Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Scientists in academia, government, and industry involved in bacterial food safety Changes/Problems:Some aspects of the project were delayed due to the SARS-CoV-2 outbreak. We were unable to hold our planned training in summer 2020 but hope to do so next year either in person or virtual, depending on Penn State policies. What opportunities for training and professional development has the project provided?Two postdoctoral fellows and one graduate student have been provided research training. How have the results been disseminated to communities of interest?Publication in scientific journals, research presentations at other academic institutions, and press releases on Penn State run web services. What do you plan to do during the next reporting period to accomplish the goals?Goal 1. Plans for last year were to pursue genome sequencing studies with Mount Nittany Medical Center and the US Geological Society to study transmission of Salmonella from wild birds to humans. This project was delayed due to the current pandemic, however;we have been able to establish it again during Fall 2020. Goal 2. We are continuing to characterize the microcin including purification in collaboration with individuals at Rowan University. We will be investigating the intriguing observation that strains producing this microcin suppress E. coli O157:H7 colonization in mice. Goal 3. We plan to hold the summer undergraduate program in 2021, either online or in person, depending on Penn State policies.
Impacts
What was accomplished under these goals?
Goal 1. Non-typhoidal Salmonella (NTS) are a significant source of foodborne illness worldwide, with disease symptoms most often presenting as self-limiting gastroenteritis; however, occasionally the infection spreads and becomes invasive, frequently requiring anti-microbial treatment. The cattle-adapted Dublin serovar of NTS has commonly been associated with invasive illness and anti-microbial resistance (AMR). Here, the enhanced resolution conferred by whole-genome sequencing was utilized to elucidate and compare the resistome and genetic relatedness of 14 multidrug-resistant (MDR) and one pan-susceptible S. Dublin, isolated primarily in Pennsylvania, from fresh retail meat (one isolate) and humans (14 isolates). Twelve different genetic AMR determinants, including both acquired and chromosomal, were identified. Furthermore, comparative plasmid analysis indicated that AMR was primarily conferred by a putative IncA/C2 plasmid. A single pan-susceptible S. Dublin isolate, collected from the same timeframe and geographical region as the MDR isolates, did not carry an IncA/C2 replicon sequence within its genome. Moreover, the pan-susceptible isolate was genetically distinct from its MDR counterparts, as it was separated by ≥267 single nucleotide polymorphisms (SNPs), whereas there was a ≤38 SNP distance between the MDR isolates. Collectively, this data set advanced our understanding of the genetic basis of the highly drug-resistant nature of S. Dublin, a serovar with significant public health implications. Goal 2. We have continued characterizing a newly discovered microcin, and how strains producing it can impact Shiga toxin production by E. coli O157:H7. We have identified CirA as the protein present on the pathogen that is responsible for recognition and initiating internalization, and two genes responsible for providing immunity against the microcin. In a mouse model, strains producing the microcin do not appear to impact Shiga toxin production by E. coli O157:H7, and to the contrary are strong inhibitors of pathogen colonization. Goal 3. The indicated training program was not held Summer 2020 due to the SARS-CoV-2 outbreak.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Eyler, A. B., M'ikanatha, N. M., Xiaoli, L., and E. G. Dudley. 2020. Whole-genome sequencing reveals resistome of highly drug-resistant retail meat and human Salmonella Dublin. Zoonoses Public Health. 67:251-262. doi: 10.1111/zph.12680
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Ouattara HG, Elias RJ, Dudley EG. 2020. Microbial synergy between Pichia kudriazevii YS201 and Bacillus subtilis BS38 improves pulp degradation and aroma production in cocoa pulp simulation medium. Heliyon. 6:e03269. doi: 10.1016/j.heliyon.2020.e03269
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Nawrocki EM, Mosso HM, Dudley EG. 2020. A toxic environment: a growing understanding of how microbial communities affect Shiga toxin expression by E. coli O157:H7. Appl Environ. Microbiol. doi: 10.1128/AEM.00509-20
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Connolly CJ, Kaminsky L, Pinto GN, Sinclair PC, Bajracharya G, Yan R, Nawrocki EM, Dudley EG, Kovac J. Whole-Genome Sequences of Salmonella Isolates from an Ecological Wastewater Treatment System. Microbiol Resour Announc 9:e00456-20. doi: 10.1128/MRA.00456-20
