Progress 06/01/19 to 05/31/23
Outputs
Target Audience:Tharget audience reached in the past year were two graduate students (one at Penn State and one at Cornell University) working on the project, as well as the scientific community reached through conference presentations. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided an opportunity to develop microbiology and bioinformatics research skills to four graduate students (Tyler Chandross-Cohen, Taejung Chung, Runan Yan, Naomi Niyah) and four undegraduate students (Cassidy Prince, Kayla Kimble, Tyler Chandross-Cohen, Mackenna Yount) at Penn State. They conducted genomic bioinformatic analyses, cytotoxicity experiments, growth experiments and statistical analyses of data. They also presented their research at multiple university, regional, and national conferences, which gave them an opportunity to expand their professional network, practice science communication skills, and get involved with the leadership of the International Association for Food Protection (IAFP). Taejung Chung used the developed bioinformatics skills to provide leadership in data analyses at the Penn State One Health Microbiome Center where he served as the Director of the Data Analysis Working Group that trained new graduate students and other interested members in bioinformatics data analyses. Tyler Chandross-Cohen is currently serving the food science community in his role as the Treasurer of the IAFP Student Professional Development Group (PDG) and the President of the Penn State Phi Tau Sigma Food Science Honorary Society. Three of the undegraduate students that had worked on this project are currently PhD students and one is applying to graduate programs. At Cornell University, this project provided professional development opportunities for a PhD student Jun Su. She presented this research at IAFP Annual Meeting 2023 in Toronto Canada. At this meeting, she networked with other food safety professionals and engaged in Dairy Quality and Safety PDG and Microbial Modelling and Risk Analysis PDG. In addition, she initiated a customer discovery process for the BRisk App, the exposure assessment tool that she developed from this research by participating in the U.S. National Science Foundation Innovation Corps Regional Program. How have the results been disseminated to communities of interest?The results from this project have been disseminated at scientific meetings and conferences, including local university meetings (Penn State Gamma Sigma Delta, Penn State Graduate Exhibition), regional meetings (Allegheny Branch of the American Society for Microbiology) and national or international conferences (International Association for Food Protection). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Obj. 1: We obtained 309 isolates of diverse rpoB or panC genotypes, isolated from diverse sources. 200 of these have not yet been whole-genome sequenced and were sequenced using an Illumina platform in this project. Sequences were checked for quality and assembled. Genomes were then annotated, taxonomically identified, classified into panC phylogenetic groups and MLST sequence types, and screened for the presence of virulence and antimicrobial resistance genes using BTyper. Single nucleotide polymorphisms (SNP) were detected and used for maximum likelihood phylogenetic analysis to confirm the the panC phylogenetic grouping. All isolates with 5 or fewer core SNPs difference were excluded from subsequent association studies to mitigate bias. This resulted in a set of 270 isolates that were further analyzed to identify associations between specific genes and cytotoxicity quantified in Obj. 2. Genomes from phylogenetic group IV, which was significantly more cytotoxic compared to groups II through VIII, and was also represented by 59 isolates, were further analyzed using a pan-genome-wide association study (pan-GWAS) with pyseer to identify genes associated with cytotoxicity. The pan-GWAS results using Scoary revealed no genes significantly associated with the binary cytotoxicity output. Similarly, no significant untigs or kmers were identified from the GWAS analysis using DBGWAS. Using the virulence genes detected in Obj. 1 and the cytotoxicity data generated in Obj. 2, we calculated the sensitivity and specificity for predicting cytotoxicity for each enterotoxin gene. All individualnheandhblgenes, as well as the entirenheABCandhblABCDoperons, had sensitivity values of 1 and specificity values of 0. In contrast tonheandhbl,cytKvariants had low sensitivity values and high specificity values (cytK-1: 0 and 1;cytK-2: 0.37 and 0.87, respectively). Furthermore, associations between nonsynonymous SNPs within these genes and cytotoxicity were assessed using a logistic regression model to assess each site in a gene alignment. We