<p>Biomimetic hydroxyapatite nanocrystals are an active carrier for <em>Salmonella</em> bacteriophages</p>

Abstract

The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute-at the same time-to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on. Materials and methods: Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen. Results: Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions. Conclusion: Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.

Plant Dynamic Metabolic Response to Bacteriophage Treatment After Xanthomonas campestris pv. campestris Infection

“…The Xcc strain was grown in Nutrient Broth (NB) at 24 • C to the concentration of 10 8 CFU/mL, as determined by measuring the optical density at 600 nm. Cells at the exponential growth phase, were infected with phage (10 3 to 10 −3 PFU/CFU) in a 96 well plates (Corning R 96 Well CellBIND R Microplates, Sigma Aldrich), then incubated at 24 • C for 48 h. CFUs were counted by standard soft agar overlay assay (Klement et al, 1990;Fulgione et al, 2019). Experiments were performed in triplicate.…”

Bacteriophages Promote Metabolic Changes in Bacteria Biofilm

“…The action of HA on the biofilm was also tested. From crystal violet measurements, we found that HA has no effect on Xcc biofilm (Papaianni et al, manuscript in preparation) and exerts its enhancing action [20] (building the phage and improving its lytic activity) only in the presence of the phage. The C20:0 was approximately equally active at 60 µg/mL, 120 µg/mL and 240 µg/mL-all reducing the amount of biofilm by ca.…”

“…Flasks were incubated overnight at 24 • C. Cultures were treated with chloroform, clarified by centrifugation, and filtered through Millipore 0.22 µm-pore-size membrane filters (MF-Millipore, Darmstadt, Germany). Filtrates were tested for the presence of Xcc-specific phages as described [20].…”

“…In bacterial infections, hydroxyapatite (HA) nanocrystals help in bacteriophage delivery and are reported to improve some of the bacteriophage biological properties [20]. In addition, although not bactericidal, C20:0 could be able to modify the microbial biofilm structure by altering the permeability of the cell [21,22,31,32].…”

“…Previous studies have demonstrated that hydroxyapatite (HA) enhances the activity of phages. The low degree of crystallinity and the presence of carbonate ions in the crystal structure make HA extremely reactive in biological systems and particularly suitable to interact and transport bacteriophages [20]. The eicosanoic acid (C20:0) weakens the bacterial biofilm structure [21][22][23], and phage Xccϕ1 can control Xcc infection in plants (submitted by Papaianni).…”