Genomic characterization and pre-clinical evaluation of a new polyvalent lytic Loughborough phage

Abstract

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a critical concern that necessitates the development of novel antimicrobial approaches. One of the most promising innovative approaches for combating CRAB infections is using effective and lytic bacteriophages (phages), known as phage therapy. Therefore, we recovered and characterized a polyvalent lytic Salmonella_phage_VB_ST-SA173, producing lytic activity against 6 CRAB clinical isolates and 3 multidrug-resistant (MDR) Salmonella serovars. Throughout pH 2-10, and thermal stability at up to 60 °C, the phage maintained its stability and lytic activity against the tested isolates. The presence of a tailed phage with a characteristic prolate head and a contractile tail was detected by the transmission electron microscope (TEM). According to the Oxford nanopore sequencing data, the genome of Salmonella_phage_VB_ST-SA173 was 53,636 bp in size, contained 45.9% G + C, and had 53 opening reading frames (ORFs). According to the TEM, ORFs, and BLASTn analysis findings, it was proved that the Salmonella_phage_VB_ST-SA173 belongs to the Loughboroughvirus genus. The efficacy of the phage-formulated Carbopol 940 hydrogel in wound healing was assessed preclinically in an infected burn wound animal model with a CRABa clinical isolate. The survival rate was enhanced in the phage-treated group compared to the untreated control groups. Histopathological analysis showed improved wound healing in the form of apparently healthy skin with apparently normal epidermal and dermis layers. In conclusion, depending on its in vitro and physicochemical traits, the phage-loaded hydrogel is expected to be a promising candidate for clinical trials against human CRAB-related skin infections. KEY POINTS: • A polyvalent Loughboroughvirus phage showed lytic activity against CRAB and Salmonella serovars. • The phage showed stability at a wide range of pH and temperature. • The phage hydrogel enhanced healing in the burn-wound animal model infected with CRABa.

Keywords: Acinetobacter baumannii; CRAB; Hydrogel; MDR Salmonella; Molecular analysisSalmonella_; Polyvalent phage; Thermal animal model.

Fig. 1

Phage lysate spot test that exhibited the most distinct lytic zones on bacterial lawns

Fig. 2

Results of the plaque assay. a A sheet of interrupted bacterial growth (net-shaped) was observed at dilutions (10⁻³, 10⁻⁴); b too numerous to be counted (TNTC) plaques were observed at dilutions (10⁻⁵, 10⁻⁶, 10⁻⁷); c well-defined, circular plaques were observed at dilution (10.⁻⁸)

Fig. 3

TEM showing a tailed phage with a characteristic prolate head and a contractile tail (myoviral morphotype)

Fig. 4

Circular genome map of Salmonella_phage_VB_ST-SA173. (NCBI GenBank Accession code PQ351918; size 53,636 bp, purple ring) and the reference phage (Salmonella_phage ZCSE2, complete genome; NCBI accession code NC_048179.1; size 53,965 bp, orange ring). The color coding of genes indicates the functional categories of putative proteins: structural proteins (blue), terminase protein (red), non-structural proteins (green), portal proteins (black), and hypothetical proteins (fuchsia). The creation of the circular image was performed using the BLAST Ring Image Generator (BRIG) tool v0.95 (https://sourceforge.net/projects/brig/, accessed on 19 September 2024)

Fig. 5

The survival rate percentage of the control animal groups and the treated animal group with Salmonella phage VB_ST-SA173–loaded hydrogel over 14 days

Fig. 6

Photographs of wounded skin on the 14th day after injury in a representative rat from each group. Groups I, II, III, and VI demonstrated poor healing responses. Group IV showed a healing response to phage hydrogel treatment superior to the control groups. Group V had a better healing response than groups I, II, III, and VI. Group VII exhibited normal skin

Fig. 7

Microscopical examination of wound healing process in various groups and skin layers histopathological characteristics (H&E X 400). I Group I displayed coagulative necrosis, scattered collagen fibers (star), epidermal layer shedding, and inflammatory cell infiltration in the dermis, primarily lymphocytes and neutrophils (red arrow). II Group II revealed mild response to healing, polymorphonuclear cells infiltrating the top layer of the dermis (star), and necrotic tissue with large numbers of inflammatory and fibroblast cells (arrow) invading the dermis layer. III Group III showed poor healing response, necrotic tissue (red star), diffuse infiltration of inflammatory cells, primarily lymphocytes and neutrophils, vacuolation (black star), and the development of connective tissue in the dermal layer. IV Group IV showed apparent healthy skin with apparent normal epidermal layer and dermis. V Group V displayed a modest response to healing, epidermal layer separation (star) and inflammatory cells infiltrating the dermis, primarily neutrophils (black arrow) and lymphocytes (red arrow). VI Group VI displayed connective tissue formation in the dermal layer, dermal edema, diffuse necrotic tissue with significant infiltration of inflammatory cells, primarily neutrophils (red arrow) and lymphocytes (black arrow), and a moderate response to healing. VII Group VII exhibited normal skin structure of epidermal and dermal layers