identified a total of 21 nonsynonymous SNPs that were associated with cytotoxicity. Of the 21 SNPs, 13 were inhblgenes and 8 were innhegenes. All identified SNPs had higher specificity values than enterotoxin gene presence. These SNPs warrant further experimental assessment of their role in cytotoxicity. Obj. 2: To assess isolates' cytotoxicity, cell-free supernatants were collected from cultures grown to early stationary phase in brain heart infusion broth at 37°C. Isolates from phylogenetic groups II and VI, which are known for psychrotolerant isolates, were tested also at 32°C. Human colorectal carcinoma cells (CaCo-2) were intoxicated with cell-free supernatants (5% v/v), and cell viability was measured as a readout for cytotoxicity using the colorimetric WST-1 assay. To control for variability among plates of CaCo-2 cells, cytotoxicity values were normalized to controls using min-max normalization. The mean normalized cytotoxicity across all isolates was 0.51 ± 0.35. Isolates with the highest mean cytotoxicity (1 ± 0.033) belonged to phylogenetic group I (B. pseudomycoides). The phylogenetic group IV (B. cereus sensu lato) contained the second most cytotoxic isolates (0.762 ± 0.362), followed by group V (0.593 ± 0.283, B. toyonensis). Isolates with the lowest mean cytotoxicity were found in phylogenetic group VII (0.187 ± 0.333, B. mycoides), followed by phylogenetic group IV (0.256 ± 0.205, B. mycoides and B. paramycoides. Isolates from phylogenetic group VI are psychrotolerant isolates known to cause milk spoilage ("B. weihenstephanensis"). Among the 74 psychrotolerant isolates from phylogenetic group II and VI tested for cytotoxicity when grown at 32°C, 11 were cytotoxic. These 11 isolates were not cytotoxic when grown at 37°C, displaying temperature dependent cytotoxicity. Cytotoxicity analyses were used to investigate genes associated with cytotoxicity in Obj. 1. Obj. 3: 17 cytotoxic isolates representative of different virulence gene profiles and six phylogenetic groups (as determined in Obj. 1) were tested for their ability to grow in skim milk broth at 22, 10 and 4°C. Additional growth experiments were performed at 6, 8, 14, and 18°C to evaluate growth/no growth. The ability of 17 tested strains to grow at different temperatures varied and was dependent on a phylogenetic group. All isolates grew at 22°C, 15 isolates grew at 10°C, and there was no detectable growth of any of the isolates at 4°C over a 24-day incubation. Growth/no growth experiments were conducted at 6°C and 8°C for the 15 isolates that grew at 10°C. No isolates grew at 6°C while one isolate from group V grew at 8°C. Growth/no growth experiments were also conducted at 14°C and 18°C for the 2 isolates from group I that did not grow at 10°C. Both isolates grew at 14°C and 18°C. The growth data for 17 selected B. cereus isolates were fitted using a Baranyi model and their growth characteristics varied by phylogenetic group. At 22°C, isolates from group VII had the longest average lag time of 17.24 h compared to isolates from other groups. This was significantly longer than the average lag time of isolates from group IV (8.2 h; p=0.0279). Isolates from group I had the largest maximum growth rate of 1.14 ln CFU/ml/h compared to isolates from other groups. This was significantly higher than the average maximum growth rate of isolates from group IV of 0.47 ln CFU/mL/h (p=0.0192). At 10°C, isolates from Group V have the shortest average lag time of 56.04 h, while isolate from Group III has the longest average lag time of 154.55 h. Growth data collected in Obj. 3 was used in exposure assessment in Obj. 4. Obj. 4: An exposure assessment model was developed by simulating the growth of 16 B. cereus strains (1 strain was excluded due to the lack of a linear secondary model) in HTST milk products when they were transported along a five-stage supply chain that includes processing plant storage, transportation from the processing plant to retail, retail storage, transportation from retail to the consumer's home, and consumer storage for up to 35 days. Each hypothetical lot of HTST milk was contaminated by one B. cereus strain. Each lot of HTST milk has 100 iterations, representing 100 half-gallon milk containers. The model predicts the percentage of simulated half-gallon milk containers contaminated with a given B. cereus isolate that had B. cereus concentrations over 5 log on consumer home storage day 14, 21, 35, respectively. A percentage of half-gallon milk containers that had B. cereus concentrations that exceeded 105 CFU/mL were computed for each lot.When the initial contamination level of the HTST milk was set at an average of 100 CFU/mL, the model predicts that on consumer home storage day 35, all 16 lots have milk containers that exceed B. cereus concentrations of 5 log (10 of