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6 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Comparative Genomics Applied to Systematically Assess Pathogenicity Potential in Shiga Toxin-Producing Escherichia coli O145:H28

Shiga toxin-producing Escherichia coli (STEC) O145:H28 can cause severe disease in humans and is a predominant serotype in STEC O145 environmental isolates. Here, comparative genomics was applied to a set of clinical and environmental strains to systematically evaluate the pathogenicity potential in environmental strains. While the core genes-based tree separated all O145:H28 strains from the non O145:H28 reference strains, it failed to segregate environmental strains from the clinical. In contrast, the accessory genes-based tree placed all clinical strains in the same clade regardless of their genotypes or serotypes, apart from the environmental strains. Loss-of-function mutations were common in the virulence genes examined, with a high frequency in genes related to adherence, autotransporters, and the type three secretion system. Distinct differences in pathogenicity islands LEE, OI-122, and OI-57, the acid fitness island, and the tellurite resistance island were detected between the O145:H28 and reference strains. A great amount of genetic variation was detected in O145:H28, which was mainly attributed to deletions, insertions, and gene acquisition at several chromosomal “hot spots”. Finally, our study demonstrated a distinct virulence gene repertoire among the STEC O145:H28 strains originating from the same geographical region and revealed unforeseen contributions of loss-of-function mutations to virulence evolution and genetic diversification in STEC.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Comparative evaluation of antimicrobial activity of human granulysin, bovine and porcine NK-lysins against Shiga toxin-producing Escherichia coli O157:H7

Shiga toxin-producing Escherichia coli (STEC) O157:H7 (O157) is a foodborne pathogen causing human disease ranging from hemorrhagic colitis and hemolytic uremic syndrome to kidney failure, while remaining harmless to cattle, its primary reservoir. The severity of the human disease associated mainly with Shiga toxin production and a global emergence of antibiotic resistant STEC highlights the need for effective non-antibiotic, pre-harvest strategies to reduce O157 in cattle, the principal source of human infection. Towards this goal three synthetic antimicrobial peptides (AMPs): human granulysin (hGRNL), bovine NK-lysin (bNK2A), and porcine NK-lysin (pNKL), were tested in vitro against O157 isolates. As expected, circular dichroism spectroscopy findings were consistent with a predominantly α-helical conformation for all three AMPs in an environment mimicking bacterial outer surface or liposaccharides. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations of hGRNL (200 μM), bNK2A (12.5 μM against strain 86–24 and 25 μM against EDL933), and pNKL (6.25 μM) were determined using the Clinical and Laboratory Standards Institute broth microdilution method in Müeller-Hinton broth (cation-adjusted). The bNK2A and pNKL AMPs did not induce Shiga toxin expression in O157 at MIC, as there was a significant decrease or no change in toxin expression following 4- or 20 h incubation with the AMPs; bNK2A p <0.0001 (4 h) and p = 0.4831 (20 h); pNKL p <0.0001 (4 h) and p = 0.0001 (20 h). Propidium iodide uptake assay revealed faster O157 membrane damage or killing kinetics with bNK2A and pNKL compared to hGRNL. Nonetheless, transmission electron microscopy demonstrated that all three AMPs mediated damage to O157 membranes. In contrast, the three AMPs showed minimal cytotoxicity (<2%) against cattle red blood cells at tested concentrations (0.39–50 μM). Overall, our results demonstrate the potential for bNK2A and pNKL to be further developed into novel non-antibiotic agents to reduce O157 shedding in cattle.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Prevalence of Listeria monocytogenes , Salmonella spp., Shiga toxin-producing Escherichia coli , and Campylobacter spp. in raw milk in the United States between 2000 and 2019: A systematic review and meta-analysis

