation between the mutant and the wild type strain and the relative biovolume of the wild-type or mutant in biofilms.