the 16 lots have 3 % milk containers exceeding 5 log). Notably, the model predicts higher percentages of containers exceeding 5 log for 1 of 6 group IV isolates and 1 of 2 group V isolates. On consumer storage day 21, all 16 lots have milk containers that exceed B. cereus concentrations of 5 log. On consumer storage day 14, 15 of 16 lots (1 isolate from group I is an exception) have milk containers that exceed B. cereus concentrations of 5 log (8 of the 16 lots have 2 % milk containers exceeding 5 log). We have integrated this growth model with genomic and cytotoxicity data to develop the BRisk App, a user-friendly digital tool that can help the dairy industry assess likely human exposure to cytotoxic B. cereus strains, facilitating food safety management decisions. The application of this digital tool can be expanded to other food products once B. cereus group growth data in those food matrices become available.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Chandross-Cohen, T., Yount, M., Su, J., Qian, C., Wiedmann, M., Kovac, J. (July 17, 2023). Growth potential of Bacillus cereus group strains from different phylogenetic groups in a dairy food model. 2023 International Association for Food Protection Annual Meeting. Toronto, Canada.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Chandross-Cohen, T., Yount, M., Su, J., Qian, C., Wiedmann, M., Kovac, J. (March 30, 2023). Growth potential of Bacillus cereus group strains from different phylogenetic groups in a dairy food model. 2023 Gamma Sigma Delta Symposium. University Park, Pennsylvania.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Su, J., Qian, C., Chandross-Cohen, T., Yount, M., Wiedmann, M., Kovac, J. (July 20, 2023). An exposure assessment of cytotoxic Bacillus cereus strains from various phylogenetic groups in HTST milk. 2023 International Association for Food Protection Annual Meeting. Toronto, Canada.
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:Tharget audience reached in the past year were a graduate and undergraduate student working on the project, as well as the scientific community reached through conference presentations and papers. Changes/Problems:Challenges: We experienced ripple effects of the pandemic (e.g., supply chain issues), which have delayed the project. Due to delays, we have requested a 1-year no-cost-extension (NCE) to complete this project. The NCE was approved. Major change: We initially planned to carry out growth curve experiements for select isolates at 4, 10, and 22 C. We have changed the strategy to carry out experiements at 6 and 22 C. Six degrees Celsius was selected as a temperature that represents a slight refrigeration storage temperature abuse that has been used in other published studies. Nevertheless, collecting experimental data at 6 C and 22 C, will allow us to transform growth-model fitted data to 4C and 10 C. We will use the Ratkowsky's square-root model for transformation to obtain the estimated lag time and estimated maximum growth rate at both 4 and 10 C, as previously reported by Buehler et al., 2018 (https://pubmed.ncbi.nlm.nih.gov/29803413/). What opportunities for training and professional development has the project provided?The project has provided an opportunity for students to practice their laboratory technical skills, as well as science communication skills by presenting their research at conferences. The latter also presented an opportunity to network and expand a professional network. How have the results been disseminated to communities of interest?The results have been disseminated in local and international conferences (see the list of conference presentations). What do you plan to do during the next reporting period to accomplish the goals?Obj. 2: Perform tissue culture assays to assess expression of toxins and other virulence characteristics in B. cereus group isolates representing different phylogenetic clades and virulence gene profiles. For isolates from phylogenetic groups II and VI, we are planning to complete isolate growing at 10 and 6 dC and use cell-free supernatants for cytotoxicity assessment. Obj. 3: Assess growth capabilities, as well as toxin expression and other virulence characteristics of diverse B. cereus group strains, using different food-relevant environmental conditions. We are planning to complete the second replicate of growth curves at 22 dC, as well as complete both replicates of growth curves at 6 C in the first half of the last project year. Obj. 4: Integrate genomic and phenotypic data into a virulence characterization and exposure assessment tool, and assess human exposure to B. cereus by consumption of food contaminated with different B. cereus group genotypes. A final risk assessment model will be built using experiemental data collected in objectives 1 through 3.