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Lacher DW, Mammel MK, Gangiredla J, Gebru ST, Barnaba TJ, Majowicz SA, Dudley EG. Draft Genome Sequences of Isolates of Diverse Host Origin from the E. coli Reference Center at Penn State University. Microbiol Resour Announc 2020 Sep 24;9(39):e01005-20. doi: 10.1128/MRA.01005-20.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Scientists in academia, government, and industry involved in bacterial food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?During the last reporting period, one Ph.D student (project 2) graduated and one postdoctoral fellow (project 1) left for a position at the US Centers for Disease Control. One of the former undergraduates from project 3 began a Ph.D program at Penn State, and an additional 3 others will apply for Fall 2020 admission in STEM graduate programs. How have the results been disseminated to communities of interest?Publications in peer-reviewed journals, and press releases on Penn State run web services. What do you plan to do during the next reporting period to accomplish the goals?1. In collaboration with the PA Department of Health, we are building links to the Department of Environmental Protection, and Mount Nittany Medical Center, to study the transmission of pathogenic Salmonella from wild birds to humans; 2. We are continuing to characterize the identified microcin, including identifying its mechanism of action, in efforts to determine whether it could be used to eliminate E. coli O157:H7 from colonized animals; 3. We plan to submit a renewal for the USDA-NIFA grant that funded this project, which ends May 2020. Additionally, a National Science Foundation training grant was submitted along with the University of Puerto Rico-Aguadilla for transitioning UPR students into STEM Ph.D programs.
Impacts
What was accomplished under these goals?
1. This project sequenced approximately 80 Escherichia coli isolates from farm animals in Puerto Rico. Sequencing and analysis was done along with project 3, below. Using genomic analyses, antimicrobial resistance genes were identified, and a correlation was observed between the number of resistance genes and use of antimicrobial compounds in the farm environment. 2. Work from this year identified a previously undescribed, small molecule that enhances Shiga toxin production by E. coli O157:H7. An E. coli strain, designated 0.1229, was identified that amplified Stx2a production when co-cultured with E. coli O157:H7 strain PA2. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with Proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2 kb region of a 12.8 kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two other E. coli strains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggest E. coli 0.1229 and other E. coli produce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced by E. coli, this study provides an additional mechanism by which stx2a expression is increased in response to the gut microflora. 3. Our second eight-week summer program was held June-July 2019, and included 12 students from undergraduate-centric colleges and universities around Pennsylvania, and the University of Puerto-Rico Aguadilla. Students received training in microbial food safety, genomics, and metagenomics, and had the opportunity to visit the US FDA in College Park, MD, for a day.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Figler, H. M., Xiaoli, L., Banerjee, K., Hoffmann, M., Yao, K., Dudley, E. G. 2019. A putative microcin amplifies Shiga toxin 2a production of Escherichia coli O157:H7. J Bacteriol. doi: 10.1128/JB.00353-19
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Denagamage TN, Wallner-Pendleton E, Jayarao BM, Xiaoli L, Dudley EG, Wolfgang D, Kariyawasam S. 2019. Detection of CTX-M-1 extended-spectrum beta-lactamase among ceftiofur-resistant Salmonella enterica clinical isolates of poultry. J. Vet Diagn Invest. 31:681-687. doi: 10.1177/1040638719864384
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Abelman RL, M'ikanatha NM, Figler HM, Dudley EG. 2019. Use of whole genome sequencing in surveillance for antimicrobial-resistant Shigella sonnei infections acquired from domestic and international sources. Microb Genom. doi: 10.1099/mgen.0.000270
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Nwanosike H, Chung T, Xiaoli L, Condello M, Dudley EG, Kovac J. 2019. Whole-Genome Sequences of Escherichia coli Isolates from Cocoa Beans Imported from Bolivia. Microbiol Resour Announc. 2019 Jan 17;8(3). pii: e01516-18. doi: 10.1128/MRA.01516-18
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Keefer AB, Xiaoli L, M'ikanatha NM, Yao K, Hoffmann M, Dudley EG. 2019. Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates. Microbiology. 2019 Mar;165(3):270-286. doi: 10.1099/mic.0.000768
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Progress 04/02/18 to 09/30/18
Outputs
Target Audience:Scientists and industry personel interested in food safety. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project trained undergraduates, graduate students, and postdoctoral fellows. How have the results been disseminated to communities of interest?Through poster presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
