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.

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% CO2 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.

AB5 Enterotoxin-Mediated Pathogenesis: Perspectives Gleaned from Shiga Toxins.

Foodborne diseases affect an estimated 600 million people worldwide annually, with the majority of these illnesses caused by Norovirus, Vibrio, Listeria, Campylobacter, Salmonella, and Escherichia coli. To elicit infections in humans, bacterial pathogens express a combination of virulence factors and toxins. AB5 toxins are an example of such toxins that can cause various clinical manifestations, including dehydration, diarrhea, kidney damage, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). Treatment of most bacterial foodborne illnesses consists of fluid replacement and antibiotics. However, antibiotics are not recommended for infections caused by Shiga toxin-producing E. coli (STEC) because of the increased risk of HUS development, although there are conflicting views and results in this regard. Lack of effective treatment strategies for STEC infections pose a public health threat during outbreaks; therefore, the debate on antibiotic use for STEC infections could be further explored, along with investigations into antibiotic alternatives. The overall goal of this review is to provide a succinct summary on the mechanisms of action and the pathogenesis of AB5 and related toxins, as expressed by bacterial foodborne pathogens, with a primary focus on Shiga toxins (Stx). The role of Stx in human STEC disease, detection methodologies, and available treatment options are also briefly discussed.

Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen.

Previously, we had demonstrated that Escherichia coli O157:H7 (O157) strain 86-24 expresses proteins involved in survival rather than virulence in vitro in rumen fluid from dairy cattle limit fed a maintenance diet. Here, we verified if this observation would be true for different O157 strains grown in vitro in rumen fluid from, and in vivo in the rumen of, animals on contrasting maintenance (high fiber) and lactation (high energy-protein) diets usually limit fed to dairy cattle. For the in vivo studies, an economical, novel, reusable and non-terminal rumen-fistulated animal model permitting simultaneous evaluation of multiple bacterial strains in the bovine rumen was developed. All experiments were conducted in duplicate using different animals to account for host-related variations. The O157 strains included, 86-24, EDL933 and the super shed SS-17. E. coli NalR (#5735), derived from a bovine intestinal commensal E. coli, was included as a control. As expected, diet influenced ruminal pH and volatile fatty acid (VFA) composition. The pH ranged from 6.2-7.0 and total VFA concentrations from 109-141 µM/ml, in animals fed the maintenance diet. In comparison, animals fed the lactation diet had a ruminal pH ranging between 5.18-6.0, and total VFA of 125-219 µM/ml. Strain dependent differences in O157 recovery from the rumen fluid of cattle fed either diet was observed, both in vitro and in vivo, with O157 strains 86-24 and EDL933 demonstrating similar survival patterns. Analysis of the O157 proteomes expressed in the rumen fluid/rumen verified previous observations of adaptive responses. Any difference in the adaptive response was mainly influenced by the animal's diet and growth conditions (in vitro and in vivo) and not the O157 strain. These new insights into the O157 responses could help formulate modalities to control O157 across strains in cattle at all stages of husbandry.

Lactoferrin as an antimicrobial against Salmonella enterica and Escherichia coli O157:H7 in raw milk.

Improper storage conditions or processing of milk leads to potential spoilage and illness, due in part to temperature abuse, allowing bacteria present to grow and spoil the product. However, certain proteins naturally found in raw milk, such as lactoferrin, have reported antibacterial properties. The levels of lactoferrin required to effectively inhibit growth of pathogens have not been investigated thoroughly. This study aimed to examine various concentrations of lactoferrin as a potential biopreservative and as an antimicrobial against the common dairy pathogens Salmonella enterica and Escherichia coli O157:H7. Minimum inhibitory concentration assays were conducted on raw bovine milk in which the bacteria were exposed to varying concentrations of lactoferrin. In the raw milk system, the growth of E. coli O157:H7 was significantly decreased at levels greater than 14.05 mg/mL lactoferrin based on the reduction of tetrazolium salts. For S. enterica, only lactoferrin concentrations at or above 112.5 mg/mL in the milk resulted in reduced growth. Taken together, these results indicate that lactoferrin may have biopreservative potential. To fully examine the practicality and effectiveness of lactoferrin as an antimicrobial additive, a similar study should be conducted using additional (gram-positive) pathogens, such as Bacillus cereus and Listeria monocytogenes. If effective, lactoferrin could prolong the shelf life of dairy products and help reduce the incidence of foodborne illnesses in developing countries with limited refrigeration capability.