Impacts
What was accomplished under these goals?
Obj. 1: Characterize food-associated and clinical B. cereus group isolates using whole genome sequencing data and comprehensive bioinformatics analyses. Whole genome sequencing and sequence analyses have been completed. Obj. 2: Perform tissue culture assays to assess expression of toxins and other virulence characteristics in B. cereus group isolates representing different phylogenetic clades and virulence gene profiles. Cytotoxicity screening was completed in two independent replicates for all 300 isolates at 32 C. Cytotoxicity screening of isolates from clade II and VI grown at 32 C and 6 C is ongoing. Obj. 3: Assess growth capabilities, as well as toxin expression and other virulence characteristics of diverse B. cereus group strains, using different food-relevant environmental conditions. Seventeen isolates have been selected for growth curve experiments at 4, 10, and 22 dC. These isolates have been selected based on a unique combination of phylogenetic grouping and virulence gene profiles. Thus far, one replicate of growth curves for all 17 isolates has been completed at 22 C. The second replicate is currently conducted. Obj. 4: Integrate genomic and phenotypic data into a virulence characterization and exposure assessment tool, and assess human exposure to B. cereus by consumption of food contaminated with different B. cereus group genotypes. The backbone of the exposure assessment tool is being set up.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Carroll LM, Cheng RA, Wiedmann M, Kovac J. Keeping up with the Bacillus cereus group: taxonomy through the genomics era and beyond. Crit Rev Food Sci Nutr. 2021 May 3:1-26. doi: 10.1080/10408398.2021.1916735. PMID: 33939559.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Kimble K, Prince K, Chung, T, Kovac J. Cytotoxicity analysis of B. cereus group isolates from diverse sources. Annual meeting of the Allegheny Branch of American Society for Microbiology. November, 2021, online.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Kimble K, Prince K, Chung, T, Johler S, Kovac J. Genetic biomarkers of Bacillus cereus sensu lato cytotoxicity in a CaCo-2 model. World Microbe Forum, June, 2021, online.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Kimble K, Prince K, Chung, T, Johler S, Kovac J. Cytotoxicity analysis of B. cereus group isolates from diverse sources. BACT2022, April 2022, Paris, France/online.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Kimble K, Prince K, Chung, T, Kovac J. Cytotoxicity analysis of B. cereus group isolates from diverse sources. Gamma Sigma Delta Research Expo, March, 2022, University Park, PA.