To utilize whole genome sequencing (WGS) to enhance surveillance capabilities for foodborne pathogens throughout Pennsylvania. Non-typhoidalSalmonellaare a leading cause of outbreak and sporadic-associated foodborne illnesses in the U.S. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibioticsusceptibility testing (AST) have been used to facilitate public health investigations ofSalmonellainfections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historicSalmonellaisolates, subdivided into three subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed the retail meat isolates withinS. Heidelberg,S. Typhimurium var. O5- subset 1, andS. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96, and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, includingfosA7, a gene only recently found in a limited number ofSalmonellaand a ≥ 95% phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated inS. Heidelberg andS. Typhimurium var. O5- subset 2; whereas, AMR was chromosomally-carried inS. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance ofSalmonellafrom multiple sources. Shigella spp.are a major cause of gastroenteritis worldwide, andS. sonneiis the most common species isolated within the United States. Previous surveillance work in Pennsylvania documented increased antimicrobial resistance (AMR) inS. sonneiassociated with reported illnesses. The present study examined a subset of these isolates by whole genome sequencing (WGS) to determine the relationship between domestic and international isolates, identify genes that could separateS. sonneiisolates by a previously described Global Lineage classification, and test the accuracy of WGS for predicting AMR phenotype. A collection of 22 antimicrobial resistant isolates from patients infected within the United States or while travelling internationally between 2009 and 2014 was chosen for WGS. Phylogenetic analysis revealed both international and domestic isolates were one of two previously defined Global Lineages ofS. sonnei, designated Lineage II and Lineage III. Seventeen alleles that could distinguish these two lineages were identified, and these correctly categorized 36 of 37 publicly availableS. sonneigenomes. Lastly, genome analysis was used to identify AMR determinants. Genotypic analysis was concordant with phenotypic resistance for six of eight antibiotic classes. For aminoglycosides and trimethoprim, resistance genes were identified in 2 and 3 phenotypically sensitive isolates, respectively. To elucidate the mechanism(s) by which non-pathogenicE. colienhance toxin expression of E.coliO157:H7. Escherichia coli serotype O157:H7 is a food-borne pathogen. Symptoms vary greatly among individuals, even those infected with the same strain. Shiga toxin (Stx), one of O157:H7's main virulence factors, is encoded on a phage and released upon induction of bacterial cell lysis (Serna & Boedeker 2008), which can be caused by DNA damage and other mechanisms. It has been shown that the production of Stx can increase depending on the other bacteria present in the environment. This research aims to identify novel secreted factors in ST73 strain 0.1229 that can induce Stx2 and worsen O157:H7 infections. A co-culture protocol was used where an O157:H7 strain, PA2, was grown with the whole cells (16 hours) or cell-free supernatant (8 hours) of each isolate at 37°C. One step recombination was used to knock out gene(s) and regions in E. coli strain 0.1229. Whole genome sequencing was done on this isolate using both the Illumina Miseq and PacBio sequencing platforms. Annotations were completed by RAST. Thirteen E. coli strains were tested as a co-culture with PA2, five were able to amplify Stx2a production. Of those five, only one strain also amplified Stx2a production in a cell independent manner. This E. coli strain designated 0.1229 produced 33.7 μg/mg of Stx2a when co-cultured with PA2 (compared to 6.1 μg/mg for PA2 alone) and 33.96 μg/mg of Stx2 when PA2 was grown in cell-free supernatant of 0.1229 (compared to 5.51 μg/mg for PA2 in LB media). Putative bacteriocin producing genes for microcin B17 (MccB17) were revealed in the E. coli 0.1229 genome, however, knockouts had no differing phenotype, suggesting molecule(s) other than MccB17 are responsible for this phenotype. Treatment of the 0.1229 supernatant with Proteinase K (1mg/ml) had 20-fold decreased Stx2a amplification, suggesting protein involvement. Through sequencing, three plasmids were identified. The 12kb plasmid (p0.1229_3) carries an ABC transporter and thus is of most interest. From the data, we conclude that 0.1229 produces a Proteinase K sensitive molecule that is able to induce Stx2a production of O157:H7. It is not uncommon for two bacteriocins to be carried on the same plasmid, and in that case, the genes for both tend to be close to each other (Cascales et al. 2007). Therefore, we postulate that p0.1229_3 houses the novel gene(s), including the annotated ABC transporter. To develop training experiences in food safety for students at undergraduate-centric colleges and universities. A cohort of 13 undergraduate students spent 8 weeks in the Department of Food Science as part of a Research Experiences for Undergraduates program, studying various aspects of food microbiology.
Publications
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