Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2–8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

The World Health Organization notes that some bacteria have been demonstrated to possess significant public health risks; they have antibiotic resistance, and there are fewer alternatives for control. The n-hexane extract and cinaguaiacin obtained from Artemisia cina show promising antibacterial activity, including against multidrug-resistant bacteria that affect animal and human health. Objective: The aim of this study was to determine the antibacterial activity of the n-hexane extract of A. cina and cinaguaiacin against multidrug-resistant bacteria. Methods:A. cina was collected in the pre-flowering period, the n-hexane extract was obtained, and chromatographic techniques and structure were used to separate the lignans, which were elucidated with nuclear magnetic resonance techniques. Four ATCC strains were used, and four strains were isolated from clinical cases with different resistance profiles. The antibacterial activity was determined by calculating the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), the time-kill kinetics assay, and the cell membrane integrity and DNA release assay. Molecular docking studies of lignans demonstrated the binding mode involved in the active site of DNA gyrase B. Results: The n-hexane extract inhibited growth against 87.5% of the strains tested (MIC 5.31 to 42.5 mg/mL) and showed bactericidal activity against 25% of the strains tested (MBC 0.62 to 85 mg/mL). Cinaguaiacin inhibited growth against 100% of the strains tested (MIC, 0.56 to 2.25 mg/mL) and exhibited bactericidal activity against 25% of the strains tested (MBC, 0.62 to 85 mg/mL). Conclusions: The mechanism of cinaguaiacin’s action may be associated with damage to the plasma membrane, as the protein and DNA levels were higher than those of the positive control. The n-hexane extract and cinaguaiacin obtained from A. cina showed a bacteriostatic or bactericidal effect, depending on the strain evaluated.

Lactic acid bacteria are components of the gastrointestinal tract microbiota in both humans and animals and are widely used as probiotics. Lactobacillus is the most closely related genus to probiotic activity. It is capable of releasing membrane microvesicles (MVs), whose primary functions include carrying and transmitting antigens to host tissues and modulating host defense responses. In the present study, MVs were isolated from Lactobacillus acidophilus resident in the ileum of free-living rats, and their immunostimulant effect was evaluated in two biological models. MVs were characterized using SDS-PAGE electrophoresis, electron microscopy, and nanoparticle tracking analysis. In the first model, the immunostimulatory effect of MVs was evaluated on ovine abomasal explants, which had been previously stimulated with MVs and then challenged with third-stage larvae of Haemonchus contortus. This resulted in a decrease in the percentage of larval association and favored the migration of inflammatory cells to the infection site. In the second model, the macrophage cell line RAW 264.7 was stimulated with MVs to evaluate the expression of transcripts encoding IL-1β and TNF-α. MVs isolated from L. acidophilus demonstrate immunostimulatory and probiotic effects in the two biological models assessed. This suggested that the MVs possess similar immunostimulatory effects as those reported for the parent bacteria.

The indiscriminate use of antibiotics has led to an increase in multidrug-resistant bacteria, necessitating the search for effective alternative therapies to reduce antimicrobial use. Lactic Acid Bacteria (LAB) have been explored as an alternative to antibiotics due to their multiple beneficial properties. These bacteria secrete membrane vesicles (MVs), key acellular components in combating pathogens. This study aimed to evaluate the effects of Lactiplantibacillus plantarum MVs co-cultured with Escherichia coli (MVsplE) or Salmonella Typhimurium (MVsplS) through inhibition assays using disk diffusion on agar plates, as well as their activation and cytokine expression in the RAW 264.7 macrophage cell line. The results showed that MVsplE and MVsplS were produced in greater quantity and size than non-co-cultured L. plantarum MVs (MVspl). Additionally, MVsplE and MVsplS exhibited a dose-dependent inhibitory effect on the growth of enteropathogenic bacteria. Furthermore, RAW 264.7 cells stimulated with these MVs demonstrated that the expression of IL-1β, TNF-α and IL-10 depended on the enteropathogenic strain with which L. plantarum was previously co-cultured. Following a challenge with enteropathogenic bacteria, the MVs induced an immunomodulatory response. These findings demonstrate that L. plantarum MVs exert bactericidal and immunomodulatory effects against enteropathogenic bacteria, suggesting their potential use as an alternative treatment to antimicrobials.

The interaction between probiotic bacteria and the innate immune system is of increasing interest due to its capacity to modulate inflammatory and antimicrobial responses. The murine macrophage cell line RAW 264.7 is widely used to investigate the immunomodulatory effects of probiotic bacteria and their cell-free derivatives, such as membrane vesicles (MVs). In this study, we evaluated whether MVs derived from Lactobacillus acidophilus promote superior modulation of cytokine production and antimicrobial signaling in RAW 264.7 macrophages compared with whole cells (WCs). Our results show that L. acidophilus MVs exhibited direct bactericidal activity against Escherichia coli and induced a more selective and balanced cytokine profile than whole cells. These findings highlight the potential of probiotic-derived membrane vesicles as acellular immunomodulatory effectors for the development of novel cell-free biotherapeutic strategies.