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:In the past year, the audiences reached were students working on the project and the audience at scientific meetings. Changes/Problems:The closure of labs due to COVID-19 in Spring 2020 and Summer 2020 impacted the work on this project. Specifically, the cytotoxicity assessment was delayed due to the lost access to the lab in Spring 2020, and the limited access to the lab due to reduced capacity in Summer and Fall 2021, as well as Spring 2021. Full access to the lab without reduced capacity was gained at the end of June 2021. Importantly, the effects of the changes due to COVID-19 impacted student's mental health in 2021. Both the closure of labs, as well as students' health challenges, have slowed down the progress of this project. This will likely result in needing more time for the completion of the proposed research. What opportunities for training and professional development has the project provided?A graduate student in Molecular, Cellular, and Integrative Biosciences, Naomi Niyah (Native American, female) worked on this project as a graduate research assistant. Naomi worked with undergraduate students Cassidy Prince (a recent graduate in Microbiology that started a PhD program in Microbiology at Cornell University in Fall 2021) and Kayla Kimble (a rising senior undergraduate student in Immunology and Infectious Disease program). Students were trained to independently carry out cytotoxicity screening of B. cereus group isolates. Students were also trained in science communication and bioinformatics analyses of whole genome sequences. In Summer 2021, two latinx undergraduate students that participated in a USDA-funded REEU program were involved in research on this project. An African American female student joined the project in August 2021 to gain research experience. How have the results been disseminated to communities of interest?Results have been disseminated in the form of research papers, presentations at scientific meetings, and by making BTyper3 program publicly and freely accessible via GitHub. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan on completing Obj. 2 - cytotoxicity evaluation and starting and completing Obj. 3 - growth and cytotoxicity evaluation of B. cereus groups strains in a food model. We also plan on starting Obj. 4 - integrating genomic and phenotypic data into a virulence characterization and exposure assessment tool, and assessing human exposure to B. cereus by consumption of food contaminated with different B. cereus group genotypes.
Impacts
What was accomplished under these goals?
Obj. 1: Characterize food-associated and clinical B. cereus group isolates using whole genome sequencing data and comprehensive bioinformatics analyses. Obj. 1 was completed in the previous project year. Obj. 2: Perform tissue culture assays to assess expression of toxins and other virulence characteristics in B. cereus group isolates representing different phylogenetic clades and virulence gene profiles. Filtered supernatants have been collected for 310 isolates at 37 dC (one independent replicate). Cytotoxicity has been screened on CaCo-2 cells using 15% v/v supernatants for 236 isolates; the remaining isolates are in the process of cytotoxicity testing. Filtered supernatants have also been collected for 236 isolates at 37 dC in a second independent replicate and are in the process of cytotoxicity testing. Additionally, supernatants were collected for 32 isolates from psychrotolerant clades (II and IV) grown at 32 dC. Cytotoxicity has been evaluated for all 30 isolates. The supernatants for the second replicate will be collected in the next year. Obj. 3: Assess growth capabilities, as well as toxin expression and other virulence characteristics of diverse B. cereus group strains, using different food-relevant environmental conditions. This objective will start in September 2021. Obj. 4: Integrate genomic and phenotypic data into a virulence characterization and exposure assessment tool, and assess human exposure to B. cereus by consumption of food contaminated with different B. cereus group genotypes. This objective will start in 2022.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
M�ndez Acevedo M, Carroll LM, Mukherjee M, Mills E, Xiaoli L, Dudley EG,
Kovac J. Novel Effective Bacillus cereus Group Species "Bacillus clarus" Is Represented by Antibiotic-Producing Strain ATCC 21929 Isolated from Soil. mSphere. 2020 Nov 4;5(6):e00882-20. doi: 10.1128/mSphere.00882-20. PMID:33148822; PMCID: PMC7643830.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Carroll LM, Cheng RA, Kovac J. No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus Group With Historical Typing Methods. Front Microbiol. 2020 Sep 22;11:580691.doi: 10.3389/fmicb.2020.580691. PMID: 33072050; PMCID: PMC7536271.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Carroll LM, Cheng RA, Kovac J. No Assembly Required: Using BTyper3 to Assess the Congruency of a Proposed Taxonomic Framework for the Bacillus cereus Group With Historical Typing Methods. Front Microbiol. 2020 Sep 22;11:580691. doi: 10.3389/fmicb.2020.580691. PMID: 33072050; PMCID: PMC7536271.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Carroll LM, Wiedmann M. Cereulide Synthetase Acquisition and Loss Events
within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens.
mBio. 2020 Aug 25;11(4):e01263-20. doi: 10.1128/mBio.01263-20. PMID: 32843545;PMCID: PMC7448271.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
C. R. Prince, N. Niyah, T. Chung, S. Johler, J. Kovac. Genetic biomarkers of Bacillus cereus sensu lato cytotoxicity in a CaCo-2 model. World Microbe Forum 2021, June 20 - 24, 2021, online conference.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
N. Niyah, C. Prince, T. Chung, J. Kovac. Correlation between enterotoxin gene presence and cytotoxicity of Bacillus cereus group isolates in a CaCo-2 model. SACNAS 2021, October 25 29, 2021, online conference.
