Characterization of a Unique Bordetella bronchiseptica vB_BbrP_BB8 Bacteriophage and Its Application as an Antibacterial Agent

Abstract

Bordetella bronchiseptica, an emerging zoonotic pathogen, infects a broad range of mammalian hosts. B. bronchiseptica-associated atrophic rhinitis incurs substantial losses to the pig breeding industry. The true burden of human disease caused by B. bronchiseptica is unknown, but it has been postulated that some hypervirulent B. bronchiseptica isolates may be responsible for undiagnosed respiratory infections in humans. B. bronchiseptica was shown to acquire antibiotic resistance genes from other bacterial genera, especially Escherichia coli. Here, we present a new B. bronchiseptica lytic bacteriophage-vB_BbrP_BB8-of the Podoviridae family, which offers a safe alternative to antibiotic treatment of B. bronchiseptica infections. We explored the phage at the level of genome, physiology, morphology, and infection kinetics. Its therapeutic potential was investigated in biofilms and in an in vivo Galleria mellonella model, both of which mimic the natural environment of infection. The BB8 is a unique phage with a genome structure resembling that of T7-like phages. Its latent period is 75 ± 5 min and its burst size is 88 ± 10 phages. The BB8 infection causes complete lysis of B. bronchiseptica cultures irrespective of the MOI used. The phage efficiently removes bacterial biofilm and prevents the lethality induced by B. bronchiseptica in G. mellonella honeycomb moth larvae.

Keywords: Galleria mellonella; animal model; antibiotic resistance; atrophic rhinitis; biofilm; emerging diseases; phage stability; phage therapy; veterinary microbiology; zoonosis.

Figure 1

Characteristics of BB8 phage virions and plaques. (A and B) TEM image of BB8 phage virions is typical of the Podoviridae family, Caudovirales order. The scale bars in A and B represent 50 nm. (C–E) The growth of phage plaques over time. Plaque phenotype observed after 8, 12, and 24 h is presented in panels C–E, respectively. The scale bars in C–E panels represent 3 mm.

Figure 2

Phage BB8 stability under various conditions. (A) Effect of pH on the phage activity. The number of active phage particles at pH 3 was below the detection limit (<400 PFU/mL). (B) Effect of temperature on the phage activity. The number of active phage particles at 60 °C and 80 °C was below the detection limit (<400 PFU/mL). (C) Effect of UV radiation on the phage activity. The presented results are average values from three experiments, with SD represented by error bars.

Figure 3

Phage BB8 development. (A) Adsorption of phage particles to the bacterial cells was assessed after 1, 2.5, 5, 10, and 15 min. (B) One-step growth curve shows that the eclipse period was a little under 60 min while latent period was around 75 min and resulted in the production of approx. 88 phage particles. (C) Collapse assay of the phage-infected bacterial culture indicated the high efficiency of host cell lysis. Regardless of the MOI used, the bacterial culture was completely lysed by the phage. The presented results are average values from three experiments, with SD represented by error bars.

Figure 4

Analysis of restriction patterns of BB8 genome. (A) Electrophoretic gel image of BB8 genomic DNA digested with various restriction enzymes shows the same pattern as (B) in silico prepared electrophoretic gel image of the restriction fragments. Lines: (1) DNA size marker, (2) NcoI + SmaI, (3) NcoI + NdeI, (4) NcoI, and (5) NdeI.

Figure 5

Genome organization and comparative genomics of BB8 phage. (A) Genome organization of BB8. A block at the top shows host-virus interaction (HVI), RNA polymerase (RNAP), DNA replication (REP), virus structure and assembly (VSA) and host cell lysis (HCL) modules, as well as transcription of genes of class i, ii, and iii. The modules were identified and described with the use, and according to, the genome organization of the model phage T7. The arrows below represent open reading frames identified in the BB8 genome and their transcriptional orientation. The coloring scheme of the arrows is as follows: grey—hypothetical proteins with unknown function, orange—proteins with predicted function, pink—RNA polymerase, blue—DNA polymerase, green—virus structure and assembly proteins. (B) The comparison of BB8 sequences with those of T7 phage of Enterobacteria (NC_001604), two most similar Ralstonia phages, i.e., RsoP1EGY (MG711516) and DU-RP-I (MF979559), and all known podoviruses infecting Bordetella, i.e., BMP-1 (AY526908), BIP-1 (AY526909), and BPP-1 (AY029185). The comparison was performed with the Circoletto tool on nucleotide (top graph) and amino acid (bottom graph) sequence levels. The blocks on the outmost ring correspond to the location of RNAP, DNA polymerase, and VSA modules. The colors of these correspond to the colors in panel A. (C) Sequence similarity network of proteins encoded by BB8 and the most closely related bacterial viruses. Each node represents a single phage genome and each edge, and its thickness, reflects the number of similar proteins encoded by two phages above the set thresholds. The coloring is based on the host’s family taxonomy. Enterobacteria T7 phage’s node is additionally indicated with a yellow star. All viruses within the presented network belong to Podoviridae family except the Bordetella phage vB_BbrM_PHB04 (MF663786) annotated as a member of Siphoviridae family—indicated by the red arrow.

Figure 6

Effect of phage BB8 treatment on B. bronchisepica biofilm. Decrease of (A) viability and (B) mass of B. bronchiseptica biofilm after 4-h treatment with BB8 phage in three different concentrations. A B. bronchiseptica biofilm without the addition of BB8 served as a control. Each dot in graphs (A) and (B) represents a biological replicate and the results were analyzed with one-way ANOVA (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05). The SEM images of B. bronchiseptica biofilm untreated and treated with phage BB8 are presented in panels C and D, respectively. Figure insets present untreated (C) and phage-treated (D) biofilms at higher magnification. The scale bars in C and D represent 200 nm.

Figure 7

Lethality of honeycomb moth larvae. (A) Influence of B. bronchiseptica cell number on larvae survivability. Twenty-five larvae per group and three groups (2.5 × 10³, 2.5 × 10⁵, and 2.5 × 10⁷ CFU/larva) were analyzed. (B) Treatment of B. bronchiseptica-infected G. mellonella larvae with BB8 phage. Control group (red line) includes Galleria larvae after treatment with B. bronchiseptica alone (approx. 2.5 × 10⁷ CFU). Treatment with phage BB8 almost completely abolished the lethality of the infection at MOIs of 1 and 10 (blue and green line, respectively). Twenty-five larvae per group and three groups (control, MOI = 1, and MOI = 10) were analyzed. Survivability in larvae treated with buffer alone or with phage alone was 100% (these lines were omitted for clarity).