Neutrophils are important effector cells of the innate immune system, traditionally regarded to have a short life span. The goal of this study was to evaluate the effect of the whole blood storage on neutrophil functions, e.g., viability, antimicrobial effect, neutrophil extracellular trap (NET) formation and phagocytosis. Therefore, fresh porcine whole blood was compared to whole blood stored for 24 h in the dark at room temperature. Different cell parameters in whole blood and in isolated neutrophils were analyzed. The following parameters were analyzed: cell count, band and segmented neutrophil count, viability, cholesterol content, release of free DNA as a marker for cell death, phagocytic activity in whole blood and in isolated neutrophils, the transmigration rate of neutrophils to IL8 stimulus, the production of reactive oxygen species (ROS), and the formation of NETs. It was observed that the number of isolated neutrophils decreased over time, indicating cell death occurs during 24 h of blood storage. However, the surviving neutrophils isolated from stored blood reacted comparably or even showed enhanced antimicrobial activity in the case of phagocytosis of Streptococcus (S.) suis, ROS production, and transmigration. The slightly altered cholesterol level of the harvested neutrophils in stored blood when compared to fresh blood partially explains some of the detected differences.
Staphylococcus aureus is an important nosocomial and community-acquired facultative intracellular pathogen. Many studies have reported that S. aureus infections are associated with intestinal symptoms, but little is known about the molecular mechanisms implicated in S. aureus-induced alterations of intestinal functions. In this study, we investigated the implication of lipid rafts in the interaction of S. aureus with Caco-2 cells. To assess potential alterations in the lipid raft structure and effects on the hydrolytic function, we utilized sucrase–isomaltase (SI) as the major intestinal α-glucosidase that is associated with and sorted to the apical membrane via lipid rafts. Seven days post-confluent, Caco-2 cells were infected with S. aureus Newman and further incubated for an additional 2 days. After 48 h, the levels of SI expression as well as the enzymatic function of this protein were assessed in the infected versus non-infected cells. Analysis of the sorting behavior of SI to the apical membrane constituted another crucial aspect in studying the effects of S. aureus on Caco-2 cells. For this purpose, the apical membranes or brush border membranes (BBMs; referred to as P2 fraction) were separated in both infected and non-infected cells from the basolateral and intracellular membranes (referred to as P1 fraction) by employing a cationic-based procedure using CaCl2. The data show that there is no significant change in the overall expression levels of SI in the infected versus non-infected cells as assessed by Western blotting analysis using monoclonal anti-SI antibodies. By contrast, a significant decrease in the localization as well as the specific hydrolytic activities of SI toward sucrose and isomaltose (Palatinose) was observed in the BBM (P2 fraction) in Caco-2 cells 48 h post-infection. Concomitantly, the specific SI activities increased in the basolateral membrane/intracellular fraction (P1). Noteworthy, the specific activity of SI in the BBM of infected cells was markedly reduced as compared with that of the non-infected counterparts. The data accumulated from this study strongly suggest that infections with S. aureus influence the final step in the lipid raft-associated trafficking of human SI and thereby may trigger secondary functional gastrointestinal disorders.
Antimicrobial therapy is the most applied method for treating and preventing bacterial infection in livestock. However, it becomes less effective due to the development of antimicrobial resistance (AMR). Therefore, there is an urgent need to find new antimicrobials to reduce the rising rate of AMR. Recently, antimicrobial peptides (AMPs) have been receiving increasing attention due to their broad-spectrum antimicrobial activity, rapid killing activities, less toxicity, and cell selectivity. These features make them potent and potential alternative antimicrobials to be used in animals. Here, we discuss and summarize the AMPs in animals, classification, structures, mechanisms of action, and their potential use as novel therapeutic alternative antimicrobials to tackle the growing AMR threat.
Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 μg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 μg/mL. This level is comparatively lower than 9.2 μg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.
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