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Progress 06/01/19 to 05/31/20
Outputs
Target Audience:Since the project started on 6/1/2019, the audiences reached were students and scientists involved in the project initiation. A graduate student in Molecular, Cellular, and Integrative Biosciences, Naomi Niyah (Native American, female) was hired to work as a graduate research assistant on this project. Naomi worked with scientists from the University of Zurich and from the Centers for Disease Control and Prevention to initiate a collaboration that involves an exchange of biological materials that will be tested in this research project. Naomi also worked with an undergraduate student Cassidy Prince on the development of outreach materials related to the research conducted as part of this project, for the general public. Changes/Problems:We were not able to obtain human clinical isolates to include in our study. We overcame this problem by reaching an agreement with the CDC for them to share filtered supernatants of their clinical isolates with us for cytotoxicity assessment. CDC also agreed to share with us the whole genome sequences of their isolates to allow for biomarker identification. By May 2020, CDC had shared with us 41 supernatants of human and food isolates, as well as whole-genome sequences of these isolates. This substantially increased the representation of human clinical isolate in a set of investigated isolates. What opportunities for training and professional development has the project provided?This project provided an opportunity for graduate training to one female Native American graduate student Naomi Niyah and a female junior undergraduate student Cassidy Prince that is working with a graduate student as part of her independent studies course. Engagement of the undergraduate student in this research project is helping her develop the scientific skills needed for graduate school, which she is planning to pursue upon completion of BSc in Microbiology. During the remote work phase (due to COVID-19), Naomi and Cassidy have worked together on whole-genome sequence analyses and outreach related to the research conducted in this project. This has allowed them to develop their science communication skills. How have the results been disseminated to communities of interest?Naomi and Cassidy developed an outreach video that has been shared on a lab website as well as social media outlets (e.g., Twitter, Facebook). The goal of their outreach video was to explain the science of whole genome sequencing and how it can be used to track and characterize foodborne pathogens, such as those from the B. cereus group. The video was made publicly available via YouTube: https://youtu.be/lfP6Wf36Blk What do you plan to do during the next reporting period to accomplish the goals?In the next period, we plan to complete cytotoxicity experiments and work towards identifying genomic biomarkers associated with determined cytotoxicity in a tissue culture model. We submitted abstracts to present the resulting work at IAFP and ASM Microbe 2020. The abstracts were accepted; however, the ASM Microbe conference was canceled due to COVID-19. The IAFP conference is still planned to be carried out in person. Furthermore, Naomi intends to present work at the Society for the Advancement of Chicanos/Hispanics and Native Americans in Science (SACNAS) meeting 2020, as well as The Annual Biomedical Research Conference for Minority Students (ABRCMS) 2020.
Impacts
What was accomplished under these goals?