Raw (unpasteurized) milk is available for sale and direct human consumption within some states in the United States (US); it cannot be sold or distributed in interstate commerce. Raw milk may contain pathogenic microorganisms that, when consumed, may cause illness and sometimes may result in death. No comprehensive review for prevalence and levels of the major bacterial pathogens in raw milk in the US exists. The objective of the present research was to systematically review the scientific literature published from 2000 to 2019 to estimate the prevalence and levels of Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), and Campylobacter spp. in raw milk in the US. Peer-reviewed studies were retrieved systematically from PubMed®, Embase®, and Web of ScienceTM. The unique complete nonduplicate references were uploaded into the Health Assessment Work Collaborative (HAWC). Based on the selection criteria, twenty studies were included in the systematic review and meta-analysis. Comprehensive Meta-Analysis (CMA) was used for statistical analyses, specifically, random effects meta-analyses were used to synthesize raw bulk tank milk (BTM) and milk filters (MF) data. Data from studies using culture and non–culture-based detection methods were included. Forest plots generated in CMA (Biostat, Englewood, NJ) were used to visualize the results. The average prevalence (event rate) of L. monocytogenes, Salmonella spp., STEC, and Campylobacter spp. in raw BTM in the US was estimated at 4.3% (95% confidence intervals [CIs], 2.8–6.5%), 3.6% (95% CIs, 2.0–6.2%), 4.3% (95% CIs, 2.4–7.4%), and 6.0% (95% CIs, 3.2–10.9%), respectively. Estimated prevalence was generally larger in MF than in BTM. There was not enough data to perform a meta-analysis for the prevalence or levels of pathogens in raw milk from retail establishments or other milk categories.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Antimicrobial resistance of enteric pathogens in the Military Health System, 2009 – 2019

Abstract Background Acute diarrhea (AD) can have significant impacts on military troop readiness. Medical providers must understand current trends of enteropathogen antimicrobial resistance (AMR) in service members (SMs) to inform proper, timely treatment options. However, little is known of enteric pathogen profiles across the Military Health System (MHS). The primary objectives of this study were to identify gaps in enteric pathogen surveillance within the MHS, describe the epidemiology of AMR in enteric pathogens, and identify trends across the MHS both within the Continental United States (CONUS) and outside of the Continental United States (OCONUS). Methods Health Level 7 (HL7)-formatted laboratory data were queried for all specimens where Salmonella , Shigella , and Campylobacter species, as well as Shiga toxin-producing Escherichia coli ( E. coli ) (STEC) were isolated and certified between 1 January 2009 - 31 December 2019. Antibiotic susceptibility testing (AST) results were queried and summarized where available. Descriptive statistics were calculated for each organism by specimen source, year, and susceptibility testing availability. Results Among a total of 13,852 enteric bacterial isolates, 11,877 (86%) were submitted from CONUS locations. Out of 1479 Shigella spp. and 6755 Salmonella spp. isolates, 1221 (83%) and 5019 (74%), respectively, reported any susceptibility results through the MHS. Overall, only 15% of STEC and 4% of Campylobacter spp. specimens had AST results available. Comparing AST reporting at CONUS versus OCONUS locations, AST was reported for 1175 (83%) and 46 (78%) of Shigella isolates at CONUS and OCONUS locations, respectively, and for 4591 (76%) and 428 (63%) of Salmonella isolates at CONUS and OCONUS locations, respectively. Conclusions This study revealed inconsistent enteropathogen AST conducted across the MHS, with differing trends between CONUS and OCONUS locations. Additional work is needed to assess pathogen-specific gaps in testing and reporting to develop optimal surveillance that supports the health of the force.

Ashbaugh, Hayley↗
DOE OSTI2023

Bovine Rectoanal Junction In Vitro Organ Culture Model System to Study Shiga Toxin-Producing Escherichia coli Adherence

Studies evaluating the interactions between Shiga toxin-producing Escherichia coli O157:H7 (O157) and the bovine recto–anal junction (RAJ) have been limited to either in vitro analyses of bacteria, cells, or nucleic acids at the RAJ, providing limited information. Alternatively, expensive in vivo studies in animals have been conducted. Therefore, our objective was to develop a comprehensive in vitro organ culture system of the RAJ (RAJ-IVOC) that accurately represents all cell types present in the RAJ. This system would enable studies that yield results similar to those observed in vivo. Pieces of RAJ tissue, obtained from unrelated cattle necropsies, were assembled and subjected to various tests in order to determine the optimal conditions for assaying bacterial adherence in a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Tissue integrity was assessed using cell viability, structural cell markers, and histopathology, while the adherence of bacteria was evaluated via microscopy and culture methods. DNA fingerprinting verified the recovered bacteria against the inoculum. When the RAJ-IVOC was assembled in Dulbecco’s Modified Eagle Medium, maintained at a temperature of 39 °C with 5% CO 2 and gentle shaking for a duration of 3–4 h, it successfully preserved tissue integrity and reproduced the expected adherence phenotype of the bacteria being tested. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

59 BASIC BIOLOGICAL SCIENCES↗