PROJECT TIMETABLE: Objs. 1 and 2: Months 1 to 18; Objs. 3 and 4: Months 19 to 36. Obj. 1: Characterize food-associated and clinical B. cereus group isolates using whole genome sequencing data and comprehensive bioinformatics analyses. Accomplishments: We whole-genome sequenced 200 B. cereus group isolates from diverse phylogenetic groups as planned for year 1. Whole-genome sequences of newly-sequenced isolates, as well as previously sequenced isolates (those sequenced and published by us as well as those provided by the CDC) were analyzed to determine phylogenetic clades to which individual isolates belong to, as well as taxonomic species for each tested isolate. Known and putative virulence genes present in individual isolates were also identified. Among 200 whole-genome sequenced isolates, there were 197 isolates that belonged to B. cereus group species and 3 that did not belong to the B. cereus group, according to the sequence analyses, and were therefore excluded from further analyses. In addition to newly-sequenced isolates, a total of 78 isolates from dairy-related and outbreak-related sources that had previously been sequenced were included in the analyses. Additionally, 31 isolates' sequences of human and food isolates, provided by the CDC, have been analyzed. Overall, 306 of the isolates belonged to the phylogenetic clades, clade I (N = 5), clade II (N = 43), clade III (N = 74), clade IV (N = 82), clade V (N = 45), clade VI (N = 45), clade VII (N = 12). In regards to taxonomic species, we identified 295 of the isolates belonging to the following species: B. albus (N = 4) B. anthracis (N = 5; isolates from CDC that are not handled in the BSL-2 lab at Penn State), B. cereus (N = 65), B. cytotoxicus (N = 11), B. mobilis (N = 15), B. mycoides (N = 34), B. pacificus (N = 19), B. paranthracis (N = 46), B. proteolyticus (N = 3), B. pseudomycoides (N =6), B. thuringiensis (N = 18), B. toyonensis (N = 40) B. tropicus (N = 2), B. weihenstephanensis (N = 4), and B. wiedmannii (N = 23). The rest of the isolates (N=11) could not be reliably identified. The following sources of isolation have been represented among the analyzed set of isolates: food (N = 226), animal (N = 1), environment (N = 16), human (N = 23), and biopesticides (N = 11); the rest did not have information on the source of isolation. Lastly, 74.1% isolates carried one or more of the hbl genes, 93.8% one or more of the nhe genes, 16.49% cytK-1 gene, 42.1% cytK-2 gene, and 29.6% isolates carried one or more of the ces genes. Clades III and VII overall had the lowest proportions of hbl genes present relative to the other clades. With 12 isolates in clade VII, the clade still had the highest proportion of isolates (75%) with the cytk-1 gene present. Obj. 2: Perform tissue culture assays to assess the expression of toxins and other virulence characteristics in B. cereus group isolates representing different phylogenetic clades and virulence gene profiles. Accomplishments: B. cereus group isolates' expression of enterotoxins is under the regulation of quorum sensing. In order to identify the time of the entry into the stationary phase, we screened the growth curves of isolates via optical density monitoring in a microtiter plate format. The growth curves were determined using optical density (OD) for a total of 306 B. cereus group isolates to identify the early stationary phase in which isolates' supernatants will be collected and tested for cytotoxicity on CaCo-2 cells. The R package "growthcurver" was used to fit a logistic growth model to the OD data. Two tangential lines were drawn, one following the exponential phase (and intersecting the maximum growth rate) and the other one across the stationary phase. The point at which the two tangential lines intersected + 2 hours was calculated and defined as the time of entry into the early stationary phase. The R package "factoextra" was then used to classify the growth rates using the k-means clustering and the elbow method. Using this approach, three clusters were identified: fast (median of 8 hours), intermediate (median of 12 hours), and slow (median of 17 hours) growers. Once appropriate growth times were identified, we began screening isolates for cytotoxicity on CaCo-2 cells using WST-1 as a viability indicator. The screening began early in February. We completed screening for 25 isolates before the COVID-19-related lab closure.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
N. Niyah, C. Prince, T. Chung, S. Johler, J. Kovac. Genotyping and Growth Rate Characterization of B. Cereus Group Isolates from Diverse Sources. Accepted for a poster presentation at Penn State Gamma Sigma Delta symposium, University Park, PA. Symposium canceled due to COVID-19.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
Taejung Chung, Cassidy Prince, Naomi Niyah, Sophia Johler, Jasna Kovac. Genomic characterization and growth rates of B. cereus group isolates from diverse sources. Accepted for a poster presentation at IAFP Annual Meeting 2020, Cleveland, OH.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2020
Citation:
N. Niyah, C. Prince, T. Chung, S. Johler, J. Kovac. Genotyping and Growth Rate Characterization of B. Cereus Group Isolates from Diverse Sources. Accepted for a poster presentation at ASM Microbe 2020, Chicago, IL. Conference canceled due to COVID-19.
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